Citation
Plant parasitic nematodes of the U.S. Virgin Islands with the description, life cycle and morphology of Meloidogyne cruciani n.sp. (Nematoda: Meloidogynidae) and its interaction with Rotylenchulus reniformis

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Title:
Plant parasitic nematodes of the U.S. Virgin Islands with the description, life cycle and morphology of Meloidogyne cruciani n.sp. (Nematoda: Meloidogynidae) and its interaction with Rotylenchulus reniformis
Creator:
Garcia Martinez, Roberto, 1946-
Publication Date:
Language:
English
Physical Description:
xi, 117 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Female animals ( jstor )
Genitalia ( jstor )
Larvae ( jstor )
Microscopes ( jstor )
Roundworms ( jstor )
Soil science ( jstor )
Soil temperature regimes ( jstor )
Soils ( jstor )
Species ( jstor )
Tomatoes ( jstor )
Dissertations, Academic -- Entomology and Nematology -- UF
Entomology and Nematology thesis Ph. D
Meloidogyne cruciani ( lcsh )
Nematode diseases of plants -- Virgin Islands of the United States ( lcsh )
Plant nematodes -- Virgin Islands of the United States ( lcsh )
Rotylenchulus reniformis
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1981.
Bibliography:
Bibliography: leaves 111-116.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Roberto Garcia Martinez.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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020730768 ( ALEPH )
07518991 ( OCLC )

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PLANT PARASITIC NEMATODES OF THE U.S. VIRGIN ISLANDS
WITH THE DESCRIPTION, LIFE CYCLE AND MORPHOLOGY
OF Meloidogyne cruciani n.sp. (NEMATODA:
MELOIDOGYNIDAE) AND ITS INTERACTION WITH
Rotylenchulus reniformis







BY

ROBERTO GARCIA MARTINEZ


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


UNIVERSITY OF FLORIDA


1981
















ACKNOWLEDGMENTS


I extend my sincere appreciation to Dr. G.C. Smart,

Jr., my committee chairman, Dr. D.W. Dickson, Dr. D.E.

Stokes and Dr. S.J. Locascio, my committee members for

their patience, understanding and valuable assistance.

I also express my appreciation to Dr. R.P. Esser,

Dr. J.R. Rich and Mr. A.L. Taylor for all their help and

encouragement during this study.
















TABLE OF CONTENTS


ACKNOWLEDGMENTS ........... ...................ii

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

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

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

CHAPTER I
SURVEY OF THE PLANT PARASITIC NEMATODES OF THE U.S.
VIRGIN ISLANDS ....... ...................1

Introduction ....... .................. 1
Materials and Methods ....... ............. 2
Results ........... .................... 3
Discussion ........ .................... 7

CHAPTER II
Meloidogyne cruciani n.sp., A ROOT-KNOT NEMATODE FROM
ST. CROIX, U.S. VIRGIN ISLANDS .. ........... 44

Introduction ...... .................. 44
Materials and Methods ... ............ ..44
Results ....... ................... 46
Meloidogyne cruciani n.sp ........... ... 46
Females ..... ............... 46
Holotype ..... ............... .. 46
Description ... ............. 47
Males ..... ................ 54
Allotype ..... ............... .. 54
Description ... ............. 55
Second stage larvae .......... 55
Description ... ............. 58
Holotype ..... ............... .. 61
Allotype ..... ............... .. 61
Paratypes .... .............. 62
Diagnosis .... .............. 62
Type host and type habitat ...... .. 63
Type locality ... ............ 63

Discussion ....... ................... 64


iii










CHAPTER III
POST-INFECTION DEVELOPMENT OF FEMALES OF Meloidogyne
cruciani n.sp ....... .................... .. 65

Introduction ...... .................. 65
Materials and Methods .... ............. 66
Results ....... .................... 67
Discussion ....... ................... 78

CHAPTER IV
ESOPHAGEAL GLANDS OF ADULT FEMALES OF Meloidogyne
cruciani n.sp ....... .................... .. 84

Introduction ...... .................. 84
Materials and Methods .... ............. 84
Results ........ .................... 86
Discussion ....... ................... 86

CHAPTER V
INTERACTION OF Rotylenchulus reniformis AND
Meloidogyne cruciani ON TOMATO .. ........... 98

Introduction ...... .................. 98
Materials and Methods .... ............. 98
Results ............................ ...100
Rotylenchulus reniformis .. ......... 101
Meloidogyne cruciani .. ........... 103
Disucssion ........ ................... 107

LITERATURE CITED ....... .................. ill

BIOGRAPHICAL SKETCH ...... ................. 117















LIST OF TABLES


Table Page

1 Plants from which soil samples were
taken through the root zones and
nematodes recovered on St. Croix and
St. Thomas, U.S. Virgin Islands...... . 4

2 Incidence and percentage frequency of
occurrence of plant parasitic nematodes
in St. Croix ........ ................ 6

3 Incidence and percentage frequency of
occurrence of plant parasitic nematodes
in St. Thomas ....... ................ 6

4 Effects of soil type and time of sampling
on populations of Rotylenchulus reniformis
when inoculated alone and simultaneously
with an equal number of Meloidogyne
cruciani ....... .................. 102

5 Effects of soil type and temperature on
populations of Rotylenchulus reniformis when
inoculated alone and simultaneously with an
equal number of Meloidogyne cruciani . . 103

6 Effects of soil type and sampling on
populations of Meloidogyne cruciani when
inoculated alone and simultaneously with an
equal number of Rotylenchulus reniformis . 105

7 Effects of soil type and temperature on
populations of Meloidogyne cruciani when
inoculated alone and simultaneously with an
equal number of Rotylenchulus reniformis . 106
















LIST OF FIGURES


Figure Page

1 St. Croix, U.S. Virgin Islands. Shading
indicates total area sampled.
Rotylenchulus spp. were found in all of
the above areas .... .............. 11

2 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Helicotylenchus spp ... ............. ... 13

3 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Tylenchorhynchus mashhoodi .. ......... 15

4 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Xiphinema
americanum ...... ................. .. 17

5 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Pratylenchus pratensis ... ........... 19

6 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Criconemoides citri ... ............ 21

7 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Meloidogyne
cruciani n.sp ..... ............... ... 23

8 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Hemicriconemoides cocophillus ....... .. 25

9 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Hoplolaimus
columbus ...... .................. 27

10 St. Thomas, U.S. Virgin Islands. Shading
indicates total area sampled.
Rotylenchulus spp. were found in all of the
above areas ..... ................ 29









11 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Helicotylenchus spp ... ............. ... 31

12 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Tylenchorhynchus mashhoodi .. ......... 33

