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The interactions of surface carbohydrates of Meloidogyne spp. with soybean roots

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Title:
The interactions of surface carbohydrates of Meloidogyne spp. with soybean roots
Added title page title:
Surface carbohydrates of Meloidogyne spp. with soybean roots
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Davis, Eric L., 1958-
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English
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vii, 118 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Agglutinins ( jstor )
Juveniles ( jstor )
Lectins ( jstor )
Nematology ( jstor )
pH ( jstor )
Plant interaction ( jstor )
Plant roots ( jstor )
Roundworms ( jstor )
Soybeans ( jstor )
Sugars ( jstor )
Cell membranes ( lcsh )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Meloidogyne incognita ( lcsh )
Meloidogyne javanica ( lcsh )
Root-knot ( lcsh )
Soybean -- Diseases and pests -- Control ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 104-117).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Eric L. Davis.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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20442730 ( OCLC )
AFP7300 ( NOTIS )
AA00004799_00001 ( sobekcm )

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THE INTERACTIONS OF SURFACE CARBOHYDRATES OF
MELOIDOGYNE SPP. WITH SOYBEAN ROOTS
By
ERIC L. DAVIS
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
1988


IN MEMORY OF
LAWRENCE DAVIS


ACKNOWLEDGEMENTS
I express my sincere appreciation to Dr. D. T.
Kaplan for serving as chairman of my supervisory committee.
His guidance, assistance, and friendship were invaluable to
this research endeavor.
Special thanks are extended to Dr. D. W. Dickson and
Dr. D. J. Mitchell for serving as members of my supervisory
committee. Their advice and support cannot be
overemphasized.
Appreciation is extended to the professional staff
and technical support, especially Diana Johnson and Janice
Rahill, of the United States Department of Agriculture,
Horticultural Research Laboratory, Orlando, Florida.
I would like to thank Dr. A. M. Golden, Beltsville,
Maryland, Dr. J. G. Baldwin, Riverside, California, and Dr.
J. D. Eisenback, Blacksburg, Virginia, for their assistance
in identification of Meloidogyne spp. Thanks are extended
to Drs. K. Hinson, Gainesville, Florida, and E. E. Hartwig,
Stoneville, Mississippi, for providing soybean seed.
Appreciation is extended to the Union Carbide
Agricultural Products Division, Research Triangle, North
Carolina, for providing financial support for this research
project.
iii
i


TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTER 1 : INTRODUCTION 1
CHAPTER 2: CHARACTERIZATION OF CARBOHYDRATES ON THE
SURFACE OF SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.. 10
Introduction 10
Materials and Methods 12
Results 18
Discussion 27
CHAPTER 3: ROOT TISSUE RESPONSE OF TWO RELATED
SOYBEAN CULTIVARS TO INFECTION BY LECTIN-TREATED
MELOIDOGYNE SPP 32
Introduction 32
Materials and Methods 34
Results 38
Discussion 47
CHAPTER 4: QUANTIFICATION OF LECTIN BINDING TO SECOND-
STAGE JUVENILES OF MELOIDOGYNE SPP 52
Introduction 52
Materials and Methods 54
Results 60
Discussion 63
IV


CHAPTER 5: REPRODUCTION OF LECTIN-TREATED MELOIDOGYNE
SPP. IN TWO RELATED SOYBEAN CULTIVARS 65
Introduction 65
Materials and Methods 67
Results 70
Discussion 78
CHAPTER 6: SUMMARY AND CONCLUSIONS 82
APPENDIX A: BINDING OF FLUORESCENT SOYBEAN AGGLUTININ
TO POSTINFECTIVE SECOND-STAGE JUVENILES OF
MELO IDOGYNE SPP 90
APPENDIX B: VIABILITY OF SECOND-STAGE JUVENILES OF
MELOIDOGYNE SPP. AFTER EXPOSURE TO GLYCOHYDROLASE
BUFFERS 94
APPENDIX C: BINDING OF FLUORESCENT LECTINS TO AXENIZED,
PREINFECTIVE MELOIDOGYNE SPP 97
APPENDIX D: EFFECT OF ACIDITY OF SIALIC ACID ON
PENETRATION OF SOYBEAN ROOTS BY SECOND-STAGE JUVENILES
OF MELO IDOGYNE SPP 100
BIBLIOGRAPHY 104
BIOGRAPHICAL SKETCH 118
v
i


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
THE INTERACTIONS OF SURFACE CARBOHYDRATES OF
MELOIDOGYNE SPP. WITH SOYBEAN ROOTS
By
Eric L. Davis
April 1988
Chairman: David T. Kaplan
Major Department: Entomology and Nematology
Fluorescent (rhodamine) conjugates of the lectins,
soybean agglutinin (SBA), Concanavalin A (CON A), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA) bound exclusively to
amphidial openings and cephalic secretions of preinfective
second-stage juveniles (32) of Meloidogyne incognita races 1
and 3 (Mil, Mi3), and M. javanica (Mj). No substantial
differences in fluorescent lectin-labeling were observed
among preinfective J2 of the Meloidogyne spp. populations
examined, and only binding of LOT and LPA was inhibited in
the presence of 0.1 M competitive sugar. Differences in
structure of amphidial carbohydrate complexes among
populations of Mil, Mi3, and Mj were revealed by
glycohydrolase treatment of preinfective J2 and subsequent
labeling with fluorescent lectins. Several glycohydrolases
vi
i


eliminated binding of LPA to the amphidial region of J2 of
the Meloidogyne spp. tested. Quantitative differences in
binding of the peroxidase-labeled lectins, SBA, CON A, LOT,
WGA, and LPA to J2 of Mil, Mi3, and Mj were determined by
microfiltration enzyme-linked lectin assay. Preinfective J2
of Mj bound the greatest amount of SBA, LOT, and WGA,
whereas preinfective J2 of Mil bound the most LPA in two
separate experiments. Preinfective J2 of Mi3 generally
bound the least amount of all lectins tested.
Treatment of J2 of Mil and Mj with purified,
unconjugated SBA, CON A, WGA, LOT, or Limax flavus
agglutinin (LFA) did not influence root tissue response of
'Centennial' and 'Pickett 71' soybean cultivars to infection
by Mil or Mj. Giant cells were usually associated with
untreated Mi3 in Centennial root tissue 20 days after
inoculation. Treatment of J2 of Mi3 with lectins or
carbohydrates caused Centennial root tissue to respond to
infection by treated Mi3 in a hypersensitive manner.
Nematodes could not be detected within soybean roots 5 days
after inoculation of root tips with J2 suspended in
solutions of LFA or sialic acid. Only treatment of J2 with
sialic acid and sialic acid plus LPA strongly reduced
reproduction of all populations of Meloidogyne spp. in
soybean roots of both cultivars. Treatment of J2 of
Meloidogyne spp. with LFA, LPA, or sialic acid was not
lethal to nematodes.
Vll


CHAPTER 1
INTRODUCTION
Most agricultural crops are vulnerable to attack by
phytoparasitic nematodes. Nematode damage has caused
estimated losses as high as 21% in major U.S. crops (111).
Dowler and Van Gundy (26) recently estimated annual world
crop losses to nematode damage to be in the order of $500
million. Root-knot nematodes (Meloidogyne spp.) are
considered to be the most economically important
phytoparasitic nematodes because of their world-wide
distribution, interaction with other phytoparasitic
organisms, and extensive host range (100). The host range
includes almost all of the plants that account for the
majority of the world's food supply (100, 101, 102, 121).
Four major species of root-knot nematode, Meloidogyne
incognita (Kofoid and White) Chitwood, Meloidogyne javanica
(Treub) Chitwood, Meloidogyne hapla Chitwood, and
Meloidogyne arenaria (Neal) Chitwood comprised over 95% of
the root-knot nematode populations detected in over 500
samples collected from agroecosystems around the world
(100).
1


2
The principal management strategies implemented to
reduce nematode-related crop damage include field
application of fumigant and nonfumigant nematicides,
cultivation of plant varieties which are resistant to
phytoparasitic nematodes, and prudent cultural practices
such as sanitation and crop rotation. Nematicides are
relied upon as the primary means of reducing nematode-
related crop losses in many crops. Their toxic properties,
water solubility, and persistence in the environment have,
however, triggered restriction of nematicide application,
making them unavailable for use or reducing their efficacy
in many economically important crops (54). Nematicides
often cannot be incorporated into agricultural pest
management strategies in developing nations because
chemicals may be inaccessible or too expensive, application
equipment may not be available, and growers may not be
properly educated in the safe and effective use of
pesticides (18, 101). To date, crop rotation is still the
most widely used pest management strategy implemented on a
world-wide basis (41, 73). Difficulties encountered in crop
rotation schemes include identification of potentially
damaging phytoparasitic nematodes and their host ranges,
selection of suitable nonhosts for polyspecific nematode
field populations or species with wide host ranges, and
!


3
production of crops that are economically beneficial for
growers and that are marketable (41).
Cultivation of crops that are resistant to diseases
induced by root-knot nematodes is appealing because it
provides an effective, economical, and environmentally safe
means of reducing nematode-related crop damage (32, 33).
Hundreds of crop cultivars are currently available that
possess resistance to one or more species of root-knot
nematode (32, 33, 103). Resistance is a term that
encompasses two main components: the ability of a plant to
tolerate nematode-related damage or to limit nematode
reproduction (58, 59). Thus, varying degrees of resistance
to phytoparasitic nematodes have been reported among
different plant cultivars (34). Most nematode-resistant
germplasm has been developed towards nematodes that are
endoparasites because, according to Roberts (94), "natural
selection of resistance genes is more likely to have
occurred in the most highly specialized host-parasite
relationships where co-evolutionary development of host and
parasite has produced a highly specific interaction in which
host and parasite compete for a genetic advantage (118)."
This dynamic interaction between host and parasite
on a microevolutionary scale threatens the durability of
resistance in plants to nematodes. Continued cultivation of
nematode-resistant crops on the same parcel of land has


4
selected for populations of phytoparasitic nematodes that
overcome plant resistance to nematode attack (34, 99, 108,
119, 123). Although the genetic basis of inheritance of
resistance to nematodes has been identified for a number of
plant cultivars (9, 34, 55, 108), little is known about how
resistance genes function (58). A greater knowledge of the
mechanisms of plant resistance to nematodes should
facilitate the application of germplasm modification and the
development of bioengineering for the transfer of nematode
resistance genes between plant genomes (34, 58, 71).
A number of studies have investigated the mechanisms
of plant resistance to nematodes and the results have been
discussed in several reviews (37, 39, 42, 58, 59, 97, 127).
The terms "incompatible" and "compatible" are used here to
designate plant-nematode interactions that inhibit nematode
development and reproduction, and interactions which promote
nematode development and reproduction in plants,
respectively (58, 59). Passively or preinfectionally
incompatible plant-nematode interactions may involve
morphological plant barriers to nematode infection or
constitutive plant factors that affect egg hatch, locating a
food source, survivability, and host suitability (32, 35,
39, 58, 59, 97, 127). Actively or postinfectionally
incompatible plant-nematode interaction involves the
elicitation and subsequent sequence of host defense


5
reactions induced by challenges of certain nematode species
or races to specific plant cultivars (32, 58, 59, 127).
The physiological sequence of events following
elicitation of active plant defenses, the "expressive phase"
(63), has been described for a number of incompatible plant-
nematode interactions (58). Inhibition of nematode
development in incompatible plant cultivars is often
associated with hypersensitive reactions (HR) in plant
tissue adjacent to nematodes and in feeding sites shortly
(hours) after nematode or stylet penetration of roots (12,
43, 57-59, 109, 112, 134). Phytoalexin accumulation has
been associated with the HR in some incompatible plant-
nematode interactions (43, 60, 92, 130), and the potential
involvement of phytoalexins in incompatible plant-microbe
interactions has been discussed (7, 42, 59, 68, 128).
The incompatible and compatible interactions of
soybean (Glycine max (L.) Merr.) cultivars with root-knot
nematodes have been examined in detail (28, 30, 56, 60, 61,
129). The introduction and incorporation of genes for
resistance to root-knot nematodes, primarily M. incognita,
into successive soybean cultivars and the development of
soybean resistance-breaking populations of Meloidogyne spp.
have been summarized (33, 105). Resistance to M. incognita
in soybean is conditioned by one major gene with at least
one modifying gene (16, 98), and the degree of resistance


6
varies with the soybean cultivar and the population of M.
incognita examined (8, 10, 15, 65, 96). The tissue reaction
of roots of two related soybean cultivars to infection by
the same population of M. incognita has been examined
progressively from one to twelve days after exposure of
soybean root tips to second-stage juveniles (J2) of M.
incognita (61). Giant cell formation in 'Pickett 71'
soybean roots infected with M. incognita (compatible)
progressed normally throughout the observation period. The
incompatibility of 'Centennial' soybean with M. incognita
was associated with an HR of soybean root tissue in the
region of invading J2 within 3 days of inoculation. The HR
was strongly correlated with the accumulation of the
phytoalexin, glyceollin, but biotic elicitors of the HR were
not identified (60).
Indeed, little is known about the "determinative
phase" (63) of plant-nematode incompatibility; factors are
involved in this phase that enable an incompatible plant
cultivar to recognize a specific potential pathogen and
invoke active plant defenses (58). Since nematodes locate
and penetrate roots of most cultivars in either compatible
or incompatible pathosystems, recognition of endoparasitic
nematodes by incompatible plants appears to occur following
penetration (1, 24, 44, 90, 116, 124). The primary
determinants in the specificity of plant-nematode
i


7
incompatibility probably include the interactions of
nematode and plant cell surfaces, nematode secretions, and
derepression of nematode and plant genomes (58).
The concept that recognition and specificity in
plant-microbe interactions may involve the interaction of
carbohydrate moieties of cell surface glycoconjugates and
corresponding receptors on the surface of cells of which
they come in contact, similar to cell to cell communication
involved in antigenicity, blood group specificity, and
mitogenesis in animal systems, was proposed by Albersheim
and Anderson-Prouty (3). Numerous investigations of this
hypothesis in different plant-microbe systems have been
conducted and reviewed (21, 23, 31, 62, 83, 106, 136). The
potential interaction of bacterial surface carbohydrates and
lectins present on the surface of plant root cells as
determinants of recognition and specificity in mutualistic
Rhizobium spp.-legume interactions has been examined (6, 87,
126). For example, the binding of soybean and clover
lectins appeared to be specific for most nodulating strains
of Rhizobium spp. (13, 25). Some evidence for the
involvement of cell surface interactions in incompatible
plant-microbe interactions includes the elicitation of
glyceollin accumulation in soybean tissue exposed to
polysaccharide (4, 5) and glycoprotein-rich (64) wall
fractions isolated from Phytophthora megasperma Drechs. f.


8
sp. glycinea, and induction of glyceollin accumulation in
soybean tissue exposed to fractions of cellular envelopes
isolated from incompatible races of Pseudomonas glycinea
Coerper (19). Whether interaction of cell surface
macromolecules is important in recognition and specificity
in plant-microbe interactions remains controversial (6, 23,
87, 126). Inconsistencies in some results are difficult to
interpret and may involve factors other than surface to
surface interactions.
Characterization of the surface carbohydrate
composition of a number of nematode species and the
potential involvement of surface carbohydrate interactions
in recognition and specificity between nematodes and other
organisms have been investigated and are discussed in
several reviews (47, 58, 80, 135, 136). Selected evidence
from some of these investigations and information gathered
since publication of the above reviews is mentioned in the
introductions to the chapters within this dissertation.
Zuckerman and Jansson (136) have postulated that specific
interactions between nematodes and other organisms may be
influenced by modification of nematode surface
carbohydrates. The objectives of this investigation were to
characterize the surface carbohydrates of second-stage
juveniles of several populations of Meloidogyne spp. and to
evaluate the potential involvement of nematode surface
i


9
carbohydrates in recognition and specificity in the
incompatible and compatible response of two related soybean
cultivars to infection by M. javanica and races 1 and 3 of
M. incognita.


CHAPTER 2
CHARACTERIZATION OF CARBOHYDRATES ON THE SURFACE OF SECOND-
STAGE JUVENILES OF MELOIDOGYNE SPP.
Introduction
The importance of surface carbohydrate biochemistry
in recognition and specificity between plants and
microorganisms has been the subject of many recent
investigations and discussions (3, 21/ 23, 31, 62, 106).
Although few investigations concerning this phenomena have
been conducted between nematodes and plants, surface
carbohydrates of nematodes have been implicated in
recognition between nematodes and nematophagous fungi (17,
47, 52, 80, 81, 135, 136). The surface carbohydrates of
some helminth parasites of animals have been characterized
and related to helminth antigenicity and chemoresponse (14,
72, 85). The involvement of surface carbohydrate
recognition in the specificity of interaction between
nematodes and Pasteuria cenetrans Sayre and Starr has also
been investigated (117).
Carbohydrates present on biological surfaces exist
primarily as glvcoconjugates such as glycolipids,
polysaccharides, and especially as glycoproteins (79). The
carbohydrate residues are often comprised of a number of
10


11
monosaccharide molecules covalently linked in various
sequences and spatial arrangements (66, 122). The
accessibility of surface carbohydrates to potential
receptors in other organisms or as receptors of chemostimuli
may be obscured by attached carbohydrate molecules, as is
sometimes the case with sialic acids in animal systems
(biological masks) (104). Enzymatic or inorganic chemical
degradation can reveal "masked" carbohydrates that may exist
on biological surfaces. Conversely, enzymes which cleave
specific carbohydrate residues from glycoconjugates,
glycohydrolases, can remove carbohydrates from biological
surfaces and potentially alter biological interactions. An
example of this latter phenomenon is the apparent loss of
chemosensory perception of culture filtrates of Escherichia
coli (Mig.) Castellani and Chambers by the nematodes
Caenorhabditis elegans (Mau.) Dougherty and Panagrellus
redivivus (L.) Goodey after treatment of these nematodes
with mannosidase or sialidase (49).
Lectins, proteins that bind to specific carbohydrate
residues, make excellent probes for the study of
carbohydrates that exist on biological surfaces (31, 69, 70
107). Several methods, including lectin probes, have been
used to characterize carbohydrates on the surface of a
number of free-living and phytoparasitic nematodes, and the
results of some of these investigations have been summarized


12
(47, 135, 136). Application of lectins to soil infested
with Meloidogyne incognita (Kofoid and White) Chitwood
reduced the number of nematode-induced galls on tomato
roots, but the function of lectins in this system was
unclear (74).
Several studies have attempted to relate nematode
surface carbohydrates to specificity in plant pathogenicity
(36, 76, 95). Differences in binding of fluorescent lectins
to pathotypes of Globodera spp. and Meloidogyne spp. were
reported (36, 76). The objective of this investigation was
to characterize surface carbohydrates of three Florida
populations of preinfective second-stage juveniles of
Meloidogyne spp. using selected lectins and glycohydrolases.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
(Mil and Mi3) and M. j avanica (Treub) Chitwood (Mj) were
maintained in greenhouse culture on roots of 'Rutgers'
tomato (Lycopersicon esculentum Mill.) and 'Black Beauty'
eggplant (Solanum melongena L.). Meloidogyne spp.
populations were typified by adult female perineal patterns,
second-stage juvenile (J2) lengths, and performance on
differential hosts (101). Species identifications were also
confirmed by three independent nematode taxonomists (A. M.
Golden, Beltsville, MD; J. G. Baldwin, Riverside, CA; J. D.
i


13
Eisenback, Blacksburg, VA). Eggs of each nematode
population were extracted from host roots with 0.53% NaOCl
for 30 seconds (46) and hatched at room temperature on a
Baermann funnel. Preinfective J2 that had hatched within 48
hours were used as test organisms in each experiment. A few
eggs of Meloidogyne spp. were present in each suspension of
J2.
Surface carbohydrates of preinfective J2
Fluorescent lectin probes were used to identify and
locate carbohydrates on the surface of preinfective J2 of
Mil, Mi3, and Mj. Tetramethylrhodamine isothiocyanate
(TRITC) conjugates of soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Concanavalin A (CON A), Lotus
tetragonolobus L. agglutinin (LOT), and Limulus polyphemus
L. agglutinin (LPA) (E-Y Labs, San Mateo, CA) were used.
The ratios of absorbance at 550 to 280 nm for SBA, WGA, CON
A, LOT, and LPA were 0.44, 0.55, 0.41, 0.57, and 0.20,
respectively. The specific sugars to which each lectin
binds are listed in Table 1-1.
A small sample of J2 (approx. 1000 J2) of each
Meloidogyne spp. population was suspended in distilled water
to serve as a control treatment. The remaining J2 of each
population were concentrated into 2.0 ml of the appropriate
buffer by centrifugation at 1000g for 3 minutes. Buffer
solutions included: 0.01 M phosphate-buffer saline (PBS) at


Table 1-1. Sugar specificity and competitive sugars of soybean
agglutinin (SBA), wheat germ agglutinin (WGA), Concanavalin A
(CON A), Lotus tetragonolobus agglutinin (LOT), and Limulus
Polyphemus agglutinin (LPA).
Lectin
Sugar Specificity
Competitive Sugar3
SBA
a-D-galactose
N-acetyl-a-D-galactosamine
D-galactose
WGA
N-acetyl-B-D-glucosamine
N-acetyl-D-glucosamine
CON A
a-D-mannose
a-D-glucose
D-mannose
LOT
a-L-fucose
L-fucose
LPA
neuraminic (sialic) acid
N-acetylneuraminic acid
a Corresponding competitive sugars (0.1 M) used for all lectin
and glycohydrolase assays.


15
pH 7.2 for SBA, WGA, and LOT; 0.05 M Tris-saline plus 0.01 M
CaC2 at pH 7.5 for CON A; 0.05 M Tris-saline plus 0.01 M
CaCl2 at pH 8.0 for LPA. Preinfective J2 (approx. 5000 J2)
of each population were incubated in lectin-TRITC conjugate
(200 jig/ml) for 2 hours at 4C. Additional treatments
included nematodes (approx. 5000 J2) incubated in lectin-
TRITC plus 0.1M corresponding competitive sugar (Table 1-1)
to inhibit lectin binding, J2 incubated in 0.1M sugar plus
buffer, and J2 incubated in buffer minus sugar. Treated J2
were washed by transferring J2 three times to
microcentrifuge tubes that contained fresh buffer or water
and allowing J2 to settle to the bottom of the tube. A
sample of J2 (approx. 500 J2) in final wash solution was
placed on a glass microscope slide and covered with a cover
glass. The edges of the cover glass were sealed with clear
fingernail polish. Approximately fifty specimens from each
treatment were immediately observed at 100x under a Zeiss
epifluorescent microscope equipped with TRITC and FITC
(fluorescein isothiocyanate) filters. Photographs of
selected nematodes were taken when nematode movement ceased
(approx. 1-3 hours after J2 were mounted on slides). Each
test was repeated twice.
The relative binding capacity of each lectin-TRITC
conjugate was determined through hemagglutination assay
(89). Twenty-five microliter volumes of lectin were
i


16
serially diluted (1:1) with the appropriate buffer in
adjacent wells across a 96-well microtiter plate. Twenty-
five microliters of a 4% suspension of trypsinized,
gluteraldehyde-stabilized, human Type 0 red blood cells
(Sigma Chemical Co., St. Louis, MO) were added to each well,
except for wells containing LPA. A 4% suspension of
gluteraldehyde-stabilized, horse red blood cells (Sigma
Chemical Co.,St. Louis, MO) was used for LPA-TRITC assays.
The greatest dilution of lectin that exhibited visible
hemagglutination (titer) was determined after 3 hours
incubation at room temperature. The titer divided by the
milligrams lectin/ml in each sample is a measure of the
specific hemagglutination activity of each lectin-TRITC
conjugate. Similar tests were conducted in the presence of
0.1 M competitive sugar to assess inhibition of lectin
binding activity.
Surface carbohydrates of glycohydrolase-treated
preinfective J2
Enzymes (glycohydrolases) that cleave specific
carbohydrate residues from glycoconjugates were assayed for
their effect on surface carbohydrates of Mil, Mi3, and Mj.
Glycohydrolases tested consisted of the following:
a-galactosidase (a-gal) EC 3.2.1.22 (20 U/mg) from
recombinant E. coli, a-L-fucosidase (a-fuc) EC 3.2.1.51 (2.0
U/mg) from beef kidney, B-N-acetyl-glucosaminidase (B-glu)


17
EC 3.2.1.30 (4.0 U/mg) from beef kidney, a-mannosidase
(a-man) EC 3.2.1.24 (10 U/mg) from Canavalia ensiformis DC.,
neuraminidase (sialidase) EC 3.2.1.18 (1.0 U/mg) from
Clostridium perfringens (Veil, and Zub.) Holland. All
glycohydrolases were obtained from Boehringer Mannheim
Biochemicals, Indianapolis, IN, and enzyme activity was
determined by reaction with the appropriate _p-nitrophenyl
conjugate of each carbohydrate substrate (information
supplied by manufacturer).
Preinfective J2 of Mil, Mi3, and Mj were each
concentrated into 2.0 ml of the appropriate buffer by
centrifugation at 1 000c[ for 3 minutes. Buffer solutions
included 0.01 M phosphate buffer (pH 7.2) for a-gal, 0.05 M
sodium citrate buffer (pH 5.0) for a-fuc, 0.05 M sodium
citrate buffer (pH 4.5) for fl-glu, 0.05 M sodium citrate
buffer plus 1.0 mM ZnS04 (pH 4.5) for a-man, 5.0 mM sodium
acetate buffer plus 72.0 mM NaCl and 7.0 mM CaCl2 (pH 5.0)
for neuraminidase. Enzyme buffers were formulated to the pH
optimum of enzyme activity as suggested by the manufacturer.
Preinfective J2 (approx. 5000 J2) of each population were
incubated in either a-gal (1.0 U/ml), a-fuc (0.25 U/ml),
B-glu (1.0 U/ml), a-man (1.0 U/ml), or sialidase (0.25 U/ml)
solution for 18 hours at 37*C. Nematodes were also
incubated in enzyme plus 0.1 M corresponding competitive
sugar (Table 1-1) to inhibit enzyme activity. Control
i


18
treatments consisted of J2 in buffer alone and J2 in buffer
plus 0.1 M sugar at 37C for 18 hours. Nematode viability
after treatment with enzyme buffers under experimental
conditions was confirmed by bioassay (Appendix B).
Glycohydrolase-treated nematodes were washed three
times with the appropriate lectin buffer and subsequently
treated with separate lectin-TRITC conjugates as described
above for untreated, preinfective J2. Specimens were
immediately mounted on glass microscope slides and observed
under epifluorescent microscopy as described above.
Results
Hemagglutination tests indicated that the binding
capacity of all lectin-TRITC conjugates, except LPA, was
relatively strong. Specific hemagglutination activities of
1024, 512, 4096, 2048, and 16 units/mg lectin were
determined for SBA, CON A, WGA, LOT, and LPA, respectively.
Hemagglutination activity of all lectin-TRITC conjugates was
completely inhibited in the presence of 0.1 M corresponding
competitive sugar.
Viable, preinfective J2 were labeled with lectin-
TRITC almost exclusively in the vicinity of the amphidial
openings (Figs. 1-1a 1-1c). Fluorescent lectin labeling
often extended outward from these openings, suggesting that
carbohydrates occur within amphidial secretions. Binding of


Fig. 1-1. Binding of fluorescent (rhodamine) lectin
conjugates to Meloidogyne spp. second-stage juveniles (J2)
and egg. a) Strong amphidial (SA) fluorescence of M.
incognita race 3 (Mi3) labeled with Lotus tetragonolobus
agglutinin, b) Binding of wheat germ agglutinin (WGA) to
amphidial secretions (AS) of M. javanica (Mj) after a-
galactosidase treatment, c) Weak amphidial fluorescence (WA)
of M. incognita race 1 (Mil) labeled with Limulus polyphemus
agglutinin, d) Fluorescence of dead (D) vs. living (L) J2 of
Mil after treatment with soybean agglutinin, e) Fluorescent
cuticle (FC; note annulation) and amphidial ducts (AD) of Mj
labeled with Concanavalin A after J2 exposure to a-
galactosidase. f) Binding of WGA to egg shell (ES) of Mi3.
(Note: Incandescent light provided to enhance J2 image in
photographs a-d results in artifactual cuticular glow. True
labeling of J2 cuticle by fluorescent lectin is presented in
plate e.)


