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

MISSING IMAGE

Material Information

Title:
The interactions of surface carbohydrates of Meloidogyne spp. with soybean roots
Uncontrolled:
Surface carbohydrates of Meloidogyne spp. with soybean roots
Physical Description:
vii, 118 leaves : ill. ; 28 cm.
Language:
English
Creator:
Davis, Eric L., 1958-
Publication Date:

Subjects

Subjects / Keywords:
Soybean -- Diseases and pests -- Control   ( lcsh )
Root-knot   ( lcsh )
Meloidogyne javanica   ( lcsh )
Meloidogyne incognita   ( lcsh )
Cell membranes   ( lcsh )
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

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

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 001147636
oclc - 20442730
notis - AFP7300
sobekcm - AA00004799_00001
System ID:
AA00004799:00001

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 Meloidoqyne 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














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















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
MELOIDOGYNE 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 MELOIDOGYNE SPP ................................... 100

BIBLIOGRAPHY ............................................. 104

BIOGRAPHICAL SKETCH....................................... 118














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 (J2) of Meloidoqyne 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








eliminated binding of LPA to the amphidial region of J2 of

the Meloidoqyne 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.


vii













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, Meloidoqyne 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).











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











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











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











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 Meloidoqyne 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











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











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.











sp. glycinea, and induction of glyceollin accumulation in

soybean tissue exposed to fractions of cellular envelopes

isolated from incompatible races of Pseudomonas qlycinea

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








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 Penetrans Sayre and Starr has also

been investigated (117).

Carbohydrates present on biological surfaces exist

primarily as glycoconjugates such as glycolipids,

polysaccharides, and especially as glycoproteins (79). The

carbohydrate residues are often comprised of a number of

10










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











(47, 135, 136). Application of lectins to soil infested

with Meloidoqyne 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 Meloidoqyne 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 Meloidoqyne incognita races 1 and 3

(Mil and Mi3) and M. javanica (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.











Eisenback, Blacksburg, VA). Eggs of each nematode

population were extracted from host roots with 0.53% NaOC1

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

Meloidocyne 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


























0o4

O *r4 H
0 -H

o tor-
m4


S0 t
3 U




ar) -




04.J 0
U C5



(4 C0 -






o a) 0p
- r-I 4
44 0-1O
rd Ctr





-i 014
S


'-- 4-C
4 |- ) C

rd )a ) i
> f< r--
3o C 01 '7
' tO 3

(* 0 m

r-i P e

S03 0
r- i-i E >A
.I0 -I
10 UO 0
E I-V- 0


a)


U
0

4

to








*-I
I




to
0


4d
r-1
tI
mt
0 I
u I

I 1


toa
II
U Z


d

-I
r-=


o











0
r-l



























(0 0
k
a)
0









I,-i
m
'.1









to















,01O
a)d


#tO


V0



ON
or
a)






C


4-1
mr -










pH 7.2 for SBA, WGA, and LOT; 0.05 M Tris-saline plus 0.01 M

CaC12 at pH 7.5 for CON A; 0.05 M Tris-saline plus 0.01 M

CaCI2 at pH 8.0 for LPA. Preinfective J2 (approx. 5000 J2)

of each population were incubated in lectin-TRITC conjugate

(200 gg/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

fluoresceinn 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











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 O 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 titerr) 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 (8-glu)











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 10002 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 ZnSO4 (pH 4.5) for a-man, 5.0 mM sodium

acetate buffer plus 72.0 mM NaC1 and 7.0 mM CaC12 (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











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-la 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.)























1----SA


a 10 pm






WA-w






10 om


AD-a-




FC-o-





10 ijrn


ASS




bSlop^ ^



D L-o-^^^^^Bw^











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

















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).



Mil Mi3 Mj


Lectin -Suga +Sug -Sug +Sug -Sug +Sug


SBAb ++c ++ + + ++ ++
WGA +++ +++ +++ +++ ++++ ++++
CON A ++ ++ ++ ++ ++ ++
LOT +++ NF +++ NF ++++ NF
LPA ++ NF ++ NF ++ NF


a J2 incubated in
competitive sugar.


lectin solution +/-
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
tetraqonobolus 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.











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-le). 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 8-glu treatment.