13 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by Xiphinema
americanum ...... ................. .. 35

14 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Pratylenchus pratensis ... ........... 37

15 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by Meloidogyne
cruciani n.sp ...... ................ .. 39

16 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Hemicriconemoides cocophillus ....... .. 41

17 St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Criconemoides citri ... ............ 43

18 A) Drawings of perineal patterns of
Meloidogyne cruciani n.sp. B) Outlines of
females in varying sizes and shapes . . 49

19 A) Anterior region of female. B) Face view
of showing cephalic framework. C) Vari-
ations in size and shape of esophageal
glands ...... .................. 51

20 Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograph showing sub-
cuticular punctations ... ........... 53

21 Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograph snowing sub-
cuticular punctations .. ........... 53

22 Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograph showing sub-
cuticular punctations .. ........... 53

23 Perineal patterns of Meloidogyne cruciani
n.sp. Scanning electron micrograph . . 53


vii









24 Male of Meloidogyne cruciani n.sp. A)
Entire specimen (curvature of specimen for
convenience in illustrating). B) Face view
showing cephalic framework. C) Anterior
portion. D) Tail (lateral view). E)
Lateral field ...... ................ 57

25 Larvae of Meloidogyne cruciani n.sp.
A) Entire specimen (curvature of specimen
for convenience of illustrating). B) Face
view showing cephalic framework. C) Tails
(lateral view). D) Lateral field at tail
region. E) Tail (ventral view) ........ .60

26 Second stage larvae of Meloidogyne cruciani
n.sp. A) Infective second stage larva. BT
Post-infective sexually undifferentiated
second stage larva. C-F) Sexually
differentiated, female second stage larvae. 69

27 Third and fourth stage larvae of Meloidogyne
cruciani n.sp. A-B) Third stage larvae
encased in the second stage cuticle. C)
Fourth stage larva encased in the second
and third stage cuticles ... .......... 73

28 Adult females of Meloidogyne cruciani n.sp.
A) Early adult female still enclosed in old
cuticles of the second, third and fourth
stages. B) Early adult female in the
process of shedding the second stage
cuticle. C-D) Fully developed females ... 77

29 Scanning electron micrograph of Meloidogyne
cruciani female internal structures crumbled. 88

30 Scanning electron micrograph of Meloidogyne
cruciani female dissected anterior region . 90

31 Enlarged portion of Fig. 30 showing
esophageal glands ..... .............. 92

32 Scanning electron micrograph of Meloidogyne
cruciani female dissected anterior region . 94

33 Enlarged portion of Fig. 32 showing
esophageal glands and metacorpus ...... 96


viii















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



PLANT PARASITIC NEMATODES OF THE U.S. VIRGIN ISLANDS
WITH THE DESCRIPTION, LIFE CYCLE AND MORPHOLOGY
OF Meloidogyne cruciani n.sp. (NEMATODA:
MELOIDOGYNIDAE) AND ITS INTERACTION WITH
Rotylenchulus reniformis



By

Roberto Garcia Martinez

March, 1981


Chairman: G. C. Smart, Jr.
Major Department: Entomology and Nematology


A nematode survey was conducted on St. Croix and St.

Thomas, U.S. Virgin Islands. Soil samples were collected

and processed from all agricultural and some non-agricul-

tural lands. The nematodes recovered were preserved and

brought to the University of Florida for identification.

Eleven species in nine genera of plant parasitic nematodes

were recovered: Rotylenchulus reniformis, R. parvus,

Helicotylenchus dihystera, H. multicinctus,

Tylenchorhynchus mashhoodi, Xiphinema americanum,

Pratylenchus pratensis, Criconemoides citri, Meloidogyne

cruciani n.sp. and Hemicriconemoides cocophillus on both

islands, and Hoplolaimus columbus only on St. Croix.

ix










Meloidogyne cruciani n.sp. differs from other species

of the genus by having punctations around the anus of the

female and by the larvae possessing extremely long tri-

lobed esophageal glands. Females, males and larvae possess

a uninucleate gland excretory system. Post-infection

developmental stages of females of M. cruciani were dissected

from tomato roots, killed and fixed in lactophenol-cotton

blue and mounted in glycerin. Eleven days after inoculation,

the procorpus, metacorpus and esophageal glands of the

second stage larva were enlarged and prominent. Two small

lobes were present just posterior to the metacorpus. The

excretory duct of the second stage larva was directed

anteriorly and seemingly connected to the cuticle of the

third stage larva opposite the procorpus. The esophagus of

the adult female appeared typical for the species, having

a prominent procorpus, metacorpus and five nucleated lobes.

The excretory pore was opposite the procorpus with the

excretory duct directed posteriorly and terminating in

a uninucleate gland.

Rotylenchulus reniformis was distributed throughout

the two islands while M. cruciani was restricted in

occurrence. The interaction between these two species

was investigated in a clay vs. a sandy soil and at two

temperatures. While R. reniformis populations developed

better in clay soils, M. cruciani developed better in sandy

soils. When M. cruciani was present, R. reniformis

populations did not increase as much as when the latter









was alone. M. cruciani populations also were suppressed

when R. reniformis was present. This indicates compe-

tition for available feeding sites. The soil texture of

the U.S. Virgin Islands and the faster reproduction rate

of R. reniformis may be reasons why R. reniformis was

found throughout those islands, while M. cruciani was

restricted in distribution.















CHAPTER I
SURVEY OF THE PLANT PARASITIC NEMATODES
OF THE U.S. VIRGIN ISLANDS

Introduction


The U.S. Virgin Islands consists of three islands, St.

Croix, St. Thomas and St. John. They are located between
17040' and 180241 latitude north, 640301 and 65004'

longitude west and have an area of 207,199; 77,699; and

51,799 square kilometers, respectively. These islands are

of volcanic origin with elevations up to 366 meters; they

have a tropical trade wind climate, with an average

temperature of 27 C and an average annual rainfall of

965 millimeters.

The tropical and subtropical regions of the world have

climates that will permit year round production of a great

variety of agricultural crops. These continuous crop

productions favor nematode pests, since they are able to

reproduce continuously, increasing their numbers and their

damage. In 1978, I conducted a survey on St. Croix and St.

Thomas Islands to determine the genera of plant parasitic

nematodes present, their relative abundance and geographic

distribution, and the crops with which they were associated.

All cultivated areas and some non-cultivated areas were

sampled on each island. St. John Island was not included

1









in the survey because most of the island is a national park

with very little agriculture on the remainder.