20


21
fluorescent lectins to any other portions of the nematode
surface was rarely observed, except as indicated below for
several glycohydrolase treatments. Nematodes that were
straightened, vacuolated, and displayed no movement
(nonviable) often exhibited strong labeling of the stylet,
esophageal lumen, and especially the gut region after
exposure to lectin-TRITC conjugates (Fig. 1-1d). No
labeling of viable J2 with unconjugated TRITC was observed.
Few differences in fluorescent lectin labeling were
observed among nonglycohydrolase-treated, preinfective J2 of
the Meloidogyne spp. tested (Table 1-2). Amphids of Mil,
Mi3, and Mj labeled weakly with SBA, CON A, and LPA and
strongly with WGA and LOT. No binding of TRITC-conjugated
Limax flavus L. agglutinin (LFA; sialic-acid specific) to J2
was observed in preliminary tests (unpublished results), and
binding of LPA-TRITC to J2 was not observed until combined
with an improved fluorescent microscope light source (50
watt mercury lamp; Carl Zeiss, West Germany). Inhibition of
lectin binding in the presence of the appropriate
competitive sugar was only observed for LOT and LPA. All
lectins tested bound to egg shells of Mil, Mi3, and Mj
(Fig. 1 1f) and binding was not inhibited in the presence of
0.1 M corresponding competitive sugar.
Differences in lectin labeling among the populations
of Meloidogyne spp. tested were observed after preinfective


22
Table 1-2. Binding of fluorescent lectins to
preinfective second-stage juveniles (J2) of
Meloidogyne incognita races 1 and 3 (Mil, Mi3),
and M. javanica (Mj).
Lectin
Mil
Mi 3
Mj
-Sug
a +Sug
-Sug
+ Sug
-Sug
+ Sug
SBAb
++c
++
+
+
++
++
WGA
+++
+++
+++
+++
+ + + +
++ + +
CON A
++
++
++
++
++
++
LOT
+++
NF
+++
NF
+ + + +
NF
LPA
++
NF
++
NF
++
NF
a J2 incubated in lectin solution +/-
competitive sugar. Competitive sugars included:
D-galactose for SBA; N-acetyl-glucosamine for
WGA; D-mannose for CON A; L-fucose for LOT; N-
acetyl-neuraminic acid for LPA.
b Soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Concanavalin A (CON A), Lotus
tetragonobolus agglutinin (LOT), and Limulus
polyphemus agglutinin (LPA).
c Epifluorescent microscope observations
included: + = very weak amphidial fluorescence;
++ = weak aphidial fluorescence; +++ = strong
amphidial fluorescence; ++++ = very strong
amphidial fluorescence; NF = no fluorescence.


23
J2 were treated with various glycohydrolases (Table 1-3).
Lectin labeling of J2 treated with glycohydrolases was
compared to labeling of J2 which were incubated in enzyme
buffer minus glycohydrolase. In most cases, enzyme activity
was inhibited in the presence of the appropriate competitive
sugar, except where indicated below.
Treatment of J2 with a-gal eliminated binding of
SBA-TRITC to the amphids of Mj and Mil, but not to amphids
of Mi3. Binding of LOT-TRITC to the amphids of Mj was
reduced by treatment of J2 with a-gal. The cuticle on the
anterior half of the body of Mj and Mil labeled weakly with
CON A-TRITC, with fluorescence of body annulation growing
weaker from head to mid-body (Fig. 1-1e). Enzyme activity
was not inhibited in the presence of 0.1M D-galactose for
Mj. Similar cuticular labeling was not observed for J2 of
Mi3 treated with a-gal, however, binding of CON A-TRITC to
the amphids of Mi3 was eliminated. Binding of LPA-TRITC to
the amphids of J2 was unchanged on Mj, increased on Mil and
eliminated on Mi3 after treatment with a-gal.
Treatment of J2 of Mil with B-glu reduced binding of
WGA-TRITC to amphids and promoted binding of WGA-TRITC to
the anterior cuticle of Mil. Binding of LPA-TRITC to the
amphids of J2 of Mj and Mil was eliminated by B-glu
treatment. However, binding of LPA-TRITC to the amphids of
Mi3 increased after B-glu treatment.


24
Table 1-3. Binding of fluorescent-lectins to preinfective second-
stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil,
Mi3), and M. javanica (Mj) after J2 treatment with different
glycohydrolases.
Mil
Mi 3
Mj
Enzyme (Lectin)
-Enza +Enz
-Enz +Enz
-Enz +Enz
a-galactosidase
SBAb
++c
NF
+
+
++
NF
WGA
+++
+++
+++
+ + +
+++
+++
CON A
++
++,WC
++
NF
++
++,WC
LOT
+++
+++
+++
+++
+++
++
LPA
+
+++
++
NF
+
+
B-N-Acetyl-
Glucosaminidase
SBA
++
++
+
+
+
+
WGA
+++
++,wc
+++
+++
+++
+++
CON A
++
++
+
+
++
++
LOT
+++
+++
+++
+++
+++
+++
LPA
++
NF
NF
++
++
NF
a-mannosidase
SBA
++
NF
++
++
++
++
WGA
+++
+++
+ + +
+++
+++
+++
CON A
++,WC
++,wc
++
NF
++
NF
LOT
+++
+++
++
++
+++
NF
LPA
++
NF
++
NF
++
NF
i


25
Table 1-3continued.
Mil
Mi3
Mj
Enzyme (Lectin)
-Enz +Enz
-Enz +Enz
-Enz +Enz
a-L-fucosidase
SBA
++
++
++ +
++ +
++
++
WGA
+++
+++
+++
+++
+++
+++
CON A
+ +,WC
++,WC
++
++
+++
+++
LOT
+++
+ +
+++
+ + +
+++
+
LPA
+++,wc
NF, WC
++
NF
++
NF
Neuraminidase
SBA
NF
NF
+
+
+
+
WGA
+ + +
+++
+ + +
+++
+++
+ + +
CON A
++,wc
++,wc
++ +
++
++'
WC +++,wc
LOT
+++
+++
+++
++
+++
+
LPA
++
NF
++
++
++
NF
a J2 incubated
in lectin
solution
+ /-
prior
treatment
with
select glycohydrolase.
k Soybean agglutinin (SBA), wheat germ agglutinin (WGA),
Concanavalin A (CON A), Lotus tetragonobolus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA).
c Epifluorescent microscope observations included: + = very weak
amphidial fluorescence; ++ = weak aphidial fluorescence; +++ =
strong amphidial fluorescence; ++++ = very strong amphidial
fluorescence; NF = no fluorescence; WC = weak fluorescence of J2
cuticle along anterior half of body.


26
Binding of SBA-TRITC to the amphids of Mil and LPA-
TRITC to the amphids of Mj, Mil, and Mi3 was eliminated by
a-man. Binding of LOT-TRITC to the amphids of Mj was
eliminated by treatment of J2 with a-man, but a-man activity
was not inhibited in the presence of 0.1 M mannose. Amphids
of Mj and Mi3 did not label with CON A-TRITC after a-man
treatment, but CON A-TRITC did bind to the anterior cuticle
of Mil after treatment with a-man buffer plus or minus
enzyme.
Binding of LOT-TRITC to the amphids of Mj and Mil
was reduced and binding of LPA-TRITC to amphids of Mj, Mil,
and Mi3 was eliminated after treatment of J2 with a-fuc.
Weak labeling of the anterior cuticle of Mil with CON A-
TRITC and LPA-TRITC occurred after incubation of J2 in a-fuc
buffer with or without the enzyme.
Treatment of J2 with neuraminidase partially
inhibited binding of LOT-TRITC to amphids of Mj and Mi3, and
completely inhibited binding of LPA-TRITC to amphids of Mj
and Mil. Neuraminidase treatment increased binding of CON
A-TRITC to amphids of Mj and Mil, but reduced binding of
CON A-TRITC to amphids of Mi3. The anterior cuticle of Mj
and Mil labeled weakly with CON A-TRITC after incubation in
neuraminidase buffer with or without enzyme.
i


27
Discussion
Fluorescein isothiocyanate (FITC)-lectin conjugates
were not used in fluorescence assays because untreated J2 of
the Meloidogyne spp. populations examined strongly
autofluoresced at the excitation wavelength of FITC.
Difficulty with autofluorescence of C. elegans and P.
redivivus at the excitation wavelength of FITC has also been
reported (48). Lectins conjugated with rhodamine (TRITC)
fluorophors were more appropriate for the study of lectin
binding to nematodes. Preinfective J2 of Meloidogyne spp.
were not visible when viewed through the TRITC microscope
filter, except for body portions labeled with lectin-TRITC
conjugates.
It is apparent from observations that nematode
viability is critical for true labeling of nematodes with
fluorescent lectins. Living (motile) J2 bound fluorescent
lectin almost exclusively in the vicinity of the amphidial
openings. The entire body of nematodes that were apparently
dead fluoresced after TRITC-lectin treatment, especially in
the gut region. The fluorescence of dead J2 was similar to
the observations of enzymatically induced fluorescence of
dead nematodes reported by Bird (11). This phenomenon may
have influenced fluorescent observation of sialyl residues
over the entire body of J2 of M. javanica as reported by
Spiegel et a_l. (114). Labeling of sialyl residues with


28
fluorescent LPA was relatively weak and confined to the
amphidial region of viable J2 of Meloidogyne spp. observed
in these studies. Since the specific hemagglutination
activity and absorbance ratio (550nm/280nm) of LPA-TRITC was
relatively low compared to the other lectins tested, it may
be possible that more sialic acid exists on the J2 surface
than can be detected with fluorescent lectin probes.
Lectin binding to the tail region of M. incognita has also
been reported (77), but it was not observed in this study.
The observed binding of fluorescent lectins to egg shells of
Meloidogyne spp. in this investigation has been reported for
eggs of M. javanica (113).
Only the binding of LOT and LPA to specific sugars
on preinfective J2 were confirmed by competitive sugar
inhibition, although all of the fluorescent lectins tested
bound to amphids of preinfective J2 of the three Meloidogyne
spp. populations examined. The binding of SBA, CON A, and
WGA to J2 was apparently not specific for a-D-galactose, a-
D-mannose, and S-N-acetylglucosamine, respectively, since
0.1 M concentrations of these sugars were insufficient to
inhibit binding of these TRITC-lectins to J2. Soybean
agglutinin, CON A, and WGA bind to other molecular forms of
galactose, mannose, and N-acetylglucosamine, respectively,
and WGA has been reported to have multiple carbohydrate
binding sites (38). It is possible that the affinity of
i


29
SBA, CON A, and WGA for carbohydrate-specific sites near
amphidial openings was too strong to be inhibited by the
competitive sugar solutions used in these assays. The
binding of SBA, CON A, and WGA to amphidial secretions of J2
of Meloidogyne spp., however, may represent binding of these
lectins to hydrophobic ligands (possibly lipids), as
reported elsewhere (93). Incubation of J2 in fluorescent
SBA, CON A, or WGA in the presence of 1,8 anilinonaphthalene
sulfonic acid plus or minus competitive sugar may confirm
the presence of hydrophobic binding since the hydrophobic
and carbohydrate binding sites are independent of each other
(93). The direct binding of unconjugated TRITC to surface
lipids of J2 did not apparently occur in these assays since
nematodes did not fluoresce after incubation in unconjugated
TRITC.
The greater intensity of fluorescent labeling by LOT
and WGA conjugates may be due to their relatively higher
binding capacities. Lack of differential lectin labeling
among nonglycohydrolase-treated, preinfective J2 of Mil,
Mi3, and Mj makes it difficult to extrapolate a potential
role of surface carbohydrates in the specificity of
pathogenicity (58). Substances to which lectins were bound
were concentrated and sometimes emanated from the amphidial
region of J2 of Meloidogyne spp., and this has also been
reported for invasive juveniles of pathotypes of potato cyst


30
nematode and other populations of Meloidogyne spp. (36, 76).
Since this is the portion of the nematode body around which
some postinfectional, incompatible plant responses occur
(43, 61 ), it may be possible that carbohydrates in amphidial
secretions of postinfectional J2 affect plant-nematode
interactions.
Results of experiments involving glycohydrolases
suggested that carbohydrates located in the amphidial region
of J2 occurred in complexes and that these complexes were
structurally different among populations of Meloidogyne spp.
The inability of enzymes to alter carbohydrate residues on
some nematode surfaces may be a reflection of the substrate
specificity exhibited by glycohydrolases (2, 125, 132).
Enzyme treatment did reveal cuticular carbohydrates,
especially mannose and/or glucose, on the anterior half of
some J2 and sialyl residues were often removed from J2
amphids by a number of different glycohydrolases. This may
indicate that sialic acids are some of the outermost
residues present in the carbohydrate complexes which
apparently exist in amphidial secretions of J2 of
Meloidogyne spp. Whether surface carbohydrate changes
similar to those reported for glycohydrolase treatments
occur once Meloidogyne spp. J2 enter plant roots is unknown.
Alteration of the surface carbohydrates of J2 of Meloidogyne


31
spp., and subsequent plant root tissue responses to
infection by treated J2, may provide insight into the
specificity of plant-nematode interactions.


CHAPTER 3
ROOT TISSUE RESPONSE OF TWO RELATED SOYBEAN CULTIVARS
TO INFECTION BY LECTIN-TREATED MELOIDOGYNE SPP.
Introduction
Plant incompatibility with nematodes often results
from active plant defense reactions to infection by
phytoparasitic nematodes (37, 39, 42, 58, 59, 97, 127).
Although several mechanisms of active incompatibility have
been proposed, little is known about nematode
characteristics that may elicit plant responses that are
incompatible with nematode development and their relation to
specifity in plant-nematode interactions (58). The
occurrence of physiological races of phytoparasitic
nematodes (108, 119) suggests that specific interactions
occur between nematodes and plant genotypes, and that
populations of phytoparasitic nematodes adapt to overcome
incompatibility.
Evidence that surface biochemistry, especially
glycoconjugates of cells and organisms, promotes specificity
in plant-microbe interactions has been the subject of
several reviews (3, 21, 23, 62, 106). Keen (62) suggested
that biochemical surface interactions were important in the
specificity of incompatibility in gene-for-gene systems
32


33
between plant cultivars and microbial pathogens. It is
unclear if surface interactions are important in plant-
nematode incompatibility; however, the existence of
carbohydrates on the surface of nematodes, and evidence that
surface carbohydrates may be important in interactions
between nematodes and microbes has been reported and
summarized (47, 135, T36). Zuckerman and Jansson (136)
proposed that interaction between nematodes and other
organisms may be altered by obliteration or blocking of
carbohydrates on the nematode surface.
To evaluate this concept with respect to specificity
in plant-nematode interactions a model system was chosen
which consisted of two related soybean cultivars,
'Centennial' and 'Pickett 71', and three Meloidogyne spp.
populations that differed in compatibility with Centennial
soybean. The incompatibility of Centennial soybean with
Meloidogyne incognita has been associated with a
hypersensitive reaction (HR) of soybean root tissue in the
region of invading second-stage juveniles (J2) (61). The HR
was strongly correlated with the accumulation of glyceollin,
but biotic elicitors of the HR were not identified (60).
The surface carbohydrates of several populations of
cyst and root-knot nematodes, including J2 of Florida
populations of M. incognita races 1 and 3 (Mil, Mi3), and M.
javanica (Mj) (see Chapter 2 above), have been characterized


34
with fluorescent lectin probes (36, 76, 95). Lectins bound
to infective J2 of Meloidogyne spp. primarily in the region
proximate to cephalic chemosensillae (see Chapter 2 above,
77). In the study reported here, surface carbohydrates of
J2 of Mil, Mi3, and Mj were "blocked" with lectins and
subsequent soybean root tissue responses to lectin-treated
J2 were observed.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
and M. javanica (Treub) Chitwood were maintained in
greenhouse culture on roots of 'Rutgers' tomato
(Lycopersicon esculentum Mill.) and 'Black Beauty' eggplant
(Solanum melongena L.). Meloidogyne spp. populations were
typified by adult female perineal patterns, second-stage
juvenile (J2) lengths, and performance on differential
hosts (101). Species identifications were also confirmed by
three independent nematode taxonomists (A. M. Golden,
Beltsvilee, MD; J. G. Baldwin, Riverside, CA; J. D.
Eisenback, Blacksburg, VA). Eggs of each nematode
population were extracted from host roots with 0.53% NaOCl
for 30 seconds (46) and hatched at room temperature on a
Baermann funnel. Preinfective J2 which had hatched within
48 hours were used as test organisms in each experiment.


35
Surface carbohydrates of Meloidogyne spp. J2 were
blocked by incubating nematodes in solutions containing
unconjugated, purified soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT),
Concanavalin A (CON A), or Limax flavus agglutinin
(LFA) (E-Y Labs, San Mateo, CA). The sugar specificity,
appropriate lectin buffers, corresponding competitive
sugars, and procedure used to determine the specific
hemagglutination activity for each lectin were described
above in Chapter 2. The sugar specificity of LFA is
N-acetyl-neuraminic (sialic) acid, and LFA assays were
conducted in buffer which contained 0.05 M Tris-saline plus
0.01 M CaCl2 at pH 7.5.
Preinfective J2 of Mil, Mi3, and Mj were
concentrated in the appropriate buffer or in distilled water
by centrifugation at 1000cj for 3 minutes. Treatments for
each lectin included incubating J2 (approx. 2000 J2) of each
population in solutions of lectin (200 ug/ml), lectin (200
jig/ml) plus 0.1M competitive sugar, and 0.1M sugar minus
lectin for 2 hours at 4C. Juveniles in these solutions
were used as direct (unwashed treatment) inoculum for
subsequent soybean root challenge. Since a 0.1 M solution
of sialic acid in LFA buffer was quite acidic (pH ~ 3.0), a
soybean root penetration bioassay was conducted to address
the effect of sialic acid and acidity on the activity of J2
i


36
of Meloidogyne spp. (see Appendix D below). In addition, J2
exposed to each lectin and sugar treatment were washed three
times in buffer and subsequently used as inoculum for
soybean root challenge. Control treatments included J2 in
buffer and J2 in distilled water.
Two related cultivars of soybean (Glycine max (L.)
Merr.), 'Pickett 71' and 'Centennial', were used for root
tissue challenge by treated J2 of Meloidogyne spp. It has
been reported that Pickett 71 was compatible and Centennial
was incompatible with M. incognita, and both soybean
cultivars were compatible with M. j avanica (61). Seeds of
each variety were dusted with Thiram 75WP (Kerr-McGee
Chemical Corp., Jacksonville, FL) and germinated in moist
germination paper which was rolled up (ragdolls) and
incubated in the dark at 27*C. Newly germinated soybeans
with roots 3-5 cm long were placed on trays containing
autoclaved Astatula fine sand (hyperthermic, uncoated typic
quartzipsamments) and the root tips covered with a small
amount of sand. Nematode suspensions (approx. 600 J2) from
each treatment were placed on separate soybean root tips
(61, 75). Trays containing inoculated soybeans in sand were
incubated in the dark at 27*C. Treatments were arranged as
a 3x5x8x2 factorial including three Meloidogyne spp.
populations, five lectins, eight treatments, and two soybean
i


37
varieties. There were seven replicates of each treatment
combination.
Soybeans were removed from trays and their roots
washed free of sand and any nematodes that had not
penetrated approximately 40 hours after inoculation. The
seedlings were then placed on moist germination paper and
the inoculated portions of the roots were marked on the
paper. The germination paper which contained inoculated
seedlings was covered with an additional piece of moist
germination paper, carefully rolled into ragdolls, and
incubated in the dark at 27C. This "pulse inoculation" was
used to ensure that nematodes observed within roots had
entered within 40 hours of inoculation.
Inoculated soybean root segments were excised and
immediately fixed in 10% alcoholic formalin (1:9,
formalin:95% EtOH, v/v) a total of 5 days after inoculation.
Additional treatments of J2 of Mi3 in water applied to
Centennial and Pickett 71 soybean roots were excised 20 days
after inoculation and processed according to these
protocols. Fixed root segments were dehydrated through a
tert-butyl alcohol series and embedded in paraffin. Serial
sections (12 urn) were mounted on glass slides, stained with
safranin-fast green, and observed under light microscopy.
Sections from seven replicates of each treatment combination
were observed and the most frequent tissue responses to
i


38
nematode infection were determined. This experiment was
repeated once.
Results
Hemagglutination assays indicated that the binding
capacity of pure lectins was relatively strong, except for
LFA. Specific hemagglutination activities of 4096, 4096,
8192, 8192, and 4 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LFA, respectively. Hemagglutination
activity of all lectins was completely inhibited in the
presence of 0.1 M corresponding competitive sugar.
The most common or primary responses of soybean root
tissue to infection by Meloidogyne spp. J2 are reported in
Tables 2-1 to 2-5. Infective juveniles incubated in buffer
or water became enlarged and induced giant cell formation in
compatible interactions but remained vermiform and were
associated with a hypersensitive reaction in incompatible
combinations (Fig. 2-1). Giant cells of normal appearance
were usually observed in roots of Pickett 71 and Centennial
soybean 5 and 20 days after roots were exposed to Mi3
incubated in buffer or water. Giant cells associated with
Mi3 controls in Centennial soybean sometimes contained
granular cytoplasm. No gall formation, evidence of
hyperplasia of pericycle cells adjacent to giant cells, or
development of Mi3 past third-stage juvenile was observed in


Table 2-1. Primary tissue reactions of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in soybean
agglutinin (SBA) solution (200 ng/ml) and (or) 0.1M galactose (gal) solution prior to
inoculation.
Treatment
Mil
Mi 3
Mj
P
C
P
C
P
C
Washed3
SBA
GC,
GRCYb
HR,
NR
GC, NR
HR,
EGC
GC,
S
GC,
S
SBA + gal
GC
HR
GC, GRCY
HR,
NR
GC,
GRCY
GC,
S
gal
GC,
GRCY
HR
GC, GRCY
HR,
NR
GC,
GRCY
GC
Unwashed
SBA
GC,
GRCY
HR,
EGC
GC, S
HR,
EGC
GC,
S
GC,
s
SBA + gal
GC,
GRCY
HR,
EGC
EGC, GRCY
HR,
EGC
GC,
EGC
GC,
s
gal
GC,
S
HR,
GRCY
GC, S
HR,
NR
GC,
S
GC,
s
Buffer
GC,
GRCY
HR,
GRCY
GC, S
GC,
GRCY
GC,
S
GC,
s
Distilled H2O
GC,
S
HR
GC, S
GC,
GRCY
GC,
S
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.


Table 2-2. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Concanavalin A
(CON A) solution (200 ng/ml) and (or) 0.1M mannose (man) solution prior to
inoculation.
Treatment
Mil
Mi3
Mj
P
c
P
C
P
C
Washed3
CON A
GC,
NRb
NR,
HR
GC,
S
HR
GC
GC,
s
CON A +
man
GC,
GRCY
HR,
NR
GC,
S
HR, NR
GC,
S
GC,
s
man
GC,
S
HR
GC
NR, GC
GC,
GRCY
GC,
GRCY
Unwashed
CON A
GC,
S
HR
GC,
EGC
HR, NR
GC,
S
GC,
S
CON A +
man
GC,
S
HR
GC,
S
HR, EGC
GC,
S
GC,
S
man
GC,
s
NR,
EGC
GC,
S
GC, GRCY
GC,
S
GC,
S
Buffer
GC,
s
HR,
NR
GC,
S
GC, GRCY
GC,
s
GC,
S
Distilled
h2o
GC,
s
HR,
GRCY
GC,
S
GC, EGC
GC,
s
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.


Table 2-3. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Lotus
tetrgonolobus agglutinin (LOT) solution (200 jig/ml) and (or) 0.1M fucose (fuc)
solution prior to inoculation.
Mil
Mi3
Mj
Treatment
P
C
P
C
P
C
Washed3
LOT
GC,
Sb
HR,
EGC
GC,
S
HR,
EGC
GC,
S
GC,
GRCY
LOT + fuc
GC,
S
HR,
NR
GC,
NR
HR,
GRCY
GC,
S
GC,
S
fuc
GC,
GRCY
HR
GC,
EGC
HR,
NR
GC,
S
GC,
S
Unwashed
LOT
GC,
GRCY
HR
GC,
GRCY
HR,
EGC
GC,
S
GC,
GRCY
LOT + fuc
GC,
GRCY
HR,
GRCY
GC,
S
HR,
EGC
GC,
EGC
GC,
NR
fuc
GC,
GRCY
HR,
GRCY
GC,
S
HR,
EGC
GC,
S
GC,
S
Buffer
GC,
S
HR
GC,
S
GC,
EGC
GC,
s
GC,
S
Distilled H2O
GC,
S
HR
GC,
S
GC,
S
GC,
s
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.


Table 2-4. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in wheat germ
agglutinin (WGA) solution (200 ug/ml) and (or) 0.1M N-acetylglucosamine (NAcGlu)
solution prior to inoculation.
Mil
Mi 3
Mj
Treatment
P
C
P
C
P
C
Washed3
WGA
GC,
EGCb
HR,
NR
GC,
NR
HR,
NR
GC,
NR
GC
WGA + NAcGlu
GC,
NR
HR,
NR
GC,
EGC
HR,
NR
GC,
S
GC,
s
NAcGlu
GC,
HR
HR,
GRCY
GC,
S
NR,
HR
GC,
S
GC,
s
Unwashed
WGA
GC,
NR
HR,
NR
GC,
S
HR,
GRCY
GC,
S
GC,
GRCY
WGA + NAcGlu
GC,
HR
NR,
HR
GC,
s
HR
GC,
S
GC,
S
NAcGlu
GC,
HR
HR
GC,
s
HR,
EGC
GC,
S
GC,
S
Buffer
GC,
S
HR,
NR
GC,
s
GC,
NR
GC,
S
GC,
S
Distilled H2O
GC,
S
HR,
NR
GC,
s
GC,
GRCY
GC,
S
GC,
S
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.


Table 2-5. Primary tissue responses of 'Pickett 71 (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Limax flavus
agglutinin (LFA) solution (200 ng/ml) and (or) 0.1M N-Acetylneuraminic (sialic) acid
solution prior to inoculation.
Mil
Mi3
Mj
Treatment
P
C
P
c
P
C
Washed3
LFA
GC, Sb
NR, HR
GC,
EGC
HR, NR
GC, S
GC,
s
LFA + sialic
GC, S
HR
NR,
EGC
HR, EGC
GC, S
GC,
s
sialic
GC, GRCY
HR, GRCY
GC,
EGC
HR
GC, S
GC,
s
Unwashed
LFA
c
_
__
_
_
LFA + sialic


-
-
--

-
-
sialic







Buffer
GC, S
HR, GRCY
GC,
S
GC, EGC
GC, S
GC,
s
Distilled H2O
GC, S
HR, NR
GC,
s
GC, GRCY
GC, S
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.
c Few or no nematodes observed within soybean root tissue.


Fig. 2-1. Response of 'Centennial' soybean root tissue
within 5 days of infection by lectin-treated second-stage
juveniles (J2) of Meloidogyne spp. a) Hypersensitive
reaction (HR) to M. incognita race 1 treated with soybean
agglutinin. b) Giant cells (GC) induced by the untreated
nematode (N), M. incognita race 3. c) Giant cells induced
by M. javanica treated with soybean agglutinin plus
galactose. d) Hypersensitive reaction induced by M.
incognita race 3 treated with N-acetylglucosamine.