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 Mi3 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











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
select glycohydrolase.


solution +/- prior treatment with


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; WC = weak fluorescence of J2
cuticle along anterior half of body.











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.











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 motilee) 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 al. (114). Labeling of sialyl residues with











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 Meloidoqyne

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











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 Meloidoqyne 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











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 Meloidoqyne 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

Meloidoqyne spp. Whether surface carbohydrate changes

similar to those reported for glycohydrolase treatments

occur once Meloidoqyne 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











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, 1-36). 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

Meloidoqyne 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











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.










Surface carbohydrates of Meloidogyne spp. J2 were

blocked by incubating nematodes in solutions containing

unconjugated, purified soybean agglutinin (SBA), wheat germ

agglutinin (WGA), Lotus tetraqonolobus 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 CaC12 at pH 7.5.

Preinfective J2 of Mil, Mi3, and Mj were

concentrated in the appropriate buffer or in distilled water

by centrifugation at 1000q for 3 minutes. Treatments for

each lectin included incubating J2 (approx. 2000 J2) of each

population in solutions of lectin (200 Ig/ml), lectin (200

lg/ml) plus 0.1M competitive sugar, and 0.1M sugar minus

lectin for 2 hours at 40C. 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











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. javanica (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 270C. 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











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 gm) 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











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















0

m- -0 14
C 0
U -"( .
-,-t



(l 1 0+
04


U -r.0 C-r
- 0 0
-0 0
G 0 C;a rtn
I -H -H

0 Q)(a) w

.a cI-J I
S4-1 -H4
P0 k 0)
Um
-4--4
07U)
V O






. 4 2:

4-t 1 4
4-U) fd i
H0 0

- 30









u ?40 0
t- rd

4U 4(-) -H(

0 12
-t 0 -> 4








I-u -I V.---/
r.Z
00 C




07

01 0

a) aO
14 0
-0 C -
m>I-0



> r



4 W 0 -
4>1 i%




I m n





-0 0I U
E-4 1 -4 -


E* 14 14 tU-,4


Cl) CO
Ca3

UUU




>4>4










u u
UU























UCC
udd



Of P


UJU0
u u u





COD

U UU





UUC







>

Di;
CO Cl

-u -
U U


O O

mu
U


0u




0'
>u
0 -


CO C








0cj
u S
CDCD


>4 >4
UUU
rz)rz1CD C
WWm3 E


.0





CDCD


H
t(
4-


>> 4
UU



UUU
CU C C
uu'u
u0 u


-I
r-4




m (
o e


x
>4
U



uu
u u




0
MV






a
(U
r14
U) -'.
4-1 4-)
4-4 In


-4

4-i
14







0
4J
:)
-4






0
4J





E
41
'4
a





















4J
0
(


4-






4
0U






-H
.-U






a)
> rq

0
0













1.4
044
ca

-el
(0
0)





14
'1
0




U).



*-r


rIn
41
i-H


m
a
04
>1





>i
An

00
0)



II



>-1







>
01
04
r-H



ON




r-4

S0

0 In



(a 1
H11
H

00








01u
CO
04




OC
4
a

rO
t^H







ae
*0
019
















00 0
-) 4I- -0
I' -4
0 1 t0


- C
0r 0
40) 0
S. 0 -0 -
rl > U


(.- 4 & $-
001 r

a1) Z14- 0
U (a -H

V M O ,
- U,--4



0 4
C i 0


-0 0 0
rO.




000
I a
0 > p
mC)



- 3


I- I -



O C1



1 $4 -

H 0 4 0




to 0' 03
I -U- 03
ar- 41 a)A za



















A 0 (0 0
Su










04 kM -
I 0



I 0

En --4





(B 0}m 3
r(IJQ1U0)Z
43 0 U 0
(0 (BU (
Ec h M ^-


U



uuu




>4
OS





0uG


u u



z u











MOo

















uuu
r20
z 4










UM
000 c


0 U u
UUU











U U U



>W M











UUU
00C0


En M


3











0 u

UU
CO




uu
u u





U




u
C/lr/


-4
4-





ra
0
0




-H
0
0





0
43
-I




0




4.1
or





4-1








$4
r-l

0

4
(d

Ca
.-4
roi


















04
aH









00

(U
0-I
$3





rt@





00






VO
-4
(0 *<-
*H~
ua


4-,
4.1


aU
0
m







0
43 *
00












II rl







0
r-4*




r-4
Ua










$45
mL






















00
r-4












ar-
4 0
II

























(P






( U

0
il
44
0)
e u
me

GO-





10







U0















a l


>
u u
0 19%
W Z 0
frz z


U u U
0 u0
000


C14




o


a
r1-
4m -4
1) -,H
4-1 4-
44 0l
a (


4-



$4
E-i4
5
E;