Materials and Methods


Large maps of St. Croix and St. Thomas Islands were

constructed with aerial photographs and the areas under

cultivation and those areas that could be cultivated were

marked on the maps for easy reference and location. The

survey extended over a period of four months covering

agricultural fields, home gardens, golf courses, nurseries,

lawns and non-cultivated areas that had a potential for

agricultural development. Soil subsamples were taken with

a cone-type soil sampler (22) and combined in the field to

form a composite sample that was used for the extraction of

nematodes. The number of subsamples comprising a composite

sample was determined by the size of the area and diversity

of crops sampled, but ranged from three from about 3 m2

garden plots to 10 from 2 hectare fields. The subsamples

were mixed thoroughly and approximately 500 cm3 of soil were

placed in plastic bags, numbered, and the number recorded on

the map of each island. The crop or plants from which a

sample was taken also was recorded. A total of 80 composite

soil samples were taken from the root zone of 30 different

plants on St. Croix and 26 samples from the root zone of 16

different plants on St. Thomas. Samples were processed on

St. Croix using a modification of the centrifugation-









flotation technique described by Caveness and Jensen (5).

The nematodes recovered from 100 cm3 of soil were killed in

hot water, fixed and preserved in 4% formalin-2% glycerin,

placed in vials and brought back to the Nematology

Laboratory, University of Florida, Gainesville, Florida,

where the plant parasitic nematodes were identified to genus

and the number per sample determined.

Twenty adult nematodes of each genus were mounted in

2% formalin on glass slides and using an Olympus Vanox

compound microscope with a Nomarski reflected light

differential interference contrast attachment, measurements

and other morphological characters of the nematodes were

recorded and the species determined.



Results


There were nine genera and 11 species of plant

parasitic nematodes recovered from soil associated with 30

different host plants (Table 1). Rotylenchulus reniformis

Linford and Oliveira, 1940 (34), R. parvus (Williams, 1960)

Sher, 1961 (48), Helicotylenchus dihystera (Cobb, 1893)

Sher, 1961 (48), H. multicinctus (Cobb, 1893) Golden, 1956

(27), Tylenchorhynchus mashhoodi Siddiqui and Basir, 1959

(51), Xiphinema americanum Cobb, 1913 (14), Pratylenchus

pratensis (de Man, 1880) Filipjev, 1936 (25), Criconemoides

citri Steiner, 1949 (58) = Macroposthonia sphaerocephala

(Taylor, 1936) De Grisse and Loof, 1965 (18), Meloidogyne











Table 1


Plants from which soil samples were taken through the root
zones and nematodes recovered on St. Croix and
St. Thomas, U.S. Virgin Islands


Host plants


Nematodes


Bahia grass (PaspaLum notatum Flgge)

Banana (Musa acwninata Colla)

Bean (Phaseolus vulgaris L.)

Bermuda grass [Cynodon dactylon (L.) Pers.]

Citrus [Citrus aurantiifolia (Christm.) Swingle]

Corn (Zea mnys L.)

Grapes (Vitis rotundigolia Michx.)

Guayaba (Psidiwn guajaba L.)

Guinea grass (Panicum maximum Jacq.)

Hairy crabgrass [ Digitaria sanguinaZis (L.)
Scop. ]

Hibiscus (Hibiscus rosa-sinensis L.)

Rurricanegrass [ Sporobolus cryptandrus (Torr.)

Gray J

Mango (Mangifera indica L.)


+ +++++




+ + ++

+ + +++

+ + +++


+

+ +

+ +


+

+


+

+ +


U) ~

*t~ 0 Ci~



~0~J




~3 0

0


++

++


++


++ +++

++ +

++ + +

+++ ++


+++ +

++


+ ++


++ +










Table 1--continued

Host plants


Nematodes


"0 0




Okra [Abe Zmoschus esculentus (L.) Moench] ++ + ++ + ++ + +
Onion (Alliucepa L.) + + + + +

Pangola grass (Digitaria decwmbens Stent) + ++ + + +
Papaya (Carica papaya L.)

Pepper ( Capsicum annum L.) + + + +

Pineapple [Ananas cormosus (L.) Merrill] + + + +++ +

Spanish bayonet ( Yucca aloif'olia L. ) + + + + + +
Sorghum [Sorghum bicolor (L.) Moench] ++ + + + + + + +

Squash (Cucurbita pepo L.) + + + + + +

Sugar apple (Annonasquamosa L. ) + + + + + +

Sugar cane (pSacchara officinarwm L.) + + + . +

Sour sop (Annona muricata L.) + + ++ + +

Sweet potato [Ipomoea batatas (L.) Lam.] + + + + +

Tomato (Lycopersicon esculentw Mill.) + + + + + +++

Watermelon [ Citrullus lanatu (Thunb.)
Matsum. and Nakai] + + + + +

Yam (Dioscorea alata L.) L.) + + + + + +


Yuca (Manihot escuZenta Crantz) + + + + + +

1Hoplolaimus columbus recovered from St. Croix only.









cruciani n.sp. and Hemicriconemoides cocophillus (Loos,

1949) Chitwood and Birchfield, 1957 (8) were found on both

St. Croix and St. Thomas (Tables 2 and 3). Hoplolaimus

columbus Sher, 1963 (49) were recovered only on St. Croix

from the three golf courses on the island (Table 2).

Rotylenchulus reniformis were present in every sample

taken from both islands while R. parvus was present in 8 and

5% respectively, of the samples taken from St. Croix and St.

Thomas (Tables 2 and 3, Figs. 1 and 10). Helicotylenchus

dihystera, H. multicinctus, Tylenchorhynchus mashhoodi,

Xiphinema americanum and Pratylenchus pratensis were

recovered from 70, 4, 65, 58 and 58%, respectively, of

the samples taken from St. Croix (Table 2, Figs. 2-5), and

from 74, 3, 45, 16 and 13%, respectively, of the samples

taken from St. Thomas (Table 3, Figs. 11-14).

Meloidogyne, an economically important nematode genus

which is widespread in most tropical regions (9), was found

in only 11 samples from St. Croix (Table 2, Fig. 7) and five

samples from St. Thomas (Table 3, Fig. 15). It was found

only in home gardens, but not in all home gardens sampled.

Criconemoides citri and Hemicriconemoides cocophillus

were recovered in relatively low numbers from both St. Croix

and St. Thomas. Criconemoides was found in 15% of the

samples from St. Croix (Table 2, Fig. 6) and only in 8% of

the samples from St. Thomas (Table 3, Fig. 17).

Hemicriconemoides was recovered from 9% of the samples from

St. Croix (Table 2, Fig. 8) and in 11% of the samples from

St. Thomas (Table 3, Fig. 16).










