The intraspecific variation of pratylenchus brachyurus

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The intraspecific variation of pratylenchus brachyurus
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Payan, Luis A., 1957-
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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 69-77).
Statement of Responsibility:
by Luis A. Payan.
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Typescript.
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Vita.

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University of Florida
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Full Text












THE INTRASPECIFIC VARIATION OF
PRATYLENCHUS BRACHYURUS















By

LUIS A. PAYAN


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


1989







































For Julie
My Adorable Wife


I















ACKNOWLEDGMENTS


I want to express my sincere appreciation to Dr. D. W.

Dickson for his guidance, his support, and especially the

friendship that he has given me during my years in

Gainesville. I also want to thank Dr. A. C. Tarjan, Dr. D.

J. Mitchell, and Dr. R. McSorley for their assistance and

for serving as members of my supervisory committee.

I would like to thank Dr. R. N. Huettel, USDA,

Beltsville, Maryland, Dr. C. E. Vallejos, Vegetable Crops,

University of Florida, and G. C. Marlow, Plant Pathology,

University of Florida for providing technical support.

Appreciation is extended to the people of the

Nematology Laboratory in Gainesville for providing

assistance during this research.


iii
















TABLE OF CONTENTS

PAGE

ACKNOWLEDGMENTS............... ......................... .. iii

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

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

ABSTRACT..... ............................................ ix

CHAPTERS

1 INTRODUCTION.......................................1

2 IDENTIFICATION, ANATOMY, AND MORPHOMETRICS
OF PRATYLENCHUS BRACHYURUS...................... 5

Introduction. ..................... ....... .... 5
Materials and Methods......................... 10
Results....................................... 13
Discussion..................................... 21

3 HOST SPECIFICITY OF FOUR PRATYLENCHUS
BRACHYURUS POPULATIONS........................... 24

Introduction................................... 24
Materials and Methods......................... 25
Results....................................... 27
Discussion ................................... 32

4 COMPARISON OF ENZYME PHENOTYPES OF FIVE
PRATYLENCHUS BRACHYURUS POPULATIONS.............. 34

Introduction.................................. 34
Materials and Methods......................... 37
Results....................................... 45
Discussions.................................... 53

5 SUMMARY AND CONCLUSIONS.......................... 61










APPENDIX ................................................. 64

LITERATURE CITED.................. ........... ............. 69

BIOGRAPHICAL SKETCH .................. ................... 78















LIST OF TABLES


Table Page

2-1 Measurements of mature females (n=15) of
Pratylenchus brachvurus 101 originating from
'Pioneer 304C' corn, Alachua County, Florida...... 15

2-2 Measurements of mature females (n=15) of
Pratylenchus brachyurus 102 originating from
'Florunner' peanut, Alachua County, Florida....... 16

2-3 Measurements of mature females (n=15) of
Pratylenchus brachyurus 103 originating from
'Florunner' peanut, Tift County, Georgia.......... 17

2-4 Measurements of mature females (n=15) of
Pratylenchus brachyurus 105 originating from
'Forrest' soybean, North Carolina................. 18

2-5 Measurements of mature females (n=15) of
Pratylenchus brachyurus 108 originating from
citrus, Polk County, Florida...................... 19

3-1 Shoot weights (g) of seven plant species 64
days after inoculation with four Pratylenchus
brachvurus populations and the uninoculated
control............................................ 28

3-2 Root weights (g) of seven plant species 64
days after inoculation with four Pratylenchus
brachyurus populations and the uninoculated
control........................................... 29

3-3 Final population densities (nematodes per
g of root) of four Pratylenchus brachyurus
populations 64 days after inoculation.............. 30

3-4 Final population densities (nematodes per
100 cm of soil) of four Pratylenchus
brachyurus populations 64 days after
inoculation....................................... 31









4-1 Enzymes examined, activity, and references to
stains and reaction mixtures used in isoelectric
focusing of Pratylenchus brachyurus mass
homogenates....................................... 43

4-2 Paired affinity indices of five Pratylenchus
brachyurus populations based on three isozyme
phenotypes........................................ 54

A-i Final population densities (nematodes per 10 g
of root) of Pratylenchus brachyurus originating
from 'Pioneer 304C' corn, Alachua County, Florida,
on selected plants 64 days after inoculation...... 65

A-2 Final population densities (nematodes per 10 g
of root) of Pratylenchus brachyurus originating
from 'Florunner' peanut, Alachua County, Florida,
on selected plants 64 days after inoculation...... 66

A-3 Final population densities (nematodes per 10 g
of root) of Pratylenchus brachyurus originating
from 'Florunner' peanut, Tift County, Georgia,
on selected plants 64 days after inoculation...... 67

A-4 Final populations densities (nematodes per 10 g
of root) of Pratvlenchus brachyurus originating
from 'Forrest' soybean, North Carolina,
on selected plants 64 days after inoculation...... 68


vii















LIST OF FIGURES


Figure Page


2-1 A cross section of the modified Baermann
funnel used for nematode extraction. The
screen was glued between two sections of
PVC pipe used for support. Inside diameter
of PVC pipe = 10.2 cm (drawing not to scale)..... 12

2-2 Stylet knob shapes of Pratylenchus brachvurus.... 20

2-3 Tail tip shapes of Pratylenchus brachyurus........ 20

4-1 Two hundred and fifty Pratylenchus brachvurus
females stored alive in a 25-g capillary tube.... 39

4-2 Protein extraction equipment..................... 40

4-3 Diagrammatic sketch (A) and photograph (B) of
patterns of malate dehydrogenase phenotypes
following isoelectric focusing of crude protein
homogenates from 250 females from each of five
Pratylenchus brachyurus populations.............. 47

4-4 Diagrammatic sketch (A) and photograph (B) of
patterns of phosphoglucomutase phenotypes
following isoelectric focusing of crude protein
homogenates from 250 females from each of five
Pratylenchus brachyurus populations.............. 49

4-5 Diagrammatic sketch (A) and photograph (B) of
patterns of phosphoglucose isomerase phenotypes
following isoelectric focusing of crude protein
homogenates from 250 females from each of five
Pratvlenchus brachyurus populations.............. 52

4-6 Polar ordination of five Pratylenchus brachyurus
populations on the first axis..................... 55


viii















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 INTRASPECIFIC VARIATION OF
PRATYLENCHUS BRACHYURUS

By

Luis A. Payan

May 1989

Chairman: Dr. D. W. Dickson
Major Department: Entomology and Nematology

Two differentiating techniques (host response and

isoelectric focusing electrophoresis) were used to study

intraspecific variation among five Pratylenchus brachyurus

populations selected from different geographical regions and

hosts. Although small morphological differences were

observed among the populations, each population was

identifiable as P. brachyurus. No nematode population had an

effect (P < 0.05) on fresh shoot or root weights of plants.

Each population reproduced similarly on each of the host

plants. Analysis of enzymes from 250 females by isoelectric

focusing electrophoresis in conjunction with enzyme-staining

systems revealed differences in protein banding patterns

among populations. Three distinct phenotypic groups were

observed in the malate dehydrogenase and phosphoglucomutase

systems. The number of bands among populations varied from









two to eight in the malate dehydrogenase system, and from

three to five in the phosphoglucomutase system. Only one

phenotype, composed of one band, was observed for all

populations in the phosphoglucose isomerase system.















CHAPTER 1
INTRODUCTION


Plant-parasitic nematodes are microscopic worms that

damage most agricultural crops. Estimated overall average

annual yield loss of the world's major crops due to damage

by plant-parasitic nematodes is 12.3% (70). Monetary losses

due to nematodes on 21 crops, 15 of which are "life

sustaining", were estimated in 1987 at $77 billion annually.

The United States' portion of these losses was $5.8 billion

(70).

Nematode management is a practice whereby plant-

parasitic nematodes are maintained at levels that do not

cause economic losses (32). Nematode management practices

generally include the application of fumigant and

nonfumigant nematicides, the use of cultural practices, and

resistant cultivars. The number of currently registered

nematicides is small and inadequate to meet the many needs

of growers. Several of these nematicides may be associated

with groundwater pollution or present a potential risk to

animal life (46). Crop rotation is one of the most important

cultural methods for managing nematodes (45), but its

usefulness is limited because before it can be properly

implemented the target nematode must be correctly identified











and its host range known. Similar constraints applied to the

successful use of plant resistance to nematodes.

Species of the genus Pratylenchus can be particularly

difficult to identify. Several species are morphologically

similar and the intraspecific variation can be great (52).

Many species are erected on weak, inadequate comparisons,

subjective criteria, a minimal number of specimens, and (or)

insufficient diagnostic data (28). Not all species are

accepted as being valid by some nematologists (28). An

understanding of the intraspecific variability among and

within species of this genus is essential in developing

proper nematode management programs.