(0
0
01
(U
S


C
(U

+


ZZ
0 0
u u















m


0 &
nO --




-C 4J0)
r-l > 0 M
-H4 0O
0- 4 .-H i 44
a) 0
4-r-lrUO Z

U 40
o14
0 Z-
O C 0


0) 0
0-4)


> >
-- 3 -- o
v- 14J -i
i ,- 5 ,
4JO 0'4J M
41) (a :
(0) 4)


a4 C .-
E) 0 Z

4-I -- 0
0 U)0 -C 0
(d 44

O4 0
0 w

4 w E-
40) Ora 0 C.
tO W










S d '- -0.
Srn- rn C 3
C4J Z -) 00
4J 0 C

Li 0)-r0
m4 $ 02
- r 44 -


0n 4 -H
0C 0


*-H
I r-


E- O4 k W
BNMM ol


M


0 oCU
000 )C
CD CD CD


>


0










C)CU'U
Ct)






000

uU 0


W4D
0








00 u
000
000 )C
CDCDC
rz0r



>4M
C)M


i:
CDC2U

C0u0)
000 CD


tJ
U u
00













CE En
fA %

00



uU



0u
MUC











00


U 0










W Z
CZ
















M 2
c oo

000
0-







uuu

u u u
CD CD C


0
044
+

00
1-1 13 4


aU

-.4
4I-
a













or


-t
0


.IJ
$4















-1
0






0
-4

'o
(0
a)
41
0)
a

0)
4
0






0)
4-

(U
C






044

0r4
aO
mr
0)


*H -
C
0)0

?
0) 0





OI-I
I M
C ^
0 *

CO

#<


-4


a
-4


4

>4







r-.
4 *c

C0)


(U:3
C 02






0)
e o




4C
0U


COi
0 1-
>i1
>4



SIM
0C

a
r-4 $4


>




-4 11




r.4
(Uz
-H

0
U0
14






0
0 0
(0


>4 >
u u
99 #

EmM MM


>4 >4

CD C

C)CJC
CDCDC


u4

+

004
000


U U




0





'04
0)


IN 44
44 0}














a u 01

Oa -00 u 00 1
to l A 03

. rU 4. P4
0 tr e- U u u u u u u C
o01-10 UUU UUU UU (1

- r M 4 0)
4U 4- m
a) fa z oa a)
- C3- O)
>4 0 t 4 0 >
0* 0 P 4
CV rO r. -H I>r-
)0 --2 UUU UUU UU 0 B
a0 to .
>> i-l0 0 M C i)
0 1 C) 00

) 0 Mo u u 0
4 3 .Z U U -m l C0
T3 U-l u P4 U 4-1 U 0)
C0 Cr Z>S w ZU r-I
(d 4) -H4- (1 r-4 .) ri
-10 00 UU % % 0 o
cuc -n (1) Q rg u* u0
-030 (3 1 4-
> 21 Z I C I
S) I 4O



+i 0 ca ra MCQ rd


) E) 0 M 0(1 0)H
4.c) a 53z z



0- to :L 1 0
Am 0 z to 4
0 i0 UUU UU UU










-I- O 01-H :DL1 W- to4( z
S40- U 0 E





40 >r-4 Z
0341) z u t o u 0i U >











S1 10 u0 a 0
W s Va) w1





('da O -) 0 Z r- 4M
m 0> S 4J 4 It







Id 0 V 12 04-a a 3H
SI 0 C t o 0 ) 04
4.4 4 + r a + 03 1 UI
43a J -H) 03 u U 0 k r-4 (1) C4 ur
S-0 l -I 03 ) 3 z :3 'H w As t4 a
t1 Ot0m0 w t 0., a