Table 2


Incidence and percentage frequency of occurrence
of plant parasitic nematodes in St. Croix


Nematode genera Incidence % Frequency
of occurrence


Rotylenchulus reniformis 80 100
Rotylenchuius parvus 6 8
Helicotylenchus dihystera 56 70
Helicotylenchus multicinctus 3 4
Tylenchorhynchus mashhoodi 52 65
Xiphinema americanum 46 58
Pratylenchus pratensis 46 58
Criconemoides citri 12 15
Meloidogyne cruciani n.sp. 11 14
Hemicriconemoides cocophillus 7 9
Hoplolaimus columbus 4 5




Table 3


Incidence and percentage frequency of occurrence
of plant parasitic nematodes in St. Thomas


Nematode genera Incidence % Frequency
of occurrence


Rotylenchulus reniformis 38 100
Rotylenchulus parvus 2 5
Helicotylenchus dihystera 28 74
Helicotylenchus multicinctus 1 3
TyVlenchorhynchus mcshhoodi 17 45
Xiphinema americanwn 6 16
Pratylenchus pratensis 5 13
Meloidogyne cruciani n.sp. 5 13
Hemicriconemoides cocophil lus 4 11
Criconemoides citri 3 8










Discussion


Rotylenchulus reniformis was found to be more

widely distributed than R. parvus. Dasgupta et al. (17)

gave an extensive list of plants with which R. reniformis

has been found associated and the localities from which they

were reported. They found R. reniformis associated with

banana, citrus, corn, papaya, sugarcane, sweet potato and

tomato. These crops also were found to be common hosts of

R. reniformis in St. Croix and St. Thomas (Table 1).

Helicotylenchus dihystera was found to be the most

widely distributed species of the genus. Sher (50) gave an

extensive list of plants with which H. dihystera has been

found associated and the localities from which they were

reported. He found H. dihystera associated with banana,

citrus, corn, mango, onion, papaya, pineapple and sugarcane.

These crops also were found to be common hosts of H.

dihystera in St. Croix and St. Thomas (Table 1).

The occurrence of Hoplolaimus columbus on golf courses

associated with bermuda grass indicates that these genera

of nematodes might have been introduced to St. Croix with

the turfgrass.

During this survey, a new species of the genus

Meloidogyne was discovered. The complete description and

a host differential test of the new species, named

Meloidogyne cruciani, are reported in Chapter II.









The extensive distribution of R. reniformis throughout

these islands indicates favorable environmental conditions

for the growth, development and reproduction of these

nematodes. The widespread distribution of this nematode

may be due to the extensive and intensive production of

sugarcane on both islands in the past (63). Contrary to

this, the low recovery rate of M. cruciani indicates the

presence of biotic or abiotic factors that are restricting

further establishment of these nematodes. With this in

mind, experiments were initiated to determine whether

certain factors favored Rotylenchulus reniformis and were

detrimental to Meloidogyne cruciani. These experiments

are reported in Chapter V.
































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CHAPTER II
Meloidogyne cruciani n.sp., A ROOT-KNOT NEMATODE
FROM ST. CROIX, U.S. VIRGIN ISLANDS

Introduction


During a survey to determine the plant-parasitic

nematode complement in the U.S. Virgin Islands, an

undescribed species of Meloidogyne was recovered from

tomato roots. Specimens of the nematode were brought to

the University of Florida and populations established on

'Rutgers' tomato plants. Single egg mass isolations were

made to establish a population originating from a single

female. One isolate was selected and used for the

taxonomic and morphologic studies reported herein.



Materials and Methods


Egg masses dislodged from 'Rutgers' tomato (Lycopersicon

esculentum Mill.) roots were teased apart to expose the eggs.

These eggs were placed in water and left overnight at 28-29

C. Newly-hatched second stage larvae were mounted in 2%

formalin on slides. The specimens were measured and drawn

immediately using a camera lucida (23). Males were dissected

from large root galls and from egg masses and prepared as

above. Females were dissected from roots, placed in 2% for-

malin and their posterior ends were excised. These sections

44









were transferred to 45% lactic acid, cleaned of debris,

trimmed to the perineal region and mounted in glycerin for

observation. Anterior ends of females were prepared by

fixing and staining whole females in lactophenol-cotton

blue (21), after which the anterior ends were excised and

mounted in glycerin. All type material was prepared using

the same technique as for the female anterior ends.

Photomicrographs of perineal patterns were made with

an automatic 35-mm camera using an interference contrast

system (Nomarski) attached to a compound microscope. In

making the scanning electron microscope (SEM) micrographs,

females were killed and fixed in 2.5% glutaraldehyde

solution with phosphate buffer for 12 hours, transferred to

2% canium tetroxide for 24 hours at 8 C, dehydrated in an

ethanol series (10-100%) for 15 minutes in each

concentration, dried to critical point, coated with gold,

and examined with a SEM.

A differential host test (59) was conducted by trans-

planting seedlings of the following plants into steam-

sterilized, sandy soil contained in 10-cm clay pots and

inoculating the plants with 5000 eggs per pot: sweet corn

(Zea mays L. var. rugosa Bonaf. cv 'Silver Queen'), cotton

Gossypium hirsutum L. cv 'Delta pine'), peanut (Arachis

hypogaea L. cv 'Florunner'), pepper (Capsicum annuum L. cv

'California Wonder'), strawberry (Fragaria ananassa Duch.

cv 'Albritton' and 'Florida 90'), sweet potato [Ipomoea

batatas (L.) Poir. cv 'Allgold' and 'Porto Rico'], tobacco









(Nicotiana tabacum L. cv 'NC 95'), watermelon [Citrullus

lanatus (Thunb.) Matsum and Nakai cv 'Charleston Gray'],

and tomato (Lycopersicon esculentum Mill. cv 'Rutgers').

Each treatment was replicated five times. Inoculum was

prepared by shaking egg masses for two minutes in a 1%

NaOCl solution to obtain a suspension of eggs and larvae

(31). Sixty days after inoculation at a greenhouse

temperature of 22-26 C, roots were removed from soil,

washed and examined for galls and egg masses.



Results


Meloidogyne cruciani n.sp.

Females. (21): Length: 426.0-1121.8 pm (mean 787.5

pm, 95% confidence interval + 86.2); body width: 315.7-770.0

Pm (505.1 Pm + 61.5); a: 1.2-2.1 (1.5 + 0.1); stylet length:

11.4-16.2 pm (14.2 pm + 0.6); stylet knob height: 2.1-2.9

pm (2.4 m + 0.1); stylet knob width: 3.8-5.1 Vm (4.5 pm +

0.2); dorsal gland orifice to base of stylet knobs: 3.2-5.1

pm (3.9 pm + 0.2); excretory pore to anterior end: 25.7-45.1

Pm (32.2 Pm + 2.2); center of median bulb to anterior end:

65.4-93.3 pm (78.3 pm + 3.4); vulva slit length: 20.0-25.7

Pm (23.2 Pm + 0.7); vulva slit to anus: 15.9-20.3 pm (18.1

pm + 0.6); interphasmidial distance: 25.7-36.8 pm (30.3

pm + 1.2).