Pratylenchus brachyurus (Godfrey, 1929) Filipjev &

Stekhoven is a migratory endoparasite of important

agricultural crops. The nematode forms tunnels and cavities

as it feeds and moves through roots. It causes extensive

damage to the cortex and in some cases to the vascular

system of the plant (51). Numerous pathogenic studies were

conducted on the nematode in a variety of economically

important crops (6, 9, 17, 18, 29, 30, 48, 51, 62, 71, 76,

79, 87).

Pratylenchus brachyurus is widely disseminated

throughout the warmer regions of the world (9, 10). The

nematode is also widely distributed throughout the southern

portion of the United States (77). It is the most ubiquitous

species of Pratvlenchus in Florida, and it is associated











with plant roots in 54 of 67 counties in the state (Division

of Plant Industry, unpubl.). In Florida, Pratvlenchus spp.

were detected in 93% of all citrus groves and P. brachyurus

was identified in 90% of them (83).

Records of intraspecific variability within P.

brachyurus are limited to morphological studies (68, 82).

Field observations, however, suggested behavioral

differences within P. brachyurus populations (D. W. Dickson

and R. A. Dunn, personal communication). The presence of P.

brachyurus biotypes has not been documented, but their

existence is suggested by variation in numbers of P.

brachyurus extracted from roots of Citrus limon (L.) Burm.

f. seedlings when inoculated with different nematode

isolates (55).

Races within species of plant-parasitic nematodes are

recognized as morphologically similar, but with different

host preferences (81). Other terms, such as biotype and

pathotype, are also used to describe such populations (15,

74). These physiological races are documented in several

species of plant-parasitic nematodes (15). Differentiation

of races is usually determined with differential host tests,

as for example with Meloidogyne incognita (Kofoid and White,

1919) Chitwood, 1949, races 1-4 (31). Separation of races

can be a complicated task, such as is the case for

Heterodera glycines Ichinohe, 1952 (66). Newer and better











methods are needed to separate races quickly and reliably in

order to design more successful nematode management

programs.

Biochemical approaches in nematode taxonomy have

considerable potential for assisting in the identification

and characterization of nematodes as well as establishing

phylogenetic relationships (39). The most common biochemical

methods include the analyses of proteins and nucleic acids.

Currently, restriction fragment analyses, DNA-RNA

hybridization, and nucleotide sequencing are being used

widely for systematic purposes (3). Phenotypic variation

among certain populations of H. glycines was detected using

isoelectric focusing electrophoresis in conjunction with

enzyme-staining systems (60). Most biochemical data are

obtained from mass homogenates of nematodes since individual

nematodes contain small quantities of protein. However,

techniques such as isoelectric focusing electrophoresis are

sensitive enough to detect proteins from a single female of

H. glycines (34, 64).

The objective of this research was to determine whether

intraspecific variability existed among five P. brachvurus

populations collected from diverse geographical regions and

hosts based on their response on seven crop plant species

and enzyme profiles obtained by isoelectric focusing

electrophoresis.















CHAPTER 2
IDENTIFICATION, ANATOMY, AND MORPHOMETRICS
OF PRATYLENCHUS BRACHYURUS



Introduction

Pratylenchus brachvurus, a migratory endoparasitic

nematode, was first described by Godfrey as the primary

cause of pineapple-root disease in Hawaii (29). The disease

is characterized by brown root lesions and in more severe

cases by the death of many of the branch rootlets and some

main roots. Godfrey named the nematode Tylenchus brachyurus

and placed it in the subgenus Chitinotylenchus (29). He made

reference to other nematodes of similar general appearance:

T. musicola Cobb, T. pratensis de Man (=T. penetrans Cobb),

T. coffeae Zimmermann, and T. sacchari Soltwedel, all of

which were later also placed in the genus Pratylenchus.

Godfrey noted that one major difference between P.

brachyurus and other Pratylenchus spp. was the absence of

males in P. brachyurus (29).

The genus Pratylenchus was first proposed by Filipjev

in 1934 with Tylenchus pratensis de Man, 1880 as the type-

species (24). According to the International Code of

Zoological Nomenclature, a new genus name must be

accompanied by a statement that purports to give characters











differentiating the new genus plus the name of the type-

species (80). Two years later when Filipjev characterized

the genus, the new name was universally accepted (25). Since

the establishment of the genus, 89 species have been

proposed (28). But not all of them were accepted by most

nematologists (28). Initially, the taxonomy of the genus was

confused chiefly because the identity of the type species,

P. pratensis (de Man), was not unambiguously established

(52).

Since Godfrey's description, the following species were

synonymized as P. brachvurus: Tylenchus brachyurus Godfrey,

1929; T. (Chitinotylenchus) brachyurus Godfrey, 1929

(Filipjev, 1934); Anquillulina brachvura (Godfrey, 1929)

Goodey, 1932; A. brachvura (Godfrey, 1929) Goodey, 1932

(Scheider, 1939); P. pratensis Thorne, 1940; P. leiocephalus

Steiner, 1949; and P. steineri Lordello, Zamith, and Book,

1954 (10).

Several reviews of the genus Pratylenchus were

published (52, 53, 73). The latest compendium reviewed 89

described species, but only 49 of them were considered valid

(28). Only a few investigators have reported studies on the

extent of variation within certain species. The degree of

intraspecific variation must be known if we are to

understand the morphology of existing species and to avoid

unnecessary creation of new ones (28).


1









7

Identification of Pratylenchus species is based

primarily on mature females. Several species are

morphologically similar, and intraspecific variation can be

great. Therefore nematode taxonomic expertise is required

when working with this genus (52, 73).

The following characters are considered to have

diagnostic value: number of annules and shape of lip region,

length of stylet, annulation and shape of tail tip, position

of vulva, length of posterior uterine branch, body length,

presence and shape of the spermatheca, number of lines in

the lateral field, and presence or absence of males (52,

53). Various degrees of intraspecific variability are

observed in several surface features of the nematode through

scanning electron microscope studies (11).

Cultures of Pratvlenchus brachyurus and P. coffeae

(Zimmermann) Filipjev & Stekhoven from individual females

revealed that the shape of the lip region had variable lip

shapes within and between the two species (82).

Intraspecific variation is observed in the number of lip

annules in P. brachyurus, P. coffeae, P. scribneri Steiner,

P. vulnus Allen & Jensen, P. zeae Graham, but not in P.

penetrans (Cobb) Filipjev & Stekhoven (68, 84). Electron

microscope studies of the lip region of Pratylenchus spp.

clearly show the existence of variation among and within

species of the genus (11).









8

The length of the stylet is one of the most reliable

characters for identifying Pratylenchus spp. (2, 27, 75,

86). Although the stylet length is one of the least variable

characters, the shape of the stylet knobs varies among some

species (68, 84).

In the original description of P. brachvurus,

variations of tail shapes were reported (29). Intraspecific

variation in the shape and length of the tails of P.

brachyurus, P. coffeae, P. hexincisus Taylor & Jenkins, P.

penetrans, and P. zeae was demonstrated using natural and

single gravid female cultures (82, 84, 86). Electron and

light microscope studies on the genus Pratylenchus reveal

that the number of annules in the tail vary within and among

species (11, 68, 82, 84). Most species have a mean number of

18 to 24 annules with overlapping ranges; therefore, this

character is of little use in differentiating species (11).

The lateral field in Pratylenchus spp. has four mutually

equidistant straight lines (68). Although errors in

interpretation and, to some extent, the intraspecific

variation in the lateral field are too great to allow this

to be a good diagnostic character, it is a major character

used in separating some species, such as P. hexincisus. (11,

68, 84).

Most species descriptions of Pratylenchus include

values for ratios of "a" (total body length / body width),

"b" (total body length / distance from anterior end to











posterior end of esophagus), and "c" (total body length /

tail length) (78), plus the distance of the vulva from the

anterior end as a percent of the body length ("V"). The

first three ratios are highly variable, whereas the "V"

value is not (68, 75). The "V" value is one of the least

variable characters in the genus, and it is regarded as a

good feature in species differentiation (2, 27, 52, 53, 68,

73, 86).

Intraspecific variability may be due, at least in part,

to the effect of the environment on the nematode. Host

nutrition, crowding, and temperature influenced nematode

size in P. brachyurus and P. zeae (56, 57).

Pratylenchus brachvurus bears the typical

characteristics of the genus. It is recognized by the

angular margins of the lip region, which is set off from the

body and bears two distinct annules. The spermatheca is

almost invariably empty. The vulva is located near the

posterior end. The tail shape is subcylindrical with smooth,

rounded, or truncated tail terminus, with no striations

around it. The female dimensions are body length = 0.39-0.75

mm, a = 15-29, b = 5-10, c = 13-18, V = 82-89%, stylet = 17-

22 Am, egg = 70-80 X 20-28 Am. Males are extremely rare, but

general appearance is similar to the females. They have a

single outstretched testis. The male dimensions are body

length = 0.46-0.56 mm, a = 27-29, b = 6, c = 21, T = 51-53%,

stylet = 19 im (52, 73).