0E-4 k 0 M 1 ;D m p
It C4 C 3 3 Q > r
E<^ (003 6< S 0l (HQ &










43




m iia me a
) 4-- -H M 23 CO CO CO -H
- C- 0 0 I I I +
H0 1 11


- a u u u u w
O# #U UU -, C
- i -


V-O 44 III a
0 0 I I II 1 I V
S-H r- 4J w

0co 4 *a a m m
- 4C 0l 0 0 0P4


-O & # I C ,I -,-O 0i X
0C q* C UO U UU ,-


( )0 -, u0u uu Ic
c q roha e c- )





w -HI-) III H 4 0
44 0 Qi) (0 r- 0


OC U0 UU 4) If 0C
0 0-# CKU -H o)





0 C -H M 0
-ri4 q I1 0lr 3


Dr- I I I O 0- 01
4 0Nc w E uun I 1O .-


4 i o 0o0 0UPu -H to4 >
3 ;? l I *C II




a- IC *H C0m 0
S0 (d Ma (a C 0 dO
E) 00* 0 0 -H o
44 (0)-H (d -4 43J 0
( 0-A U U I U 01 1
) 4- (d MM U m k-
m) a -- 0 I 0) d





o 1i s o # CII 0
m Oa *(d u >




00 >C ) 0 k 1



$HO-H-I II 0i0 -HII t0 0
S0-HU U 0 4




41 m (a- C
-o 02 B 0u z u HI H r0
-H C 40 0 H 1
C U) V 3- U 02 I 2 I I-I r,- 0)








O4I X: V 0 0 O 0
N4 to +H 0 + H 0 U
4-- 0 HH (D H mU 0
rd Wao 0 S-l rz )
C- ( +4 >4 4) 01 a 0 II-



- 4 M0 U uB# 0 0 O




00 4 0 H ei 0 4J 0-- C U 0
(-a-i -4 i C U i p4Hn C r. r
4 ( CI U fI I I I 1C 0 U O z U
0 ^f UUU (1) >0H 0 4




4 mm-- UU 0oHn r-H 2
4(0 ra *r 4 2 r I 0 u r. e4
0r 4J 7 ra) r-4 0 da ar (a H
.0 a 0 ( aq 4) m 1. m1W R
0 Lo c0 a -0 tp 0t4 ISo :j IH f a
E) P 0 1 4- 4 (U E -40 0 <3 (- M U O Ma


^k m 3 Hf lE- CQQ 3 H


























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.










45






11 E




L
I II



























i a











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 Meloidoqyne 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;











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











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 Meloidoqyne 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 secretary 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 Meloidoqyne 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











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 composition 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

Meloidoqyne 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











incubated in CON A solution (200 gg/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











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

Meloidoqyne spp.











Materials and Methods

Populations of Meloidoqyne incognita (Kofoid &

White) Chitwood race 3 (Mi3), M.incognita race 1 (Mil), and

Meloidogyne javanica (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

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 tetragonolobus agglutinin

(LOT), Concanavalin A (CONA), and Limulus polyphemus

agglutinin (LPA). The sugar specificity, corresponding











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 gm pore size were incubated

with 200 gl of 1.0% bovine serum albumin (BSA) in phosphate

buffer saline (PBS), pH 7.2, at 370C 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 gl of

lectin-HRP solution (200 gg/ml) for 2 hours at 4"C. Control

treatments included J2 in buffer (untreated J2) and 500 il

lectin solution (200gg/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, 100-4l 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 4l of buffer and transferred to separate microcentrifuge









































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











tubes. The volume of each microcentrifuge tube was

increased to 250 4l with buffer, and 50 41 of suspension

were withdrawn from each tube to quantify the number of J2

per 50-4l sample. Four, 50-4l 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-pl volumes of

lectin-HRP solution across a 96-well EIA plate and adding

100 gl 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,
R = 0.957; Soybean agglutinin (SBA) = 11,388.817x+12.636,
R2 = 0.995; Lotus tetragonobolus agglutinin (LOT) =
11,791.765x-31.613, RI = 0.995; Limulus polyphemus
agglutinin (LPA) = 7069.594x-459.472, R = 0.977; Wheat germ
agglutinin (WGA) = 6282.532x-428.531, R2= 0.967.










LD

E
c



SW
CJ


LCm
CD M CD n cO C)
cU c __j 3: CD cf)




0OD tO OJ C OC CD t \ C\J C


NI133l SNVO90NVN











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 Meloidoqyne 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











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 Meloidoqyne 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.