46
Centennial root tissue 20 days after inoculation with J2 of
Mi3.
The primary tissue response of both soybean
cultivars to J2 of Mil or Mj exposed to any lectin or sugar
treatment was essentially unchanged from that of tissues
infected by J2 incubated in buffer or water. Lectin or
sugar treatment of J2 of Mi3 did not influence host response
in Pickett 71, but in Centennial soybean roots, treatment of
J2 of Mi3 with any lectin or sugar stimulated a
hypersensitive response rather than giant cells.
Soybean tissue responses to infection by J2 that
were atypical of the primary tissue response sometimes
occurred frequently enough to warrant report. The
occurrence of early giant cells in Centennial tissue
challenged by Mil treated with SBA and SBA plus galactose,
unwashed (Table 2-1), was in contrast to the primary
response of hypersensitivity. Early giant cell formation
was also observed in Centennial challenged by Mil treated
with mannose, unwashed (Table 2-2), and Mil treated with
LOT, washed (Table 2-3).
Second-stage juveniles of Meloidogyne spp. were
observed in soybean roots with no apparent plant tissue
reaction. This was the primary observation in Centennial
root tissue with Mil treated with CON A, washed; Mil treated
with mannose, unwashed; Mi3 treated with mannose, washed;
i


47
Mil treated with WGA plus N-Acetyl-D-glucosamine (NAcGlu),
unwashed; Mi3 treated with NAcGlu, washed; and Mil treated
with LFA, washed (Tables 2-2, 2-4, 2-5). No tissue response
was observed in Pickett 71 to Mi3 treated with LFA plus
sialic acid, washed (Table 2-5). A hypersensitive response
was sometimes observed in Pickett 71 challenged by Mil
treated with NAcGlu, washed; Mil treated with WGA plus
NAcGlu, unwashed; and Mil treated with NAcGlu, unwashed.
Almost no J2 were observed in soybean roots challenged by
any population of Meloidogyne spp. that received unwashed
LFA and sialic acid treatments. When J2 of these treatments
were occasionally observed in soybean roots, tissue response
was similar to comparable treatments with SBA.
Discussion
It appears that incubation of J2 of Mil in the
lectins or sugars tested had little effect on soybean root
tissue response in the incompatible pathosystem. If the
concept that preformed sites (carbohydrate moieties) on the
surface of Mil J2 are responsible for recognition by plant
cell surface receptors and subsequent plant defense reaction
were valid, blockage of these sites with lectins should have
prevented the HR observed in Centennial root tissue. In a
few instances, early giant cell formation in Centennial by
Mil was noted, but the HR was much more common. The more


48
frequent occurrence of HR in Pickett 71 exposed to Mil
treated with N-acetyl-D-glucosamine may indicate an
alteration of nematode surface carbohydrates which promoted
incompatibility.
The fate of lectin bound to J2 of Meloidoayne spp.
once the nematode entered soybean root tissue is
questionable. Unwashed treatments were included in this
study to ensure that J2 were present in an environment of
lectin and (or) sugar until they penetrated roots. No
treatments, however, had an effect on the compatible
interaction between Mj and root tissue of either soybean
cultivar. Differences in the quantity, balance, or
accessibility of secretory carbohydrates to potential plant
receptors, however, may promote incompatibility or
compatibility. Previous reports indicated that lectins
bound to amphidial secretions of J2 of Meloidogyne spp. (see
Chapter 2 above, 77), but the rates of production of
amphidial secretions by J2 of Meloidogyne spp. and the
quantities of this material that are sloughed off in plant
tissue or the soil environment are unknown.
The occurrence of normal giant cells in Centennial
20 days after inoculation of root tips with J2 of Mi3
incubated in water was unexpected. This host-parasite
relationship was apparently incomplete compared to Mi3 in
Pickett 71, however, since pericyclic hyperplasia and


49
nematode development were strongly inhibited 20 days after
inoculation. Differences in the degree of incompatibility
have been reported for several soybean cultivars and M.
incognita populations (105). Treatment of J2 of Mi3 with
any lectin or sugar promoted active incompatibility (HR) in
Centennial soybean, and may actually have facilitated
recognition of invasive Mi3 and subsequent defense response
by the plant. The lack of specificity of lectin or sugar
effects in the Mi3-Centennial interaction makes it seem
unlikely that alteration of surface carbohydrate composition
of preinfective J2 was responsible for promoting
incompatibility. If one considers the interaction of
nematode surface carbohydrates with potential plant cell
surface receptors as a "lock and key" phenomenon, however,
it may be feasible that a slight alteration in surface
carbohydrate compostion was sufficient to promote
incompatibility to Mi3 in Centennial soybean roots.
Possibly a greater alteration of the carbohydrates examined
here on Mil and Mj, or alteration of surface carbohydrates
not examined in these studies, would influence their host-
parasite interactions.
Treatment of J2 of Mi3 with lectin or sugar may have
stimulated the production of a substance by the nematode
that induces HR in Centennial soybean roots. Juveniles of
Mil may inherently have the capacity to induce HR while J2


50
of Mj cannot promote incompatibility in soybean no matter
what the treatment. For some populations, such as in the
case of Mi3, incompatibility may be a process that can be
stimulated. Conversely, substances produced by J2 of
Meloidogyne spp. (ie. amphidial or stylet secretions) may be
essential to induce compatibility between host and parasite
(82), and these substances were altered sufficiently in Mi3
to inhibit compatibility in Centennial soybean roots.
The ability of J2 of Mil, Mi3, and Mj to penetrate
roots of both soybean varieties was apparently strongly
impaired when J2 were introduced to roots in a solution that
contained LFA, sialic acids, or combination of the two. It
was not determined if J2 penetrated and exited from roots
within the 40 hour "pulse inoculation." Inhibition of
soybean root penetration by J2 of Meloidogyne spp. treated
with sialic acid occurred in similar tests (see Appendix D
below), and this appeared to be more than an effect of the
low pH of a 0.1 M sialic acid solution. It has been
reported that a single soil application of CON A, and LFA at
relatively higher concentrations, significantly reduced
galling of tomato roots induced by M. incognita (74), but
contact and effect of active lectins to nematodes in this
system was unclear. Little effect of CON A on soybean root
penetration by J2 of any Meloidogyne spp. population was
observed in this investigation, even though J2 were


51
incubated in CON A solution (200 ng/ml) before their
application to roots in soil. Sialic acids have been
reported to be important in the adhesion of conidia of Meria
coniospora to nematode surfaces, especially at the
chemosensory organs (50-52). In the studies reported
herein, nematode viability after LFA and sialic acid
treatment was confirmed by infectivity of washed J2, and
also viable J2 of Meloidogyne spp. have been observed
microscopically after similar treatment (see Chapter 2
above). Removal of sialic acids from amphidial secretions
by a number of selective glycohydrolases (see Chapter 2
above) suggests that sialic acids are some of the outermost
carbohydrate residues of amphidial glycoconjugates. Whether
sialic acid residues proximate to nematode chemosensillae
have a masking or regulatory effect similar to those
observed in other animal systems (104) should be the subject
of further investigation.


CHAPTER 4
QUANTIFICATION OF LECTIN BINDING TO SECOND-STAGE JUVENILES
OF MELOIDOGYNE SPP.
Introduction
Lectins, proteins that bind to specific carbohydrate
residues, are excellent probes for the study of the
carbohydrate chemistry of biological surfaces. Lectins, and
their involvement in recognition and specificity in plant-
microbe interactions, have been the subject of several
reviews (3, 21, 23, 31, 70, 106, 107). The surface
carbohydrate chemistry of nematodes of various parasitic
habits has been characterized by several techniques,
including lectin probes. Some of this work has been
summarized by Zuckerman and Jansson (136), who postulated
that blockage or obliteration of specific carbohydrates on
nematode surfaces may alter interactions between nematodes
and other organisms. Examples include the association of
surface carbohydrates in antigenicity and chemoresponse of
nematodes parasitic to animals (14, 72, 85) and the
interaction of nematodes with nematophagous fungi (17, 47,
80).
The potential involvement of nematode surface
carbohydrates in recognition and specificity in plant-
52


53
nematode interactions, however, has not been clearly
demonstrated (47, 58, 135, 136). Application of
fluorescent-lectin probes to differentiate nematode species
and pathotypes on the basis of their surface carbohydrates
has been attempted (see Chapter 2 above, 36, 76, 95).
However, the intensity of fluorescence of rhodamine-labeled
lectins that bound to cephalic sensory structures of
Meloidogyne (Goeldi, 1887) spp. second-stage juveniles (J2)
differed among the lectins tested (see Chapter 2 above, 76).
Quantification of lectin binding to nematode pathotypes may
reveal differences in relative carbohydrate content that
were not detectable in fluorescent-lectin assays. Lectins
labeled with hemocyanin and with tritium have been used to
quantify relative amounts of carbohydrates on the surface of
nematodes and bacteria, respectively (77, 82). The
difficulty in production and handling of radiolabeled
lectins, and the sophisticated equipment required to observe
hemocyanin conjugates on the nematode surface, limits the
practicality of these methods. We have developed a
microfiltration enzyme-linked lectin assay (27, 78, 84, 120)
to quantify the amount of lectin that binds to J2 of
Meloidogyne spp.
i


54
Materials and Methods
Populations of Meloidogyne incognita (Kofoid &
White) Chitwood race 3 (Mi3), M.incognita race 1 (Mil), and
Meloidogvne javanica (Treub) Chitwood (Mj) were maintained
in greenhouse culture on roots of 'Rutgers' tomato
(Lvcooersicon asculentum Mill.) and 'Black Beauty' eggplant
(Solarium melongena L.). Meloidogyne spp. populations were
identified by adult female perineal pattern, second-stage
juvenile (J2) length, and development on differential hosts
(101). Species identifications were also confirmed by three
independent nematode taxonomists (A. M. Golden, Beltsville,
MD; J. G. Baldwin, Riverside, CA; J. D. Eisenback,
Blacksburg, VA). Eggs of each nematode population were
extracted from host roots in 0.53% NaOCl solution for 30
seconds (46) and hatched at room temperature on a Baermann
pan. Preinfective J2 which had hatched within 48 hours were
used as test organisms in each experiment.
The quantity of lectin binding to preinfective J2 of
Mil, Mi3, and Mj was determined using a microfiltration
enzyme-linked lectin assay (78, 84). Lectins conjugated
with horseradish peroxidase (HRP) were purchased from E-Y
Labs (San Mateo, CA) and included soybean agglutinin (SBA),
wheat germ agglutinin (WGA), Lotus tetraaonolobus agglutinin
(LOT), Concanavalin A (COMA), and Limulus Polyphemus
agglutinin (LPA). The sugar specificity, corresponding


55
buffer solutions, and procedure for determination of the
specific hemagglutination activity of each lectin were
presented in Chapter 2.
Ninety six-well microfilter plates (SV-96, Millipore
Corp., Bedford, MA) with a 5 um pore size were incubated
with 200 ul of 1.0% bovine serum albumin (BSA) in phosphate
buffer saline (PBS), pH 7.2, at 37C for 2 hours. These
plates were washed three times with PBS on a microfiltration
apparatus (Millipore Corp., Bedford, MA) prior to their use
in the following assay (Fig. 3-1).
Preinfective J2 of each Meloidogyne spp. population
were concentrated into the appropriate buffer for each
lectin-HRP conjugate. Nematodes were incubated in 500 jil of
lectin-HRP solution (200 ug/ml) for 2 hours at 4C. Control
treatments included J2 in buffer (untreated J2) and 500 y.1
lectin solution (200y.g/ml) minus J2 (lectin wash).
Competitive sugar controls similar to ones described in
Chapter 2 were omitted due to insufficient numbers of
freshly-hatched Meloidogyne spp. J2. Five, 100-ul samples
from each treatment combination (approx. 2000 J2 suspended
in lectin solution) were placed in separate wells on a
microfilter plate. Each well was washed five times with the
appropriate lectin buffer on a microfiltration apparatus.
The treated J2 or lectin wash in each well were suspended in
100 jil of buffer and transferred to separate microcentrifuge


56
Figure 3-1.
apparatus (Millipore
Microfiltration plates and suction
Corp., Bedford, MA).


57
tubes. The volume of each microcentrifuge tube was
increased to 250 jil with buffer, and 50 nl of suspension
were withdrawn from each tube to quantify the number of J2
per 50-m.I sample. Four, 50-ul suspensions of treated J2 or
lectin wash were transferred from each tube to separate
wells on a fresh 96-well microfilter plate. One hundred
microliters of peroxidase substrate,([2, 2'-azino-di-(3-
ethyl-benzthiazoline) sulfonic acid]) (ABTS, Sigma Chemical
Corp., St. Louis, MO) were added to each of these wells and
allowed to incubate at room temperature in the dark for 30
minutes (110). The solution from each well was transferred
to corresponding wells on a 96-well enzyme immunoassay plate
using a microfiltration apparatus to remove J2. Absorbance
(414 nm) of solution in each well was determined on an
automated microplate reader (Model EL309, Bio-Tek
Instruments, Winooski, VT). Twenty separate absorbance
values were determined for each treatment combination.
The absorbance values determined in this assay were
compared to the linear portion of a standard curve prepared
for each lectin-HRP conjugate (Fig. 3-2). Standard curves
were prepared by diluting (1:1, v/v) 50-nl volumes of
lectin-HRP solution across a 96-well EIA plate and adding
100 m.1 of ABTS solution per well. The quantity of lectin
which adsorbed to a single J2 was determined by dividing the


Figure 3-2. Standard curves of lectin-peroxidase
conjugates. Concanavalin A (CON A) = 6258.651x-468.338,
= 0.957; Soybean agglutinin (SBA) = 11,388.817x+12.636,
= 0.995; Lotus tetragonobolus agglutinin (LOT) =
11,791.765x-31.613, R^ = 0.995;Limulus polyphemus
agglutinin (LPA) = 7069.594x-459.472, R^ = 0.977; Wheat germ
agglutinin (WGA) = 6282.532x-428.531, R 0.967.
i


ABSORBANCE (414nm)
NANOGRAMS LECTIN
oru-t^cncDoroj^cncD
6S
- CON


60
observed lectin value by the number of J2 estimated for that
sample (approx. 500-2000 J2). The test was repeated once.
Results
Hemagglutination tests indicated that the binding
capacity of all HRP-lectins, except LPA, was relatively
strong. Specific hemagglutination activities of 512, 512,
4096, 2048, and 8 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LPA, respectively. Hemagglutination
activity of all lectin-HRP conjugates was completely
inhibited in the presence of 0.1 M corresponding competitive
sugar.
It was critical to incubate the first plate in BSA
and transfer the treated J2 to a clean microfilter plate
before addition of peroxidase substrate to reduce background
to negligible levels. The reduction in background levels
allowed the detection of differential amounts of lectin in
microplate wells which contained lectin-treated nematodes.
No peroxidase activity above background levels was detected
among untreated, preinfective Meloidogyne spp. J2 and
lectin wash treatments.
Relatively high numbers of J2 per sample (approx.
2000 J2 per microplate well) were required at the initiation
of each experiment. As many as 75% of the J2/well remained
in the first microfilter plate after transfer of washed


61
nematodes to a fresh microfilter plate for peroxidase
substrate reaction. No J2 were observed in the wash
solution which had passed through the first microfilter
plate. Thus, estimation of the number of nematodes in a
sample was made upon transfer of washed nematodes to the
second microfilter plate.
Approximately 500-2000 J2 per well were used to
quantify the amount of lectin bound to nematodes after
addition of peroxidase substrate. Lectins were most likely
bound to the surface of the Meloidogyne spp. J2 examined,
since microscopic observation of lectin-treated J2 from
aliquots transferred to peroxidase substrate reaction plates
indicated that J2 were intact and viable. Different amounts
of lectin bound to J2 of Mil, Mi3, and Mj (Table 3-1).
Preinfective J2 of Mj bound more SBA, LOT, and WGA than Mil
or Mi3 J2, and preinfective J2 of Mil bound more LPA than Mj
and Mi3 in two experiments. Populations of Mi3 bound less
lectin than J2 of Mil and Mj in all tests except experiment
2 with LOT. Preinfective J2 of Mil bound the most CON A in
experiment 1, and J2 of Mj bound the most CON A in
experiment 2. Considerable differences in the relative
amount of lectin which bound to J2 within a single lectin-
nematode combination were detected between experiment 1 and
experiment 2.
i


62
Table 3-1. Binding of peroxidase-labeled lectins to second-
stage juveniles (J2) of Meloidogyne incognita race 1 (Mil),
M. incognita race 3 (Mi3), and M. javanica (Mj) as
determined by microfiltration assay in two separate
experiments.
Picograms lectin/J2a
Lectin
Mj
Mil
Mi 3
SBAb
Exp. 1
4.20

0.35c
1 .36

0.07
0.63

0.03
Exp. 2
3.63

0.21
1 .30

0.09
0.55
+
0.03
CON A
Exp. 1
0.66

0.04
0.81

0.03
0.62

0.04
Exp. 2
1 .04

0.07
0.86

0.06
0.70

0.04
LOT
Exp. 1
9.07

1 .01
4.79

0.35
4.40

0.49
Exp. 2
6.18

0.43
4.17

0.15
4.33

0.13
WGA
Exp. 1
3.42

0.25
0.39

0.11
0.21

0.03
Exp. 2
1 .92

0.10
0.78

0.07
0.54

0.07
LPA
Exp. 1
1.18

0.05
2.31

0.07
0.65

0.05
Exp. 2
1 .25

0.07
3.13

0.13
0.83

0.05
a Nanograms lectin divided by the number of J2 (ca. 500-
2000 J2) estimated for each sample.
b Soybean agglutinin (SBA); Concanavalin A (CON A); Lotus
tetragonolobus agglutinin (LOT); wheat germ agglutinin
(VIGA); Limulus polyphemus agglutinin (LPA).
c Mean of 20 observations standard error.


63
Discussion
Differences in lectin binding among populations of
Meloidogyne spp. that were not detected in assays with
fluorescent lectins were detected by microfiltration enzyme-
linked lectin assay. Variability in estimation of the
number of J2 per sample most likely influenced the
quantitative differences in lectin binding to J2 determined
among Mil, Mi3, and Mj and between experiments 1 and 2. If
this were a major influence, however, standard errors should
have been greater than those calculated for each experiment.
The quantitative differences in lectin binding detected in
these experiments could have been due to the production of
carbohydrates in amphidial secretions of J2 of Meloidogyne
spp. as reported above and elsewhere (76). Differences
between experiments 1 and 2 may be due to the handling and
relative age of the groups of J2 used in separate
experiments. The rate of production of amphidial secretions
by J2 of Meloidogyne spp. and the amount of amphidial
secretion lost (if any) through the initial centrifugation
or microfiltration wash remains unknown. Loss of excess
amphidial secretion was noted in preliminary lectin-TRITC
assays when centrifugation was used for all nematode washes,
but this occurrence was inconsistent (unpublished
results).
i


64
The technique described here for quantifying lectin
binding to Meloidogyne spp. is useful because it is rapid
and provides ease of handling compared to techniques used
previously to quantify lectin binding to nematodes and
bacteria (77, 88). The assay is very sensitive (nanogram
level), but a considerable number of nematodes (several
hundred) was necessary to achieve measurements above
background levels. The potential use of this technique for
quantification of lectin binding to other microorganisms or
detection of nematode surface antigens by immunoassay should
be considered.


CHAPTER 5
REPRODUCTION OF LECTIN-TREATED MELOIDOGYNE SPP.
IN TWO RELATED SOYBEAN CULTIVARS
Introduction
Interactions between nematodes and other organisms
are influenced by chemosensory stimuli (22, 29, 40, 45, 86,
131, 136). Researchers have postulated that intervention in
host finding and recognition of nematodes may be achieved by
blockage or obliteration of carbohydrates on nematode
surfaces (135, 136). Proteins (lectins) that bind mannose,
glucose, and sialic acids, and enzymes (glycohydrolases)
which may cleave these carbohydrates from nematode surfaces
impaired nematode chemotaxis toward source attractants (49,
53). Adhesion of conidia of Meria coniospora Drechs. to
nematode chemosensory organs, nematode attraction to the
fungus, and infection of nematodes by adhering conidia were
inhibited by sialic acids, sialidase, or limulin (50, 51,
52). A lectin specific for mannose appeared to inhibit
chemoreception necessary for the feeding and sexual
attraction of males of Trichostrongylus columbriformis Giles
(14). Capture of nematodes by Arthrobotrys oligospora Fres,
appeared to involve interaction of lectin on fungal traps
65


66
with N-acetylgalactosamine moieties present on the nematode
surface (17, 81).
Other studies have reported the presence of
carbohydrates on the head region and surface of some
phvtoparasitic nematodes (see Chapter 2 above, 36, 76, 77,
95, 113, 115). Various lectins bound to carbohydrates
present in amphidial exudates of second-stage juveniles (J2)
of potato cyst and root-knot nematodes (see Chapter 2 above,
36, 76). Differences in structure and relative amounts of
specific carbohydrates in amphidial carbohydrate complexes
of several populations of Meloidogyne spp. have been
reported above in Chapters 2 and 4.
Nematode surface carbohydrates may be involved in
plant-nematode interactions (see Chapter 3 above, 74). Soil
applications of Concanavalin A (Con A; mannose and glucose-
specific lectin), and relatively higher concentrations of
Limax flavus agglutinin (LFA; sialic acid-specific lectin),
significantly suppressed galling of tomato roots induced by
Meloidogyne incognita (Kofoid & White) Chitwood (74).
Treatment of J2 from a population of race 3 of M. incognita
with various lectins and carbohydrates promoted
hypersensitivity in an apparently compatible soybean-M.
incognita interaction (see Chapter 2 above). Nematodes
could not be detected within soybean roots after inoculation
of root tips with J2 of Meloidogyne spp. suspended in


67
solutions of LFA or sialic acid. The objective of this
research was to determine the quantitative effect of several
lectins and carbohydrates on establishment and reproduction
of three populations of Meloidogyne spp. in two related
soybean (Glycine max (L.) Merr.) cultivars.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
(Mil and Mi3) and M. javanica (Treub) Chitwood (Mj) were
maintained in greenhouse culture on roots of 'Rutgers'
tomato (Lycooersicon esculentum Mill.) and 'Black Beauty'
eggplant (Solarium melongena L.). Meloidogyne spp.
populations were identified by adult female perineal
patterns, second-stage juvenile (J2) lengths, and
performance on differential hosts (101). Species
identifications were also confirmed by three independent
nematode taxonomists (A. M. Golden, Beltsville, MD; J. G.
Baldwin, Riverside, CA; J. D. Eisenback, Blacksburg, VA).
Eggs of each nematode population were extracted from host
roots with 0.53% NaOCl for 30 seconds (46) and hatched at
room temperature on a Baermann funnel. Preinfective J2
which had hatched within 48 hours were used as test
organisms in each experiment.
Surface carbohydrates of Meloidogyne spp. J2 were
blocked by incubating nematodes in solutions containing


68
unconjugated, purified soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), CON
A, or Limulus polvphemus agglutinin (LPA) (E-Y Labs, San
Mateo, CA). The sugar specificity of each lectin,
corresponding competitive sugars, and procedure used to
determine the specific hemagglutination activity for each
lectin were described in Chapter 2. Buffer solutions
included: 0.01 M phosphate-buffer saline (PBS) at pH 7.2 for
SBA, WGA, and LOT; 0.05 M Tris-saline plus 0.01 M CaCl2 at
pH 7.5 for CON A; 0.05 M Tris-saline plus 0.01 M CaCl2 at pH
8.0 for LPA.
Preinfective J2 of Mil, Mi3, and Mj were
concentrated in the appropriate buffer or in distilled water
by centrifugation at 1 000 each lectin included incubating J2 (approx. 2000 J2) of each
population in lectin solution (200 ug/ml), lectin (200
ng/ml) plus 0.1M competitive sugar, and 0.1M sugar solution
minus lectin for 2 hours at 4C. Control treatments
included J2 in buffer and J2 in distilled water incubated
for 2 hours at 4C. Suspensions of J2 in each treatment
(1.0 ml total volume per treatment) were diluted to 16 ml
(12.5 ng/ml lectin and/or 6.25mM sugar) immediately before
being added to soil in which soybeans were grown as
described below.
i


69
Since a 0.1 M solution of sialic acid in 0.05 M
Tris-saline buffer was quite acidic (pH ~ 3.0), a soybean
root penetration bioassay was conducted to evaluate the
effects of sialic acid neutralized with NaOH (pH 7.0) and of
an acidic buffer (pH 3.0) on activity of J2 of Meloidogyne
spp. (see Appendix D below). Solutions containing J2 of
Mil, Mi3, and Mj were incubated for 2 hours at 4C, diluted
1:16 with Tris-saline buffer, placed on soybean roots, and
the number of 32 within roots was determined after 24 hours.
Two related cultivars of soybean (Glycine max cv.
Pickett 71 and Centennial) were used for root challenge by
J2 of Meloidogyne spp. Pickett 71 was compatible and
Centennial was incompatible with M. incognita, and both
soybean cultivars were compatible with M. javanica (61).
Individual soybean seedlings were grown in a greenhouse in
150-cm^ Conetainers (Leach Nursery, Canby, OR) containing
steam-pasteurized Astatula fine sand (hyperthermic, uncoated
typic quartzipsamments). Two-milliliter suspensions
(approx. 2000 32) of each treatment combination were added
to the soil in each Conetainer using a syringe fitted with a
10-cm-long canulus (24). There were four replicates of each
treatment combination. Test plants were maintained in a
glasshouse at 27 3C, watered daily, and fertilized once a
week with a solution containing 10-6-10 (N-P-K) plus


70
microelements. Experiment 1 was conducted in the spring and
experiment 2 was conducted in the summer.
Soybean plants were removed from Conetainers 60 days
after soil was infested with J2 and the roots were rinsed
free of soil. The number of Meloidogyne spp. egg masses per
root system was rated on a 0-5 scale (101 ). Data were
subjected to analysis of variance procedure and treatment
differences were determined by the Waller-Duncan k-ratio t-
test with k=100 (P £ 0.05). This experiment was repeated
once.
Results
Hemagglutination assays indicated that the binding
capacity of pure lectins was relatively strong, except for
LPA. Specific hemagglutination activities of 4096, 4096,
8192, 8192, and 16 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LPA, respectively. Hemagglutination
activity of all lectins was completely inhibited in the
presence of 100 mM corresponding competitive sugar.
Pickett 71 soybean was highly compatible with Mil in
two experiments, as indicated by the high egg mass ratings
in buffer and water controls (Table 4-1). Little reduction
in Mi 1 reproduction in Pickett 71 compared to controls was
demonstrated by any lectin and sugar treatment except LPA
plus sialic acid and sialic acid alone in the second test.
This was not, however, verified by the results of the first


Table 4-1. Reproduction of Meloidogyne incognita race 1 in 'Pickett 71' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass rating3/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment
Lectin
1
4.50
*
a
3.50
abede
3.75
abed
4.50
a
3.00
ede
Lectin +
sugar
4.25
ab
3.50
abede
3.00
ede
3.75
abed
2.75
de
Sugar
3.25
bcde
3.75
abed
3.25
bcde
4.25
ab
2.50
e
Buffer
4.00
abc
3.50
abede
3.50
abede
4.00
abc
2.50
e
Distilled
water
4.00
abc
3.75
abed
4.25
ab
4.25
ab
3.00
ede
Experiment
Lectin
2
4.50
abc
4.25
bed
4.50
abc
4.00
cd
4.50
abc
Lectin +
sugar
4.25
bed
4.75
ab
4.75
ab
5.00
a
1 .00
e
Sugar
4.75
ab
3.75
d
4.50
abc
4.50
abc
3.75
d
Buffer
4.50
abc
4.00
cd
4.50
abc
5.00
a
4.75
ab
Distilled
water
5.00
a
4.75
ab
4.25
bed
4.75
ab
5.00
a
3 Scale;
0 = 0; 1
CM
1
II
2 = 3
i
o
^
u>
II
1
u>
o
^
4 =
31-100;
5 = >100 egg
masses
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.