In this chapter the objective was to provide

morphometric data on the identification of five Pratylenchus

brachyurus populations selected from different geographical

locations and hosts, and to determine the extent of

intraspecific morphological variation within each

population.



Materials and Methods

The origins and sources of the different Pratylenchus

brachyurus populations are 101 from corn (Zea mavs L. cv.

Pioneer 304C) in Alachua County, Florida; 102 from peanut

(Arachis hypogaea L. cv. Florunner) in Alachua County,

Florida; 103 from peanut cultivar Florunner in Tift County,

Georgia; 105 from soybean (Glycine max (L.) Merr. cv.

Forrest) in North Carolina; and 108 from citrus (Citrus sp.)

in Polk County, Florida.

Each population was established by inoculating snap

bean (Phaseolus vulgaris L. cv. Harvester) seedlings with

200 nematodes (mixed life stages). The inoculated seedlings

were maintained in a greenhouse at 25 C. Nematodes were

extracted using a modified Baermann funnel (17). The

infected roots were washed and cut into pieces about 1 cm

long, and blended with 150 ml water for 30 seconds. The

sample was washed from the blender onto a 38-im-pore sieve,

rinsed with tap water, and washed off onto a nylon screen

with 1.5-mm-openings that stretched over a 11.5-cm-diameter









11
PVC pipe. The nylon screen was covered with Scotties tissue

paper (Scott Paper Company, Philadelphia, Pennsylvania) to

retain the blended roots, then placed in a plastic sandwich

box (13 X 13 X 4 cm). Finally, the box was filled with tap

water to cover the nylon screen forming a thin film of water

around the root pieces. The nylon screen plus the tissue

retained the roots and allowed the nematodes to migrate into

the water contained in the box. After 72 hours of

incubation, the nematodes were collected by sieving the box

contents through a 38-Am-pore sieve (Figure 2-1).

Fifteen females from each nematode population were

measured using a microscope fitted with a drawing tube.

Measurements from live females were made within 30 minutes

after mounting the specimens. Females that had the

appearance of being well-fed were randomly picked and placed

in a small drop of water on a microscope slide. A ring of

Zut was placed around the drop with a small painting brush,

and a coverslip was gently lowered onto the ring. The excess

water and air were slowly forced out by gently pressing the

coverslip with a dissecting needle. Five minutes later the

nematodes were measured as they lay motionless.

Specimens were measured with the use of a drawing tube.

A line was drawn through the middle of the female body from

the head to the tail at 20X magnification. The excretory

pore, the vulva, and the anus measurements were taken from

the center of each orifice and a mark was placed onto the








































Blended roots


-4-r
1.0 cm



1.5 cm


Screen Water level


Figure 2-1. A cross section of the modified Baermann funnel
used for nematode extraction. The screen was
glued between two sections of PVC pipe used for
support. Inside diameter of PVC pipe = 10.2 cm
(drawing not to scale).


Tissue paper


PVC support









13

line drawn for the body length. The stylet length, the width

of the nematode, the shape of the lip region, and the shape

of the tail were drawn at 100X magnification using an oil

immersion objective. The nematode line was measured with a

map reader and redrawn on a separate straight line which

subsequently was measured with a ruler and converted into

micrometers. The length of the stylet and the width of the

nematode were taken directly from the drawings.

The following measurements, ratios, and characters were

recorded: body length, body width, stylet length, distance

from excretory pore to anterior body end as % of total body

length, distance from vulva to anterior body end as % of

total body length, "a" and "c" ratios, and the number of

lines in the lateral field. The ratios "a" (total body

length / body width) and "c" (total body length / tail

length) were calculated using de Man indices (78). The

extent of variability in the characters was expressed by

calculating the mean, range, standard deviation, and

coefficient of variation.



Results

All measurements obtained from all specimens were

within the reported ranges for P. brachyurus (52). There

were no significant morphological differences among any of

the populations studied. It is concluded that all of the

populations were P. brachyurus, and that they do not differ









14

morphologically from each other (Tables 2-1 to 2-5). All of

the specimens had the typical truncated head composed of two

lip annules, three large amalgamated stylet knobs, and four

lines in the lateral field.

The shape of the amalgamated stylet knobs was rounded,

but one-third of the specimens of the peanut population from

Florida had a cup-shaped anteriorly directed ventral knob

(Figure 2-2). The shape of the tail tip was smooth and

bluntly rounded, but it also varied from flat in two

specimens to indented in two others (Figure 2-3).

Among the measurements obtained, the stylet length and

the position of the vulva as a percent of the total body

length were the least variable characters and thus the most

reliable ones in the identification of the species. The

stylet length varied from 17 to 20 Am with a mean of

18.4 Am, and a coefficient of variability (CV) of 3.0% among

all populations. The position of the vulva varied from 80 to

88.2% with a mean of 85% and a CV of 1.7% among all

populations.

All the other characters measured, such as body length,

body width, tail length, vulva-anus distance, "a" and "c"

ratios, and the excretory pore as a percent of total body

length had relatively high coefficients of variability

across all populations.












Table 2-1: Measurements of mature females (n=15) of
Pratylenchus brachyurus 101 originating from
'Pioneer 304C' corn, Alachua County, Florida.



Morphological
characters Range Mean SDa CV(%)b



Measurements in Am:
Body length 521.9-687.5 585.6 48.9 8.4
Body width 20.0-30.6 24.9 3.2 12.9
Stylet length 17.1-18.8 18.0 0.6 3.0
Tail length 25.0-40.6 30.4 3.7 12.2
Vulva-anus distance 43.8-70.3 56.0 7.2 12.9

Measurements as %:
Position of the
excretory pore
as % of total
body length (n=ll) 9.2-16.8 14.4 2.1 14.6
Position of the
vulva as % of
total body length 83.2-87.7 85.2 1.4 1.6


Ratios:
a 20.4-27.3 23.8 2.4 10.0
c 16.7-22.9 19.4 2.0 10.3


aSD = Standard deviation.
CV = Coefficient of variation.












Table 2-2: Measurements of mature females (n=15) of
Pratylenchus brachyurus 102 originating from
'Florunner' peanut, Alachua County, Florida.



Morphological
characters Range Mean SDa CV(%)b



Measurements in Am:
Body length 553.1-712.5 646.7 47.7 7.4
Body width 26.5-35.3 30.9 2.3 7.4
Stylet length 17.0-20.0 18.5 0.8 4.3
Tail length 25.0-37.5 30.8 3.5 11.4
Vulva-anus distance 46.9-100.0 70.2 13.7 19.5

Measurements as %:
Position of the
excretory pore
as % of total
body length (n=14) 14.9-18.5 16.2 0.9 5.5
Position of the
vulva as % of
total body length 80.0-87.6 84.4 2.0 2.4


Ratios:
a 19.1-22.9 20.9 1.2 5.7
c 17.7-27.2 21.2 2.6 12.3


aSD = Standard deviation.
bCV = Coefficient of variation.












Table 2-3: Measurements of mature females (n=15) of
Pratylenchus brachyurus 103 originating from
'Florunner' peanut, Tift County, Georgia.



Morphological
characters Range Mean SDa CV(%)b



Measurements in jm:
Body length 606.2-712.5 657.4 28.4 4.3
Body width 28.2-32.9 30.4 1.5 4.9
Stylet length 17.6-19.4 18.7 0.6 3.2
Tail length 31.3-37.5 33.5 2.4 7.2
Vulva-anus distance 46.9-75.0 64.9 7.1 10.9

Measurements as %:
Position of the
excretory pore
as % of total
body length (n=ll) 13.9-17.7 15.6 1.0 6.4
Position of the
vulva as % of
total body length 82.6-88.2 85.0 1.4 1.6


Ratios:
a 19.9-23.7 21.6 1.0 4.6
c 16.9-22.8 19.7 1.6 8.1


aSD = Standard deviation.
bCV = Coefficient of variation.












Table 2-4: Measurements of mature females (n=15) of
Pratylenchus brachyurus 105 originating from
'Forrest' soybean, North Carolina.



Morphological
characters Range Mean SDa CV(%)b


Measurements in gm:
Body length 521.9-687.5 618.8 44.8 7.2
Body width 23.5-31.2 27.6 2.4 8.7
Stylet length 17.0-20.0 18.5 0.7 3.8
Tail length 28.1-40.6 32.9 3.1 9.4
Vulva-anus distance 53.1-68.8 61.0 4.7 7.7

Measurements as %:
Position of the
excretory pore
as % of total
body length (n=10) 13.4-15.9 14.9 0.9 6.0
Position of the
vulva as % of
total body length 82.0-87.3 84.7 1.5 1.8

Ratios:
a 20.3-26.0 22.5 1.5 6.7
c 15.8-22.3 18.9 1.8 9.5


aSD = Standard deviation.
bCV = Coefficient of variation.