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 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 t 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
(WGA); Limulus polyphemus agglutinin (LPA).


c Mean of 20 observations standard error.











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 Meloidoqyne

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).











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











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

phytoparasitic 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

Meloidocyne 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











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 (Lycopersicon esculentum Mill.) and 'Black Beauty'

eggplant (Solanum 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 Meloidoqyne spp. J2 were

blocked by incubating nematodes in solutions containing











unconjugated, purified soybean agglutinin (SBA), wheat germ

agglutinin (WGA), Lotus tetraqonolobus agglutinin (LOT), CON

A, or Limulus polyphemus 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 CaC12 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 1000g for 3 minutes. Treatments for

each lectin included incubating J2 (approx. 2000 J2) of each

population in lectin solution (200 jg/ml), lectin (200

gg/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 gg/ml lectin and/or 6.25mM sugar) immediately before

being added to soil in which soybeans were grown as

described below.











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 46C, diluted

1:16 with Tris-saline buffer, placed on soybean roots, and

the number of J2 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-cm3 Conetainers (Leach Nursery, Canby, OR) containing

steam-pasteurized Astatula fine sand (hyperthermic, uncoated

typic quartzipsamments). Two-milliliter suspensions

(approx. 2000 J2) 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 30C, watered daily, and fertilized once a

week with a solution containing 10-6-10 (N-P-K) plus












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 s 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

























~0
rr
#ts
4-

4.J -H4

go


-v
0)
4 .
Q)
- *
0)
0) 4

0 )
rl
4-1




C 0)
0-

> (3
an
1



O-f
> 0)


0
O 41







O 4
M0)
4C
0 I
O






044
00
1-H







0 (3
0)





P4 4.)

r4 0)
>0) 0
1- 4J4 41
40
E-1 -f


m4

0

01




0)0)


-4
0





1-4
r4 0) *r-
(T 441 4)
014-4 M
3 3-rH


0) 0)
OV O) 03




0 0








0 n Ln Lo Ln
Lin r- oN oa
9 *
V 0








a)
ro a) ra
o a) a 0
S(aUO a a




(0 Ua 0 0 rt







0 a 0 0U
LALAAN
omm0 U
















* .3 ..3..
0(00 00









Ln (N CN 00
a)oo
*****
wwm P


S 0)4
(0 41

0

+ 0)
1 -
QC C; Mr-
.-4 .r. 540) .9.
4J 4-) 03B4 41J
0 0 4-40 2
0U 0) 7 ..


0












o o no
a o in o
u30 i-.m <3- 0












U l0 O0















I U! 4430




waoa
Nm mc r- o








* 0 9 4 *
V)C4P4
ammoo J~U


0
O-


It








II
a



























If










0
a,
o






























I
o







































0
II
o










0








I
0*
































48
o













I

It



0



II
0
**


U 6


>
m


1:3
- )
02
0(0





>4 Ou
C 0
c -




0)





0( .4
.000
04-
0)-



>m E
00)-




40
3 ri.


0 00 -rq






U 0) d
0 I) (m to

-- 0 0r-




04.1 r.







0a0) H
0) 4-)
0 0
400)




0 MO-t 3

(n cV.. &


U43 t.






10D-
H 3
Ur
a509




0000 d
04> Ilj


-4-1


Q00)
Q;







1-4
.0
-t o






rOO
0)
3O-
0



(4
0 -




O.5
lo

r-



0J
Or





0-
EU

* '













41,
CO

















() 4- ,
4 14J
4.)4
U 0


5 1l
-H









r--I
0k (

C t
0054



P 4
(00








(DC
<0 9



A-!