72
test. Reproduction of Mil on Pickett 71 in experiment 2 was
significantly lower for LPA plus sialic acid compared to all
other lectin plus sugar treatments. The rating for LPA plus
sialic acid in the first experiment, however, was only
significantly lower than that of SBA plus galactose.
Treatment with LPA alone in the first experiment,
significantly reduced reproduction of MI1 in Pickett 71
compared to SBA and CON A alone. Sialic acid significantly
reduced the egg mass rating in experiment 1 compared to N-
acetylglucosamine and mannose. Both buffer and water
controls for LPA produced relatively low egg mass ratings,
however, compared to all other treatments in experiment 1.
Centennial soybean was highly incompatible with Mil,
as indicated by poor nematode reproduction among all lectin,
sugar, and control treatments (Table 4-2). Significantly
lower egg mass ratings were produced in Centennial by Mil
for several treatments in experiment 2; however the ratings
were very low overall, and the few lower ratings in
experiment 2 were not verified by results from the first
experiment.
Race 3 of M. incognita was highly compatible with
Pickett 71; almost all treatments and controls had high egg
mass ratings (Table 4-3). Treatment with sialic acid alone
and sialic acid plus LPA significantly reduced reproduction
of Mi3 in Pickett 71 over that in the controls in both
i


Table 4-2. Reproduction of Meloidogyne Incognita race 1 in 'Centennial' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
1 .00
*
a
0.50
a
0.75
a
0.75
a
0.75
a
Lectin + sugar
1 .50
a
1 .00
a
1 .25
a
1 .25
a
0.50
a
Sugar
1 .75
a
1 .25
a
1 .00
a
1 .00
a
0.50
a
Buffer
1 .25
a
1 .25
a
0.75
a
1 .00
a
1 .00
a
Distilled water
1.25
a
1 .50
a
1 .25
a
1 .75
a
1 .25
a
Experiment 2
Lectin
1 .25
abcde
1 .75
abc
1 .75
abc
1 .00
bede
1 .50
abed
Lectin + sugar
1 .50
abed
1 .75
abc
1 .25
abcde
1 .50
abed
0.75
ede
Sugar
1 .75
abc
1 .25
abcde
1 .50
abed
1 .25
abcde
0.25
e
Buffer
1 .50
abed
2.00
ab
1 .50
abed
1 .75
abc
1 .50
abed
Distilled water
2.25
a
1 .50
abed
1 .75
abc
1.25
abcde
0.50
de
a Scale: 0 = 0; 1
= 1-2;
2 = 3-
10; 3 .
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
-j
u>


Table 4-3. Reproduction of Meloidogyne incognita race 3 in 'Pickett 71* soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
4.75
ab*
4.75
ab
4.75
ab
5.00
a
4.00
bed
Lectin + sugar
4.50
abc
3.75
cd
4.50
abc
3.25
de
2.50
ef
Sugar
5.00
a
3.75
cd
5.00
a
4.50
abc
2.25
f
Buffer
4.50
abc
4.00
bed
4.75
ab
4.75
ab
4.75
ab
Distilled water
4.75
ab
4.75
ab
4.75
ab
4.50
abc
4.50
abc
Experiment 2
Lectin
4.50
ab
4.25
be
5.00
a
4.50
ab
4.50
ab
Lectin + sugar
5.00
a
4.75
ab
4.50
ab
4.50
ab
1 .25
d
Sugar
4.75
ab
4.50
ab
5.00
a
4.75
ab
3.75
c
Buffer
5.00
a
4.75
ab
4.75
ab
5.00
a
4.50
ab
Distilled water
5.00
a
4.75
ab
5.00
a
5.00
a
5.00
a
a Scale: 0 = 0; 1
CN
1
r
II
2 =
3-10; 3 :
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (VIGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
5|C
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
-j


75
experiments. Moderate reduction in Mi3 reproduction in
Pickett 71 was observed for CON A plus mannose treatment, as
compared to the controls, in experiment 1 but not in
experiment 2.
Centennial soybean was relatively incompatible with
Mi3, as indicated by low egg mass ratings for the majority
of treatments in two experiments (Table 4-4). Significant
differences in Mi3 reproduction in Centennial were observed
for some treatments, but ratings were low overall and none
of the reductions in one of the tests was verified by a
similar reduction in the other test.
Pickett 71 soybean was compatible with Mj, as
indicated by high egg mass ratings for many lectin, sugar,
and control treatments (Table 4-5). Reproduction of Mj in
Pickett 71 was significantly reduced by sialic acid and
sialic acid plus LPA treatment compared to all other
treatments in the second experiment. Treatment with sialic
acid alone and LPA plus sialic acid significantly reduced
reproduction of Mj in Pickett 71 compared to all other
sugar, and lectin plus sugar treatments, respectively, in
experiment 1. Ratings for sialic acid and LPA plus sialic
acid were not significantly lower, however, than those for
buffer and water controls of LPA in the first experiment.
Relatively high egg mass ratings for many lectin,
sugar, and control treatments indicate that Centennial


Table 4-4. Reproduction of Meloidogyne incognita race 3 in 'Centennial' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
1 .00
*
c
1 .00
c
1 .00
c
2.00
abc
2.00
abc
Lectin + sugar
1 .00
c
2.00
abc
1 .25
be
2.25
ab
1 .25
be
Sugar
1.50
abc
1 .75
abc
1 .25
be
1 .75
abc
1 .50
abc
Buffer
2.50
a
1 .50
abc
1 .00
c
2.25
ab
1 .75
abc
Distilled water
2.25
ab
1 .75
abc
1 .50
abc
1 .25
be
1 .25
be
Experiment 2
Lectin
2.50
ab
1 .75
bed
2.00
abc
2.00
abc
1 .50
cd
Lectin + sugar
2.75
a
2.50
ab
2.00
abc
2.50
ab
1 .00
d
Sugar
2.50
ab
2.50
ab
2.25
abc
2.00
abc
1 .75
bed
Buffer
2.50
ab
2.75
a
2.00
abc
2.25
abc
2.75
a
Distilled water
2.75
a
2.50
ab
2.25
abc
1 .75
bed
2.50
ab
a Scale: 0 = 0; 1
= 1-2;
2 =
3-10; 3 :
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
o\


Table 4-5. Reproduction of Meloidogyne javanica in 'Pickett 71' soybean roots after
treatment of second-stage juveniles with selected lectins and their competitive
sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment
Lectin
1
3.75
,*
ab
2.50
edefg
3.25
abed
2.00
ef g
2.75
bedef
Lectin +
sugar
3.00
abcde
3.75
ab
4.00
a
3.00
abcde
1 .50
g
Sugar
3.00
abcde
3.25
abed
3.75
ab
3.75
ab
1 .75
fg
Buffer
3.25
abed
3.50
abc
3.00
abcde
2.25
defg
2.00
ef g
Distilled
water
3.25
abed
3.50
abc
2.25
def g
3.00
abcde
1 .50
g
Experiment
Lectin
2
4.00
bed
4.50
abc
4.50
abc
3.00
e
4.25
abed
Lectin +
sugar
3.50
de
4.75
ab
4.75
ab
4.50
abc
1 .25
f
Sugar
4.00
bed
3.75
ede
5.00
a
4.50
abc
1 .00
f
Buffer
4.00
bed
4.25
abed
4.25
abed
4.50
abc
4.25
abed
Distilled
water
4.75
ab
4.50
abc
4.25
abed
4.25
abed
4.50
abc
a Scale:
0 = 0; 1
= 1-2;
2 = 3-
o

U)
II
1
OJ
o
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
*
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.


78
soybean was strongly compatible with Mj (Table 4-6).
Reproduction of Mj in Centennial was significantly reduced
by sialic acid and LPA plus sialic acid compared to all
other treatments in experiment 2. Treatment with LPA plus
sialic acid significantly reduced egg mass ratings in the
first experiment compared to SBA plus galactose and WGA plus
N-acetylglucosamine. Reproduction of Mj in Centennial was
significantly reduced by treatment with sialic acid in
experiment 1, compared to egg mass ratings for galactose, N-
acetylglucosamine, and fucose. Ratings for sialic acid
alone and sialic acid plus LPA, however, were not
significantly lower than those for buffer and water controls
of LPA in the first experiment.
Discussion
Results from this research generally agree with the
histological observations reported above in Chapter 3. The
inhibition of reproduction of untreated Mi3 in Centennial
soybean roots, however, contrasts with the apparently
compatible response of Centennial root tissue to untreated
Mi3 observed in histological tests. Intact giant cells were
associated with untreated Mi3 in Centennial soybean roots 20
days after exposure of roots to infective J2 of Mi3.
However, no gall formation or development of Mi3 past third-
stage juvenile was observed 20 days after inoculation.


Table 4-6. Reproduction of Meloidogyne javanica in 'Centennial' soybean roots after
treatment of second-stage juveniles with selected lectins and their competitive
sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA
LOT
CON
A
LPA
Experiment
1
Lectin
4.50
ab*
3.00
edef
3.75
abed
2.25
fg
4.00
abc
Lectin +
sugar
3.75
abed
4.00
abc
3.00
edef
2.75
def g
1 .75
fg
Sugar
3.50
bede
4.00
abc
4.25
ab
3.00
edef
2.00
fg
Buffer
4.50
ab
4.75
a
4.50
ab
2.50
ef g
3.00
edef
Distilled
water
3.50
bede
3.50
bede
4.50
ab
3.50
bede
2.50
ef g
Experiment
2
Lectin
4.25
abc
4.50
abc
4.75
ab
4.00
be
4.00
be
Lectin +
sugar
4.00
be
4.75
ab
4.50
abc
4.00
be
1 .25
d
Sugar
4.25
abc
4.75
ab
4.25
abc
3.75
c
1 .75
d
Buffer
4.75
ab
5.00
a
4.50
abc
4.75
ab
4.50
abc
Distilled
water
4.75
ab
4.25
abc
4.75
ab
4.25
abc
4.00
be
a Scale:
0 = 0; 1
= 1-2;
2 = 3
-10; 3 =
= 11-30;
4 =
31-100;
5 = >100 egg
masses,
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P £ 0.05) according the the
Waller-Duncan k-ratio t-test.


80
Strong reduction in the rate of Mi3 development, or a
possible nutritional deficiency which culminated in nematode
death, may have occurred in this host-parasite relationship
(91, 133) since no evidence of active plant defense
(ie. hypersensitivity) occurred in histological tests.
Differences in the degree of incompatibility of "M.
incognita-resistant" soybean cultivars with several M.
incognita populations have also been reported (105).
Any effect of lectin or sugar on successful nematode
infection of soybean roots most likely occurred at initial
infection; however, environmental conditions and duration of
the experiment were conducive to at least two generations of
root-knot nematode reproduction. One investigation has
indicated that soil application of CON A significantly
reduced galling of tomato roots by M. incognita (74), but
the activity of CON A in soil was difficult to interpret.
Although moderate reductions in egg mass ratings were
occasionally associated with CON A, sialic acid appeared to
have the greatest and most consistent adverse effect on
successful nematode infection. These results are supported
by the apparent inability of several Meloidogyne spp. J2 to
penetrate soybean roots in "unwashed" sialic acid and LFA
treatments (see Chapter 3 above). Hemagglutination tests
determined that the binding capacity of LPA was relatively
weak, and it was completely inhibited in the presence of 100


81
mM sialic acid. This may indicate that concentrations of
LPA when mixed with sialic acid were insufficient to inhibit
(and may have acted in combination with) the activity of
sialic acid on root-knot nematode infection of soybean
roots. Threshold levels of sialic acid that significantly
inhibit nematode infection need to be determined.
Microscopic observation of J2 treated with sialic acid and
LPA, and penetration of soybean roots by J2 treated with
sialic acid and LFA and "washed", indicated that these
treatments are not lethal to J2 of Meloidogyne spp. (see
Chapters 2 and 3 above). The inhibition of soybean root
penetration after treatment of J2 of Meloidogyne spp. with
sialic acid was apparently more than just an adverse effect
of low pH (see Appendix D below). The adverse effect of
sialic acids on Meloidogyne spp. reproduction in soybean may
be manifested in impairment of host-finding and penetration
by treated J2. Perhaps sialic acids act as "biological
masks" similar to those found in other animal systems (104).
Subsequent investigations of these phenomena may provide
information valuable to the development of novel means of
nematode management.
i


CHAPTER 6
SUMMARY AND CONCLUSIONS
Proteins (lectins) which bind to specific
carbohydrates were used as probes to characterize
carbohydrates on the surfaces of second-stage juveniles (J2)
of the root-knot nematodes Meloidogyne incognita races 1 and
3 (Mil and Mi3) and Meloidogyne j avanica (Mj). The binding
of the fluorescent (rhodamine conjugated) lectins, soybean
agglutinin (SBA), Concanavalin A (CON A), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA) to freshly-hatched,
preinfective J2 of root-knot nematodes was comparable among
the populations of Melodogyne spp. examined. It was
apparent from these experiments that nematode viability was
critical for accurate detection of fluorescent lectin
binding to nematodes, and that rhodamine (TRITC) conjugates
of lectin were preferable to fluorescein (FITC) conjugates
since J2 of Meloidogyne spp. autofluoresced at the
excitation wavelength of FITC. Viable, preinfective J2
bound fluorescent lectin almost exclusively in the vicinity
of the amphidial (cephalic chemosensillae) openings.
Substances to which lectins bound were concentrated and
82


83
sometimes emanated from the amphidial region of J2 of
Meloidogyne spp. Amphids of J2 of Mil. Mi3, and Mj labeled
weakly with SBA, CON A, and LPA and strongly with WGA and
LOT. The greater intensity of fluorescent labeling by WGA
and LOT conjugates may have been due to their relatively
higher binding capacities as indicated by hemagglutination
assays. The presence of fucosyl and sialyl residues in
amphidial secretions was supported by the inhibition of LOT
and LPA binding, respectively, in the presence of 0.1 M
corresponding competitive sugars. The binding of SBA, CON
A, and WGA was not inhibited in the presence of 0.1 M
corresponding competitive sugars. This probably indicates
nonspecific binding of the lectins to nematodes, or possibly
a strong affinity of SBA, CON A,and WGA for carbohydrate-
specific sites in amphidial secretions. Different molecular
forms of the competitive sugars chosen may have provided
greater inhibition of lectin binding activity. Soybean
agglutinin, CON A, and WGA may have bound to hydrophobic
sites (ie. lipids) on the nematode surface, and would not
have been influenced by competitive sugars. All fluorescent
lectins tested bound to egg shells of Mil, Mi3, and Mj and
were not inhibited in the presence of competitive sugars.
Results from treatment of J2 with enzymes
(glycohydrolases) that cleave specific carbohydrate residues
from glycoconjugates, including a-galactosidase, a-
i


84
mannosidase, S-N-acetylglucosaminidase, a-fucosidase, and
sialidase (neuraminidase), and subsequent binding of
fluorescent lectins to treated J2 suggested that differences
in the sequence and spatial arrangement of amphidial
carbohydrate complexes exist among Mil, Mi3, and Mj. A
number of different glycohydrolases eliminated binding of
LPA-TRITC to amphids of J2 which suggested that sialic acids
were some of the outermost carbohydrate moieties present in
J2 amphidial carbohydrate complexes. Several
glycohydrolases promoted binding of CON A to the anterior
cuticle of Mil and Mj and WGA to the anterior cuticle of
Mil .
Quantitative differences in lectin binding to J2 of
Mil, Mi3, and Mj were determined by a modified
microfiltration enzyme immunoassay developed for use with
peroxidase-labeled lectins instead of antibody probes.
Preinfective J2 of Mj bound the greatest amount of SBA, LOT,
and WGA while J2 of Mil bound the most LPA in two separate
experiments. Preinfective J2 of Mi3 consistently bound the
least amount of all lectins tested. This may indicate that
preformed carbohydrates in amphidial secretions of J2 differ
quantitatively, as well as in configuration, among different
populations of Meloidogyne spp. The rate of production of
amphidial secretions by J2 of Meloidogyne spp. has not been
determined. The microfiltration assay using peroxidase-


85
labeled lectins was rapid and relatively easy to conduct.
Although the assay was sensitive (nanogram level), it
required relatively high numbers (approx. 2000) of J2 per
sample to achieve final levels of lectin binding that were
sufficiently higher than background levels.
Differential lectin binding to the head region of
preinfective J2 suggested that carbohydrates were
concentrated in this portion of invasive juveniles. Since
the anterior end of these nematodes appears to stimulate
hypersensitive incompatible (resistant) plant responses, it
was hypothesized that blocking of preformed amphidial
carbohydrates with lectins might influence recognition and
specificty in incompatible and compatible Meloidogyne spp.-
soybean interactions. The response of root tissue was
examined histologically 5 days after exposure of M.
incognita-compatible 'Pickett 71' and M. incognijta-
incompatible 'Centennial' soybean roots to lectin and/or
sugar-treated J2 of Mil, Mi3, and Mj. Untreated J2 of all
three root-knot nematode populations induced the formation
of feeding sites (giant cells) in Pickett 71 soybean roots,
and untreated J2 of Mi3 and Mj induced giant cell formation
in Centennial soybean roots 5 days after inoculation. Giant
cells were maintained in Centennial 20 days after
inoculation with untreated J2 of Mi3, but no gall formation,
hyperplasia of pericycle cells adjacent to giant cells, or
i


86
development of Mi3 past third-stage juvenile was observed at
day 20. Untreated J2 of Mil induced a hypersensitive
response (HR; localized plant tissue necrosis) in Centennial
soybean roots 5 days after inoculation. Treatment of J2 of
Mil and Mj with SBA, CON A, WGA, LOT, LPA and/or their
corresponding competitive sugars did not influence the root
tissue response of either soybean cultivar to infection by
these nematodes. Hence, the surface carbohydrates of Mil
and Mj did not appear to be involved in plant-nematode
interactions, but the fate of lectin bound to J2 once they
have entered the plant root remains unknown. Treatment of
J2 of Mi3 with any lectin and/or sugar tested, however,
induced the formation of HR in Centennial soybean root
tissue 5 days after inoculation. Treatment of J2 of Mi3 in
this manner may actually have facilitated recognition of
invasive J2 and subsequent defense response by the plant.
The lack of specificty of lectin or sugar effects in the
Mi3-Centennial interaction, however, makes it seem unlikely
that alteration of surface carbohydrate composition of
preinfective J2 and recognition by a carbohydrate-specific
plant receptor was responsible for promoting
incompatibility. If one considers the interaction of
nematode surface carbohydrates with potential plant cell
surface receptors as a "lock and key" phenomenon, however,
it may be feasible that a slight alteration in surface


87
carbohydrate compostion was sufficient to promote
incompatibility to Mi3 in Centennial soybean roots.
Possibly a greater alteration of the carbohydrates examined
here on Mil and Mj, or alteration of surface carbohydrates
not examined in these studies, would influence their host-
parasite interactions.
Treatment of J2 of Mi3 with lectin or sugar may have
stimulated the production of a substance by the nematode
that induces HR in Centennial soybean roots. Juveniles of
Mil may inherently have the capacity to induce HR while J2
of Mj cannot promote incompatibility in soybean no matter
what the treatment. For some populations, such as in the
case of Mi3, incompatibility may be a process that can be
stimulated. Conversely, substances produced by J2 of
Meloidogyne spp. (ie. amphidial or stylet secretions) may be
essential to induce compatibility between host and parasite,
and these substances were altered sufficiently in Mi3 to
inhibit compatibility in Centennial soybean roots.
The ability of J2 of Mil, Mi3, and Mj to penetrate
the roots of either soybean cultivar was apparently strongly
impaired when J2 were introduced to roots in a solution that
contained Limax flavus agglutinin (LFA; sialic acid-
specific), sialic acid, or combination of the two. The
inhibition of soybean root penetration after sialic acid
treatment of J2 of Meloidogyne spp. was apparently more than


88
just an adverse effect of low pH. These treatments were not
lethal to J2 of Meloidogyne spp. since J2 that were treated
with LFA and/or sialic acid and rinsed regained their
infectivity to both soybean cultivars. In addition, J2
treated with LPA-TRITC and/or sialic acid and rinsed were
viable when observed microscopically.
The results of histological experiments were
generally confirmed by greenhouse assays that were designed
to quantify the effects of SBA, CON A, WGA, LOT, LPA, and
their competitive sugars on Meloidogyne-soybean
interactions. The reproduction of lectin and/or sugar-
treated J2 of Mil, Mi3, and Mj in Centennial and Pickett 71
soybean roots was determined by rating the number of root-
knot nematode egg masses per root system 60 days after
inoculation with treated J2. The reproduction of untreated
Mi3 in Centennial soybean was extremely poor, in contrast to
the apparent compatibility (giant cells) observed in
Centennial root tissue after exposure of soybean roots to
untreated J2 of Mi3. Only treatment of J2 of Mil, Mi3, and
Mj with sialic acid, and especially sialic acid plus LPA,
significantly reduced reproduction of these nematodes in
compatible soybean cultivars. This may have been an
indirect result of reduced initial root penetration by J2 of
Meloidogyne spp. that were exposed to these treatments. It
is possible that sialic acid residues proximate to nematode


89
cheraosensillae have a masking or regulatory effect similar
to that observed in other animal systems. Modification of
sialic residues in nematode chemosensory organs may strongly
impair host finding and penetration of plant roots by root-
knot nematodes.
i


APPENDIX A
BINDING OF FLUORESCENT SOYBEAN AGGLUTININ TO POSTINFECTIVE
SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.
Alteration of nematode surface or secretory
carbohydrates may influence plant compatibility with phyto-
parasitic nematodes. Changes in surface carbohydrates of
second-stage juveniles (J2) of Meloidogyne spp. which may
occur once J2 enter plant roots have never been studied.
Nematode penetration and subsequent interaction with plant
roots may stimulate or alter production of secretory or
surface molecules by the nematode. Conversely, plant
products such as lectins or carbohydrates may bind to sites
which exist on infective J2 and influence plant-nematode
interactions. We have conducted preliminary experiments to
monitor potential changes in surface carbohydrates of J2 of
several Meloidogyne spp. populations after they have entered
roots of compatible and incompatible soybean cultivars.
Seeds of 'Pickett 71' and 'Centennial' soybean were
germinated in ragdolls and placed on trays of autoclaved
sand as described for histology experiments (see Chapter 3
above). One hundred-microliter suspensions (approx. 2000 J2
hatched within 48 hours of inoculation) of Meloidogyne
incognita races 1 and 3 (Mil, Mi3), and M. javanica (Mj) in
90
i


91
tap water were placed on separate soybean root tips of both
cultivars. Soybean roots were washed free of sand and
inoculated segments of roots were excised and placed in
phosphate-buffer saline (PBS), pH 7.2, approximately 40
hours after inoculation.
Excised root segments of each Meloidogyne spp.
population-soybean cultivar combination were immediately
decorticated under a 40x dissecting microscope. Soybean
root steles and cortices containing Meloidogyne spp. J2 were
placed in separate BPI dishes containing PBS. The root
tissue was incubated at room temperature overnight to allow
J2 to emerge from the plant tissue. These "postinfective"
J2 were washed three times with PBS and subsequently
incubated in fluorescent, soybean agglutinin-
tetramethylrhodamine isothiocyanate (SBA-TRITC) solution
(200 ug/ml), 0.1 M D-galactose solution plus SBA-TRITC (200
ng/ml), 0.1M D-galactose solution minus SBA-TRITC, or
distilled water for 2 hours at 4C (see Chapter 2 above).
Treated postinfective J2 were washed three times with PBS,
mounted on glass slides, and immediately observed under
epifluorescent microscopy.
Little difference in labeling of postinfective J2
with fluorescent SBA was observed among Meloidogyne spp.
populations, soybean cultivars, and J2 from root cortices or
steles. No fluorescence was observed on any postinfective
i


92
J2 treated with only 0.1M D-galactose or distilled water.
Postinfective J2 of all Meloidogyne spp. populations tested
labeled weakly with SBA-TRITC in the amphidial region,
similar to results obtained with preinfective J2 (see
Chapter 2 above). The cuticles of postinfective J2 of Mil,
Mi3, and Mj labeled strongly with SBA-TRITC, however, and
this binding was greatly inhibited in the presence of 0.1M
D-galactose. Binding of SBA-TRITC to cuticles of
postinfective Meloidogyne spp. J2 was confined to the
anterior half of the body of most nematodes observed (Fig.
A-1). Fluorescent SBA did bind to the cuticle on the
posterior half of some postinfective J2, but SBA-labeling
was often discontinuous ("patchy") along the posterior
cuticle (Fig. A-1).
The binding of SBA to cuticles of postinfective J2
of Meloidogyne spp. and the inhibition of SBA binding in the
presence of D-galactose indicated the presence of galactosyl
residues on the body wall of J2 that had penetrated soybean
roots. The cuticles of preinfective J2 of Meloidogyne spp.
did not bind SBA-TRITC in this manner. It is not known
whether galactosyl residues on the body wall of
postinfective J2 of Meloidogyne spp.are of plant or nematode
origin.


Full Text



THE INTERACTIONS OF SURFACE CARBOHYDRATES OF
MELOIDOGYNE SPP. WITH SOYBEAN ROOTS
By
ERIC L. DAVIS
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
1988

IN MEMORY OF
LAWRENCE DAVIS

ACKNOWLEDGEMENTS
I express my sincere appreciation to Dr. D. T.
Kaplan for serving as chairman of my supervisory committee.
His guidance, assistance, and friendship were invaluable to
this research endeavor.
Special thanks are extended to Dr. D. W. Dickson and
Dr. D. J. Mitchell for serving as members of my supervisory
committee. Their advice and support cannot be
overemphasized.
Appreciation is extended to the professional staff
and technical support, especially Diana Johnson and Janice
Rahill, of the United States Department of Agriculture,
Horticultural Research Laboratory, Orlando, Florida.
I would like to thank Dr. A. M. Golden, Beltsville,
Maryland, Dr. J. G. Baldwin, Riverside, California, and Dr.
J. D. Eisenback, Blacksburg, Virginia, for their assistance
in identification of Meloidogyne spp. Thanks are extended
to Drs. K. Hinson, Gainesville, Florida, and E. E. Hartwig,
Stoneville, Mississippi, for providing soybean seed.
Appreciation is extended to the Union Carbide
Agricultural Products Division, Research Triangle, North
Carolina, for providing financial support for this research
project.
iii
i

TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS iii
ABSTRACT vi
CHAPTER 1 : INTRODUCTION 1
CHAPTER 2: CHARACTERIZATION OF CARBOHYDRATES ON THE
SURFACE OF SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.. 10
Introduction 10
Materials and Methods 12
Results 18
Discussion 27
CHAPTER 3: ROOT TISSUE RESPONSE OF TWO RELATED
SOYBEAN CULTIVARS TO INFECTION BY LECTIN-TREATED
MELOIDOGYNE SPP 32
Introduction 32
Materials and Methods 34
Results 38
Discussion 47
CHAPTER 4: QUANTIFICATION OF LECTIN BINDING TO SECOND-
STAGE JUVENILES OF MELOIDOGYNE SPP 52
Introduction 52
Materials and Methods 54
Results 60
Discussion 63
IV

CHAPTER 5: REPRODUCTION OF LECTIN-TREATED MELOIDOGYNE
SPP. IN TWO RELATED SOYBEAN CULTIVARS 65
Introduction 65
Materials and Methods 67
Results 70
Discussion 78
CHAPTER 6: SUMMARY AND CONCLUSIONS 82
APPENDIX A: BINDING OF FLUORESCENT SOYBEAN AGGLUTININ
TO POSTINFECTIVE SECOND-STAGE JUVENILES OF
MELO IDOGYNE SPP 90
APPENDIX B: VIABILITY OF SECOND-STAGE JUVENILES OF
MELOIDOGYNE SPP. AFTER EXPOSURE TO GLYCOHYDROLASE
BUFFERS 94
APPENDIX C: BINDING OF FLUORESCENT LECTINS TO AXENIZED,
PREINFECTIVE MELOIDOGYNE SPP 97
APPENDIX D: EFFECT OF ACIDITY OF SIALIC ACID ON
PENETRATION OF SOYBEAN ROOTS BY SECOND-STAGE JUVENILES
OF MELO IDOGYNE SPP 100
BIBLIOGRAPHY 104
BIOGRAPHICAL SKETCH 118
v
i

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
THE INTERACTIONS OF SURFACE CARBOHYDRATES OF
MELOIDOGYNE SPP. WITH SOYBEAN ROOTS
By
Eric L. Davis
April 1988
Chairman: David T. Kaplan
Major Department: Entomology and Nematology
Fluorescent (rhodamine) conjugates of the lectins,
soybean agglutinin (SBA), Concanavalin A (CON A), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA) bound exclusively to
amphidial openings and cephalic secretions of preinfective
second-stage juveniles (32) of Meloidogyne incognita races 1
and 3 (Mil, Mi3), and M. javanica (Mj). No substantial
differences in fluorescent lectin-labeling were observed
among preinfective J2 of the Meloidogyne spp. populations
examined, and only binding of LOT and LPA was inhibited in
the presence of 0.1 M competitive sugar. Differences in
structure of amphidial carbohydrate complexes among
populations of Mil, Mi3, and Mj were revealed by
glycohydrolase treatment of preinfective J2 and subsequent
labeling with fluorescent lectins. Several glycohydrolases
vi
i

eliminated binding of LPA to the amphidial region of J2 of
the Meloidogyne spp. tested. Quantitative differences in
binding of the peroxidase-labeled lectins, SBA, CON A, LOT,
WGA, and LPA to J2 of Mil, Mi3, and Mj were determined by
microfiltration enzyme-linked lectin assay. Preinfective J2
of Mj bound the greatest amount of SBA, LOT, and WGA,
whereas preinfective J2 of Mil bound the most LPA in two
separate experiments. Preinfective J2 of Mi3 generally
bound the least amount of all lectins tested.
Treatment of J2 of Mil and Mj with purified,
unconjugated SBA, CON A, WGA, LOT, or Limax flavus
agglutinin (LFA) did not influence root tissue response of
'Centennial' and 'Pickett 71' soybean cultivars to infection
by Mil or Mj. Giant cells were usually associated with
untreated Mi3 in Centennial root tissue 20 days after
inoculation. Treatment of J2 of Mi3 with lectins or
carbohydrates caused Centennial root tissue to respond to
infection by treated Mi3 in a hypersensitive manner.
Nematodes could not be detected within soybean roots 5 days
after inoculation of root tips with J2 suspended in
solutions of LFA or sialic acid. Only treatment of J2 with
sialic acid and sialic acid plus LPA strongly reduced
reproduction of all populations of Meloidogyne spp. in
soybean roots of both cultivars. Treatment of J2 of
Meloidogyne spp. with LFA, LPA, or sialic acid was not
lethal to nematodes.
Vll

CHAPTER 1
INTRODUCTION
Most agricultural crops are vulnerable to attack by
phytoparasitic nematodes. Nematode damage has caused
estimated losses as high as 21% in major U.S. crops (111).
Dowler and Van Gundy (26) recently estimated annual world
crop losses to nematode damage to be in the order of $500
million. Root-knot nematodes (Meloidogyne spp.) are
considered to be the most economically important
phytoparasitic nematodes because of their world-wide
distribution, interaction with other phytoparasitic
organisms, and extensive host range (100). The host range
includes almost all of the plants that account for the
majority of the world's food supply (100, 101, 102, 121).
Four major species of root-knot nematode, Meloidogyne
incognita (Kofoid and White) Chitwood, Meloidogyne javanica
(Treub) Chitwood, Meloidogyne hapla Chitwood, and
Meloidogyne arenaria (Neal) Chitwood comprised over 95% of
the root-knot nematode populations detected in over 500
samples collected from agroecosystems around the world
(100).
1

2
The principal management strategies implemented to
reduce nematode-related crop damage include field
application of fumigant and nonfumigant nematicides,
cultivation of plant varieties which are resistant to
phytoparasitic nematodes, and prudent cultural practices
such as sanitation and crop rotation. Nematicides are
relied upon as the primary means of reducing nematode-
related crop losses in many crops. Their toxic properties,
water solubility, and persistence in the environment have,
however, triggered restriction of nematicide application,
making them unavailable for use or reducing their efficacy
in many economically important crops (54). Nematicides
often cannot be incorporated into agricultural pest
management strategies in developing nations because
chemicals may be inaccessible or too expensive, application
equipment may not be available, and growers may not be
properly educated in the safe and effective use of
pesticides (18, 101). To date, crop rotation is still the
most widely used pest management strategy implemented on a
world-wide basis (41, 73). Difficulties encountered in crop
rotation schemes include identification of potentially
damaging phytoparasitic nematodes and their host ranges,
selection of suitable nonhosts for polyspecific nematode
field populations or species with wide host ranges, and
!