Table 2-5: Measurements of mature females (n=15) of
Pratylenchus brachyurus 108 originating from
citrus, Polk County, Florida.



Morphological
characters Range Mean SDa CV(%)b


Measurements in Am:
Body length 515.6-659.4 583.5 42.1 7.2
Body width 20.6-25.3 22.5 1.4 6.2
Stylet length 18.2-18.8 18.5 0.1 0.5
Tail length 25.0-37.5 31.9 2.9 9.1
Vulva-anus distance 37.5-59.4 49.2 4.8 9.8

Measurements as %:
Position of the
excretory pore
as % of total
body length (n=14) 9.2-16.8 14.4 2.1 5.5
Position of the
vulva as % of
total body length 84.7-87.9 86.1 0.9 1.0

Ratios:
a 21.9-29.3 26.8 2.1 8.0
c 15.0-24.9 18.5 2.5 13.5


aSD = Standard deviation.
CV = Coefficient of variation.

































Figure 2-2. Stylet knob shapes of Pratvlenchus brachyurus:
a = rounded amalgamated knobs which are the
typical shape for the species, b = cup-shaped,
anteriorly directed knob.


Figure 2-3. Tail tip shapes of Pratvlenchus brachvurus:
a = bluntly rounded (typical shape for the
species), b = flat, c = slightly indented.











Discussion

Morphological studies confirmed the identity of the

five nematode populations as P. brachyurus. Although slight

morphological variations were observed among populations,

they could not be separated morphologically.

The lip region of all specimens was similar to that

described for P. brachyurus (68, 73, 82). Variations

reported include rounded lips and the presence of two

annules on one side and three on the other side. There was

no apparent variation in shape or number of lip annules in

specimens from each population used in this study.

Stylet length was reported as one character with

limited variation in P. brachyurus (68, 82). In one study,

stylet length of 50 specimens ranged from 17.4-19.2 Am with

a mean of 18.4 Am and a CV of 2.6% (68). In another case,

the range of 216 specimens was 17-19 pm with a mean of 17.9

Am and a CV of 2.4% (82). In this study, combined data from

all nematode populations showed a range of 17-20 jm with a

mean of 18.4 jm and a CV of 3.0%. The variation in stylet

knob shapes observed was similar to those reported

previously for P. penetrans and P. zeae (68, 84).

Vulva position was the morphological character with the

least variation. In previous studies vulva position of P.

brachyurus ranged from 80-87% with a mean of 85% and a CV of

1.7% (68). Combined data from all nematode populations

studied showed a range of 80-88.2% with a mean of 85% and a

CV of 1.7%.









22

Most of the specimens observed in this study had

smooth, bluntly rounded tails with a hyaline portion at the

terminus as described previously for the species (68). The

tail length was more variable than the shape. In one study,

tail length of P. brachyurus ranged from 20-40 pm with a

mean of 29.3 Am and a CV of 11% (82). In another study it

ranged from 25.2-37.8 Am with a mean of 31.5 gm and a CV of

7.4% (68). Combined data from all populations studied here

showed a range of 25.0-40.6 Am with a mean of 31.9 pm and a

CV of 9.8%.

The distance between the vulva and the anus is a good

diagnostic character to differentiate between P. coffeae and

P. brachyurus (82). The distance between the vulva and the

anus in P. brachyurus ranges from 42-75 Pm with a mean of

56.5 Am and a CV of 10.9% (82). Another study reports a

range of 39-64.2 Am with a mean of 50.6 im and a CV of 10%

(68). Similar results were obtained in this study. The

combined data from all populations showed a range of 37.5-

100 jm with a mean of 60.3 Am and a CV of 12.2%.

The degree of intraspecific morphological variability

among P. brachyurus populations depended on the

morphological characters studied. Morphometric data is

necessary in the identification of Pratylenchus spp. but it

should be used with caution. In addition to considering the

normal intraspecific variation that can occur, care must be

exercised in obtaining measurements. Differences between










23

measurements were obtained when the same specimen was

measured by several experienced researchers, and even when

one specimen was measured by the same researcher on

different days (27).















CHAPTER 3
HOST SPECIFICITY OF FOUR PRATYLENCHUS
BRACHYURUS POPULATIONS



Introduction

Pratylenchus brachyurus causes damage to many tropical

and subtropical crops (29, 76). This nematode is widely

distributed throughout the tropics and it is found in

association with many plant species (see Chapter 1).

Taxonomic separation of the species of Pratylenchus is

difficult because they exhibit little morphological

diversity. Difficulties often arise from underestimation of

intraspecific variability of certain morphological

characters currently used for distinguishing species (see

Chapter 2, 68). The validity of some species is questionable

(28).

Populations of plant-parasitic nematodes that are

designated as biological or physiological races are

recognized as morphologically similar, but with different

host preferences (81). Physiological races are documented in

certain species of plant-parasitic nematodes, such as those

from the genus Meloidogyne (85), but the presence or absence

of races within P. brachyurus has not been documented. The

presence of P. brachyurus races is suggested by variation in











numbers of P. brachyurus extracted from roots of Citrus

limon (L.) Burm. f. seedlings when inoculated with different

nematode populations (55). Field observations suggest

possible behavioral differences within P. brachvurus

populations (D. W. Dickson and R. A. Dunn, personal

communication). The morphometrics of P. brachyurus vary

considerably in response to geographical locations,

unfavorable hosts, overcrowding, and high temperatures (56,

57). Intraspecific morphological variation is demonstrated

using populations that originated from single gravid females

(82, 84).

In this chapter the objectives were to determine the

usefulness of different host plants in separating four P.

brachvurus populations obtained from different geographical

locations and to study host response to each population.



Materials and Methods

The designation and sources of the four populations of

lesion nematode, P. brachyurus, are given in Chapter 2.

Seven plant species, representing a wide diversity of plant

types and reported as hosts for P. brachvurus, were

evaluated in a greenhouse. The differential plants were

alfalfa (Medicago sativa L. cv. Florida 77); snap bean

(Phaseolus vulgaris L. cv. Harvester); citrus cultivar Rough

Lemon; corn cultivar Pioneer 304C; peanut cultivar









26
Florunner; soybean cultivar Braxton; and tomato

(Lycopersicon esculentum Mill. cv. Rutgers).

Nematode inoculum was extracted as described in

Chapter 2. Germinated seedlings of the different plant types

with 3-cm-long radicles were transplanted into 900 cm3 of

steam-pasteurized sandy loam soil in individual 15-cm-

diameter clay pots. When the seedlings were 1 week old, the

soil was infested separately with 300 nematodes (mixed life

stages) per pot (approximately 0.33 nematodes/cm3 soil). The

nematodes in an aqueous suspension were poured into two

small holes, each 5-cm deep, near the base of the seedling.

The experiment was conducted in the summer of 1986 with

six replications and repeated in the fall of 1987 with five

replications. Plants were harvested 64 days after

inoculation and shoot fresh weight, root fresh weight, and

the nematode density levels were determined. Densities of

nematodes in the soil were determined by processing 100 cm3

of soil by the sugar-flotation-centrifugation method (44).

Root densities were determined by processing 10 g of roots

by the method described in Chapter 2.

A factorial experiment was conducted using a split-plot

design. The plants were blocked by replicate using plant

type as a main plot and populations as a subplot on a

greenhouse bench. The greenhouse temperature was maintained

at 25 + 5 C. The effects by the plant, by the population,

and interactions of the two were determined by analysis of

variance.











Results

No nematode population affected fresh shoot or root

weight when compared to the uninoculated controls in either

experiment (Tables 3-1 and 3-2). Final population densities

were different (P < 0.05) on 'Harvester' snap bean in the

two experiments and on 'Braxton' soybean in the second

experiment (Tables 3-3 and 3-4). In the first experiment,

populations 102 and 103, both from peanut, had higher final

population densities on 'Harvester' snap bean than the other

two populations (P < 0.05); however, there were no

differences in population densities between the two

populations from peanut or between the other two

populations, which originated from soybean or corn. These

observations were the same whether the root or the soil

nematode population densities were compared (Tables 3-3 and

3-4). In the second test, population 101 from Florida had

higher population density in 'Harvester' snap bean and in

'Braxton' soybean than the other three populations (P <

0.05). There were no differences in population densities

among the other three populations in either of the two hosts

(Tables 3-3 and 3-4). Nematodes were also extracted from 100

cm3 of soil in 1987; however, since the numbers were very

low (5/100 cm3 of soil) no data is shown here.












Table 3-1: Shoot weights (g) of seven plant species 64 days
after inoculation with four Pratylenchus
brachyurus populations and the uninoculated
control.