Holotype. (female): Length: 949.9 pm; body width

536.4 Pm; a: 1.8; stylet length: 12.7 pm; stylet knob









height: 2.7 pm; stylet knob width: 4.4 pm; dorsal gland

orifice to base of stylet knobs: 4.1 pm; excretory pore to

anterial end: 31.7 pm; center of median bulb to anterior

end: 93.3 pm; vulva slit length: 23.8 Um; vulva slit to

anus: 20.0 pim; interphasmidial distance: 31.7 pm.

Description. Females white, pear-shaped to globular,

without prominent posterior protuberance (Fig. 18B). Neck

tapers, curving gently (Figs. 18B, 19A). Head offset

slightly with labial cap and one or two cephalic annules.

Labial or cephalic sensillae not observed. Amphidial

openings oval, inconspicuous, obscured by labial cap.

Cephalic framework with lateral sectors larger than ventral

or dorsal sectors. Stylet robust, with rounded knobs.

Excretory pore about one stylet length from base of stylet

knobs, variable in exact position; excretory duct seen

easily throughout anterior region terminating in a

uninucleate gland (Fig. 28C). Esophageal lumen between base

of stylet knobs and valve of median bulb well sclerotized

with an average width of 2.4 pm. Prominent metacorpus with

strongly sclerotized valve. Esophageal glands consisting

of five distinct nucleated lobes (Fig. 19A, C). One lobe

always larger than the other four. Perineal pattern (Figs.

18A, 20-23) with subcuticular punctations (stippling) almost

surrounding the anus on lateral and posterior sides (Figs.

18A, 20-23). Striae deep, wavy, sometimes broken. Lateral

field fairly deep with distinct phasmids. Phasmidial ducts

often visible. Vulva lips faintly serrated, margins with

very fine striae.









Figure 18

A) Drawings of perineal patterns of
Meloidogyne cruciani n.sp.

B) Outlines of females in varying
sizes and shapes.



















'I





10




I
ZUIp










Figure 19

A) Anterior region of female.

B) Face view showing cephalic
framework.

C) Variations in size and shape
of esophageal glands.






















20un









Figures 20-23

Perineal patterns of Meloidogyne
cruciana n.sp.

20-22) Photomicrographs showing
subcuticular punctations.

23) Scanning electron micrograph.






53









Males. (25): Length: 1160-1620 pm (mean 1378.8 pm,

95% confidence interval + 52.3); body width: 23.2-46.3 pm

(33.8 pm + 2.4); stylet length: 19.4-24.1 pm (22.0 pm +

0.5); stylet base to anterior end: 21.2-26.3 pm (24.4 um

+ 0.5); stylet knob height: 2.7-3.8 pm (3.3 pm + 0.1);

stylet knob width: 4.1-6.0 pm (5.2 pm + 0.2); dorsal

esophageal gland orifice to base of stylet knobs: 3.2-7.9

pm (4.9 pm + 0.4); center of metacorpus valve to anterior

end: 66.7-118.1 pm (88.9 pm + 4.1); excretory pore to head

end: 127.3-189.5 pm (149.2 pm + 5.8); anterior end of

testis to posterior end: 489.4-1127.0 pm (823.4 pm +

60.1); spicule length: 28.7-38.0 pm (31.3 pm + 0.9);

gubernaculum: 6.7-11.1 pm (8.8 pm + 0.5); phasmid to

posterior end: 8.3-22.9 pm (16.7 pm + 1.4); a: 31.9-

71.2 (43.2 + 3.7); c: 89.2-238.2 (132.6 + 13.1); c':

0.3-0.7 (0.5 + 0.1); 0 (distance from dorsal esophageal

gland orifice to base of stylet knobs, expressed as % of

stylet length): 14.3-36.8 (22.6 + 1.9); T% (distance from

anterior end of testis to posterior end, expressed as % of

body length): 39.8-79.7 (60.1 + 4.7).

Allotype. (male): Length: 1440 pm; body width:

29.9 pm; stylet length: 22.8 pm; stylet base to anterior

end: 24.8 pm; stylet knob height: 3.5 pm; stylet knob

width: 4.8 pm; dorsal esophageal gland orifice to base of

stylet knobs: 4.9 pm; center of metacorpus valve to anterior

end: 97.4 pm; excretory pore to anterior end: 139.2 pm;

anterior end of testis to posterior end: 933.8 pm; spicule









length: 28.7 pm; gubernaculum: 8.1 Um; phasmid to

posterior end: 18.4 pm; a: 48.0; c: 151.3; c': 0.4;

0: 21.5; T% 64.9%.

Description. Body long, vermiform, tapering at both

ends (Fig. 24A). Head offset with two annules and distinct

head cap. Labial or cephalic sensillae not observed.

Cephalic framework with lateral sectors larger than ventral

or dorsal sectors; ends of framework slightly forked when

viewed laterally. Stylet robust, with rounded knobs.

Amphidial glands prominent posterior to stylet knobs.

Cephalids not observed. Metacorpus poorly developed,

slightly larger than procorpus with well sclerotized valve.

Esophageal glands consisting of three distinct nucleated

lobes. Excretory pore prominent (139.1 pm from anterior

end). Hemizonid 3.5 annules anterior to excretory pore.

Excretory duct long, terminating in a sac-like gland.

Lateral fields begin anteriorly as two lateral lines

opposite stylet knobs and become four near metacorpus.

There is anastamosis of the lateral lines in the posterior

end of the body. Testis predominantly one, two occasionally.

Spicules slightly arcuate, their tips rounded (Fig. 24D).

Gubernaculum with fine serrations on the cuneus. Phasmids

5.9 vm anterior to cloaca.
Second stage larvae. (20): Length: 418.6-479.8 vm

(mean 435.3 vm, 95% confidence interval + 8.7 vm); width:

14.6-18.7 vm (17.2 vm + 0.5); stylet length: 9.8-12.1 vm









Figure 24

Male of Meloidogyne cruciani n.sp.

A) Entire specimen (curvature of
specimen for convenience in
illustrating).

B) Face view showing cephalic
framework.

C) Anterior portion.

D) Tail (lateral view).