3
production of crops that are economically beneficial for
growers and that are marketable (41).
Cultivation of crops that are resistant to diseases
induced by root-knot nematodes is appealing because it
provides an effective, economical, and environmentally safe
means of reducing nematode-related crop damage (32, 33).
Hundreds of crop cultivars are currently available that
possess resistance to one or more species of root-knot
nematode (32, 33, 103). Resistance is a term that
encompasses two main components: the ability of a plant to
tolerate nematode-related damage or to limit nematode
reproduction (58, 59). Thus, varying degrees of resistance
to phytoparasitic nematodes have been reported among
different plant cultivars (34). Most nematode-resistant
germplasm has been developed towards nematodes that are
endoparasites because, according to Roberts (94), "natural
selection of resistance genes is more likely to have
occurred in the most highly specialized host-parasite
relationships where co-evolutionary development of host and
parasite has produced a highly specific interaction in which
host and parasite compete for a genetic advantage (118)."
This dynamic interaction between host and parasite
on a microevolutionary scale threatens the durability of
resistance in plants to nematodes. Continued cultivation of
nematode-resistant crops on the same parcel of land has

4
selected for populations of phytoparasitic nematodes that
overcome plant resistance to nematode attack (34, 99, 108,
119, 123). Although the genetic basis of inheritance of
resistance to nematodes has been identified for a number of
plant cultivars (9, 34, 55, 108), little is known about how
resistance genes function (58). A greater knowledge of the
mechanisms of plant resistance to nematodes should
facilitate the application of germplasm modification and the
development of bioengineering for the transfer of nematode
resistance genes between plant genomes (34, 58, 71).
A number of studies have investigated the mechanisms
of plant resistance to nematodes and the results have been
discussed in several reviews (37, 39, 42, 58, 59, 97, 127).
The terms "incompatible" and "compatible" are used here to
designate plant-nematode interactions that inhibit nematode
development and reproduction, and interactions which promote
nematode development and reproduction in plants,
respectively (58, 59). Passively or preinfectionally
incompatible plant-nematode interactions may involve
morphological plant barriers to nematode infection or
constitutive plant factors that affect egg hatch, locating a
food source, survivability, and host suitability (32, 35,
39, 58, 59, 97, 127). Actively or postinfectionally
incompatible plant-nematode interaction involves the
elicitation and subsequent sequence of host defense

5
reactions induced by challenges of certain nematode species
or races to specific plant cultivars (32, 58, 59, 127).
The physiological sequence of events following
elicitation of active plant defenses, the "expressive phase"
(63), has been described for a number of incompatible plant-
nematode interactions (58). Inhibition of nematode
development in incompatible plant cultivars is often
associated with hypersensitive reactions (HR) in plant
tissue adjacent to nematodes and in feeding sites shortly
(hours) after nematode or stylet penetration of roots (12,
43, 57-59, 109, 112, 134). Phytoalexin accumulation has
been associated with the HR in some incompatible plant-
nematode interactions (43, 60, 92, 130), and the potential
involvement of phytoalexins in incompatible plant-microbe
interactions has been discussed (7, 42, 59, 68, 128).
The incompatible and compatible interactions of
soybean (Glycine max (L.) Merr.) cultivars with root-knot
nematodes have been examined in detail (28, 30, 56, 60, 61,
129). The introduction and incorporation of genes for
resistance to root-knot nematodes, primarily M. incognita,
into successive soybean cultivars and the development of
soybean resistance-breaking populations of Meloidogyne spp.
have been summarized (33, 105). Resistance to M. incognita
in soybean is conditioned by one major gene with at least
one modifying gene (16, 98), and the degree of resistance

6
varies with the soybean cultivar and the population of M.
incognita examined (8, 10, 15, 65, 96). The tissue reaction
of roots of two related soybean cultivars to infection by
the same population of M. incognita has been examined
progressively from one to twelve days after exposure of
soybean root tips to second-stage juveniles (J2) of M.
incognita (61). Giant cell formation in 'Pickett 71'
soybean roots infected with M. incognita (compatible)
progressed normally throughout the observation period. The
incompatibility of 'Centennial' soybean with M. incognita
was associated with an HR of soybean root tissue in the
region of invading J2 within 3 days of inoculation. The HR
was strongly correlated with the accumulation of the
phytoalexin, glyceollin, but biotic elicitors of the HR were
not identified (60).
Indeed, little is known about the "determinative
phase" (63) of plant-nematode incompatibility; factors are
involved in this phase that enable an incompatible plant
cultivar to recognize a specific potential pathogen and
invoke active plant defenses (58). Since nematodes locate
and penetrate roots of most cultivars in either compatible
or incompatible pathosystems, recognition of endoparasitic
nematodes by incompatible plants appears to occur following
penetration (1, 24, 44, 90, 116, 124). The primary
determinants in the specificity of plant-nematode
i

7
incompatibility probably include the interactions of
nematode and plant cell surfaces, nematode secretions, and
derepression of nematode and plant genomes (58).
The concept that recognition and specificity in
plant-microbe interactions may involve the interaction of
carbohydrate moieties of cell surface glycoconjugates and
corresponding receptors on the surface of cells of which
they come in contact, similar to cell to cell communication
involved in antigenicity, blood group specificity, and
mitogenesis in animal systems, was proposed by Albersheim
and Anderson-Prouty (3). Numerous investigations of this
hypothesis in different plant-microbe systems have been
conducted and reviewed (21, 23, 31, 62, 83, 106, 136). The
potential interaction of bacterial surface carbohydrates and
lectins present on the surface of plant root cells as
determinants of recognition and specificity in mutualistic
Rhizobium spp.-legume interactions has been examined (6, 87,
126). For example, the binding of soybean and clover
lectins appeared to be specific for most nodulating strains
of Rhizobium spp. (13, 25). Some evidence for the
involvement of cell surface interactions in incompatible
plant-microbe interactions includes the elicitation of
glyceollin accumulation in soybean tissue exposed to
polysaccharide (4, 5) and glycoprotein-rich (64) wall
fractions isolated from Phytophthora megasperma Drechs. f.

8
sp. glycinea, and induction of glyceollin accumulation in
soybean tissue exposed to fractions of cellular envelopes
isolated from incompatible races of Pseudomonas glycinea
Coerper (19). Whether interaction of cell surface
macromolecules is important in recognition and specificity
in plant-microbe interactions remains controversial (6, 23,
87, 126). Inconsistencies in some results are difficult to
interpret and may involve factors other than surface to
surface interactions.
Characterization of the surface carbohydrate
composition of a number of nematode species and the
potential involvement of surface carbohydrate interactions
in recognition and specificity between nematodes and other
organisms have been investigated and are discussed in
several reviews (47, 58, 80, 135, 136). Selected evidence
from some of these investigations and information gathered
since publication of the above reviews is mentioned in the
introductions to the chapters within this dissertation.
Zuckerman and Jansson (136) have postulated that specific
interactions between nematodes and other organisms may be
influenced by modification of nematode surface
carbohydrates. The objectives of this investigation were to
characterize the surface carbohydrates of second-stage
juveniles of several populations of Meloidogyne spp. and to
evaluate the potential involvement of nematode surface
i

9
carbohydrates in recognition and specificity in the
incompatible and compatible response of two related soybean
cultivars to infection by M. javanica and races 1 and 3 of
M. incognita.

CHAPTER 2
CHARACTERIZATION OF CARBOHYDRATES ON THE SURFACE OF SECOND-
STAGE JUVENILES OF MELOIDOGYNE SPP.
Introduction
The importance of surface carbohydrate biochemistry
in recognition and specificity between plants and
microorganisms has been the subject of many recent
investigations and discussions (3, 21/ 23, 31, 62, 106).
Although few investigations concerning this phenomena have
been conducted between nematodes and plants, surface
carbohydrates of nematodes have been implicated in
recognition between nematodes and nematophagous fungi (17,
47, 52, 80, 81, 135, 136). The surface carbohydrates of
some helminth parasites of animals have been characterized
and related to helminth antigenicity and chemoresponse (14,
72, 85). The involvement of surface carbohydrate
recognition in the specificity of interaction between
nematodes and Pasteuria cer.etrans Sayre and Starr has also
been investigated (117).
Carbohydrates present on biological surfaces exist
primarily as glvcoconjugates such as glycolipids,
polysaccharides, and especially as glycoproteins (79). The
carbohydrate residues are often comprised of a number of
10

11
monosaccharide molecules covalently linked in various
sequences and spatial arrangements (66, 122). The
accessibility of surface carbohydrates to potential
receptors in other organisms or as receptors of chemostimuli
may be obscured by attached carbohydrate molecules, as is
sometimes the case with sialic acids in animal systems
(biological masks) (104). Enzymatic or inorganic chemical
degradation can reveal "masked" carbohydrates that may exist
on biological surfaces. Conversely, enzymes which cleave
specific carbohydrate residues from glycoconjugates,
glycohydrolases, can remove carbohydrates from biological
surfaces and potentially alter biological interactions. An
example of this latter phenomenon is the apparent loss of
chemosensory perception of culture filtrates of Escherichia
coli (Mig.) Castellani and Chambers by the nematodes
Caenorhabditis elegans (Mau.) Dougherty and Panagrellus
redivivus (L.) Goodey after treatment of these nematodes
with mannosidase or sialidase (49).
Lectins, proteins that bind to specific carbohydrate
residues, make excellent probes for the study of
carbohydrates that exist on biological surfaces (31, 69, 70
107). Several methods, including lectin probes, have been
used to characterize carbohydrates on the surface of a
number of free-living and phytoparasitic nematodes, and the
results of some of these investigations have been summarized

12
(47, 135, 136). Application of lectins to soil infested
with Meloidogyne incognita (Kofoid and White) Chitwood
reduced the number of nematode-induced galls on tomato
roots, but the function of lectins in this system was
unclear (74).
Several studies have attempted to relate nematode
surface carbohydrates to specificity in plant pathogenicity
(36, 76, 95). Differences in binding of fluorescent lectins
to pathotypes of Globodera spp. and Meloidogyne spp. were
reported (36, 76). The objective of this investigation was
to characterize surface carbohydrates of three Florida
populations of preinfective second-stage juveniles of
Meloidogyne spp. using selected lectins and glycohydrolases.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
(Mil and Mi3) and M. j avanica (Treub) Chitwood (Mj) were
maintained in greenhouse culture on roots of 'Rutgers'
tomato (Lycopersicon esculentum Mill.) and 'Black Beauty'
eggplant (Solanum melongena L.). Meloidogyne spp.
populations were typified by adult female perineal patterns,
second-stage juvenile (J2) lengths, and performance on
differential hosts (101). Species identifications were also
confirmed by three independent nematode taxonomists (A. M.
Golden, Beltsville, MD; J. G. Baldwin, Riverside, CA; J. D.
i

13
Eisenback, Blacksburg, VA). Eggs of each nematode
population were extracted from host roots with 0.53% NaOCl
for 30 seconds (46) and hatched at room temperature on a
Baermann funnel. Preinfective J2 that had hatched within 48
hours were used as test organisms in each experiment. A few
eggs of Meloidogyne spp. were present in each suspension of
J2.
Surface carbohydrates of preinfective J2
Fluorescent lectin probes were used to identify and
locate carbohydrates on the surface of preinfective J2 of
Mil, Mi3, and Mj. Tetramethylrhodamine isothiocyanate
(TRITC) conjugates of soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Concanavalin A (CON A), Lotus
tetragonolobus L. agglutinin (LOT), and Limulus polyphemus
L. agglutinin (LPA) (E-Y Labs, San Mateo, CA) were used.
The ratios of absorbance at 550 to 280 nm for SBA, WGA, CON
A, LOT, and LPA were 0.44, 0.55, 0.41, 0.57, and 0.20,
respectively. The specific sugars to which each lectin
binds are listed in Table 1-1.
A small sample of J2 (approx. 1000 J2) of each
Meloidogyne spp. population was suspended in distilled water
to serve as a control treatment. The remaining J2 of each
population were concentrated into 2.0 ml of the appropriate
buffer by centrifugation at 1000g for 3 minutes. Buffer
solutions included: 0.01 M phosphate-buffer saline (PBS) at

Table 1-1. Sugar specificity and competitive sugars of soybean
agglutinin (SBA), wheat germ agglutinin (WGA), Concanavalin A
(CON A), Lotus tetragonolobus agglutinin (LOT), and Limulus
Polyphemus agglutinin (LPA).
Lectin
Sugar Specificity
Competitive Sugar3
SBA
a-D-galactose
N-acetyl-a-D-galactosamine
D-galactose
WGA
N-acetyl-B-D-glucosamine
N-acetyl-D-glucosamine
CON A
a-D-mannose
a-D-glucose
D-mannose
LOT
a-L-fucose
L-fucose
LPA
neuraminic (sialic) acid
N-acetylneuraminic acid
a Corresponding competitive sugars (0.1 M) used for all lectin
and glycohydrolase assays.

15
pH 7.2 for SBA, WGA, and LOT; 0.05 M Tris-saline plus 0.01 M
CaCÍ2 at pH 7.5 for CON A; 0.05 M Tris-saline plus 0.01 M
CaCl2 at pH 8.0 for LPA. Preinfective J2 (approx. 5000 J2)
of each population were incubated in lectin-TRITC conjugate
(200 jig/ml) for 2 hours at 4°C. Additional treatments
included nematodes (approx. 5000 J2) incubated in lectin-
TRITC plus 0.1M corresponding competitive sugar (Table 1-1)
to inhibit lectin binding, J2 incubated in 0.1M sugar plus
buffer, and J2 incubated in buffer minus sugar. Treated J2
were washed by transferring J2 three times to
microcentrifuge tubes that contained fresh buffer or water
and allowing J2 to settle to the bottom of the tube. A
sample of J2 (approx. 500 J2) in final wash solution was
placed on a glass microscope slide and covered with a cover
glass. The edges of the cover glass were sealed with clear
fingernail polish. Approximately fifty specimens from each
treatment were immediately observed at 100x under a Zeiss
epifluorescent microscope equipped with TRITC and FITC
(fluorescein isothiocyanate) filters. Photographs of
selected nematodes were taken when nematode movement ceased
(approx. 1-3 hours after J2 were mounted on slides). Each
test was repeated twice.
The relative binding capacity of each lectin-TRITC
conjugate was determined through hemagglutination assay
(89). Twenty-five microliter volumes of lectin were
i

16
serially diluted (1:1) with the appropriate buffer in
adjacent wells across a 96-well microtiter plate. Twenty-
five microliters of a 4% suspension of trypsinized,
gluteraldehyde-stabilized, human Type 0 red blood cells
(Sigma Chemical Co., St. Louis, MO) were added to each well,
except for wells containing LPA. A 4% suspension of
gluteraldehyde-stabilized, horse red blood cells (Sigma
Chemical Co.,St. Louis, MO) was used for LPA-TRITC assays.
The greatest dilution of lectin that exhibited visible
hemagglutination (titer) was determined after 3 hours
incubation at room temperature. The titer divided by the
milligrams lectin/ml in each sample is a measure of the
specific hemagglutination activity of each lectin-TRITC
conjugate. Similar tests were conducted in the presence of
0.1 M competitive sugar to assess inhibition of lectin
binding activity.
Surface carbohydrates of glycohydrolase-treated
preinfective J2
Enzymes (glycohydrolases) that cleave specific
carbohydrate residues from glycoconjugates were assayed for
their effect on surface carbohydrates of Mil, Mi3, and Mj.
Glycohydrolases tested consisted of the following:
a-galactosidase (a-gal) EC 3.2.1.22 (20 U/mg) from
recombinant E. coli, a-L-fucosidase (a-fuc) EC 3.2.1.51 (2.0
U/mg) from beef kidney, B-N-acetyl-glucosaminidase (B-glu)

17
EC 3.2.1.30 (4.0 U/mg) from beef kidney, a-mannosidase
(a-man) EC 3.2.1.24 (10 U/mg) from Canavalia ensiformis DC.,
neuraminidase (sialidase) EC 3.2.1.18 (1.0 U/mg) from
Clostridium perfringens (Veil, and Zub.) Holland. All
glycohydrolases were obtained from Boehringer Mannheim
Biochemicals, Indianapolis, IN, and enzyme activity was
determined by reaction with the appropriate _p-nitrophenyl
conjugate of each carbohydrate substrate (information
supplied by manufacturer).
Preinfective J2 of Mil, Mi3, and Mj were each
concentrated into 2.0 ml of the appropriate buffer by
centrifugation at 1 000c[ for 3 minutes. Buffer solutions
included 0.01 M phosphate buffer (pH 7.2) for a-gal, 0.05 M
sodium citrate buffer (pH 5.0) for a-fuc, 0.05 M sodium
citrate buffer (pH 4.5) for S-glu, 0.05 M sodium citrate
buffer plus 1.0 mM ZnS04 (pH 4.5) for a-man, 5.0 mM sodium
acetate buffer plus 72.0 mM NaCl and 7.0 mM CaCl2 (pH 5.0)
for neuraminidase. Enzyme buffers were formulated to the pH
optimum of enzyme activity as suggested by the manufacturer.
Preinfective J2 (approx. 5000 J2) of each population were
incubated in either a-gal (1.0 U/ml), a-fuc (0.25 U/ml),
B-glu (1.0 U/ml), a-man (1.0 U/ml), or sialidase (0.25 U/ml)
solution for 18 hours at 37*C. Nematodes were also
incubated in enzyme plus 0.1 M corresponding competitive
sugar (Table 1-1) to inhibit enzyme activity. Control
i

18
treatments consisted of J2 in buffer alone and J2 in buffer
plus 0.1 M sugar at 37®C for 18 hours. Nematode viability
after treatment with enzyme buffers under experimental
conditions was confirmed by bioassay (Appendix B).
Glycohydrolase-treated nematodes were washed three
times with the appropriate lectin buffer and subsequently
treated with separate lectin-TRITC conjugates as described
above for untreated, preinfective J2. Specimens were
immediately mounted on glass microscope slides and observed
under epifluorescent microscopy as described above.
Results
Hemagglutination tests indicated that the binding
capacity of all lectin-TRITC conjugates, except LPA, was
relatively strong. Specific hemagglutination activities of
1024, 512, 4096, 2048, and 16 units/mg lectin were
determined for SBA, CON A, WGA, LOT, and LPA, respectively.
Hemagglutination activity of all lectin-TRITC conjugates was
completely inhibited in the presence of 0.1 M corresponding
competitive sugar.
Viable, preinfective J2 were labeled with lectin-
TRITC almost exclusively in the vicinity of the amphidial
openings (Figs. 1-1 a - 1-1c). Fluorescent lectin labeling
often extended outward from these openings, suggesting that
carbohydrates occur within amphidial secretions. Binding of

Fig. 1-1. Binding of fluorescent (rhodamine) lectin
conjugates to Meloidogyne spp. second-stage juveniles (J2)
and egg. a) Strong amphidial (SA) fluorescence of M.
incognita race 3 (Mi3) labeled with Lotus tetragonolobus
agglutinin, b) Binding of wheat germ agglutinin (WGA) to
amphidial secretions (AS) of M. javanica (Mj) after a-
galactosidase treatment, c) Weak amphidial fluorescence (WA)
of M. incognita race 1 (Mil) labeled with Limulus polyphemus
agglutinin, d) Fluorescence of dead (D) vs. living (L) J2 of
Mil after treatment with soybean agglutinin, e) Fluorescent
cuticle (FC; note annulation) and amphidial ducts (AD) of Mj
labeled with Concanavalin A after J2 exposure to a-
galactosidase. f) Binding of WGA to egg shell (ES) of Mi3.
(Note: Incandescent light provided to enhance J2 image in
photographs a-d results in artifactual cuticular glow. True
labeling of J2 cuticle by fluorescent lectin is presented in
plate e.)

Lurl OU
\
lunoi-
0
oz

21
fluorescent lectins to any other portions of the nematode
surface was rarely observed, except as indicated below for
several glycohydrolase treatments. Nematodes that were
straightened, vacuolated, and displayed no movement
(nonviable) often exhibited strong labeling of the stylet,
esophageal lumen, and especially the gut region after
exposure to lectin-TRITC conjugates (Fig. 1-1d). No
labeling of viable J2 with unconjugated TRITC was observed.
Few differences in fluorescent lectin labeling were
observed among nonglycohydrolase-treated, preinfective J2 of
the Meloidog-yne spp. tested (Table 1-2). Amphids of Mil,
Mi3, and Mj labeled weakly with SBA, CON A, and LPA and
strongly with WGA and LOT. No binding of TRITC-conjugated
Limax flavus L. agglutinin (LFA; sialic-acid specific) to J2
was observed in preliminary tests (unpublished results), and
binding of LPA-TRITC to J2 was not observed until combined
with an improved fluorescent microscope light source (50
watt mercury lamp; Carl Zeiss, West Germany). Inhibition of
lectin binding in the presence of the appropriate
competitive sugar was only observed for LOT and LPA. All
lectins tested bound to egg shells of Mil, Mi3, and Mj
(Fig. 1 —1f) and binding was not inhibited in the presence of
0.1 M corresponding competitive sugar.
Differences in lectin labeling among the populations
of Meloidogyne spp. tested were observed after preinfective

22
Table 1-2. Binding of fluorescent lectins to
preinfective second-stage juveniles (J2) of
Meloidogyne incognita races 1 and 3 (Mil, Mi3),
and M. javanica (Mj).
Lectin
Mil
Mi 3
Mj
-Sug
a +Sug
-Sug
+ Sug
-Sug
+ Sug
SBAb
++c
++
+
+
++
++
WGA
+++
+++
+++
+++
+ + + +
++++
CON A
++
++
++
++
++
++
LOT
+++
NF
+++
NF
+ + + +
NF
LPA
++
NF
++
NF
++
NF
a J2 incubated in lectin solution +/-
competitive sugar. Competitive sugars included:
D-galactose for SBA; N-acetyl-glucosamine for
WGA; D-mannose for CON A; L-fucose for LOT; N-
acetyl-neuraminic acid for LPA.
b Soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Concanavalin A (CON A), Lotus
tetragonobolus agglutinin (LOT), and Limulus
polyphemus agglutinin (LPA).
c Epifluorescent microscope observations
included: + = very weak amphidial fluorescence;
++ = weak aphidial fluorescence; +++ = strong
amphidial fluorescence; ++++ = very strong
amphidial fluorescence; NF = no fluorescence.

23
J2 were treated with various glycohydrolases (Table 1-3).
Lectin labeling of J2 treated with glycohydrolases was
compared to labeling of J2 which were incubated in enzyme
buffer minus glycohydrolase. In most cases, enzyme activity
was inhibited in the presence of the appropriate competitive
sugar, except where indicated below.
Treatment of J2 with a-gal eliminated binding of
SBA-TRITC to the amphids of Mj and Mil, but not to amphids
of Mi3. Binding of LOT-TRITC to the amphids of Mj was
reduced by treatment of J2 with a-gal. The cuticle on the
anterior half of the body of Mj and Mil labeled weakly with
CON A-TRITC, with fluorescence of body annulation growing
weaker from head to mid-body (Fig. 1-1e). Enzyme activity
was not inhibited in the presence of 0.1M D-galactose for
Mj. Similar cuticular labeling was not observed for J2 of
Mi3 treated with a-gal, however, binding of CON A-TRITC to
the amphids of Mi3 was eliminated. Binding of LPA-TRITC to
the amphids of J2 was unchanged on Mj, increased on Mil , and
eliminated on Mi3 after treatment with a-gal.
Treatment of J2 of Mil with B-glu reduced binding of
WGA-TRITC to amphids and promoted binding of WGA-TRITC to
the anterior cuticle of Mil. Binding of LPA-TRITC to the
amphids of J2 of Mj and Mil was eliminated by B-glu
treatment. However, binding of LPA-TRITC to the amphids of
Mi3 increased after B-glu treatment.

24
Table 1-3. Binding of fluorescent-lectins to preinfective second-
stage juveniles (J2) of Meloidogyne incognita races 1 and 3 (Mil,
Mi3), and M. javanica (Mj) after J2 treatment with different
glycohydrolases.
Mil
Mi 3
Mj
Enzyme (Lectin)
-Enza +Enz
-Enz +Enz
-Enz +Enz
a-galactosidase
SBAb
++c
NF
+
+
++
NF
WGA
+++
+++
+++
+ + +
+++
+++
CON A
++
++,WC
++
NF
++
++,WC
LOT
+++
+++
+++
+++
+++
++
LPA
+
+++
++
NF
+
+
B-N-Acetyl-
Glucosaminidase
SBA
++
++
+
+
+
+
WGA
+++
++,wc
+++
+++
+++
+++
CON A
++
++
+
+
++
++
LOT
+++
+++
+++
+++
+++
+++
LPA
++
NF
NF
++
++
NF
a-mannosidase
SBA
++
NF
++
++
++
++
WGA
+++
+++
+ + +
+++
+++
+++
CON A
++,WC
++,wc
++
NF
++
NF
LOT
+++
+++
++
++
+++
NF
LPA
++
NF
++
NF
++
NF
i

25
Table 1-3—continued.
Mil
Mi3
Mj
Enzyme (Lectin)
-Enz +Enz
-Enz +Enz
-Enz +Enz
a-L-fucosidase
SBA
+ +
++
++ +
++ +
++
++
WGA
+++
+++
+++
+++
+++
+++
CON A
+ +,WC
++,WC
++
++
+++
+++
LOT
+++
+ +
+++
+ + +
+++
+
LPA
+++,WC
NF, WC
++
NF
++
NF
Neuraminidase
SBA
NF
NF
+
+
+
+
WGA
+ + +
+++
+ + +
+++
+++
+ + +
CON A
++,wc
++,wc
++ +
++
++»'
WC +++,wc
LOT
+++
+++
+++
++
+++
+
LPA
++
NF
++
++
++
NF
a J2 incubated
in lectin
solution
+ /-
prior
treatment
with
select glycohydrolase.
k Soybean agglutinin (SBA), wheat germ agglutinin (WGA),
Concanavalin A (CON A), Lotus tetragonobolus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA).
c Epifluorescent microscope observations included: + = very weak
amphidial fluorescence; ++ = weak aphidial fluorescence; +++ =
strong amphidial fluorescence; ++++ = very strong amphidial
fluorescence; NF = no fluorescence; WC = weak fluorescence of J2
cuticle along anterior half of body.