P. brachyurus population

Plant
species Year 101 102 103 105 Control


Alfalfa 1986 34b 33 33 30 39
1987 49 47 43 46 50

Snap bean 1986 55 47 45 50 60
1987 64 91 70 80 65

Citrus 1986 46 51 56 50 43
1987 10 9 9 11 10

Corn 1987 172 188 177 169 182
1986 144 128 117 176 149

Peanut 1986 69 74 70 69 67
1987 63 63 57 53 61

Soybean 1986 101 93 93 110 99
1987 23 27 28 22 26

Tomato 1986 132 125 131 129 131
1987 101 87 112 102 100


aPopulation origins: 101- 'Pioneer 304C' corn, Alachua
County, Florida; 102- 'Florunner' peanut, Alachua County,
Florida; 103- 'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina.

bData are the means of six (1986) and five (1987)
replications. No significant (P < 0.05) differences were
found among means in any row, according to least significant
difference test.











Table 3-2:


Root weights (g) of seven plant species 64 days
after inoculation with four Pratylenchus
brachyurus populations and the uninoculated
control.


P. brachyurus population

Plant
species Year 101 102 103 105 Control


Alfalfa 1986 35b 28 27 21 36
1987 43 35 39 42 40

Snap bean 1986 20 18 13 21 19
1987 36 31 34 42 37

Citrus 1986 34 48 49 46 42
1987 5 4 5 5 4

Corn 1987 171 149 139 135 142
1986 77 60 66 84 72

Peanut 1986 24 18 17 19 15
1987 18 21 17 17 21

Soybean 1986 82 62 56 61 60
1987 20 26 26 17 22

Tomato 1986 32 26 29 26 28
1987 46 49 59 42 40


aPopulation origins: 101- 'Pioneer 304C' corn, Alachua
County, Florida; 102- 'Florunner' peanut, Alachua County,
Florida; 103- 'Florunner peanut', Tift County, Georgia; 105-
'Forrest' soybean, North Carolina.

Data are the means of six (1986) and five (1987)
replications. No significant differences (P < 0.05) were
found among means in any row, according to least significant
difference test.











Table 3-3: Final population densities (nematodes per g
of root) of four Pratylenchus brachyurus
populations 64 days after inoculation.



P. brachyurus populationa

Plant
species Year 101 102 103 105


Alfalfa 1986 21 a 48 a 29 a 14 a
1987 1 a 3 a 1 a 0 a

Snap bean 1986 158 b 253 a 266 a 125b
1987 22 a 9 b 9 b 4 b

Citrus 1986 0 a 0 a 0 a 0 a
1987 1 a 1 a 0 a 0 a

Corn 1986 23 a 25 a 30 a 18 a
1987 12 a 5 a 4 a 4 a

Peanut 1986 6 a 9 a 16 a 7 a
1987 6 a 4 a 5 a 3 a

Soybean 1986 23 a 29 a 30 a 11 a
1987 23 a 8 b 9 b 7 b

Tomato 1986 89 a 41 a 44 a 36 a
1987 19 a 2 a 2 a 2 a



aPopulation origins: 101- 'Pioneer 304C' corn, Alachua
County, Florida; 102- 'Florunner' peanut, Alachua County,
Florida; 103- 'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina.

bData are the means of six (1986) and five (1987)
replications. Means within rows followed by the same letter
are not different (P < 0.05) according to least significant
difference test.











Table 3-4: Final population densities (nematodes per 100 cm3
of soil) of four Pratylenchus brachyurus
populations 64 days after inoculation.



P. brachyurus population

Plant
species Year 101 102 103 105


Alfalfa 1986 24ba 32 a 16 a 8 a

Snap bean 1986 37 b 192 a 200 a 42 b

Citrus 1986 0 a 0 a 0 a 0 a

Corn 1986 139 a 82 a 84 a 96 a

Peanut 1986 1 a 2 a 1 a 1 a

Soybean 1986 5 a 5 a 7 a 2 a

Tomato 1986 138 a 73 a 71 a 41 a



aPopulation origins: 101- 'Pioneer 304C' corn, Alachua
County, Florida; 102- 'Florunner' peanut, Alachua County,
Florida; 103- 'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina.

bData are the means of six replications. Means within
rows followed by the same letter are not different (P <
0.05) according to least significant difference test.









32

None of the nematode populations were able to reproduce on

citrus in either of the two experiments before the

experiment was terminated. Data from the final population

densities of the four P. brachyurus populations on selected

hosts are given in the Appendix.



Discussion

This attempt to separate populations of P. brachvurus

failed to discern behavioral differences on seven species of

crop plants. The patterns observed in the final population

densities in the two experiments were not consistent,

suggesting that none of the populations studied were

different from each other. The smaller numbers obtained in

the second experiment were due, in part, to the time of the

year it was conducted.

Pratylenchus brachyurus is pathogenic to at least one

type of citrus, Citrus aurantium, 'Sour Orange' (6), and can

be found associated with 'Rough Lemon' roots (82). Although

no pathogenicity was observed on citrus here, the lack of

reproduction of the nematode in citrus cultivar 'Rough

Lemon' under the conditions of the two experiments indicates

a possible nonhost relationship. Other factors that might be

involved include unsuitability of greenhouse cultural

conditions and insufficient time for nematode development.

Data from these tests provide no support for

postulating the presence of races within P. brachvurus, but









33

the possibility cannot be excluded either until citrus

populations and other populations are tested with additional

host plants.















CHAPTER 4
COMPARISON OF ENZYME PHENOTYPES OF FIVE
PRATYLENCHUS BRACHYURUS POPULATIONS


Introduction

The biochemical approach to nematode taxonomy has

considerable potential for assisting in the identification

and characterization of these organisms as well as

establishing phylogenetic relationships (39). The

application of comparative biochemistry to problems of

systematics has taken a variety of approaches. Serology (38,

54), analysis of lipids and fatty acids (49), analysis of

proteins (13, 14, 20, 23, 35, 36, 40, 61, 88), and analysis

of nucleic acids (4, 5, 12, 42, 47, 59, 60, 63) are used on

nematodes.

Proteins are a manifestation of the sequence of

nucleotides in a gene, and analysis of these macromolecules

provides a reliable approach for comparing genotypes of

organisms (39). Biochemical data can be used to

differentiate sibling nematode species that are

morphologically indistinguishable with routine morphological

examination, but are physiologically distinct (33).

The primary problem in biochemical systematics is to

find those chemical characters that will be most valuable in









35

providing information for taxonomy (39). Data that

contribute to the characterization of species and races of

nematodes are particularly valuable because of the important

role of plant-parasitic nematodes in agriculture.

The protein extraction technique used, the source, and

the general handling of the protein could be determining

factors in the success of protein analysis. It is

particularly important in the case of certain types of

proteins, such as isozymes, because they are so sensitive to

manipulation. Variation in protein and enzyme profiles of

Meloidogyne spp. were affected by the host plant and by the

growing conditions of the host (43). Protein profiles of

nematodes extracted from frozen roots were different than

those from nematodes extracted from unfrozen roots (13). The

use of a single developmental stage is critical since

differences can be obtained in profiles from different life

stages (8, 13). Plant-parasitic nematodes are microscopic

eelworm-shaped animals; consequently the amount of protein

present in each individual is very small.

The term electrophoresis is used to describe the

migration of charged particles under the influence of an

electric field (1). Isoelectric focusing (IEF) can be

regarded as electrophoresis within a hydrogen ion gradient

(1). In IEF a stable hydrogen ion gradient that decreases

progressively from anode to cathode is established by

carrier ampholytes. When proteins or other amphoteric









36

molecules are introduced into this system, they will migrate

to their corresponding surface charge in the electric field.

Eventually, they will reach their isoelectric point, a zone

where the net electrical charge is zero (67).

Isoelectric focusing electrophoresis in conjunction

with certain enzyme-specific stain systems successfully

detected intraspecific variability among populations of H.

glycines and was sensitive enough to detect polymorphisms

within populations (65). Analyses of mass homogenates of

protein from 12 H. qlycines populations using eight enzyme

systems showed consistent groupings among populations, but

in no case did isozyme analysis of populations correlate

with the conventional race scheme based on quantitative

reproduction on a set of soybean differentials (65). Four

Meloidoqyne spp. were distinguishable from each other by IEF

of nematode egg protein profiles (50). Enzyme phenotypes

from a single female soybean cyst nematode were resolved

using IEF in conjunction with enzyme-specific stains (34,

64).

In this section a new technique is presented that

allows the use of small numbers of vermiform nematodes for

protein extraction and permits studies of a single

developmental stage. This new technique was combined with

ultra-thin IEF and with enzyme-stain systems in an attempt

to determine similarities and differences among enzyme

phenotypes from mass homogenates of females and juveniles of











five populations of P. brachyurus. The investigation

reported herein is intended to add biochemical data to the

morphological, ecological, and behavioral data already

available for P. brachyurus.