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



















S0 9 (0
n ooo n











rctioor-










LNOLC IL

O -O-


0



a
S,
R1



uH


r-













0 H
0
CO





0
a
0
Sco


4 .-




He


0 r



0
Si-H
C







40


W4-a
m













.0 0
S0
O-4l
C u
00





05 0



a aoa)





m0o


S0 S 0Q 9

Om>INN


c-

E)

S 0
") 4)
4
W
B


4
k m


a,
+ a)


4-1 d 4-14-1
0 t U)-4 m

i-lCO Q Q


ra V
00 0)
S.0 a)




I- r- 0- 0- -0



a) ror
00000





ramma





(a ls ( to (d

ro r LAn Ln r-
C)
00 00 0








r-r c n n--




a)
lAO U TOTr)



Ininnoo
* 9 9 0 9



0000

(MNom nvn
* ***
# uOu
NU) V)


Q4
0 4j


a
+ a)
S -4


0 I-4 W
a) 3 : -I
1.4 () m a


o Ln Ln o
1 1
rroo


4-
0
O
0

a)
m





0)
Vr






"I-



C,
II







C
o
o













I-"
i





C',

0
r)
II
--






o
I





II
mr

c
I









o

an

0


-
m
0 0)
Q m

C 0

Oct





rino


Hi- m
> ma








4I ..
3 -






0 1
amo

0ON 0 m
- -l




1I 00


m 00
I' (B o
.fluc
C <

4m H
ram u


o) :3 C
0 4-. r a0
,0 0)


- W 4 F4

C U (O'O
I (m U



0 4-.--
*a o

a) I-

>d m




OH 0 U


*.I :i ( 0
CtM 0C
COO ChIH
OBOW (0
#MY H
C>< 6 i
0CM <-
Q- 0)C -
o) & O


4-



S)

(UO C
E43
M 4-)




1O
0

43-H

a)
4lO



o
4 .



>i o
00
4-

0 U
00



VI
a,4

mO
$4
(oc
4-) 4-
H *

o-r
Qr-4
4H 4


0
O l
4-1) 4
-'-I


4-1
to 0

W 4-) 4-
C 4J
(1) I


4> c0
mrm
r-4
0 (d
H0 3






01
4I1


OM
w3


4-
a)
(D
4-

a0
o






















.0
c

04
OC

a
C
4-)









0

( 4J
i4 0




M
4.




00
C- V



O)
0







) M

cii




00
r. 44












-H
4J 4





-i-,








0

C (1
*4.4
00



E4U
O i $4
o oi(








(U4J4
$C40
3
mo e0
0 4J ^


* *0 *

L "!r
IV t o


$4

(4
0'
m


CC
4-1
00


Ou
mi 1d
S-


00)
1-1-




$2 -i 4
$4 U)-9-41
3 3-44
U"- aU)


aO 0 (oa
o Ln n aO o

4)N 1: m1 4 in


V 0
,Q (1 4-4 (d (0

00 n Ln 0








0 0
(a (U 0 0 CC(
o n ao in o
0-c.JL r-L









on in Ln) in
w mmm W"W




0














A A A 0 a
<< 4 ( n^


Xl
S

mo o
*. V
f in


04 .0

000 0

t0(a 0








n n oL Ln

* V *
^* ^ ^


Im0.

n oS
* **n'a


m(U
r 00
o o
o o
00
LALA)


to a.

U)
'O
a
+ 0
r-l
CC 4 r-
4J 4J (04-4 4.1
0 0 '4-4 U
U) (1) 0 0 -4
1.4 $4M m I


00
Ln nL

44


4-J

$4
0


ci
U4)
a)
M
0)



0<


0






0
o

IC
a
11
rn






0




I
V


0

II

0
m






II.
02


NM


II

II



0


0





U)
(>
(d (
>t


-
4
0 o

C 0
C4 Cl








00-0
a (




oo 4-






(U >
(11-

(0 *




( 0 ,
o 0














m As
U00






S r
1-44I) (U





> 0 0U
H -
OO>*

pa
Mc c
( ,a 0 0


-U 4 *l

41 E





S- a) (U 0
04J 1-
0 3->--


0 $ 4




4 (0-I



V (0 E




0 0] 0r-1
(U UOQ
A 3
Oa o4
C M


OHO t
##U mc

a If IE
C Q)*4
49 +I


$4
4-)1


S0)







00
aO
0


,4)




m
ac








'-4.
0 *E
4-H

Uo














4
km






(d 4
'0





-u-I
0-)















4-1 k
0 0

$4-








l-4(
r-44
4a-














S-4 Qa)
r-4









a0
O
*m*






Q)U


0) -


r4U1

> 0
clU











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

















0 00
4U 04 4 0
(04 (0 (a0 Q



4 2


0



Lr4 .:


0 4





0 J
0-






0
(ci
-H 02
C -H
-i 4J





*4 0










44
03 I
C 4-l
00
-H

4)













C 4J
0



(U



H0



04



O-I

k(0
S0)

01
u a







-) 0

00 (






eW
*0 I
d 00








E-1 $40
0O




a c
3 (D *
0 4J ^
^4 0 (
d ) T
0) ^1 3
c 4J 0

* 00
Er4 -ck


00
00
oo

*






4.