26
Binding of SBA-TRITC to the amphids of Mil and LPA-
TRITC to the amphids of Mj, Mil, and Mi3 was eliminated by
a-man. Binding of LOT-TRITC to the amphids of Mj was
eliminated by treatment of J2 with a-man, but a-man activity
was not inhibited in the presence of 0.1 M mannose. Amphids
of Mj and Mi3 did not label with CON A-TRITC after a-man
treatment, but CON A-TRITC did bind to the anterior cuticle
of Mil after treatment with a-man buffer plus or minus
enzyme.
Binding of LOT-TRITC to the amphids of Mj and Mil
was reduced and binding of LPA-TRITC to amphids of Mj, Mil,
and Mi3 was eliminated after treatment of J2 with a-fuc.
Weak labeling of the anterior cuticle of Mil with CON A-
TRITC and LPA-TRITC occurred after incubation of J2 in a-fuc
buffer with or without the enzyme.
Treatment of J2 with neuraminidase partially
inhibited binding of LOT-TRITC to amphids of Mj and Mi3, and
completely inhibited binding of LPA-TRITC to amphids of Mj
and Mil. Neuraminidase treatment increased binding of CON
A-TRITC to amphids of Mj and Mil, but reduced binding of
CON A-TRITC to amphids of Mi3. The anterior cuticle of Mj
and Mil labeled weakly with CON A-TRITC after incubation in
neuraminidase buffer with or without enzyme.
i

27
Discussion
Fluorescein isothiocyanate (FITC)-lectin conjugates
were not used in fluorescence assays because untreated J2 of
the Meloidogyne spp. populations examined strongly
autofluoresced at the excitation wavelength of FITC.
Difficulty with autofluorescence of C. elegans and P.
redivivus at the excitation wavelength of FITC has also been
reported (48). Lectins conjugated with rhodamine (TRITC)
fluorophors were more appropriate for the study of lectin
binding to nematodes. Preinfective J2 of Meloidogyne spp.
were not visible when viewed through the TRITC microscope
filter, except for body portions labeled with lectin-TRITC
conjugates.
It is apparent from observations that nematode
viability is critical for true labeling of nematodes with
fluorescent lectins. Living (motile) J2 bound fluorescent
lectin almost exclusively in the vicinity of the amphidial
openings. The entire body of nematodes that were apparently
dead fluoresced after TRITC-lectin treatment, especially in
the gut region. The fluorescence of dead J2 was similar to
the observations of enzymatically induced fluorescence of
dead nematodes reported by Bird (11). This phenomenon may
have influenced fluorescent observation of sialyl residues
over the entire body of J2 of M. javanica as reported by
Spiegel et a_l. (114). Labeling of sialyl residues with

28
fluorescent LPA was relatively weak and confined to the
amphidial region of viable J2 of Meloidogyne spp. observed
in these studies. Since the specific hemagglutination
activity and absorbance ratio (550nm/280nm) of LPA-TRITC was
relatively low compared to the other lectins tested, it may
be possible that more sialic acid exists on the J2 surface
than can be detected with fluorescent lectin probes.
Lectin binding to the tail region of M. incognita has also
been reported (77), but it was not observed in this study.
The observed binding of fluorescent lectins to egg shells of
Meloidogyne spp. in this investigation has been reported for
eggs of M. javanica (113).
Only the binding of LOT and LPA to specific sugars
on preinfective J2 were confirmed by competitive sugar
inhibition, although all of the fluorescent lectins tested
bound to amphids of preinfective J2 of the three Meloidogyne
spp. populations examined. The binding of SBA, CON A, and
WGA to J2 was apparently not specific for a-D-galactose, a-
D-mannose, and B-N-acetylglucosamine, respectively, since
0.1 M concentrations of these sugars were insufficient to
inhibit binding of these TRITC-lectins to J2. Soybean
agglutinin, CON A, and WGA bind to other molecular forms of
galactose, mannose, and N-acetylglucosamine, respectively,
and WGA has been reported to have multiple carbohydrate
binding sites (38). It is possible that the affinity of
i

29
SBA, CON A, and WGA for carbohydrate-specific sites near
amphidial openings was too strong to be inhibited by the
competitive sugar solutions used in these assays. The
binding of SBA, CON A, and WGA to amphidial secretions of J2
of Meloidogyne spp., however, may represent binding of these
lectins to hydrophobic ligands (possibly lipids), as
reported elsewhere (93). Incubation of J2 in fluorescent
SBA, CON A, or WGA in the presence of 1,8 anilinonaphthalene
sulfonic acid plus or minus competitive sugar may confirm
the presence of hydrophobic binding since the hydrophobic
and carbohydrate binding sites are independent of each other
(93). The direct binding of unconjugated TRITC to surface
lipids of J2 did not apparently occur in these assays since
nematodes did not fluoresce after incubation in unconjugated
TRITC.
The greater intensity of fluorescent labeling by LOT
and WGA conjugates may be due to their relatively higher
binding capacities. Lack of differential lectin labeling
among nonglycohydrolase-treated, preinfective J2 of Mil,
Mi3, and Mj makes it difficult to extrapolate a potential
role of surface carbohydrates in the specificity of
pathogenicity (58). Substances to which lectins were bound
were concentrated and sometimes emanated from the amphidial
region of J2 of Meloidogyne spp., and this has also been
reported for invasive juveniles of pathotypes of potato cyst

30
nematode and other populations of Meloidogyne spp. (36, 76).
Since this is the portion of the nematode body around which
some postinfectional, incompatible plant responses occur
(43, 61 ), it may be possible that carbohydrates in amphidial
secretions of postinfectional J2 affect plant-nematode
interactions.
Results of experiments involving glycohydrolases
suggested that carbohydrates located in the amphidial region
of J2 occurred in complexes and that these complexes were
structurally different among populations of Meloidogyne spp.
The inability of enzymes to alter carbohydrate residues on
some nematode surfaces may be a reflection of the substrate
specificity exhibited by glycohydrolases (2, 125, 132).
Enzyme treatment did reveal cuticular carbohydrates,
especially mannose and/or glucose, on the anterior half of
some J2 , and sialyl residues were often removed from J2
amphids by a number of different glycohydrolases. This may
indicate that sialic acids are some of the outermost
residues present in the carbohydrate complexes which
apparently exist in amphidial secretions of J2 of
Meloidogyne spp. Whether surface carbohydrate changes
similar to those reported for glycohydrolase treatments
occur once Meloidogyne spp. J2 enter plant roots is unknown.
Alteration of the surface carbohydrates of J2 of Meloidogyne

31
spp., and subsequent plant root tissue responses to
infection by treated J2, may provide insight into the
specificity of plant-nematode interactions.

CHAPTER 3
ROOT TISSUE RESPONSE OF TWO RELATED SOYBEAN CULTIVARS
TO INFECTION BY LECTIN-TREATED MELOIDOGYNE SPP.
Introduction
Plant incompatibility with nematodes often results
from active plant defense reactions to infection by
phytoparasitic nematodes (37, 39, 42, 58, 59, 97, 127).
Although several mechanisms of active incompatibility have
been proposed, little is known about nematode
characteristics that may elicit plant responses that are
incompatible with nematode development and their relation to
specifity in plant-nematode interactions (58). The
occurrence of physiological races of phytoparasitic
nematodes (108, 119) suggests that specific interactions
occur between nematodes and plant genotypes, and that
populations of phytoparasitic nematodes adapt to overcome
incompatibility.
Evidence that surface biochemistry, especially
glycoconjugates of cells and organisms, promotes specificity
in plant-microbe interactions has been the subject of
several reviews (3, 21, 23, 62, 106). Keen (62) suggested
that biochemical surface interactions were important in the
specificity of incompatibility in gene-for-gene systems
32

33
between plant cultivars and microbial pathogens. It is
unclear if surface interactions are important in plant-
nematode incompatibility; however, the existence of
carbohydrates on the surface of nematodes, and evidence that
surface carbohydrates may be important in interactions
between nematodes and microbes has been reported and
summarized (47, 135, T36). Zuckerman and Jansson (136)
proposed that interaction between nematodes and other
organisms may be altered by obliteration or blocking of
carbohydrates on the nematode surface.
To evaluate this concept with respect to specificity
in plant-nematode interactions a model system was chosen
which consisted of two related soybean cultivars,
'Centennial' and 'Pickett 71', and three Meloidogyne spp.
populations that differed in compatibility with Centennial
soybean. The incompatibility of Centennial soybean with
Meloidogyne incognita has been associated with a
hypersensitive reaction (HR) of soybean root tissue in the
region of invading second-stage juveniles (J2) (61). The HR
was strongly correlated with the accumulation of glyceollin,
but biotic elicitors of the HR were not identified (60).
The surface carbohydrates of several populations of
cyst and root-knot nematodes, including J2 of Florida
populations of M. incognita races 1 and 3 (Mil, Mi3), and M.
javanica (Mj) (see Chapter 2 above), have been characterized

34
with fluorescent lectin probes (36, 76, 95). Lectins bound
to infective J2 of Meloidogyne spp. primarily in the region
proximate to cephalic chemosensillae (see Chapter 2 above,
77). In the study reported here, surface carbohydrates of
J2 of Mil, Mi3, and Mj were "blocked" with lectins and
subsequent soybean root tissue responses to lectin-treated
J2 were observed.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
and M. javanica (Treub) Chitwood were maintained in
greenhouse culture on roots of 'Rutgers' tomato
(Lycopersicon esculentum Mill.) and 'Black Beauty' eggplant
(Solanum melongena L.). Meloidogyne spp. populations were
typified by adult female perineal patterns, second-stage
juvenile (J2) lengths, and performance on differential
hosts (101). Species identifications were also confirmed by
three independent nematode taxonomists (A. M. Golden,
Beltsvilee, MD; J. G. Baldwin, Riverside, CA; J. D.
Eisenback, Blacksburg, VA). Eggs of each nematode
population were extracted from host roots with 0.53% NaOCl
for 30 seconds (46) and hatched at room temperature on a
Baermann funnel. Preinfective J2 which had hatched within
48 hours were used as test organisms in each experiment.

35
Surface carbohydrates of Meloidogyne spp. J2 were
blocked by incubating nematodes in solutions containing
unconjugated, purified soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT),
Concanavalin A (CON A), or Limax flavus agglutinin
(LFA) (E-Y Labs, San Mateo, CA). The sugar specificity,
appropriate lectin buffers, corresponding competitive
sugars, and procedure used to determine the specific
hemagglutination activity for each lectin were described
above in Chapter 2. The sugar specificity of LFA is
N-acetyl-neuraminic (sialic) acid, and LFA assays were
conducted in buffer which contained 0.05 M Tris-saline plus
0.01 M CaCl2 at pH 7.5.
Preinfective J2 of Mil, Mi3, and Mj were
concentrated in the appropriate buffer or in distilled water
by centrifugation at 1000cj for 3 minutes. Treatments for
each lectin included incubating J2 (approx. 2000 J2) of each
population in solutions of lectin (200 ug/ml), lectin (200
jig/ml) plus 0.1M competitive sugar, and 0.1M sugar minus
lectin for 2 hours at 4°C. Juveniles in these solutions
were used as direct (unwashed treatment) inoculum for
subsequent soybean root challenge. Since a 0.1 M solution
of sialic acid in LFA buffer was quite acidic (pH ~ 3.0), a
soybean root penetration bioassay was conducted to address
the effect of sialic acid and acidity on the activity of J2
i

36
of Meloidogyne spp. (see Appendix D below). In addition, J2
exposed to each lectin and sugar treatment were washed three
times in buffer and subsequently used as inoculum for
soybean root challenge. Control treatments included J2 in
buffer and J2 in distilled water.
Two related cultivars of soybean (Glycine max (L.)
Merr.), 'Pickett 71' and 'Centennial', were used for root
tissue challenge by treated J2 of Meloidogyne spp. It has
been reported that Pickett 71 was compatible and Centennial
was incompatible with M. incognita, and both soybean
cultivars were compatible with M. j avanica (61). Seeds of
each variety were dusted with Thiram 75WP (Kerr-McGee
Chemical Corp., Jacksonville, FL) and germinated in moist
germination paper which was rolled up (ragdolls) and
incubated in the dark at 27*C. Newly germinated soybeans
with roots 3-5 cm long were placed on trays containing
autoclaved Astatula fine sand (hyperthermic, uncoated typic
quartzipsamments) and the root tips covered with a small
amount of sand. Nematode suspensions (approx. 600 J2) from
each treatment were placed on separate soybean root tips
(61, 75). Trays containing inoculated soybeans in sand were
incubated in the dark at 27#C. Treatments were arranged as
a 3x5x8x2 factorial including three Meloidogyne spp.
populations, five lectins, eight treatments, and two soybean
i

37
varieties. There were seven replicates of each treatment
combination.
Soybeans were removed from trays and their roots
washed free of sand and any nematodes that had not
penetrated approximately 40 hours after inoculation. The
seedlings were then placed on moist germination paper and
the inoculated portions of the roots were marked on the
paper. The germination paper which contained inoculated
seedlings was covered with an additional piece of moist
germination paper, carefully rolled into ragdolls, and
incubated in the dark at 27®C. This "pulse inoculation" was
used to ensure that nematodes observed within roots had
entered within 40 hours of inoculation.
Inoculated soybean root segments were excised and
immediately fixed in 10% alcoholic formalin (1:9,
formalin:95% EtOH, v/v) a total of 5 days after inoculation.
Additional treatments of J2 of Mi3 in water applied to
Centennial and Pickett 71 soybean roots were excised 20 days
after inoculation and processed according to these
protocols. Fixed root segments were dehydrated through a
tert-butyl alcohol series and embedded in paraffin. Serial
sections (12 urn) were mounted on glass slides, stained with
safranin-fast green, and observed under light microscopy.
Sections from seven replicates of each treatment combination
were observed and the most frequent tissue responses to
i

38
nematode infection were determined. This experiment was
repeated once.
Results
Hemagglutination assays indicated that the binding
capacity of pure lectins was relatively strong, except for
LFA. Specific hemagglutination activities of 4096, 4096,
8192, 8192, and 4 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LFA, respectively. Hemagglutination
activity of all lectins was completely inhibited in the
presence of 0.1 M corresponding competitive sugar.
The most common or primary responses of soybean root
tissue to infection by Meloidogyne spp. J2 are reported in
Tables 2-1 to 2-5. Infective juveniles incubated in buffer
or water became enlarged and induced giant cell formation in
compatible interactions but remained vermiform and were
associated with a hypersensitive reaction in incompatible
combinations (Fig. 2-1). Giant cells of normal appearance
were usually observed in roots of Pickett 71 and Centennial
soybean 5 and 20 days after roots were exposed to Mi3
incubated in buffer or water. Giant cells associated with
Mi3 controls in Centennial soybean sometimes contained
granular cytoplasm. No gall formation, evidence of
hyperplasia of pericycle cells adjacent to giant cells, or
development of Mi3 past third-stage juvenile was observed in

Table 2-1. Primary tissue reactions of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in soybean
agglutinin (SBA) solution (200 ng/ml) and (or) 0.1M galactose (gal) solution prior to
inoculation.
Treatment
Mil
Mi 3
Mj
P
C
P
C
P
C
Washed3
SBA
GC,
GRCYb
HR,
NR
GC, NR
HR,
EGC
GC,
S
GC,
s
SBA + gal
GC
HR
GC, GRCY
HR,
NR
GC,
GRCY
GC,
s
gal
GC,
GRCY
HR
GC, GRCY
HR,
NR
GC,
GRCY
GC
Unwashed
SBA
GC,
GRCY
HR,
EGC
GC, S
HR,
EGC
GC,
S
GC,
s
SBA + gal
GC,
GRCY
HR,
EGC
EGC, GRCY
HR,
EGC
GC,
EGC
GC,
s
gal
GC,
S
HR,
GRCY
GC, S
HR,
NR
GC,
S
GC,
s
Buffer
GC,
GRCY
HR,
GRCY
GC, S
GC,
GRCY
GC,
S
GC,
s
Distilled H2O
GC,
S
HR
GC, S
GC,
GRCY
GC,
S
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.

Table 2-2. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Concanavalin A
(CON A) solution (200 ng/ml) and (or) 0.1M mannose (man) solution prior to
inoculation.
Treatment
Mil
Mi3
Mj
P
c
P
C
P
C
Washed3
CON A
GC,
NRb
NR,
HR
GC,
S
HR
GC
GC,
s
CON A +
man
GC,
GRCY
HR,
NR
GC,
s
HR, NR
GC,
S
GC,
s
man
GC,
S
HR
GC
NR, GC
GC,
GRCY
GC,
GRCY
Unwashed
CON A
GC,
S
HR
GC,
EGC
HR, NR
GC,
S
GC,
S
CON A +
man
GC,
s
HR
GC,
S
HR, EGC
GC,
S
GC,
S
man
GC,
s
NR,
EGC
GC,
S
GC, GRCY
GC,
S
GC,
S
Buffer
GC,
s
HR,
NR
GC,
S
GC, GRCY
GC,
S
GC,
S
Distilled
h2o
GC,
s
HR,
GRCY
GC,
S
GC, EGC
GC,
S
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.

Table 2-3. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Lotus
tetrágonolobus agglutinin (LOT) solution (200 jig/ml) and (or) 0.1M fucose (fuc)
solution prior to inoculation.
Mil
Mi3
Mj
Treatment
P
C
P
C
P
C
Washed3
LOT
GC,
Sb
HR,
EGC
GC,
S
HR,
EGC
GC,
S
GC,
GRCY
LOT + fuc
GC,
S
HR,
NR
GC,
NR
HR,
GRCY
GC,
S
GC,
S
fuc
GC,
GRCY
HR
GC,
EGC
HR,
NR
GC,
S
GC,
S
Unwashed
LOT
GC,
GRCY
HR
GC,
GRCY
HR,
EGC
GC,
S
GC,
GRCY
LOT + fuc
GC,
GRCY
HR,
GRCY
GC,
S
HR,
EGC
GC,
EGC
GC,
NR
fuc
GC,
GRCY
HR,
GRCY
GC,
S
HR,
EGC
GC,
S
GC,
S
Buffer
GC,
S
HR
GC,
S
GC,
EGC
GC,
s
GC,
S
Distilled H2O
GC,
S
HR
GC,
S
GC,
S
GC,
s
GC,
S
3 Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.

Table 2-4. Primary tissue responses of 'Pickett 71' (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in wheat germ
agglutinin (WGA) solution (200 ug/ml) and (or) 0.1M N-acetylglucosamine (NAcGlu)
solution prior to inoculation.
Mil
Mi 3
Mj
Treatment
P
C
P
C
P
C
Washed3
WGA
GC,
EGCb
HR,
NR
GC,
NR
HR,
NR
GC,
NR
GC
WGA + NAcGlu
GC,
NR
HR,
NR
GC,
EGC
HR,
NR
GC,
S
GC,
S
NAcGlu
GC,
HR
HR,
GRCY
GC,
S
NR,
HR
GC,
S
GC,
s
Unwashed
WGA
GC,
NR
HR,
NR
GC,
S
HR,
GRCY
GC,
S
GC,
GRCY
WGA + NAcGlu
GC,
HR
NR,
HR
GC,
S
HR
GC,
s
GC,
S
NAcGlu
GC,
HR
HR
GC,
S
HR,
EGC
GC,
s
GC,
S
Buffer
GC,
S
HR,
NR
GC,
S
GC,
NR
GC,
s
GC,
S
Distilled H2O
GC,
S
HR,
NR
GC,
S
GC,
GRCY
GC,
s
GC,
S
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.

Table 2-5. Primary tissue responses of 'Pickett 71’ (P) and 'Centennial' (C) soybean
roots 5 days after their exposure to second-stage juveniles of Meloidogyne incognita
races 1 and 3 (Mil, Mi3), and M. javanica (Mj) that were incubated in Limax flavus
agglutinin (LFA) solution (200 ug/ml) and (or) 0.1M N-Acetylneuraminic (sialic) acid
solution prior to inoculation.
Mil
Mi3
Mj
Treatment
P
C
P
c
P
C
Washed3
LFA
GC, Sb
NR, HR
GC,
EGC
HR, NR
GC, S
GC,
s
LFA + sialic
GC, S
HR
NR,
EGC
HR, EGC
GC, S
GC,
s
sialic
GC, GRCY
HR, GRCY
GC,
EGC
HR
GC, S
GC,
s
Unwashed
LFA
c
_
__
_
_
LFA + sialic
—
—
-
-
--
—
-
-
sialic
— “
— —
—
— “
—
—
—
Buffer
GC, S
HR, GRCY
GC,
S
GC, EGC
GC, S
GC,
s
Distilled H2O
GC, S
HR, NR
GC,
s
GC, GRCY
GC, S
GC,
s
a Second-stage juveniles were incubated in treatment solution and washed three times
with buffer before inoculation.
b GC= giant cells; GRCY= granular cytoplasm within giant cells; HR= hypersensitive
response; NR= no response; EGC= early giant cells; S= swollen juvenile.
c Few or no nematodes observed within soybean root tissue.

Fig. 2-1. Response of 'Centennial' soybean root tissue
within 5 days of infection by lectin-treated second-stage
juveniles (J2) of Meloidogyne spp. a) Hypersensitive
reaction (HR) to M. incognita race 1 treated with soybean
agglutinin. b) Giant cells (GC) induced by the untreated
nematode (N), M. incognita race 3. c) Giant cells induced
by M. javanica treated with soybean agglutinin plus
galactose. d) Hypersensitive reaction induced by M.
incognita race 3 treated with N-acetylglucosamine.


46
Centennial root tissue 20 days after inoculation with J2 of
Mi3.
The primary tissue response of both soybean
cultivars to J2 of Mil or Mj exposed to any lectin or sugar
treatment was essentially unchanged from that of tissues
infected by J2 incubated in buffer or water. Lectin or
sugar treatment of J2 of Mi3 did not influence host response
in Pickett 71, but in Centennial soybean roots, treatment of
J2 of Mi3 with any lectin or sugar stimulated a
hypersensitive response rather than giant cells.
Soybean tissue responses to infection by J2 that
were atypical of the primary tissue response sometimes
occurred frequently enough to warrant report. The
occurrence of early giant cells in Centennial tissue
challenged by Mil treated with SBA and SBA plus galactose,
unwashed (Table 2-1), was in contrast to the primary
response of hypersensitivity. Early giant cell formation
was also observed in Centennial challenged by Mil treated
with mannose, unwashed (Table 2-2), and Mil treated with
LOT, washed (Table 2-3).
Second-stage juveniles of Meloidogyne spp. were
observed in soybean roots with no apparent plant tissue
reaction. This was the primary observation in Centennial
root tissue with Mil treated with CON A, washed; Mil treated
with mannose, unwashed; Mi3 treated with mannose, washed;
i

47
Mil treated with WGA plus N-Acetyl-D-glucosamine (NAcGlu),
unwashed; Mi3 treated with NAcGlu, washed; and Mil treated
with LFA, washed (Tables 2-2, 2-4, 2-5). No tissue response
was observed in Pickett 71 to Mi3 treated with LFA plus
sialic acid, washed (Table 2-5). A hypersensitive response
was sometimes observed in Pickett 71 challenged by Mil
treated with NAcGlu, washed; Mil treated with WGA plus
NAcGlu, unwashed; and Mil treated with NAcGlu, unwashed.
Almost no J2 were observed in soybean roots challenged by
any population of Meloidogyne spp. that received unwashed
LFA and sialic acid treatments. When J2 of these treatments
were occasionally observed in soybean roots, tissue response
was similar to comparable treatments with SBA.
Discussion
It appears that incubation of J2 of Mil in the
lectins or sugars tested had little effect on soybean root
tissue response in the incompatible pathosystem. If the
concept that preformed sites (carbohydrate moieties) on the
surface of Mil J2 are responsible for recognition by plant
cell surface receptors and subsequent plant defense reaction
were valid, blockage of these sites with lectins should have
prevented the HR observed in Centennial root tissue. In a
few instances, early giant cell formation in Centennial by
Mil was noted, but the HR was much more common. The more

48
frequent occurrence of HR in Pickett 71 exposed to Mil
treated with N-acetyl-D-glucosamine may indicate an
alteration of nematode surface carbohydrates which promoted
incompatibility.
The fate of lectin bound to J2 of Meloidoayne spp.
once the nematode entered soybean root tissue is
questionable. Unwashed treatments were included in this
study to ensure that J2 were present in an environment of
lectin and (or) sugar until they penetrated roots. No
treatments, however, had an effect on the compatible
interaction between Mj and root tissue of either soybean
cultivar. Differences in the quantity, balance, or
accessibility of secretory carbohydrates to potential plant
receptors, however, may promote incompatibility or
compatibility. Previous reports indicated that lectins
bound to amphidial secretions of J2 of Meloidogyne spp. (see
Chapter 2 above, 77), but the rates of production of
amphidial secretions by J2 of Meloidogyne spp. and the
quantities of this material that are sloughed off in plant
tissue or the soil environment are unknown.
The occurrence of normal giant cells in Centennial
20 days after inoculation of root tips with J2 of Mi3
incubated in water was unexpected. This host-parasite
relationship was apparently incomplete compared to Mi3 in
Pickett 71, however, since pericyclic hyperplasia and

49
nematode development were strongly inhibited 20 days after
inoculation. Differences in the degree of incompatibility
have been reported for several soybean cultivars and M.
incognita populations (105). Treatment of J2 of Mi3 with
any lectin or sugar promoted active incompatibility (HR) in
Centennial soybean, and may actually have facilitated
recognition of invasive Mi3 and subsequent defense response
by the plant. The lack of specificity of lectin or sugar
effects in the Mi3-Centennial interaction makes it seem
unlikely that alteration of surface carbohydrate composition
of preinfective J2 was responsible for promoting
incompatibility. If one considers the interaction of
nematode surface carbohydrates with potential plant cell
surface receptors as a "lock and key" phenomenon, however,
it may be feasible that a slight alteration in surface
carbohydrate compostion was sufficient to promote
incompatibility to Mi3 in Centennial soybean roots.
Possibly a greater alteration of the carbohydrates examined
here on Mil and Mj, or alteration of surface carbohydrates
not examined in these studies, would influence their host-
parasite interactions.
Treatment of J2 of Mi3 with lectin or sugar may have
stimulated the production of a substance by the nematode
that induces HR in Centennial soybean roots. Juveniles of
Mil may inherently have the capacity to induce HR while J2

50
of Mj cannot promote incompatibility in soybean no matter
what the treatment. For some populations, such as in the
case of Mi3, incompatibility may be a process that can be
stimulated. Conversely, substances produced by J2 of
Meloidogyne spp. (ie. amphidial or stylet secretions) may be
essential to induce compatibility between host and parasite
(82), and these substances were altered sufficiently in Mi3
to inhibit compatibility in Centennial soybean roots.
The ability of J2 of Mil, Mi3, and Mj to penetrate
roots of both soybean varieties was apparently strongly
impaired when J2 were introduced to roots in a solution that
contained LFA, sialic acids, or combination of the two. It
was not determined if J2 penetrated and exited from roots
within the 40 hour "pulse inoculation." Inhibition of
soybean root penetration by J2 of Meloidogyne spp. treated
with sialic acid occurred in similar tests (see Appendix D
below), and this appeared to be more than an effect of the
low pH of a 0.1 M sialic acid solution. It has been
reported that a single soil application of CON A, and LFA at
relatively higher concentrations, significantly reduced
galling of tomato roots induced by M. incognita (74), but
contact and effect of active lectins to nematodes in this
system was unclear. Little effect of CON A on soybean root
penetration by J2 of any Meloidogyne spp. population was
observed in this investigation, even though J2 were

51
incubated in CON A solution (200 ng/ml) before their
application to roots in soil. Sialic acids have been
reported to be important in the adhesion of conidia of Meria
coniospora to nematode surfaces, especially at the
chemosensory organs (50-52). In the studies reported
herein, nematode viability after LFA and sialic acid
treatment was confirmed by infectivity of washed J2, and
also viable J2 of Meloidogyne spp. have been observed
microscopically after similar treatment (see Chapter 2
above). Removal of sialic acids from amphidial secretions
by a number of selective glycohydrolases (see Chapter 2
above) suggests that sialic acids are some of the outermost
carbohydrate residues of amphidial glycoconjugates. Whether
sialic acid residues proximate to nematode chemosensillae
have a masking or regulatory effect similar to those
observed in other animal systems (104) should be the subject
of further investigation.