Materials and Methods

Nematode cultures. The identification and description

of the nematode populations used are given in Chapter 2.

Nematodes were raised using a modified root explant

technique (37). They were maintained on the corn cultivar

lochief grown in plastic petri dishes containing Gamborg's

B-5 medium without auxins or cytokinins (Gibco Chem., Grand

Island, New York). The cultures were incubated in the dark

at 29 C. Nematodes were extracted from the culture dishes by

removing a block of agar with roots and placing it in a

modified Baermann funnel as described for nematode

extraction from roots in Chapter 2.

Protein extraction. Two hundred and fifty females, or

450 juveniles were pipetted with the use of a controlled

vacuum aspirator (26). They were placed into a depression

slide coated previously with Repel-Silane (LKB, Bromma,

Sweden) to produce a hydrophobic surface. The volume of

water was reduced to about 20 pl by pipetting out excess

water. The nematodes were stored in a 20-Al droplet of

deionized water in a 25-1l capillary tube and stored in a

refrigerator at 5 C. The nematodes were either used











immediately or stored overnight (Figure 4-1). The contents

of the capillary tubes were placed in a 1.5- X 1.5-mm well

made previously on a micro culture slide (75 X 25 mm, molded

glass, with well 3 mm deep X 15 mm diameter) (Figure 4-2).

The surface of the slide was coated with Repel-Silane to

obtain a hydrophobic surface that prevented the spreading of

the droplet, and allowed the nematodes to fall to the bottom

of the well. Nematodes that did not fall were pushed into

the well. The glass slide was then placed in a plastic

sandwich box that contained an ice block and a mirror

(Figure 4-2). The mirror, which was placed directly under

the glass slide, enhanced visibility since the entire

process was done under the dissecting microscope at 30X

magnification. After placing the nematodes in the well, most

of the water was removed with a syringe and filter paper.

The nematodes, which remained attached to the walls of the

well by a thin film of water, were macerated for 30 seconds

with a ground glass rod made to fit the well snugly. After

maceration, 2 il of a solution containing 20% sucrose and 2%

Triton X-100 (20) were added to the well and mixed

thoroughly. The contents of the well were absorbed onto a 5-

X 10-mm filter paper (LKB, Bromma, Sweden) and applied

directly to the surface of the gel 1 cm from the anode.

Electrophoresis. The protein from 250 macerated females

were subjected to isoelectric focusing in thin layer (1-mm)

gels (67). The gel concentration consisted of T= 5%, C= 3%,






















































Figure 4-1. Two hundred and fifty Pratylenchus brachyurus
females stored alive in a 25-A capillary tube.































Figure 4-2. Protein extraction equipment. A) Photograph of a
micro culture slide (75 X 25 mm, molded glass,
with well 3 mm deep X 15 mm diameter) showing
the wells and the glass rod constructed to
macerate vermiform nematodes for protein
extraction. B) Cross-section of the microscope
slide (not to scale) used for macerating
vermiform nematodes. The dimension of each well
is 1.5 mm wide at the bottom, 2.5 mm wide at the
surface, and 1.5 mm deep. The slide was placed
over a glass mirror that lay over an ice block
contained in a plastic sandwich box.













































Microscope slide


Plastic box Ice block Mirror











and 2.4% ampholytes with a pH range of 3.5-9.5. The LKB

ultrophor system was used with 1 M H3PO4 in the anode and 1

M NaOH in.the cathode (67). Gels were maintained at a

constant temperature of 5 C. The power was applied from a

LKB macrodrive 5 constant power supply set at 1,500 volts,

50 milliamps, and 30 watts. The settings of the power supply

were reduced proportionally with the size of the gel. Each

gel was prefocused for 10 minutes, samples were applied, and

the gel was run for 1.5 hours (final voltage 1,500 volts).

The pH gradient in the gel was measured with a surface

electrode.

Staining. After isoelectric focusing, the gels were

submerged in buffer (the same buffer used in the stain-

mixture) for 3 minutes to remove excess electrolytes that

may have been on the surface of the gel. The gels were

submerged in a plastic box previously lined with plastic

film and filled with the reaction mixtures. A list of the

enzymes studied, and references to the stain and reaction

mixtures are given in Table 4-1. The gels were incubated in

the dark at 25 C until bands appeared. After staining, the

gels were photographed and allowed to air dry. The dried

gels were covered with a plastic preserving sheet (LKB,

Bromma, Sweden) for future reference.

Number of replications. Replications varied with the

enzyme system and the populations tested. Data reported here

include results obtained after the system was tested












Table 4-1.


Enzymes examined, activity, and references to
stains and reaction mixtures used in isoelectric
focusing of Pratylenchus brachvurus mass
homogenates.


E.C.
Enzyme number Activityb Reference


Oxidoreductases
Aldehyde oxidase
a-Glycerophosphate
dehydrogenase
Isocitrate dehydrogenase
Malate dehydrogenase
Malic enzyme
Octanol dehydrogenase
Phosphogluconate
dehydrogenase
Superoxide dismutase
Xanthine dehydrogenase

Transferases
Glutamate oxaloacetate
transaminase
Hexokinase
Phosphoglucomutase

Hydrolases
Acid phosphatase
Alkaline phosphatase
Esterase

Lyases
Fumarase

Isomerases
Manose phosphate
isomerase
Phosphoglucose isomerase


1.2.3.1

1.2.1.12
1.1.1.42
1.1.1.37
1.1.1.40
1.1.1.73

1.1.1.44
1.15.1.1
1.2.1.37



2.6.1.1
2.7.1.1
2.7.5.1


3.1.3.2
3.1.3.1
3.1.1.8


4.2.1.2



5.3.1.8
5.3.1.9


aEnzyme commission number. Enzyme Committee Union of
Biochemistry Classification.
+ = activity detected; = no activity detected.













thoroughly, and results were obtained at least twice. For

example, 11 replicates in seven different runs were done for

population 101 in testing the malate dehydrogenase (MDH)

system. Populations 101, 102, 103, and 105 were tested

together twice with two replications each for the MDH,

phosphoglucomutase (PGM), and phosphoglucose isomerase (PGI)

systems. Results from population 108 were obtained by

comparing it to one of the other four populations.

Paired affinity indices. To compare shared enzymes

among the populations, paired affinity (PA) indices (16)

were calculated for each population for each of the isozymes

used. The PA index was obtained as follows:

Bands in common for pop. A and pop. B
PA =

Total bands in pop. A + pop. B

Affinities among the five P. brachyurus populations

were visualized by plotting PA indices in polar ordination

on the first axis (58). The two populations comprising the

most dissimilar population pair (the ones with the lowest PA

index) were chosen as the end points of the first axis. The

length of the line between the end points is D= 100-PA. All

the other paired comparisons were located in relation to the

end points. To determine the position of a specific

population in relation to the end points, the D value for

each of the two end points was calculated. Using the scale











of the axis, a compass was used to mark off an arc of D

units from each of the two end points. The intersection of

the two arcs determined the position of the population.



Results

Of the 18 enzyme systems studied, only the MDH, PGM,

and PGI systems were resolved efficiently (Figures 4-3, 4-4,

4-5). The isocitrate dehydrogenase (ICD) and

phosphogluconate dehydrogenase (PGD) systems showed faint

bands. Only MDH, PGM, and PGI were resolved for all five

populations, whereas ICD and PGD were resolved for only

population 105. No other enzyme system was detected (Table

4-1).

The MDH system contained three distinct phenotypic

groups (Figure 4-3). Two electromorphs (electromorph is a

single resolved band) were resolved for populations 101 and

105 at pH 5.5 and 5.9. Populations 103 and 108 shared the

two electromorphs from group one, but had two others at pH

6.4 and 6.8. Population 102 contained eight electromorphs.

Two were shared with all four populations, two others were

shared with populations 103 and 108 at pH 6.4 and 6.8. The

remaining four electromorphs at pH 5.9, 6.2, 6.4, and 6.5

were unique to population 102.

Three distinct phenotypes were detected in the PGM

system (Figure 4-4). Population 101 had three electromorphs

located at pH 5.5, 6.1, and 6.7. Populations 103, 105, and




































Figure 4-3. Diagrammatic sketch (A) and photograph (B)
of patterns of malate dehydrogenase phenotypes
following isoelectric focusing of crude protein
homogenates from 250 females from each of
five Pratylenchus brachyurus populations.
Population origins: 101- 'Pioneer 304C' corn,
Alachua County, Florida; 102- 'Florunner'
peanut, Alachua County, Florida; 103-
'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina; 108- Citrus
sp., Polk County, Florida.











pH 7.0





6.5





6.0






5.5





5.0


101 101


102 102 103
Populations
(B)


103 105 105


101 102 103 105 108
Populations


ilri



































Figure 4-4.