(0
dm

+0
C -


00


0



LnA Ln N






$4
(mmU







a)
4J



$'
r-i
*r*l
(C 4- 4J
4- 02
M m Q


ro U
o n
n 0

100L
ciO-


'no
. .


0 0 0
cd O c c!
oooV)V
Oc)omm
omomb U


0000
(0 (C0 (" C

0o 0 C
**** ~P


4-
0


(0

C
*l
4-



02
02

dl
rz


0


o0 c4 4 0 (0







0000



0 o 0- in r-
* i 1


oo'no
o o n o,









( 0 D0 U)








a 0
0 4
0
+ 4)
1-I



U) 3 34-H


41
C
4)
E-



0


-.
4-
vi *-

C 0
-H a

c-I Ov





a) r
z>i ro4


>0 0




Smo -
OW>
i CO





$ 4 E
00






02 *-)
0 r- -H
mZ 0
3 C C






-Cr(3 U


r)*H v w

4-H 9

Z4J 0



O 4 0
0 5 -- 3





a I



00 0




*-H 0



Cm


a)
4- (

Q)




0


44
Ei



41




4J 44
o-H
-ri
430







$4
02




(U -




44
>-i



0


1-H













0 -
4-14
-t










-H


0)
0WO











*MI







0 4-I-
0 a




ciO
a*







W$4
>x0
0 E

(0







'C4


0( 0
Q) il
k 4J -
(3C (
(1)




ric )
(0 a}
> x
Q~r) C
<0 kr
r- 4
43 U


4J-
C

*4
Q4
,-
M

















a)
4.1
4-4





(00
a >
41-iH
o-






Se4
a)
4 6





a)
.u


C o
0 w






-0
a)
tu.



00)
U+ G
Or
U 4

a>
mto

0 c



O
* 0



40)
C T


O

44 -m


0 4

aC
a (
4 )




O
* rc




C
> #
0




0 C
1) 1
3- (
E- 4-)


$4

0'


4. +
C
a) CC
g -4.3H
$4 00
a) a) )
x
W


$4
0)
ra
43



a)
r-
,4--4
$4 a)-
0 4-4 1
S
o aQ


4-4
()
Stt 4
0
0 O l!-4
.0 tP44d 0)
in q Ln oa
r- Ln r- a Ln
-5--cm




a) a)
to coto
4 0 4- 0
a) ( (ato ou
00 L0 C4O






co aa
0 U04-









44 000
(D XL) Ud m










oin n o q






Sa)
to( Is to
* 0000

ULA 0 l nLA
r4 0 00 0
X!X>)X1
(0 44 (Q( C
mno om in
1coN o M(
** *** *
m mm ror or


to 'o



Ln Ln o in o







0 00
0 AA LA






U (a it (d A
o o o n






+ 3
0 000






(0 (0 (0 (0
Ln r-(- c'.J
44 a 0





0 d 0 I0



in r- r. C in







.Q 0 .)0













+ aa



40 0 (4-4 4J
I) E) t3 t
mcooo


) w
0 m
rC


101 .rc M a Q


41
0
0


m
01




U)
a)





0(
a
o
o




un

o
0



1
I-
II



m
o










I


1








II
o







'.

I-
e




'-




02


0

0)


CO


CQ4

a)
C O
m

c 0

.4-1
04.)



(0 -


0 0
m0 0


a) 0)
3 ro .4-
Ia ri



-rl 013 -r4

(a 0 r0



) (0 r
a 4)ra 0

> U 0

-H 4-4 (0



0 41 14
0 0--
0r-4 -


StP -

0 $4-r 3

p (a r-l

0 a) O4.0w

$ (a5e
Ca a)







01 4 # 1
H r0L 0-
u r-l 0


)0 (0 rl

( 04- 0
MMa


41
a)
a-) 4
H 41

E4r:


Ca



>90
0
a)



Oo
0 u



4-
XI
o m)
a)






4-1
a)
0a
0)







,-H

O44 0
-HI-
4(0J

OW

0 W

4.-

C



3 0
4-r4





$4
a) I

0 0
$44 4:3
a o










O- 4
0(0
rne
3 >-
i-l ) !