E) Lateral field.





























A Hi.m

1,C,D-E 20gm


E









(10.6 pm + 0.2); stylet base to anterior end: 14.3-17.6 pm

(15.2 pm + 0.4); stylet knob height: 1.1-1.6 pm (1.4 pm +

0.1); stylet knob width: 2.1-2.7 pm (2.3 pm + 0.1); dorsal

esophageal gland orifice to base of stylet knobs: 3.2-3.9

pm (3.5 pm + 0.1); center of metacorpus valve to anterior

end: 51.7-61.9 pm (57.8 m + 1.4); distance from cardia to

anterior end: 69.5-86.7 pm (76.3 pm + 2.1); distance from

posterior end of glands to anterior end: 190.4-250.4 pm

(202.0 Pm + 6.4); excretory pore to anterior end: 74.6-

103.2 pm (88.1 pm + 3.4); genital primordium to posterior

end: 148.2-175.5 pm (163.5 pm + 4.3); phasmid to posterior

end: 34.9-43.8 pm (39.2 pm + 1.2); tail length (anus to

posterior end): 41.3-51.7 pm (46.6 pm + 1.3); tail width

(at anus): 9.8-13.0 pm (11.2 pm + 0.4); a: 22.9-29.8

(25.4 + 0.8); b: 5.0-7.1 (5.8 + 0.2); b': 1.8-2.4 (2.2 +

0.1); c: 8.6-10.5 (9.4 + 0.3); c': 3.7-4.6 (4.2 + 0.1);

0 (distance from dorsal esophageal gland orifice to base

of stylet knobs, expressed as % of stylet length): 28.9-

37.9 (33.1 + 0.9).

Description. Body vermiform, tapering slightly

anteriorly and much more posteriorly (Fig. 25A). Head

offset slightly with one annule; head cap with weakly

visible cephalic framework with lateral sectors larger than

ventral or dorsal sectors. Labial or cephalic sensillae not

observed. Stylet robust, rounded knobs slanting poste-

riorly. Cephalids not observed. Amphidial glands prominent,

posterior to stylet knobs. Esophagus extremely long, from









Figure 25

Larvae of Meloidogyne cruciani n.sp.

A) Entire specimen (curvature of specimen
for convenience in illustrating).

B) Face view showing cephalic framework.

C) Tails (lateral view).

D) Lateral field at tail region.

E) Tail (ventral view).













B


20 m


D









tip of head to posterior extremity of glands averaging 46.4%

of the total body length. Metacorpus well developed with

well sclerotized valve. Esophageal glands contained in

three distinct nucleated lobes, each with a smaller

satellite nuclear body. Excretory pore position variable;

always posterior to esophago-intestinal valve. Hemizonid

2-4 annules anterior to excretory pore. Excretory duct

long, terminating in a sac-like gland. Lateral fields

originate as two lines one stylet length posterior to base

of knobs, becoming four near metacorpus. Two inner lateral

lines terminate at phasmids and outer two terminate

posteriorly. Genital primordium in the two-cell stage,

seen easily. Rectum dilated. Phasmids small and difficult

to see; one anal body width posterior to level of anus.

Tail gradually tapering, with annules disappearing near

hyaline area. Tail terminus notched, with smooth, bluntly

conoid tip.

Holotype. (whole female): Originally recovered in

tomato roots from the Agricultural Community Gardens, St.

Croix, U.S. Virgin Islands in September 1977. It was grown

subsequently on 'Rutgers' tomato in an isolated green-

house. [Slide T-333t, USDA Nematode Collection, (USDANC)],

Beltsville, Maryland, USA.

Allotype. (male): Isolated from 'Rutgers' tomato

roots cultured in a greenhouse and established from type

locality. Slide T-334t, USDANC, Beltsville, Maryland, USA.









Paratypes. Females (whole mounts, perineal patterns),

males and larvae. Same data as allotype. USDANC,

Beltsville, Maryland; Laboratorie voor Nematologie,

Binnehaven, Wageningen, The Netherlands; Nematology

Department, Rothamsted Experimental Station, Harpenden,

Herts., England; Canadian National Collection of Nematodes,

Ottawa, Canada; Division of Plant Industry, Florida

Department of Agriculture and Consumer Services, Gainesville,

Florida; and Entomology and Nematology Department,

University of Florida, Gainesville, Florida.

Diagnosis. Meloidogyne cruciani differs from other

published descriptions of species of the genus by its

perineal pattern with punctations around the anus. The only

other species with punctations in the perineal area is M.

hapla Chitwood, 1949 (8) but the punctations of M. hapla are

around the tail terminus. The larvae of M. cruciani differ

from most other species of the genus in possessing extremely

long, distinctly tri-lobed esophageal glands.

Other morphological characters found in this species

and not reported for other members of the genus are: 1)

The presence of a guiding ring around the stylet shaft of

males and larvae (Figs. 24C, 25A). (These rings are often

associated with the order Dorylaimida, class Adenophorea

(1), but have not been reported in the class Secernentea.)

2) Three distinct lobes of the esophagi of males and larvae

of this species (Figs. 24A, 25A). Original descriptions

and illustrations of 29 other species of this genus examined










report one single esophageal lobe with three nuclei (23),

but post-infection studies of Meloidogyne naasi (52) and

M. incognita (61) illustrate the parasitic second stage

larvae as having 3-lobed esophageal glands. 3) Inside

each nucleus, a small chromocenter is present beside the

nucleolus in each lobe of the esophagi of the second stage

larvae (Figs. 25A). This has not been reported for other

species of this genus. 4) The esophageal glands of the

females (Fig. 19A, C) consist of five separate and distinct

lobes. 6) A uninucleate gland (renette-type) excretory

system. 7) The gubernaculum of the males (Fig. 24D) has

fine serrations on the cuneus; this condition is also

present in males of Verutus volvingentis (23).

In the host-differential test, peanut, strawberry, and

cotton were not hosts. Tomato, watermelon, sweet potato,

tobacco, corn and pepper were hosts. Based on these

results, Meloidogyne cruciani seems to have a similar host

range as that of M. incognita Race 2. One other plant,

cabbage (Brassica oleracea L. cv 'Greenback') also was found

to be a suitable host.

Type host and type habitat. tomato, Lycopersicon

esculentum Mill., roots

Type locality. Agricultural Community Gardens, College

of the U.S. Virgin Islands, St. Croix, U.S. Virgin Islands









Discussion


Perineal patterns of Meloidogyne cruciani differ from

perineal patterns of other species of the genus in having

punctations around the anus. Some variations of the

perineal patterns (Figs. 18A, 20-23) resemble perineal

patterns of Meloidogyne javanica Chitwood, 1949 (7) in

their pronounced lateral fields; however, length of larvae

easily separate the two species.