Diagrammatic sketch (A) and photograph (B)
of patterns of phosphoglucomutase phenotypes
following isoelectric focusing of crude protein
homogenates from 250 females from each of
five Pratylenchus brachyurus populations.
Population origins: 101- 'Pioneer 304C' corn,
Alachua County, Florida; 102- 'Florunner'
peanut, Alachua County, Florida; 103-
'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina; 108- Citrus
sp., Polk County, Florida.













pH 7.5


7.0



6.5


6.0



5.5


5.0


101 101 102 102 103 103
Populations
(B)


101 102 103 105 108
Populations


105 105









50

108 shared two electromorphs with populations 101 (pH 6.1

and 6.7), but had two others located at pH 7.0 and 7.6.

Population 102 contained five electromorphs. Two were shared

with all populations (pH 6.1 and 6.7), one with population

101 (pH 5.5), and two with the other three populations (pH

7.0 and 7.6). The PGI system exhibited only one migrating

electromorph at pH 4.6 for all populations (Figure 4-5).

Population 105 showed one faint band at pH 7.0 in the

ICD system, and one at pH 5.6 in the PGD system. When

comparing females and juveniles of population 105 using the

PGI stain system, no phenotypic differences were observed.

Phenotypes obtained in the MDH system were the same for

nematodes extracted from greenhouse cultures on 'Harvester'

snap bean and nematodes from excised root cultures of

'lochief' corn.



































Figure 4-5. Diagrammatic sketch (A) and photograph (B)
of patterns of phosphoglucose isomerase
phenotypes following isoelectric focusing of
crude protein homogenates from 250 females
from each of five Pratylenchus brachyurus
populations. Population origins: 101- 'Pioneer
304C' corn, Alachua County, Florida; 102-
'Florunner' peanut, Alachua County, Florida;
103- 'Florunner' peanut, Tift County, Georgia;
105- 'Forrest' soybean, North Carolina; 108-
Citrus sp., Polk County, Florida.


































































101 102 103 105
Populations


5.5





5.0


4.5 1


101 102 103 105 108
Populations


4.0


(A)


























(B)









53

The populations with the most similarity among the

enzyme systems tested were 103 from Georgia and 108 from

Florida (Table 4-2). Their paired affinity (PA) was 100%.

These two P. brachyurus populations had the same phenotypes

in all enzyme systems. The populations with the least

similarity among the enzyme systems tested were 101 and 102

from Florida with a PA index of 43%. Population 105 from

North Carolina was more closely related to 103 and 108 than

to 101 or 102 (Figure 4-6).



Discussion

The amount of protein from a single female nematode was

insufficient for the detection of enzymes using isoelectric

focusing electrophoresis in conjunction with certain enzyme-

staining systems. Consequently homogenates were prepared

from 250 females. Of the 18 enzyme systems studied, only the

MDH, PGM, and PGI enzyme systems were resolved efficiently

with 250 females per test. Although the ICD and PGD systems

were detected, the bands were faint and more work will be

necessary to improve the resolution. Extensive work was done

in an attempt to detect esterases, but although excellent

resolution was repeatedly obtained with a single female

Meloidogyne javanica (Treub, 1885) Chitwood, 1949, no

esterase bands were obtained with any Pratylenchus species

tested. The lack of resolution of some enzymes in this

system may be related to their age because it was learned













Table 4-2: Paired affinity indices of five
brachyurus populations based on
phenotypes.


Pratylenchus
three enzyme


Populationa 101 102 103 105 108


101


102


103


105


6/14b
43 C


5/10
50

9/14


5/8
62

7/14
50


5/10
50

9/14
64


7/9


9/9
100

7/9


Enzymes: malate dehydrogenase, phosphoglucomutase, and
phosphoglucose isomerase.
aPopulation origins: 101- 'Pioneer 304C' corn, Alachua
County, Florida; 102- 'Florunner' peanut, Alachua County,
Florida; 103- 'Florunner' peanut, Tift County Georgia; 105-
'Forrest' soybean, North Carolina; 108- Citrus sp., Polk
County, Florida.
Paired affinity index (PA) is defined as the number of
bands in common / total number of bands.
cPA%
























Population
105











58 50


7$


Population 103
Population 105


*--- Population 102


I


Population


11 -38
101 ---,


50 58


Figure 4-6.


Polar ordination of five Pratvlenchus
brachvurus populations on the first axis.
Population origins: 101- 'Pioneer 304C' corn,
Alachua County, Florida; 102- 'Florunner'
peanut, Alachua County, Florida; 103-
'Florunner' peanut, Tift County, Georgia; 105-
'Forrest' soybean, North Carolina; 108- Citrus
sp., Polk County, Florida. Units above the line
correspond to population 102, and units below
the line correspond to population 101.









56

that, in at least some cases, nematodes used for protein

extraction must be fresh (less than 3 days following

extraction from roots)for enzyme bands to be detected.

Nematodes stored in water more than 4 days had missing or

faint electromorphs (personal observation). Unfortunately,

these observations were discovered at a later date and

experiments could not be repeated before this report was

completed.

Protein extraction from small numbers of vermiform

nematodes (less than 250) presents a technique problem.

Techniques such as sonication and freezing (liquid nitrogen

or -31 C) were attempted without success. It was clear that

to detect enzymes from vermiform nematodes, it was necessary

to increase the number of nematodes or to reduce the volume

of liquid in which they were homogenized. Since the

objective of this work was to use the smallest number of

nematodes possible, the amount of liquid was reduced to a

film of water. Excellent PGI phenotypic resolution was

obtained using 100 juveniles (third and fourth stages) from

P. brachyurus population 105 from North Carolina. However,

for consistency 250 nematodes were used throughout all test.

The MDH and PGM systems indicated distinct levels of

genetic variability among populations from various

geographical locations. Pratylenchus brachyurus is a

monosexual species and reproduces by mitotic

parthenogenesis. It has a relatively high chromosome number









57
(30-32) which also indicates polyploidy (69). To understand

the genes involved in the different enzyme systems in plant-

parasitic nematodes, it is necessary to design studies with

controlled single-pair crosses and identifiable mutant forms

that serve as markers. This type of study is difficult with

a parthenogenic species such as P. brachyurus. Esterase

polymorphism was studied in H. glycines using controlled

single-pair crosses (22).

The number of electromorphs detected in the MDH systems

during this study is similar to the number observed in other

nematodes. From 291 Meloidogyne spp. populations tested

using disc-electrophoresis, eight bands of MDH activity were

detected (20). One migrating electromorph was characteristic

of M. hapla, whereas M. arenaria (Neal, 1889) Chitwood 1949,

M. incognita, and M. javanica had similar MDH phenotypes

that varied from one to five bands (20). The MDH phenotypes

do not differ in females extracted from different hosts

(20). Four migrating bands were detected for M. arenaria,

and three each for M. hapla, M. incognita, and M. javanica

(13). There are three bands for M. incognita females, and

four for juvenile stages and eggs (13). Three Florida

populations of M. javanica have similar MDH phenotypes with

three electromorphs in each population (19). The four-banded

MDH phenotype of Meloidogyne incognita separates it from M.

arenaria which has only three bands (41). The two races

(two species now) of Radopholus similis can be separated











through MDH phenotypes. The banana race (R. similis) has one

band and the citrus race (R. citrophilus) has two bands

(36).

Several interpretations are possible for the distinct

phenotypic groups found in the MDH system in P. brachyurus.

First, the multiple bands may represent isozymes, that is,

gene products of slightly variant genes. In population 102

there may be eight isozymes involved. Whereas four isozymes

may be associated with populations 103 and 108, and two

isozymes with populations 101 and 105. It is possible that

some of these isozymes were either not present or not

expressed in populations with fewer bands. Secondly, since

MDH is a diameric enzyme, it is possible that the monomeric

units may react enzymatically and break down due to the

extraction procedure. Whether or not some of these bands

represent artifacts is unknown.

Distinct phenotypic groups were also found in the PGM

systems in P. brachyurus. In addition to the interpretations

given for MDH systems, it is possible that there were two

distinct groups of populations with distinct phenotypes. One

group included populations 101, and the other group included

populations 103, 105, and 108. Population 102 may be a

mixture of the two groups. Thirty populations of Meloidogyne

spp. showed seven different PGM phenotypes ranging from four

to six bands following starch electrophoresis (21). The two









59

races (species) of R. similis have identical PGM phenotypes

with two bands each (35).