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.

















$4



4,4



4J
(0


O
*a -r4





8-










kcW

U 4-J
0





( 0
! 0











>1
a0











01 -'4














O0n
Oa


) a
-440







0C)

















44
cI













0




















kO
*











0 r-
a

















E 41
040
m c
0a)


0
r0)















*s (
R-l (
c3 0
fl3 ^


$4
(0


+

c c
-4 -4
.4-4--
00
0303
(U (


(4


ra

03

nr-
4H.
$4 0) -H
(d 4-4 4-.
04-4 02
30 -r
En PQ a


, 0 4
M 44 01
) 4- a 0 0








0LA000


o in o o a
4 **V
4* N4 (4 0






U a)

d rU d (a
CV 4-4
00)
(000(l0(0

Ln 0 LA0 0o
o- o c Ln c


0* 0 a *0 0
















on o c ) a
0) 00 0
000 L o






VO) 0





m^ m nr

$
tz 4J
$4 03
(0 4-
N 02

4- + a)

E 4- 40) *-r
-1 4- 4-) 1(0 44 4-)
0 0 0 4 01-


0
0 O

o LaLn
oa r- ru ro






0
00 aa

..Q ..0 ...0(








O.OA4044
00 c-(i Lo r






000






L oLn Ln Ln







LA (4 LA -'-
0 0


onmmom







0 0




v I I
NO CJ ^' ^
***** '~


4-1
0
0



m
cn

02






03
a0)






1>



I!
m
o
II
(-v



o



II
II




o
e,






('





II
0

II










m 0

.1


0


- 0

H O
CO -C
m (0
(0.-
0 0








040
a) in C



(0 H
>m
w 4













WH-





() H
i-l m 0 V.











0 (0











$4 (0 r-4
> 0)1
04-)
00 0
20 O













4 )
H 04 0
Q 4C
S0a3 -















(044J 4-
Or c


Ca 4b1 en
*a a)














UC r


( 401 U1
a,9 ( 1
SCe< rl-


0
H4J 4








$4
ad
00



Cn
(U0









4..
En I
0





00










4-4
,-4
Ol
CO





(03

p 41






4-
0
















-H
0)










(0 4 -







034-J 40



-$4
S1-













0 -4
4










(4 (0
X 0
0 tI
S *r









0
4O-
in e



o)
m A Q
d~XI
ff (






4-(


CI2











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











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 Meloidoqyne spp. (see

Chapters 2 and 3 above). The inhibition of soybean root

penetration after treatment of J2 of Meloidoqyne 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 Meloidoqyne 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.














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 javanica (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 cephalicc chemosensillae) openings.

Substances to which lectins bound were concentrated and











sometimes emanated from the amphidial region of J2 of

Meloidoqyne 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-












mannosidase, B-N-acetylglucosaminidase, a-fucosidase, and

sialidase neuraminidasee), 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 Meloidoqyne spp. The rate of production of

amphidial secretions by J2 of Meloidogyne spp. has not been

determined. The microfiltration assay using peroxidase-












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. incognita-

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











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











carbohydrate composition 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












just an adverse effect of low pH. These treatments were not

lethal to J2 of Meloidoqyne 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


chemosensillae 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.














APPENDIX A
BINDING OF FLUORESCENT SOYBEAN AGGLUTININ TO POSTINFECTIVE
SECOND-STAGE JUVENILES OF MELOIDOGYNE SPP.


Alteration of nematode surface or secretary

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 secretary 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 Meloidoqyne 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 Meloidoqyne

incognita races 1 and 3 (Mil, Mi3), and M. javanica (Mj) in

90











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 Meloidoqyne 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 gg/ml), 0.1 M D-galactose solution plus SBA-TRITC (200

gg/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











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 Meloidoqyne 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 Meloidoqyne 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 Meloidoqyne spp.are of plant or nematode

origin.





















































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 J2 of M. incognita race 3
that was removed from Centennial soybean root cortex. FC =
fluorescent cuticle (note body annulation); PF = "patchy"
fluorescence.


a





FC-o










10 UM