Andrassy (1) stated that as far as the triradial

symmetry of the esophagus is concerned, the 5-gland

condition in nematodes represents a more advanced

evolutionary stage. This condition would certainly aid the

feeding process of highly advanced sedentary parasites such

as members of the genus Meloidogyne. I have found that

females of M. incognita and M. arenaria also have 5-lobed

esophageal glands.

Andrassy (1) further stated that the uninucleate gland

(renette-type) excretory system is considered typical of

Torquentia and Penetrantia and does not occur in the

Secernentea (1), but this excretory system which was first

called a renette cell-type by Cobb (13), has been shown in

other genera of the Secernentea (11, 12, 14-16, 41).

The development of five glands in the esophagi of

females of Meloidogyne cruciani and the type of excretory

system are reported in Chapter III.
















CHAPTER III
POST-INFECTION DEVELOPMENT OF FEMALES
OF Meloidogyne cruciani n.sp.

Introduction


In most genera of nematodes, the adult females

resemble the larval stages in many of their morphological

characters. In the genus Meloidogyne, the developing

larvae undergo a series of morphological changes and the

mature females appear very different than the second stage

larvae. The former have a saccate pear-shaped body, a

very large robust stylet, very prominent esophageal glands

and an enlarged metacorpus. The larvae are filiform with

a small stylet and small esophageal glands.

Before Chitwood (7) placed what was known as

Heterodera marioni into the genus Meloidogyne and created

four species and one subspecies, the life cycle and

morphological studies of unknown species of Meloidogyne

(Heterodera marioni) were carried out by Nagakura (37) and

Christie and Cobb (10). Nagakura described three molts

taking place within the plant roots and the existence of

third and fourth larval stages. Christie and Cobb disagreed

with Nagakura, stating there is no third larval stage

and that the fourth is just theoretical.









More recently, Bird (3), Triantaphyllou and Hirschmann

(61) and Siddiqui and Taylor (52) studied the morphology

and developmental stages of females of M. javanica, M.

incognita and M. nassi, respectively. They all agree with

the early studies of Nagakura and report the presence of

third and fourth larval stages.

The work reported herein is an attempt to determine

the initiation, development and formation of morphological

structures in the developmental stages of Meloidogyne

cruciani n.sp. with emphasis on the esophageal region,

excretory system and genital region.



Materials and Methods


Seeds of 'Rutgers' tomato (Lycopersicon esculentum

Mill.) which is susceptible to M. cruciani, were germinated

in sterile vermiculite at 30 C in a water bath. When

seedlings were two weeks old, they were removed from the

vermiculite, their roots washed and trimmed to 1 cm in

length and the seedlings placed in sterile water for two

days. These seedlings were exposed to freshly hatched

second stage larvae for 24 hours. (Larvae were obtained by

placing egg masses in distilled water at 30 C overnight.)

After the 24 hour exposure period, roots of the seedlings

were washed to remove any larvae that had not penetrated

and the seedlings were transplanted into sterile white sand
3
in 33 cm plastic cups with a small hole punched in the

bottom for drainage.









The experiment was carried out at a constant temper-

ature of 28 + 1 C in a temperature controlled growth

chamber. The seedlings were fertilized twice a week with

2 ml of a 390 ppm NutrisolR (12-10-20) nutrient solution.

Every 24 hours, five seedlings were removed and the roots

washed and fixed using De Guiran's (29) method.

After fixation, the developmental stages were recovered

by dissecting them from roots. The nematodes then were

mounted in glycerin on glass slides, a cover glass applied

and sealed with Zut.

Morphological observations and drawings were made with

the aid of a camera lucida attached to an Olympus Vanox

compound microscope equipped with a Nomarski reflected

light differential interference contrast attachment.



Results


For the first 7-8 days, the post-infective second

stage larva underwent very few changes but generally

decreased in length when compared with the pre-infective

stage and increased in diameter (Fig. 26A, B). After 7-8

days, the genital primordium in the four-cell stage began

to migrate posteriorly, the esophageal glands became

shorter in length but larger in diameter and volume, and

there was a slight increase in the width of the metacorpus

and an increase in the body width around the esophageal

region (Fig. 26B). Inside the nuclear envelopes of the









Figure 26

Second stage larvae of Meloidogyne cruciani n.sp.

A) Infective second stage larva.

B) Post-infective sexually
undifferentiated second stage
larva.

C-F) Sexually differentiated, female
second stage larvae.






A(t\ S









esophageal glands, the nucleoli and the chromocenters

enlarged. At this point, the rectal glands were not seen,

but six irregularly arranged nuclei were present near the

anal region (Fig. 26B).

Eleven days after inoculation the second stage larva

had enlarged considerably in size (Fig. 26C). At this

time, the esophagus had decreased in length, but increased

in width and volume; the procorpus and metacorpus were

enlarged and more prominent. For the first time, two small

lobes were seen connected to the esophagus just posterior

to the metacorpus; chromocenters of the esophageal glands

were very prominent. The dorsal gland had enlarged more

than the subventral esophageal glands. The genital

primordium was in the six-cell stage and had enlarged and

migrated further posteriorly. The rectal glands were

visible in this stage. Triantaphyllou and Hirschmann

(61) and Siddiqui and Taylor (52) refer to this stage as

the "developed but sexually undifferentiated" second stage

larva based on the genital primordium. The genital

primordium had not reached the V-shape characteristic

of the developing female gonad, but the presence of

rectal glands indicated the sex as a developing female.

The "developed and sexually differentiated" second

stage larva continued to enlarge. By fourteen days after

inoculation (Fig. 26D) the procorpus, metacorpus and

esophageal glands decreased still further in length, but









became wider and more voluminous. The two esophageal lobes

seen first at day 11 were larger and nuclei could be seen

for the first time. The nucleoli and chromocenters inside

the nuclei of the other three esophageal glands were

prominent. The genital primordium in this stage had

assumed the V-shape characteristic of the developing female

gonad. The genital primordium was associated closely with

the now visible and prominent rectal glands but had not

attached to the body wall.

Sixteen days after inoculation the early second stage

female larva (Figs. 26E, F) showed further enlargement of

the esophagus. The genital primordium now assumed a

definite V-shape with two branches directed anteriad.

The branches grew in length as the gonad moved towards the

anal region and attached to the body wall (Fig. 26F); the

female second stage larva possessed six well-developed

rectal glands. At this point, a new cuticle was evident

posteriorly as the second stage cuticle began to separate

from it.