CHAPTER 4
QUANTIFICATION OF LECTIN BINDING TO SECOND-STAGE JUVENILES
OF MELOIDOGYNE SPP.
Introduction
Lectins, proteins that bind to specific carbohydrate
residues, are excellent probes for the study of the
carbohydrate chemistry of biological surfaces. Lectins, and
their involvement in recognition and specificity in plant-
microbe interactions, have been the subject of several
reviews (3, 21, 23, 31, 70, 106, 107). The surface
carbohydrate chemistry of nematodes of various parasitic
habits has been characterized by several techniques,
including lectin probes. Some of this work has been
summarized by Zuckerman and Jansson (136), who postulated
that blockage or obliteration of specific carbohydrates on
nematode surfaces may alter interactions between nematodes
and other organisms. Examples include the association of
surface carbohydrates in antigenicity and chemoresponse of
nematodes parasitic to animals (14, 72, 85) and the
interaction of nematodes with nematophagous fungi (17, 47,
80).
The potential involvement of nematode surface
carbohydrates in recognition and specificity in plant-
52

53
nematode interactions, however, has not been clearly
demonstrated (47, 58, 135, 136). Application of
fluorescent-lectin probes to differentiate nematode species
and pathotypes on the basis of their surface carbohydrates
has been attempted (see Chapter 2 above, 36, 76, 95).
However, the intensity of fluorescence of rhodamine-labeled
lectins that bound to cephalic sensory structures of
Meloidogyne (Goeldi, 1887) spp. second-stage juveniles (J2)
differed among the lectins tested (see Chapter 2 above, 76).
Quantification of lectin binding to nematode pathotypes may
reveal differences in relative carbohydrate content that
were not detectable in fluorescent-lectin assays. Lectins
labeled with hemocyanin and with tritium have been used to
quantify relative amounts of carbohydrates on the surface of
nematodes and bacteria, respectively (77, 82). The
difficulty in production and handling of radiolabeled
lectins, and the sophisticated equipment required to observe
hemocyanin conjugates on the nematode surface, limits the
practicality of these methods. We have developed a
microfiltration enzyme-linked lectin assay (27, 78, 84, 120)
to quantify the amount of lectin that binds to J2 of
Meloidogyne spp.
i

54
Materials and Methods
Populations of Meloidogyne incognita (Kofoid &
White) Chitwood race 3 (Mi3), M.incognita race 1 (Mil), and
Meloidogvne javanica (Treub) Chitwood (Mj) were maintained
in greenhouse culture on roots of 'Rutgers' tomato
(Lvcooersicon asculentum Mill.) and 'Black Beauty' eggplant
(Solarium melongena L.). Meloidogyne spp. populations were
identified by adult female perineal pattern, second-stage
juvenile (J2) length, and development on differential hosts
(101). Species identifications were also confirmed by three
independent nematode taxonomists (A. M. Golden, Beltsville,
MD; J. G. Baldwin, Riverside, CA; J. D. Eisenback,
Blacksburg, VA). Eggs of each nematode population were
extracted from host roots in 0.53% NaOCl solution for 30
seconds (46) and hatched at room temperature on a Baermann
pan. Preinfective J2 which had hatched within 48 hours were
used as test organisms in each experiment.
The quantity of lectin binding to preinfective J2 of
Mil, Mi3, and Mj was determined using a microfiltration
enzyme-linked lectin assay (78, 84). Lectins conjugated
with horseradish peroxidase (HRP) were purchased from E-Y
Labs (San Mateo, CA) and included soybean agglutinin (SBA),
wheat germ agglutinin (WGA), Lotus tetraaonolobus agglutinin
(LOT), Concanavalin A (COMA), and Limulus Polyphemus
agglutinin (LPA). The sugar specificity, corresponding

55
buffer solutions, and procedure for determination of the
specific hemagglutination activity of each lectin were
presented in Chapter 2.
Ninety six-well microfilter plates (SV-96, Millipore
Corp., Bedford, MA) with a 5 um pore size were incubated
with 200 ul of 1.0% bovine serum albumin (BSA) in phosphate
buffer saline (PBS), pH 7.2, at 37°C for 2 hours. These
plates were washed three times with PBS on a microfiltration
apparatus (Millipore Corp., Bedford, MA) prior to their use
in the following assay (Fig. 3-1).
Preinfective J2 of each Meloidogyne spp. population
were concentrated into the appropriate buffer for each
lectin-HRP conjugate. Nematodes were incubated in 500 jil of
lectin-HRP solution (200 ug/ml) for 2 hours at 4°C. Control
treatments included J2 in buffer (untreated J2) and 500 y.1
lectin solution (200y.g/ml) minus J2 (lectin wash).
Competitive sugar controls similar to ones described in
Chapter 2 were omitted due to insufficient numbers of
freshly-hatched Meloidogyne spp. J2. Five, 100-ul samples
from each treatment combination (approx. 2000 J2 suspended
in lectin solution) were placed in separate wells on a
microfilter plate. Each well was washed five times with the
appropriate lectin buffer on a microfiltration apparatus.
The treated J2 or lectin wash in each well were suspended in
100 jil of buffer and transferred to separate microcentrifuge

56
Figure 3-1.
apparatus (Millipore
Microfiltration plates and suction
Corp., Bedford, MA).

57
tubes. The volume of each microcentrifuge tube was
increased to 250 jil with buffer, and 50 nl of suspension
were withdrawn from each tube to quantify the number of J2
per 50-m.I sample. Four, 50-ul suspensions of treated J2 or
lectin wash were transferred from each tube to separate
wells on a fresh 96-well microfilter plate. One hundred
microliters of peroxidase substrate,([2, 2'-azino-di-(3-
ethyl-benzthiazoline) sulfonic acid]) (ABTS, Sigma Chemical
Corp., St. Louis, MO) were added to each of these wells and
allowed to incubate at room temperature in the dark for 30
minutes (110). The solution from each well was transferred
to corresponding wells on a 96-well enzyme immunoassay plate
using a microfiltration apparatus to remove J2. Absorbance
(414 nm) of solution in each well was determined on an
automated microplate reader (Model EL309, Bio-Tek
Instruments, Winooski, VT). Twenty separate absorbance
values were determined for each treatment combination.
The absorbance values determined in this assay were
compared to the linear portion of a standard curve prepared
for each lectin-HRP conjugate (Fig. 3-2). Standard curves
were prepared by diluting (1:1, v/v) 50-nl volumes of
lectin-HRP solution across a 96-well EIA plate and adding
100 m.1 of ABTS solution per well. The quantity of lectin
which adsorbed to a single J2 was determined by dividing the

Figure 3-2. Standard curves of lectin-peroxidase
conjugates. Concanavalin A (CON A) = 6258.651x-468.338,
= 0.957; Soybean agglutinin (SBA) = 11,388.817x+12.636,
= 0.995; Lotus tetragonobolus agglutinin (LOT) =
11,791.765x-31.613, R^ = 0.995;Limulus polyphemus
agglutinin (LPA) = 7069.594x-459.472, R^ = 0.977; Wheat germ
agglutinin (WGA) = 6282.532x-428.531, R - 0.967.
i

ABSORBANCE (414nm)
NANOGRAMS LECTIN
of\)jicj)03oruj\(no3
6S
0- CON

60
observed lectin value by the number of J2 estimated for that
sample (approx. 500-2000 J2). The test was repeated once.
Results
Hemagglutination tests indicated that the binding
capacity of all HRP-lectins, except LPA, was relatively
strong. Specific hemagglutination activities of 512, 512,
4096, 2048, and 8 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LPA, respectively. Hemagglutination
activity of all lectin-HRP conjugates was completely
inhibited in the presence of 0.1 M corresponding competitive
sugar.
It was critical to incubate the first plate in BSA
and transfer the treated J2 to a clean microfilter plate
before addition of peroxidase substrate to reduce background
to negligible levels. The reduction in background levels
allowed the detection of differential amounts of lectin in
microplate wells which contained lectin-treated nematodes.
No peroxidase activity above background levels was detected
among untreated, preinfective Meloidogyne spp. J2 and
lectin wash treatments.
Relatively high numbers of J2 per sample (approx.
2000 J2 per microplate well) were required at the initiation
of each experiment. As many as 75% of the J2/well remained
in the first microfilter plate after transfer of washed

61
nematodes to a fresh microfilter plate for peroxidase
substrate reaction. No J2 were observed in the wash
solution which had passed through the first microfilter
plate. Thus, estimation of the number of nematodes in a
sample was made upon transfer of washed nematodes to the
second microfilter plate.
Approximately 500-2000 J2 per well were used to
quantify the amount of lectin bound to nematodes after
addition of peroxidase substrate. Lectins were most likely
bound to the surface of the Meloidogyne spp. J2 examined,
since microscopic observation of lectin-treated J2 from
aliquots transferred to peroxidase substrate reaction plates
indicated that J2 were intact and viable. Different amounts
of lectin bound to J2 of Mil, Mi3, and Mj (Table 3-1).
Preinfective J2 of Mj bound more SBA, LOT, and WGA than Mil
or Mi3 J2, and preinfective J2 of Mil bound more LPA than Mj
and Mi3 in two experiments. Populations of Mi3 bound less
lectin than J2 of Mil and Mj in all tests except experiment
2 with LOT. Preinfective J2 of Mil bound the most CON A in
experiment 1, and J2 of Mj bound the most CON A in
experiment 2. Considerable differences in the relative
amount of lectin which bound to J2 within a single lectin-
nematode combination were detected between experiment 1 and
experiment 2.
i

62
Table 3-1. Binding of peroxidase-labeled lectins to second-
stage juveniles (J2) of Heloidogyne incognita race 1 (Mil),
M. incognita race 3 (Mi3), and M. javanica (Mj) as
determined by microfiltration assay in two separate
experiments. • •
Picograms lectin/J2a
Lectin
Mj
Mil
Mi3
SBAb
Exp. 1
4.20
±
0.35c
1 .36
±
0.07
0.63
±
0.03
Exp. 2
3.63
±
0.21
1 .30
±
0.09
0.55
+
0.03
CON A
Exp. 1
0.66
±
0.04
0.81
±
0.03
0.62
±
0.04
Exp. 2
1 .04
±
0.07
0.86
±
0.06
0.70
±
0.04
LOT
Exp. 1
9.07
±
1 .01
4.79
±
0.35
4.40
±
0.49
Exp. 2
6.18
±
0.43
4.17
±
0.15
4.33
±
0.13
WGA
Exp. 1
3.42
±
0.25
0.39
±
0.11
0.21
±
0.03
Exp. 2
1 .92
±
0.10
0.78
±
0.07
0.54
±
0.07
LPA
Exp. 1
1.18
±
0.05
2.31
±
0.07
0.65
±
0.05
Exp. 2
1 .25
±
0.07
3.13
±
0.13
0.83
±
0.05
a Nanograms lectin divided by the number of J2 (ca. 500-
2000 J2) estimated for each sample.
b Soybean agglutinin (SBA); Concanavalin A (CON A); Lotus
tetragonolobus agglutinin (LOT); wheat germ agglutinin
(VIGA); Limulus polyphemus agglutinin (LPA).
c Mean of 20 observations ± standard error.

63
Discussion
Differences in lectin binding among populations of
Meloidogyne spp. that were not detected in assays with
fluorescent lectins were detected by microfiltration enzyme-
linked lectin assay. Variability in estimation of the
number of J2 per sample most likely influenced the
quantitative differences in lectin binding to J2 determined
among Mil, Mi3, and Mj and between experiments 1 and 2. If
this were a major influence, however, standard errors should
have been greater than those calculated for each experiment.
The quantitative differences in lectin binding detected in
these experiments could have been due to the production of
carbohydrates in amphidial secretions of J2 of Meloidogyne
spp. as reported above and elsewhere (76). Differences
between experiments 1 and 2 may be due to the handling and
relative age of the groups of J2 used in separate
experiments. The rate of production of amphidial secretions
by J2 of Meloidogyne spp. and the amount of amphidial
secretion lost (if any) through the initial centrifugation
or microfiltration wash remains unknown. Loss of excess
amphidial secretion was noted in preliminary lectin-TRITC
assays when centrifugation was used for all nematode washes,
but this occurrence was inconsistent (unpublished
results).
i

64
The technique described here for quantifying lectin
binding to Meloidogyne spp. is useful because it is rapid
and provides ease of handling compared to techniques used
previously to quantify lectin binding to nematodes and
bacteria (77, 88). The assay is very sensitive (nanogram
level), but a considerable number of nematodes (several
hundred) was necessary to achieve measurements above
background levels. The potential use of this technique for
quantification of lectin binding to other microorganisms or
detection of nematode surface antigens by immunoassay should
be considered.

CHAPTER 5
REPRODUCTION OF LECTIN-TREATED MELOIDOGYNE SPP.
IN TWO RELATED SOYBEAN CULTIVARS
Introduction
Interactions between nematodes and other organisms
are influenced by chemosensory stimuli (22, 29, 40, 45, 86,
131, 136). Researchers have postulated that intervention in
host finding and recognition of nematodes may be achieved by
blockage or obliteration of carbohydrates on nematode
surfaces (135, 136). Proteins (lectins) that bind mannose,
glucose, and sialic acids, and enzymes (glycohydrolases)
which may cleave these carbohydrates from nematode surfaces
impaired nematode chemotaxis toward source attractants (49,
53). Adhesion of conidia of Meria coniospora Drechs. to
nematode chemosensory organs, nematode attraction to the
fungus, and infection of nematodes by adhering conidia were
inhibited by sialic acids, sialidase, or limulin (50, 51,
52). A lectin specific for mannose appeared to inhibit
chemoreception necessary for the feeding and sexual
attraction of males of Trichostrongylus columbriformis Giles
(14). Capture of nematodes by Arthrobotrys oligospora Fres,
appeared to involve interaction of lectin on fungal traps
65

66
with N-acetylgalactosamine moieties present on the nematode
surface (17, 81).
Other studies have reported the presence of
carbohydrates on the head region and surface of some
phvtoparasitic nematodes (see Chapter 2 above, 36, 76, 77,
95, 113, 115). Various lectins bound to carbohydrates
present in amphidial exudates of second-stage juveniles (J2)
of potato cyst and root-knot nematodes (see Chapter 2 above,
36, 76). Differences in structure and relative amounts of
specific carbohydrates in amphidial carbohydrate complexes
of several populations of Meloidogyne spp. have been
reported above in Chapters 2 and 4.
Nematode surface carbohydrates may be involved in
plant-nematode interactions (see Chapter 3 above, 74). Soil
applications of Concanavalin A (Con A; mannose and glucose-
specific lectin), and relatively higher concentrations of
Limax flavus agglutinin (LFA; sialic acid-specific lectin),
significantly suppressed galling of tomato roots induced by
Meloidogyne incognita (Kofoid & White) Chitwood (74).
Treatment of J2 from a population of race 3 of M. incognita
with various lectins and carbohydrates promoted
hypersensitivity in an apparently compatible soybean-M.
incognita interaction (see Chapter 2 above). Nematodes
could not be detected within soybean roots after inoculation
of root tips with J2 of Meloidogyne spp. suspended in
i

67
solutions of LFA or sialic acid. The objective of this
research was to determine the quantitative effect of several
lectins and carbohydrates on establishment and reproduction
of three populations of Meloidogyne spp. in two related
soybean (Glycine max (L.) Merr.) cultivars.
Materials and Methods
Populations of Meloidogyne incognita races 1 and 3
(Mil and Mi3) and M. javanica (Treub) Chitwood (Mj) were
maintained in greenhouse culture on roots of 'Rutgers'
tomato (Lycooersicon esculentum Mill.) and 'Black Beauty'
eggplant (Solarium melongena L.). Meloidogyne spp.
populations were identified by adult female perineal
patterns, second-stage juvenile (J2) lengths, and
performance on differential hosts (101). Species
identifications were also confirmed by three independent
nematode taxonomists (A. M. Golden, Beltsville, MD; J. G.
Baldwin, Riverside, CA; J. D. Eisenback, Blacksburg, VA).
Eggs of each nematode population were extracted from host
roots with 0.53% NaOCl for 30 seconds (46) and hatched at
room temperature on a Baermann funnel. Preinfective J2
which had hatched within 48 hours were used as test
organisms in each experiment.
Surface carbohydrates of Meloidogyne spp. J2 were
blocked by incubating nematodes in solutions containing

68
unconjugated, purified soybean agglutinin (SBA), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), CON
A, or Limulus polvphemus agglutinin (LPA) (E-Y Labs, San
Mateo, CA). The sugar specificity of each lectin,
corresponding competitive sugars, and procedure used to
determine the specific hemagglutination activity for each
lectin were described in Chapter 2. Buffer solutions
included: 0.01 M phosphate-buffer saline (PBS) at pH 7.2 for
SBA, WGA, and LOT; 0.05 M Tris-saline plus 0.01 M CaCl2 at
pH 7.5 for CON A; 0.05 M Tris-saline plus 0.01 M CaCl2 at pH
8.0 for LPA.
Preinfective J2 of Mil, Mi3, and Mj were
concentrated in the appropriate buffer or in distilled water
by centrifugation at 1 000 each lectin included incubating J2 (approx. 2000 J2) of each
population in lectin solution (200 ug/ml), lectin (200
ng/ml) plus 0.1M competitive sugar, and 0.1M sugar solution
minus lectin for 2 hours at 4°C. Control treatments
included J2 in buffer and J2 in distilled water incubated
for 2 hours at 4*C. Suspensions of J2 in each treatment
(1.0 ml total volume per treatment) were diluted to 16 ml
(12.5 ug/ml lectin and/or 6.25mM sugar) immediately before
being added to soil in which soybeans were grown as
described below.
i

69
Since a 0.1 M solution of sialic acid in 0.05 M
Tris-saline buffer was quite acidic (pH ~ 3.0), a soybean
root penetration bioassay was conducted to evaluate the
effects of sialic acid neutralized with NaOH (pH 7.0) and of
an acidic buffer (pH 3.0) on activity of J2 of Meloidogyne
spp. (see Appendix D below). Solutions containing J2 of
Mil, Mi3, and Mj were incubated for 2 hours at 4°C, diluted
1:16 with Tris-saline buffer, placed on soybean roots, and
the number of 32 within roots was determined after 24 hours.
Two related cultivars of soybean (Glycine max cv.
Pickett 71 and Centennial) were used for root challenge by
J2 of Meloidogyne spp. Pickett 71 was compatible and
Centennial was incompatible with M. incognita, and both
soybean cultivars were compatible with M. javanica (61).
Individual soybean seedlings were grown in a greenhouse in
150-cm^ Conetainers (Leach Nursery, Canby, OR) containing
steam-pasteurized Astatula fine sand (hyperthermic, uncoated
typic quartzipsamments). Two-milliliter suspensions
(approx. 2000 32) of each treatment combination were added
to the soil in each Conetainer using a syringe fitted with a
10-cm-long canulus (24). There were four replicates of each
treatment combination. Test plants were maintained in a
glasshouse at 27 ± 3°C, watered daily, and fertilized once a
week with a solution containing 10-6-10 (N-P-K) plus

70
microelements. Experiment 1 was conducted in the spring and
experiment 2 was conducted in the summer.
Soybean plants were removed from Conetainers 60 days
after soil was infested with J2 and the roots were rinsed
free of soil. The number of Meloidogyne spp. egg masses per
root system was rated on a 0-5 scale (101 ). Data were
subjected to analysis of variance procedure and treatment
differences were determined by the Waller-Duncan k-ratio t-
test with k=100 (P £ 0.05). This experiment was repeated
once.
Results
Hemagglutination assays indicated that the binding
capacity of pure lectins was relatively strong, except for
LPA. Specific hemagglutination activities of 4096, 4096,
8192, 8192, and 16 units/mg lectin were determined for SBA,
CON A, WGA, LOT, and LPA, respectively. Hemagglutination
activity of all lectins was completely inhibited in the
presence of 100 mM corresponding competitive sugar.
Pickett 71 soybean was highly compatible with Mil in
two experiments, as indicated by the high egg mass ratings
in buffer and water controls (Table 4-1). Little reduction
in Mi 1 reproduction in Pickett 71 compared to controls was
demonstrated by any lectin and sugar treatment except LPA
plus sialic acid and sialic acid alone in the second test.
This was not, however, verified by the results of the first

Table 4-1. Reproduction of Meloidogyne incognita race 1 in 'Pickett 71' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass
: rating3/lectin
Treatment
SBAb
WGA
LOT
CON
A
LPA
Experiment
1
Lectin
4.50
*
a
3.50
abede
3.75 abed
4.50
a
3.00
ede
Lectin +
sugar
4.25
ab
3.50
abede
3.00 ede
3.75
abed
2.75
de
Sugar
3.25
bcde
3.75
abed
3.25 bcde
4.25
ab
2.50
e
Buffer
4.00
abc
3.50
abede
3.50 abede
4.00
abc
2.50
e
Distilled
water
4.00
abc
3.75
abed
4.25 ab
4.25
ab
3.00
ede
Experiment
2
Lectin
4.50
abc
4.25
bed
4.50 abc
4.00
cd
4.50
abc
Lectin +
sugar
4.25
bed
4.75
ab
4.75 ab
5.00
a
1 .00
e
Sugar
4.75
ab
3.75
d
4.50 abc
4.50
abc
3.75
d
Buffer
4.50
abc
4.00
cd
4.50 abc
5.00
a
4.75
ab
Distilled
water
5.00
a
4.75
ab
4.25 bed
4.75
ab
5.00
a
a Scale;
0 = 0; 1
1
II
2 = 3
-10; 3 =
II
I
u>
o
^ •
4 = 31-100;
5 = >100 egg
masses
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.

72
test. Reproduction of Mil on Pickett 71 in experiment 2 was
significantly lower for LPA plus sialic acid compared to all
other lectin plus sugar treatments. The rating for LPA plus
sialic acid in the first experiment, however, was only
significantly lower than that of SBA plus galactose.
Treatment with LPA alone in the first experiment,
significantly reduced reproduction of MI1 in Pickett 71
compared to SBA and CON A alone. Sialic acid significantly
reduced the egg mass rating in experiment 1 compared to N-
acetylglucosamine and mannose. Both buffer and water
controls for LPA produced relatively low egg mass ratings,
however, compared to all other treatments in experiment 1.
Centennial soybean was highly incompatible with Mil,
as indicated by poor nematode reproduction among all lectin,
sugar, and control treatments (Table 4-2). Significantly
lower egg mass ratings were produced in Centennial by Mil
for several treatments in experiment 2; however the ratings
were very low overall, and the few lower ratings in
experiment 2 were not verified by results from the first
experiment.
Race 3 of M. incognita was highly compatible with
Pickett 71; almost all treatments and controls had high egg
mass ratings (Table 4-3). Treatment with sialic acid alone
and sialic acid plus LPA significantly reduced reproduction
of Mi3 in Pickett 71 over that in the controls in both
i

Table 4-2. Reproduction of Meloidogyne Incognita race 1 in 'Centennial' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
1 .00
*
a
0.50
a
0.75
a
0.75
a
0.75
a
Lectin + sugar
1 .50
a
1 .00
a
1 .25
a
1 .25
a
0.50
a
Sugar
1 .75
a
1 .25
a
1 .00
a
1 .00
a
0.50
a
Buffer
1 .25
a
1 .25
a
0.75
a
1 .00
a
1 .00
a
Distilled water
1.25
a
1 .50
a
1 .25
a
1 .75
a
1 .25
a
Experiment 2
Lectin
1 .25
abcde
1 .75
abc
1 .75
abc
1 .00
bede
1 .50
abed
Lectin + sugar
1 .50
abed
1 .75
abc
1 .25
abcde
1 .50
abed
0.75
ede
Sugar
1 .75
abc
1 .25
abcde
1 .50
abed
1 .25
abcde
0.25
e
Buffer
1 .50
abed
2.00
ab
1 .50
abed
1 .75
abc
1 .50
abed
Distilled water
2.25
a
1 .50
abed
1 .75
abc
1.25
abcde
0.50
de
a Scale: 0 = 0; 1
= 1-2;
2 = 3-
10; 3 .
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
«-j
u>

Table 4-3. Reproduction of Meloidogyne incognita race 3 in 'Pickett 71* soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
4.75
ab*
4.75
ab
4.75
ab
5.00
a
4.00
bed
Lectin + sugar
4.50
abc
3.75
cd
4.50
abc
3.25
de
2.50
ef
Sugar
5.00
a
3.75
cd
5.00
a
4.50
abc
2.25
f
Buffer
4.50
abc
4.00
bed
4.75
ab
4.75
ab
4.75
ab
Distilled water
4.75
ab
4.75
ab
4.75
ab
4.50
abc
4.50
abc
Experiment 2
Lectin
4.50
ab
4.25
be
5.00
a
4.50
ab
4.50
ab
Lectin + sugar
5.00
a
4.75
ab
4.50
ab
4.50
ab
1 .25
d
Sugar
4.75
ab
4.50
ab
5.00
a
4.75
ab
3.75
c
Buffer
5.00
a
4.75
ab
4.75
ab
5.00
a
4.50
ab
Distilled water
5.00
a
4.75
ab
5.00
a
5.00
a
5.00
a
a Scale: 0 = 0; 1
CN
1
II
2 =
3-10; 3 :
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
-j

75
experiments. Moderate reduction in Mi3 reproduction in
Pickett 71 was observed for CON A plus mannose treatment, as
compared to the controls, in experiment 1 but not in
experiment 2.
Centennial soybean was relatively incompatible with
Mi3, as indicated by low egg mass ratings for the majority
of treatments in two experiments (Table 4-4). Significant
differences in Mi3 reproduction in Centennial were observed
for some treatments, but ratings were low overall and none
of the reductions in one of the tests was verified by a
similar reduction in the other test.
Pickett 71 soybean was compatible with Mj, as
indicated by high egg mass ratings for many lectin, sugar,
and control treatments (Table 4-5). Reproduction of Mj in
Pickett 71 was significantly reduced by sialic acid and
sialic acid plus LPA treatment compared to all other
treatments in the second experiment. Treatment with sialic
acid alone and LPA plus sialic acid significantly reduced
reproduction of Mj in Pickett 71 compared to all other
sugar, and lectin plus sugar treatments, respectively, in
experiment 1. Ratings for sialic acid and LPA plus sialic
acid were not significantly lower, however, than those for
buffer and water controls of LPA in the first experiment.
Relatively high egg mass ratings for many lectin,
sugar, and control treatments indicate that Centennial

Table 4-4. Reproduction of Meloidogyne incognita race 3 in 'Centennial' soybean
roots after treatment of second-stage juveniles with selected lectins and their
competitive sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment 1
Lectin
1 .00
*
c
1 .00
c
1 .00
c
2.00
abc
2.00
abc
Lectin + sugar
1 .00
c
2.00
abc
1 .25
be
2.25
ab
1 .25
be
Sugar
1.50
abc
1 .75
abc
1 .25
be
1 .75
abc
1 .50
abc
Buffer
2.50
a
1 .50
abc
1 .00
c
2.25
ab
1 .75
abc
Distilled water
2.25
ab
1 .75
abc
1 .50
abc
1 .25
be
1 .25
be
Experiment 2
Lectin
2.50
ab
1 .75
bed
2.00
abc
2.00
abc
1 .50
cd
Lectin + sugar
2.75
a
2.50
ab
2.00
abc
2.50
ab
1 .00
d
Sugar
2.50
ab
2.50
ab
2.25
abc
2.00
abc
1 .75
bed
Buffer
2.50
ab
2.75
a
2.00
abc
2.25
abc
2.75
a
Distilled water
2.75
a
2.50
ab
2.25
abc
1 .75
bed
2.50
ab
a Scale: 0 = 0; 1
= 1-2;
2 =
3-10; 3 =
= 11-30;
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.
a\

Table 4-5. Reproduction of Meloidogyne javanica in 'Pickett 71' soybean roots after
treatment of second-stage juveniles with selected lectins and their competitive
sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA LOT CON A
LPA
Experiment
Lectin
1
3.75
,*
ab
2.50
edefg
3.25
abed
2.00
ef g
2.75
bedef
Lectin +
sugar
3.00
abcde
3.75
ab
4.00
a
3.00
abcde
1 .50
g
Sugar
3.00
abcde
3.25
abed
3.75
ab
3.75
ab
1 .75
fg
Buffer
3.25
abed
3.50
abc
3.00
abcde
2.25
defg
2.00
efg
Distilled
water
3.25
abed
3.50
abc
2.25
def g
3.00
abcde
1 .50
g
Experiment
Lectin
2
4.00
bed
4.50
abc
4.50
abc
3.00
e
4.25
abed
Lectin +
sugar
3.50
de
4.75
ab
4.75
ab
4.50
abc
1 .25
f
Sugar
4.00
bed
3.75
ede
5.00
a
4.50
abc
1 .00
f
Buffer
4.00
bed
4.25
abed
4.25
abed
4.50
abc
4.25
abed
Distilled
water
4.75
ab
4.50
abc
4.25
abed
4.25
abed
4.50
abc
a Scale:
0 = 0; 1
= 1-2;
2 = 3-
o
•
U)
II
1
UJ
o
4 =
31-100;
5 = >100 egg
masses
/ root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
*
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P s 0.05) according the the
Waller-Duncan k-ratio t-test.

78
soybean was strongly compatible with Mj (Table 4-6).
Reproduction of Mj in Centennial was significantly reduced
by sialic acid and LPA plus sialic acid compared to all
other treatments in experiment 2. Treatment with LPA plus
sialic acid significantly reduced egg mass ratings in the
first experiment compared to SBA plus galactose and WGA plus
N-acetylglucosamine. Reproduction of Mj in Centennial was
significantly reduced by treatment with sialic acid in
experiment 1, compared to egg mass ratings for galactose, N-
acetylglucosamine, and fucose. Ratings for sialic acid
alone and sialic acid plus LPA, however, were not
significantly lower than those for buffer and water controls
of LPA in the first experiment.
Discussion
Results from this research generally agree with the
histological observations reported above in Chapter 3. The
inhibition of reproduction of untreated Mi3 in Centennial
soybean roots, however, contrasts with the apparently
compatible response of Centennial root tissue to untreated
Mi3 observed in histological tests. Intact giant cells were
associated with untreated Mi3 in Centennial soybean roots 20
days after exposure of roots to infective J2 of Mi3.
However, no gall formation or development of Mi3 past third-
stage juvenile was observed 20 days after inoculation.