There was only one phenotype for P. brachvurus

populations in the PGI system; it could be the product of a

single locus. A three-band phenotype was observed for races

3, 4, and 5 of H. glycines, and a one-band phenotype was

observed for race 1 after IEF of single females protein

(34). Intraspecific variability was detected in 12 H.

glycines populations; three different phenotypic groups were

obtained (65). Four populations had a single electromorph at

pH 4.6, one had none, and seven populations had two

electromorphs at pH 4.6 and 4.4. With the use of starch-gel

electrophoresis, 10 different phenotypes ranging from one to

seven bands were obtained in 30 Meloidogyne spp. populations

(21). Also, with starch electrophoresis, six populations of

the citrus race and seven populations of the banana race

were shown to have two bands. On the other hand, two

populations of the banana race had only one band (35).

Isoelectric focusing in conjunction with certain

enzyme-staining systems was successful in detecting

intraspecific variability within P. brachyurus populations.

This technique was sensitive enough to detect polymorphism

within populations, as was reported from studies on

populations of Heterodera glycines (65), and it provides a

useful tool for studying protein polymorphism and genetic

diversity among nematode populations (65).









60

Although more data will be necessary to determine a

more conclusive relationship among P. brachyurus

populations, it is observed that the two populations with

the least affinity were collected from areas that were only

2.4 km apart. Population 103 from peanut in Georgia had the

same enzyme phenotypes as population 108 from citrus in

Florida.















CHAPTER 5
SUMMARY AND CONCLUSIONS




Morphological studies plus two differentiating

techniques (host response and isoelectric focusing

electrophoresis) were used to study intraspecific variation

among Pratylenchus brachyurus populations selected from

different geographical locations and hosts. Different

results obtained from the two techniques do not support the

hypothesis of similarity among the techniques. There were no

extant morphological differences nor behavioral differences,

but there were differences in isozyme phenotypes resolved by

isoelectric focusing electrophoresis.

The use of host differentials revealed no behavioral

differences among the four P. brachyurus populations studied

on seven different crop plants. No nematode population

affected fresh or root weight when compared to the

noninoculated controls. Final nematode population densities

showed that none of the populations were different from each

other. Although P. brachyurus is pathogenic to citrus (6),

none of the populations reproduced in Citrus limon under the

conditions of this study.









62

Isoelectric focusing electrophoresis was investigated

to obtain enzyme phenotypes of females from the five P.

brachyurus populations. Although there were differences in

enzyme phenotypes among the nematode populations in the

malate dehydrogenase (MDH) and in the phosphoglucomutase

(PGM) systems, the phosphoglucose isomerase (PGI) system

showed only one phenotype for all populations. Three

distinct phenotypic groups for MDH and PGM systems were

resolved. In the MDH system, populations 101 and 105 had two

migrating electromorphs. Populations 103 and 108 had two

electromorphs from group one plus two others. Population

102, in addition to the two electromorphs shared with all

four populations and the two shared with the second group,

had four more electromorphs. In the PGM system, population

101 had three electromorphs. Populations 103, 105, and 108

shared two electromorphs with population 101, but had two

others. Population 102 had all five electromorphs. Two were

shared with all populations, one with population 101, and

two with the other three populations. The PGI system showed

only one migrating electromorph for all populations.

Based on enzyme phenotypes, population 103 from North

Carolina and population 108 from Florida were similar

because both populations had the same enzyme phenotype for

all three enzyme systems. On the other hand, populations 101

and 102 from Florida were the least similar even though they

were collected only 2.4 km apart. This study showed that









63

isoelectric focusing in conjunction with enzyme-specific

stain systems provided a good tool to detect intraspecific

variations within P. brachyurus populations. More enzyme

systems must be studied before a more conclusive

relationship can be determined among P. brachvurus

populations. Also, the role of enzyme systems is unclear and

warrants further studies.
















APPENDIX
FINAL POPULATION DENSITIES OF FOUR
PRATYLENCHUS BRACHYURUS POPULATIONS ON
SELECTED HOSTS 64 DAYS AFTER INOCULATION












Table A-i. Final population densities (nematodes per 10 g of
root) of Pratylenchus brachvurus originating from
'Pioneer 304C' corn, Alachua County, Florida, on
selected plants 64 days after inoculation.



Replicate

Plant
species Year I II III IV V VI


Alfalfa 1986 344 130 70 22 676 26
1987 3 11 3 8 4

Snap bean 1986 1,888 2,149 444 883 3,742 239
1987 157 99 157 537 141

Citrus 1986 0 4 1 0 0 2
1987 9 5 5 8 7

Corn 1987 871 186 190 13 385 90
1986 106 61 272 148 29

Peanut 1986 36 77 129 79 41 10
1987 41 93 64 70 48

Soybean 1986 169 523 118 7 458 131
1987 203 667 93 123 70

Tomato 1986 772 1337 777 716 1,344 251
1987 60 384 37 25 9












Table A-2. Final population densities (nematodes per 10 g of
root) of Pratylenchus brachyurus originating from
'Florunner' peanut, Alachua County, Florida, on
selected plants 64 days after inoculation.



Replicate

Plant
species Year I II III IV V VI


Alfalfa 1986 120 684 1268 78 255 469
1987 4 35 0 92 3

Snap bean 1986 1,831 5,229 293 4,127 --a 1,178
1987 78 105 124 63 87

Citrus 1986 0 0 0 0 0 0
1987 7 7 5 5 6

Corn 1987 537 247 85 347 230 76
1986 53 96 34 54 8

Peanut 1986 24 105 225 91 57 24
1987 26 57 56 42 21

Soybean 1986 221 531 149 128 90 0
1987 38 193 40 85 26

Tomato 1986 328 322 491 274 270 785
1987 31 42 4 25 3


aMissing value.












Table A-3. Final population densities (nematodes per 10 g of
root) of Pratylenchus brachyurus originating from
'Florunner' peanut, Tift County, Georgia, on
selected plants 64 days after inoculation.



Replicate

Plant
species Year I II III IV V VI


Alfalfa 1986 628 150 153 343 41 410
1987 1 9 10 25 1

Snap bean 1986 3,663 3,564 3,936 2,551 1,323 904
1987 83 186 132 50 14

Citrus 1986 0 0 1 3 0 3
1987 2 3 2 2 1

Corn 1987 503 431 102 313 177 258
1986 29 26 121 11 8

Peanut 1986 174 252 213 58 151 100
1987 38 24 125 37 19

Soybean 1986 266 171 576 152 183 479
1987 83 98 81 71 117

Tomato 1986 668 371 419 114 493 602
1987 25 36 14 21 16












Table A-4. Final population densities (nematodes per 10 g of
root) of Pratylenchus brachvurus originating from
'Forrest' soybean, North Carolina, on selected
plants 64 days after inoculation.



Replicate

Plant
species Year I II III IV V VI


Alfalfa 1986 94 428 96 64 70 82
1987 4 5 2 6 2

Snap bean 1986 1,313 747 199 2,632 1,837 744
1987 25 16 21 50 73

Citrus 1986 0 0 2 1 0 0
1987 1 2 2 0 0

Corn 1987 40 295 297 120 148 156
1986 42 27 32 106 9

Peanut 1986 136 41 70 83 44 20
1987 21 46 23 24 10

Soybean 1986 114 149 31 296 84 0
1987 73 86 100 63 27

Tomato 1986 115 378 797 211 421 250
1987 7 21 16 25 5















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BIOGRAPHICAL SKETCH


Luis A. Payan was born on March 26, 1957, in Cali,

Colombia. He attended San Luis Gonzaga High School in Cali,

Colombia, and graduated in July 1974. After graduation, he

attended the University of Colombia, Palmira, Colombia. In

1978 he attended Abraham Baldwin Agricultural College,

Tifton, Georgia, on a tennis scholarship. In January 1980 he

enrolled at the University of Georgia, Athens, Georgia,

where he earned a B.S. degree in agronomy with a double

major in pest management. Before enrolling in graduate

school, he played professional tennis tournaments in Eastern

Europe during 1981. In August 1982 he began graduate studies

in the Department of Plant Pathology where he received a

M.S. degree from the University of Georgia in March 1985.

His research was conducted at the Coastal Plain Experiment

Station, Tifton, Georgia, under the direction of Dr. A. W.

Johnson and Dr. R. H. Littrell. His thesis was entitled

"Effect of nematicides and herbicides alone and in

combination on hatching, penetration, development and

reproduction of Meloidogyne incognita." In January 1985 he

moved to Gainesville, Florida, to conduct research for this

dissertation under the direction of Dr. D. W. Dickson.








I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



Donald W. Dickson, Chairman
Professor of Entomology and Nematology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



(rmen C. Tarjan /
Professor of Entomology and Nematology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



David J. $itchell
Professor of Plant Pathology


I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.



Robert T. McSorTey
Professor of Entomology and Nematology









This dissertation was submitted to the graduate faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial fulfilment of the requirements for
the degree of Doctor ofpPhils phy.

May 1989 .
Dean, College of Agriculture


Dean, Graduate School






































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