Table 4-6. Reproduction of Meloidogyne javanica in 'Centennial' soybean roots after
treatment of second-stage juveniles with selected lectins and their competitive
sugars.
Egg mass ratinga/lectin
Treatment
SBAb
WGA
LOT
CON
A
LPA
Experiment
1
Lectin
4.50
ab*
3.00
edef
3.75
abed
2.25
fg
4.00
abc
Lectin +
sugar
3.75
abed
4.00
abc
3.00
edef
2.75
def g
1 .75
fg
Sugar
3.50
bede
4.00
abc
4.25
ab
3.00
edef
2.00
fg
Buffer
4.50
ab
4.75
a
4.50
ab
2.50
ef g
3.00
edef
Distilled
water
3.50
bede
3.50
bede
4.50
ab
3.50
bede
2.50
ef g
Experiment
2
Lectin
4.25
abc
4.50
abc
4.75
ab
4.00
be
4.00
be
Lectin +
sugar
4.00
be
4.75
ab
4.50
abc
4.00
be
1 .25
d
Sugar
4.25
abc
4.75
ab
4.25
abc
3.75
c
1 .75
d
Buffer
4.75
ab
5.00
a
4.50
abc
4.75
ab
4.50
abc
Distilled
water
4.75
ab
4.25
abc
4.75
ab
4.25
abc
4.00
be
a Scale:
0 = 0; 1
= 1-2;
2 = 3
-10; 3 =
= 11-30;
4 =
31-100;
5 = >100 egg
masses,
/root
system.
b Lectins and corresponding competitive sugars included soybean agglutinin (SBA)
and galactose; wheat germ agglutinin (WGA) and N-acetylglucosamine; Lotus
tetragonolobus agglutinin (LOT) and fucose; Concanavalin A (CON A) and mannose;
Limulus polyphemus agglutinin (LPA) and sialic acid.
Table values are the mean of four relicates. Means followed by the same letter
for each experiment are not significantly different (P £ 0.05) according the the
Waller-Duncan k-ratio t-test.

80
Strong reduction in the rate of Mi3 development, or a
possible nutritional deficiency which culminated in nematode
death, may have occurred in this host-parasite relationship
(91, 133) since no evidence of active plant defense
(ie. hypersensitivity) occurred in histological tests.
Differences in the degree of incompatibility of "M.
incognita-resistant" soybean cultivars with several M.
incognita populations have also been reported (105).
Any effect of lectin or sugar on successful nematode
infection of soybean roots most likely occurred at initial
infection; however, environmental conditions and duration of
the experiment were conducive to at least two generations of
root-knot nematode reproduction. One investigation has
indicated that soil application of CON A significantly
reduced galling of tomato roots by M. incognita (74), but
the activity of CON A in soil was difficult to interpret.
Although moderate reductions in egg mass ratings were
occasionally associated with CON A, sialic acid appeared to
have the greatest and most consistent adverse effect on
successful nematode infection. These results are supported
by the apparent inability of several Meloidogyne spp. J2 to
penetrate soybean roots in "unwashed" sialic acid and LFA
treatments (see Chapter 3 above). Hemagglutination tests
determined that the binding capacity of LPA was relatively
weak, and it was completely inhibited in the presence of 100

81
mM sialic acid. This may indicate that concentrations of
LPA when mixed with sialic acid were insufficient to inhibit
(and may have acted in combination with) the activity of
sialic acid on root-knot nematode infection of soybean
roots. Threshold levels of sialic acid that significantly
inhibit nematode infection need to be determined.
Microscopic observation of J2 treated with sialic acid and
LPA, and penetration of soybean roots by J2 treated with
sialic acid and LFA and "washed", indicated that these
treatments are not lethal to J2 of Meloidogyne spp. (see
Chapters 2 and 3 above). The inhibition of soybean root
penetration after treatment of J2 of Meloidogyne spp. with
sialic acid was apparently more than just an adverse effect
of low pH (see Appendix D below). The adverse effect of
sialic acids on Meloidogyne spp. reproduction in soybean may
be manifested in impairment of host-finding and penetration
by treated J2. Perhaps sialic acids act as "biological
masks" similar to those found in other animal systems (104).
Subsequent investigations of these phenomena may provide
information valuable to the development of novel means of
nematode management.
i

CHAPTER 6
SUMMARY AND CONCLUSIONS
Proteins (lectins) which bind to specific
carbohydrates were used as probes to characterize
carbohydrates on the surfaces of second-stage juveniles (J2)
of the root-knot nematodes Meloidogyne incognita races 1 and
3 (Mil and Mi3) and Meloidogyne j avanica (Mj). The binding
of the fluorescent (rhodamine conjugated) lectins, soybean
agglutinin (SBA), Concanavalin A (CON A), wheat germ
agglutinin (WGA), Lotus tetragonolobus agglutinin (LOT), and
Limulus polyphemus agglutinin (LPA) to freshly-hatched,
preinfective J2 of root-knot nematodes was comparable among
the populations of Melodogyne spp. examined. It was
apparent from these experiments that nematode viability was
critical for accurate detection of fluorescent lectin
binding to nematodes, and that rhodamine (TRITC) conjugates
of lectin were preferable to fluorescein (FITC) conjugates
since J2 of Meloidogyne spp. autofluoresced at the
excitation wavelength of FITC. Viable, preinfective J2
bound fluorescent lectin almost exclusively in the vicinity
of the amphidial (cephalic chemosensillae) openings.
Substances to which lectins bound were concentrated and
82

83
sometimes emanated from the amphidial region of J2 of
Meloidogyne spp. Amphids of J2 of Mil. Mi3, and Mj labeled
weakly with SBA, CON A, and LPA and strongly with WGA and
LOT. The greater intensity of fluorescent labeling by WGA
and LOT conjugates may have been due to their relatively
higher binding capacities as indicated by hemagglutination
assays. The presence of fucosyl and sialyl residues in
amphidial secretions was supported by the inhibition of LOT
and LPA binding, respectively, in the presence of 0.1 M
corresponding competitive sugars. The binding of SBA, CON
A, and WGA was not inhibited in the presence of 0.1 M
corresponding competitive sugars. This probably indicates
nonspecific binding of the lectins to nematodes, or possibly
a strong affinity of SBA, CON A,and WGA for carbohydrate-
specific sites in amphidial secretions. Different molecular
forms of the competitive sugars chosen may have provided
greater inhibition of lectin binding activity. Soybean
agglutinin, CON A, and WGA may have bound to hydrophobic
sites (ie. lipids) on the nematode surface, and would not
have been influenced by competitive sugars. All fluorescent
lectins tested bound to egg shells of Mil, Mi3, and Mj and
were not inhibited in the presence of competitive sugars.
Results from treatment of J2 with enzymes
(glycohydrolases) that cleave specific carbohydrate residues
from glycoconjugates, including a-galactosidase, a-
i

84
mannosidase, S-N-acetylglucosaminidase, a-fucosidase, and
sialidase (neuraminidase), and subsequent binding of
fluorescent lectins to treated J2 suggested that differences
in the sequence and spatial arrangement of amphidial
carbohydrate complexes exist among Mil, Mi3, and Mj. A
number of different glycohydrolases eliminated binding of
LPA-TRITC to amphids of J2 which suggested that sialic acids
were some of the outermost carbohydrate moieties present in
J2 amphidial carbohydrate complexes. Several
glycohydrolases promoted binding of CON A to the anterior
cuticle of Mil and Mj , and WGA to the anterior cuticle of
Mil .
Quantitative differences in lectin binding to J2 of
Mil, Mi3, and Mj were determined by a modified
microfiltration enzyme immunoassay developed for use with
peroxidase-labeled lectins instead of antibody probes.
Preinfective J2 of Mj bound the greatest amount of SBA, LOT,
and WGA while J2 of Mil bound the most LPA in two separate
experiments. Preinfective J2 of Mi3 consistently bound the
least amount of all lectins tested. This may indicate that
preformed carbohydrates in amphidial secretions of J2 differ
quantitatively, as well as in configuration, among different
populations of Meloidogyne spp. The rate of production of
amphidial secretions by J2 of Meloidoavne spp. has not been
determined. The microfiltration assay using peroxidase-

85
labeled lectins was rapid and relatively easy to conduct.
Although the assay was sensitive (nanogram level), it
required relatively high numbers (approx. 2000) of J2 per
sample to achieve final levels of lectin binding that were
sufficiently higher than background levels.
Differential lectin binding to the head region of
preinfective J2 suggested that carbohydrates were
concentrated in this portion of invasive juveniles. Since
the anterior end of these nematodes appears to stimulate
hypersensitive incompatible (resistant) plant responses, it
was hypothesized that blocking of preformed amphidial
carbohydrates with lectins might influence recognition and
specificty in incompatible and compatible Meloidogyne spp.-
soybean interactions. The response of root tissue was
examined histologically 5 days after exposure of M.
incognita-compatible 'Pickett 71' and M. incognijta-
incompatible 'Centennial' soybean roots to lectin and/or
sugar-treated J2 of Mil, Mi3, and Mj. Untreated J2 of all
three root-knot nematode populations induced the formation
of feeding sites (giant cells) in Pickett 71 soybean roots,
and untreated J2 of Mi3 and Mj induced giant cell formation
in Centennial soybean roots 5 days after inoculation. Giant
cells were maintained in Centennial 20 days after
inoculation with untreated J2 of Mi3, but no gall formation,
hyperplasia of pericycle cells adjacent to giant cells, or
i

86
development of Mi3 past third-stage juvenile was observed at
day 20. Untreated J2 of Mil induced a hypersensitive
response (HR; localized plant tissue necrosis) in Centennial
soybean roots 5 days after inoculation. Treatment of J2 of
Mil and Mj with SBA, CON A, WGA, LOT, LPA and/or their
corresponding competitive sugars did not influence the root
tissue response of either soybean cultivar to infection by
these nematodes. Hence, the surface carbohydrates of Mil
and Mj did not appear to be involved in plant-nematode
interactions, but the fate of lectin bound to J2 once they
have entered the plant root remains unknown. Treatment of
J2 of Mi3 with any lectin and/or sugar tested, however,
induced the formation of HR in Centennial soybean root
tissue 5 days after inoculation. Treatment of J2 of Mi3 in
this manner may actually have facilitated recognition of
invasive J2 and subsequent defense response by the plant.
The lack of specificty of lectin or sugar effects in the
Mi3-Centennial interaction, however, makes it seem unlikely
that alteration of surface carbohydrate composition of
preinfective J2 and recognition by a carbohydrate-specific
plant receptor was responsible for promoting
incompatibility. If one considers the interaction of
nematode surface carbohydrates with potential plant cell
surface receptors as a "lock and key" phenomenon, however,
it may be feasible that a slight alteration in surface

87
carbohydrate compostion was sufficient to promote
incompatibility to Mi3 in Centennial soybean roots.
Possibly a greater alteration of the carbohydrates examined
here on Mil and Mj, or alteration of surface carbohydrates
not examined in these studies, would influence their host-
parasite interactions.
Treatment of J2 of Mi3 with lectin or sugar may have
stimulated the production of a substance by the nematode
that induces HR in Centennial soybean roots. Juveniles of
Mil may inherently have the capacity to induce HR while J2
of Mj cannot promote incompatibility in soybean no matter
what the treatment. For some populations, such as in the
case of Mi3, incompatibility may be a process that can be
stimulated. Conversely, substances produced by J2 of
Meloidogyne spp. (ie. amphidial or stylet secretions) may be
essential to induce compatibility between host and parasite,
and these substances were altered sufficiently in Mi3 to
inhibit compatibility in Centennial soybean roots.
The ability of J2 of Mil, Mi3, and Mj to penetrate
the roots of either soybean cultivar was apparently strongly
impaired when J2 were introduced to roots in a solution that
contained Limax flavus agglutinin (LFA; sialic acid-
specific), sialic acid, or combination of the two. The
inhibition of soybean root penetration after sialic acid
treatment of J2 of Meloidogyne spp. was apparently more than

88
just an adverse effect of low pH. These treatments were not
lethal to J2 of Meloidogyne spp. since J2 that were treated
with LFA and/or sialic acid and rinsed regained their
infectivity to both soybean cultivars. In addition, J2
treated with LPA-TRITC and/or sialic acid and rinsed were
viable when observed microscopically.
The results of histological experiments were
generally confirmed by greenhouse assays that were designed
to quantify the effects of SBA, CON A, WGA, LOT, LPA, and
their competitive sugars on Meloidogyne-soybean
interactions. The reproduction of lectin and/or sugar-
treated J2 of Mil, Mi3, and Mj in Centennial and Pickett 71
soybean roots was determined by rating the number of root-
knot nematode egg masses per root system 60 days after
inoculation with treated J2. The reproduction of untreated
Mi3 in Centennial soybean was extremely poor, in contrast to
the apparent compatibility (giant cells) observed in
Centennial root tissue after exposure of soybean roots to
untreated J2 of Mi3. Only treatment of J2 of Mil, Mi3, and
Mj with sialic acid, and especially sialic acid plus LPA,
significantly reduced reproduction of these nematodes in
compatible soybean cultivars. This may have been an
indirect result of reduced initial root penetration by J2 of
Meloidogyne spp. that were exposed to these treatments. It
is possible that sialic acid residues proximate to nematode

89
cheraosensillae have a masking or regulatory effect similar
to that observed in other animal systems. Modification of
sialic residues in nematode chemosensory organs may strongly
impair host finding and penetration of plant roots by root-
knot nematodes.
i

APPENDIX A
BINDING OF FLUORESCENT SOYBEAN AGGLUTININ TO POSTINFECTIVE
SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.
Alteration of nematode surface or secretory
carbohydrates may influence plant compatibility with phyto-
parasitic nematodes. Changes in surface carbohydrates of
second-stage juveniles (J2) of Meloidogyne spp. which may
occur once J2 enter plant roots have never been studied.
Nematode penetration and subsequent interaction with plant
roots may stimulate or alter production of secretory or
surface molecules by the nematode. Conversely, plant
products such as lectins or carbohydrates may bind to sites
which exist on infective J2 and influence plant-nematode
interactions. We have conducted preliminary experiments to
monitor potential changes in surface carbohydrates of J2 of
several Meloidogyne spp. populations after they have entered
roots of compatible and incompatible soybean cultivars.
Seeds of 'Pickett 71' and 'Centennial' soybean were
germinated in ragdolls and placed on trays of autoclaved
sand as described for histology experiments (see Chapter 3
above). One hundred-microliter suspensions (approx. 2000 J2
hatched within 48 hours of inoculation) of Meloidogyne
incognita races 1 and 3 (Mil, Mi3), and M. javanica (Mj) in
90
i

91
tap water were placed on separate soybean root tips of both
cultivars. Soybean roots were washed free of sand and
inoculated segments of roots were excised and placed in
phosphate-buffer saline (PBS), pH 7.2, approximately 40
hours after inoculation.
Excised root segments of each Meloidogyne spp.
population-soybean cultivar combination were immediately
decorticated under a 40x dissecting microscope. Soybean
root steles and cortices containing Meloidogyne spp. J2 were
placed in separate BPI dishes containing PBS. The root
tissue was incubated at room temperature overnight to allow
J2 to emerge from the plant tissue. These "postinfective"
J2 were washed three times with PBS and subsequently
incubated in fluorescent, soybean agglutinin-
tetramethylrhodamine isothiocyanate (SBA-TRITC) solution
(200 ug/ml), 0.1 M D-galactose solution plus SBA-TRITC (200
ng/ml), 0.1M D-galactose solution minus SBA-TRITC, or
distilled water for 2 hours at 4°C (see Chapter 2 above).
Treated postinfective J2 were washed three times with PBS,
mounted on glass slides, and immediately observed under
epifluorescent microscopy.
Little difference in labeling of postinfective J2
with fluorescent SBA was observed among Meloidogyne spp.
populations, soybean cultivars, and J2 from root cortices or
steles. No fluorescence was observed on any postinfective
i

92
J2 treated with only 0.1M D-galactose or distilled water.
Postinfective J2 of all Meloidogyne spp. populations tested
labeled weakly with SBA-TRITC in the amphidial region,
similar to results obtained with preinfective J2 (see
Chapter 2 above). The cuticles of postinfective J2 of Mil,
Mi3, and Mj labeled strongly with SBA-TRITC, however, and
this binding was greatly inhibited in the presence of 0.1M
D-galactose. Binding of SBA-TRITC to cuticles of
postinfective Meloidogyne spp. J2 was confined to the
anterior half of the body of most nematodes observed (Fig.
A-1). Fluorescent SBA did bind to the cuticle on the
posterior half of some postinfective J2, but SBA-labeling
was often discontinuous ("patchy") along the posterior
cuticle (Fig. A-1).
The binding of SBA to cuticles of postinfective J2
of Meloidogyne spp. and the inhibition of SBA binding in the
presence of D-galactose indicated the presence of galactosyl
residues on the body wall of J2 that had penetrated soybean
roots. The cuticles of preinfective J2 of Meloidogyne spp.
did not bind SBA-TRITC in this manner. It is not known
whether galactosyl residues on the body wall of
postinfective J2 of Meloidogyne spp.are of plant or nematode
origin.

93
Fig. A-1. a) Binding of fluorescent (rhodamine conjugated)
soybean agglutinin (SBA) to anterior cuticle of second-stage
juvenile (J2) of Meloidogyne javanica that was removed from
'Centennial' soybean root stele; b) Discontinuous binding of
fluorescent SBA to tail region of 32 of M. incognita race 3
that was removed from Centennial soybean root cortex. FC =
fluorescent cuticle (note body annulation); PF = "patchy"
fluorescence.

APPENDIX B
VIABILITY OF SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.
AFTER EXPOSURE TO GLYCOHYDROLASE BUFFERS
The importance of nematode viability for accurate
detection of Meloidogyne spp. surface carbohydrates with
fluorescent lectins has been emphasized above in Chapter 2.
The relatively low pH of most glycohydrolase buffers, the
presence of ammonium sulfate in solution, and the incubation
of second-stage juveniles (J2) in solutions at 37°C for
extended periods of time may have threatened nematode
viability in the glycohydrolase experiments presented above
in Chapter 2. To determine the effect of experimental
conditions (minus glycohydrolase) on viability of
Meloidogyne spp. J2, the following bioassay was conducted.
Preinfective J2 of Meloidogyne incognita race 1
(Mil) hatched within 48 hours were concentrated separately
in the buffers described for glycohydrolase assays (see
Chapter 2 above) or in distilled water. Treatments included
Mil J2 incubated at 37°C for 2 hours and for 24 hours in one
of the following solutions:
1. a-mannosidase buffer + 0.2M (NH4)2SO^
2. a-galactosidase buffer
94

95
3. S-N-acetylglucosaminidase buffer + 0.2M
(nh4)2so4
4. a-fucosidase buffer + 0.2M (NH4)2S04
5. Neuraminidase (sialidase) buffer
6. a-mannosidase buffer + 0.2M (NH4)2S04 + 0.1M D-
mannose
7. a-galactosidase buffer + 0.1M D-galactose
8. Distilled H20
Control treatments include Mil J2 in a-man buffer minus
(NH4)2S04 or J2 in distilled H20 at room temperature for 2
and 24 hours. Nematodes incubated in every treatment
solution except distilled water appeared slightly vacuolated
under a dissecting microscope.
Seedlings of 'Rutgers' tomato and 'California
Wonder' pepper (5-10 cm tall) were grown in a greenhouse in
small plastic cups containing Astatula fine sand.
Suspensions of Mil J2 in incubation solutions were placed in
the soil of individual tomato and pepper plants. Root
systems were washed free of sand 10 days after inoculation
with treated J2, and root systems were observed for the
presence of nematode-induced galls.
Little difference in Mil infectivity was
demonstrated among the treatments tested compared to
untreated controls. The number of galls on pepper roots of
all treatments was less than the number of galls observed on

96
tomato roots, but the relative amount of galling was
consistent among treatments. The number of galls on tomato
roots induced by J2 incubated in solutions 1 and 3 for 24
hours was slightly lower than controls. However, initial
inoculum and gall number per root system were not
quantified. Results of this assay indicate that the
experimental conditions of the glycohydrolase assays above
in Chapter 2 had little effect on viability of J2 of
Meloidogyne spp.

APPENDIX C
BINDING OF FLUORESCENT LECTINS TO AXENIZED, PREINFECTIVE
MELOIDOGYNE SPP.
Aseptic technique was not used in the fluorescent
lectin assays presented above in Chapter 2. Since microbes,
especially bacteria, produce extracellular carbohydrates
that may label with fluorescent lectins, it was necessary to
determine if microbial contaminants may have affected
results reported above in Chapter 2. The following assay
was conducted to observe fluorescent lectin binding to
surface- disinfested (axenized) preinfective second-stage
juveniles (J2) of Meloidogyne spp.
Second-stage juveniles (J2) of Meloidogyne incognita
race 3 (Mi3) and M. javanica (Mj) were axenized according to
the procedure of Krusberg and Sardanelli (67).
Chromatography columns were packed with 2-6 mm diameter
glass beads and filled with a solution containing 0.05%
kanomycin sulfate and 0.01% chlorhexidine digluconate (Sigma
Chemical Co., St. Louis, MO) in sterile tap water.
Juveniles of each Meloidogyne spp. population that had
hatched within 48 hours were placed in antibiotic solution
at the top of separate columns. Nematodes migrated down the
column overnight and were aseptically removed from the
97
i

98
bottom of the column the following morning. A small
subsample of the axenized Meloidogyne spp. J2 was placed on
potato dextrose agar (PDA) to check for microbial
contamination. The remainder of the axenized J2 were
immediately washed three times in appropriate lectin buffers
and incubated for 2 hours at 4°C in the fluorescent lectin
solutions described for preinfective Meloidogyne spp. J2
(see Chapter 2 above). Surface disinfested J2 treated with
fluorescent lectin were washed, mounted on glass slides, and
specimens were immediately observed under epifluorescent
microscopy.
Binding of fluorescent lectins to axenized,
preinfective J2 of Mi3 and Mj was almost identical to the
results obtained with nonaxenized, preinfective J2 presented
above in Chapter 2. The only exception was that the cuticle
of almost all axenized J2 of Mi3 and Mj also labeled
strongly with Concanavalin A (CON A) along the entire body
surface. The binding of CON A to Meloidogyne spp. was not
inhibited in the presence of 0.1M D-mannose. The reason for
the binding of CON A to cuticles of axenized J2 of
Meloidogyne spp. is unknown.
The subsample of axenized J2 of Mi3 and Mj that was
placed on PDA proved negative for viable bacterial
contamination. The presence or absence of dead bacteria on
nematode surfaces was not confirmed. Results of this

99
axenizing assay suggest that active microbial contamination
did not influence binding of fluorescent lectins to
preinfective J2 of Meloidogyne spp. in experiments reported
above in Chapter 2.
i

APPENDIX D
EFFECT OF ACIDITY OF SIALIC ACID ON PENETRATION OF SOYBEAN
ROOTS BY SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.
Since a 100 mM solution of sialic acid in 50 mM
Tris-saline resulted in a solution pH of approximately 3.0,
the potential effect of low pH on soybean root penetration
by second-stage juveniles (J2) of Meloidogyne incognita
races 1 and 3 (Mil , Mi3) and M. javanica (Mj) was
bioassayed. Second-stage juveniles of Mil, Mi3, and Mj were
obtained and seeds of 'Pickett 71' soybean were germinated
as described above in Chapter 3. Approximately 2000 J2 from
each population of root-knot nematode were incubated for 2
hours at 4°C in 1.0 ml of either 100 mM sialic acid at pH
3.0, 100 mM sialic acid neutralized to pH 7.0 with NaOH, or
50 mi-1 Tris-saline acidified to pH 3.0 with HC1.
Approximately 32,000 J2 of each population were also
incubated in identical solutions for 2 hours at 4°C, and the
solution volume was increased from 1.0 ml to 16.0 ml with
distilled water as described above in Chapter 5. Control
treatments included aprroximately 2000 J2 incubated in 1.0
ml Tris-saline at pH 8.0 and approximately 2000 J2 incubated
in 1.0 ml distilled water for 2 hours at 4°C.
100

101
One-hundred microliter suspensions of J2 (approx.
200 J2) in test solutions were applied to soybean roots
growing on trays of autoclaved sand. Soybean root segments
(1.0 cm long, including root tip) were excised 24 hours
after inoculation with treated J2 and washed free of sand
and external nematodes. Nematodes within excised roots were
stained with acid fuchsin (20), and the number of J2 within
each root segment was counted. Each treatment combination
was replicated six times.
Both 100 mM sialic acid at pH 3.0 and 100 mM sialic
acid at pH 3.0 diluted to 6.25 mM before inoculation caused
a significantly greater reduction in penetration of soybean
roots by J2 of Mil, Mi3, and Mj than any other treatment
(Table D-1). One hundred millimolar sialic acid neutralized
to pH 7.0 with NaOH significantly reduced root penetration
by J2 of Meloidogyne spp. compared to controls, but this
effect was apparently lost when neutralized sialic acid was
diluted to 6.25 mM. Undiluted 50 mM Tris-saline at pH 3.0
slightly reduced penetration of soybean roots by J2 of Mil,
Mi3 and Mj, but neither this treatment nor diluted Tris-
saline at pH 3.0 significantly lowered J2 penetration of
roots compared to controls. It seems apparent from this
study that the adverse effect of 100 mM sialic acid on
soybean root penetration by J2 of Meloidogyne spp. was more
i

Table D-1. Effect of 100 mM sialic acid at pH 3.0, 100 mM
sialic acid neutralized to pH 7.0 with NaOH, and 50 mM Tris-
saline at pH 3.0 on penetration of 'Pickett 71' soybean
roots by second-stage juveniles (J2) of Meloidogyne spp.
J2/root
segment
Treatment
Mj a
Mil
Mi 3
1 ) Sialic acid
(100 mM, pH 3.0)
1.67
± 0.96b
0.17
±
0.15
1 .67
±
0.69
2) Sialic acid
(100 mM, pH 7.0)
3.67
± 1 .22
5.50
±
1 .78
7.00
±
1 .03
3) Tris-saline
(50 mM, pH 3.0)
9.00
±1.76
9.33
±
2.09
13.50
±
2.14
4) Sialic acidc
(6.25 inM, pH 3.0)
1 .00
± 0.47
1 .17
±
0.60
0.67
±
0.45
5) Sialic acid
(6.25 mM, pH 7.0)
12.83
±1.83
13.67
±
2.87
14.33
±
1 .80
6) Tris-saline
(3.13 mM, pH 3.0)
12.33
± 1 .74
7.17
±
1 .56
11 .00
±
1 .65
7) Tris-saline
(50 mM, pH 3.0)
10.83
±1.92
6.67
±
1 .73
14.00
±
2.40
8) Distilled water
12.17
± 1 .72
12.33
i
1 .49
18.67
±
3.30
a Meloidogyne javanica (Mj) and M. incognita races 1 and 3
(Mil and Mi3).
b Mean of six observations ± standard error.
c Treatments 4, 5, and 6 were identical to treatments 1,
2, and 3, respectively, except that suspensions of J2 in
incubation solutions were diluted from 1.0 to 16.0 ml to
provide ca. 200 J2/100 p-1 for inoculation of soybean roots.

103
than just an effect of low pH, and that this effect is
reduced when sialic acid solution is raised to pH 7.0 with
NaOH.
i

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BIOGRAPHICAL SKETCH
Eric L. Davis was born on March 18, 1958, in Long
Branch, New Jersey. He attended Shore Regional High School
in West Long Branch, New Jersey, and graduated in June,
1976. In September, 1976 he enrolled at the University of
Rhode Island, Kingston, Rhode Island, and received a B.S.
degree in plant science in June, 1980. He was chosen as the
outstanding student in agronomy by the College of Resource
Development and he also received the Presidential Student
Excellence Award in Plant Science from the university
president. In August, 1981 he enrolled in the University of
Florida, Gainesville, Florida, as a graduate student in the
Department of Entomology and Nematology. He received the
M.S. degree in December, 1984 under the guidance of Dr. J.
R. Rich, IFAS-AREC, Live Oak, Florida. His thesis was
entitled "The role of nicotine in the resistance of tobacco
to Meloidogyne incognita." In October, 1985 he moved to
Orlando, Florida, to conduct reserach for this dissertation
under the supervision of Dr. D. T. Kaplan, USDA-ARS, USHRL,
Orlando, Florida.
11 8

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree^of Doctor of Philosophy.
ilL
David T. Kaplan, Chairman
Research Plant Pathologist,
USDA-ARS, and Assistant
Professor of Entomology and
Nematology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
)onald W. Dickson, Cochairman
Professor of Entomology and
Nematology
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
cip
David J. M/tchell
Professorv/of Plant
Pathology
This dissertation was submitted to the Graduate
Faculty of the College of Agriculture and to the Graduate
School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
April, 1988
ccJ? J1/
Dean, CoLliáge of Agriculture
Dean, Graduate School

UNIVERSITY OF FLORIDA
3 1262 08556 7757

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