The third stage larva (Figs. 27A, B) could be recognized

enclosed in the second stage larval cuticle; it did not

possess a stylet and the posterior end was round. The

second stage larval stylet remained attached to the old

cuticle. During the molt the cone and shaft of the stylet

and the lumen of the stylet knobs were shed with the

second stage cuticle, the style knobs disappeared and

there was a void at the anterior part of the body where the

stylet and stylet knobs normally would be.









Figure 27

Third and fourth stage larvae
of Meloidogyne cruciani n.sp.

A-B) Third stage larvae encased
in the second stage cuticle.

C) Fourth stage larva encased
in the second and third stage
cuticles.























40m









The esophagus compressed, with the procorpus and

metacorpus enlarging. The esophageal lumen and metacorpus

valve were visible but very faint. The esophageal glands

lost their chromocenters but the nucleoli remained very

prominent. In the early third stage (Fig. 27A) the

excretory pore was located opposite the esophageal glands

with the duct pointing anteriorly. In the late third stage

larva (Fig. 27B) the excretory pore was located opposite the

esophageal glands with the anteriorly directed duct

penetrating the body opposite the procorpus. The ovaries

of the third stage larva continued to elongate and the

uterus and vagina began to develop.

With the onset of the third molt, the body of the

fourth stage larva formed and separated from the third

stage cuticle but was still enveloped in the third and

second stage cuticles. The early fourth stage larva (Fig.

27C) differed from the late third stage larva (Fig. 27B) in

being enclosed in the old second and third larval cuticles.

There was no stylet visible, and a void was seen at the

anterior end where the stylet would have been. The

esophageal lumen and metacorpus valve were very faint; the

valve was now located in the posterior portion of the

metacorpus. The excretory duct was similar to that of the

third stage larva; it ran anteriorly and penetrated the body

opposite the procorpus. At the posterior end, the gonads

continued to elongate, the uterus and vagina formed

completely.










In the early stages of the adult female, shortly

after the fourth molt (Fig. 28A) and while still enclosed

in the second, third and fourth larval cuticles, the

stylet could be seen. The esophagus appeared typical of

adult females with the procorpus and metacorpus enlarged

and prominent. The lumen of the esophagus and valve of the

metacorpus were reformed and appeared faintly at first.

Inside the nuclear envelope, the chromocenters had

disappeared but the nucleoli remained prominent. On the

old cuticle of the second stage larva the excretory pore

could be seen below the metacorpus with its duct leading

anteriorly where it appeared to penetrate the female body

opposite the procorpus. From here the excretory duct was

seen leading posteriorly, as normally found in adult

females. At the posterior end, the gonads continued to

elongate. The uterus, vagina and vulva were prominent,

and the perineal pattern could be detected. Nineteen days

after inoculation all organs of the adult female were

developed, and molting of the second, third and fourth

cuticles occurred simultaneously (Fig. 28B).

Immediately after molting, feeding was resumed and the

female enlarged from a sausage-shape (Fig. 28C) to the pear-

shape typical of the genus (Fig. 28D). The stylet was

robust and well-developed. The enlarged procorpus had a

prominent lumen. The massive metacorpus had a strong well-

sclerotized valve. The esophageal glands consisted of five

distinct lobes with prominent nuclei and nucleoli. The









Figure 28

Adult females of Meloidogyne cruciani n.sp.

A) Early adult female still enclosed
in old cuticles of the second, third
and fourth stages.

B) Early adult female in the process of
shedding the second stage cuticle.

C-D) Fully developed females.


















A S C


(.7




7*) **













I



4

~74~



~

< ;. .7
)
.7 ;. -.3





'7,















( 461.







I





/









excretory pore was located opposite the procorpus with the

duct leading posteriorly to a unicellular gland. The

gonads elongated with one branch extending anteriorly close

to the esophageal region. The rectal glands were large,

with prominent nuclei and nucleoli.



Discussion


The post-infection development of Meloidogyne cruciani

agrees in general with the studies done by Bird (3), Tri-

antaphyllou and Hirschmann (61) and Siddiqui and Taylor (52).

The first noticeable changes that the post-infection

larvae underwent were in body length and in the esophageal

region (Fig. 26B). The esophagus increased in volume and the

body around the esophageal region had a noticeable increase

in width. These changes in the esophageal region may have

been due to the intense feeding activity of the second

stage larvae. The post-infection stage had a slight decrease

in body length when compared to the pre-infective stage.

Bird (3) also found a decrease in size of the infective

second stage larvae after root penetration; he attributed

the decrease in size to the depletion of food reserves

used during penetration and migration into the roots.

Triantaphyllou and Hirschmann (61) reported the shape

of the genital primordium (V-shape for females; straight

cylindrical shape for males) could be used to differentiate

sex in the early second stage larva. I found that sex could









be determined in the second stage larva of M. cruciani as

early as 11 days after inoculation and before the genital

primordium had assumed the V-shape typical of developing

females (Fig. 26C). This determination was based on the

presence of rectal glands in second stage immature females

and the absence of rectal glands in males (61).

I found that the stylet cone, shaft and lining of the

lumen through the stylet knobs are molted. This differs

from previous reports on other species of Meloidogyne.

According to Christie and Cobb (10), Bird (3) and

Siddiqui and Taylor (52) only the anterior conical portion

of the stylet is shed, with the basal portion of the stylet

and stylet knobs disappearing. However, my findings agree

with the illustrations of M. incognita presented by

Triantaphyllou and Hirschmann (61).

The formation of two extra esophageal lobes in females

but not in males indicates that females have different

digestive requirements than do males or larvae. (There is

no evidence that adult males feed.) Original descriptions

of other Meloidogyne females illustrate the esophagi as

tri-lobed glands or as an amorphous mass below the

metacorpus. Bird (3) made careful examination of the

esophageal region of M. javanica throughout larval

development, and reported only one lobe in the esophagus.

Chitwood's (7) illustrations showed three lobes.

Andrassy (1) stated that the five-gland condition

represents a more advanced evolutionary state. The









sedentary parasite feeding habit of Meloidogyne sp. can be

interpreted as one of the most advanced evolutionary states

of parasitism. This high degree of specialization in

feeding habit of these nematodes would undoubtedly require

also a highly specialized digestive system. A five-gland

esophageal condition should aid in the digestive process by

increasing the quantity of digestive enzymes secreted by

those glands. The two extra glands described in this study

have always been found in close association with the dorsal

esophageal gland. Baldwin and Sasser (2) and Eisenback et

al. (19) found that the dorsal gland orifice of various

species of Meloidogyne branched into three channels. That

indicates that species other than M. cruciani may have five

glands and that the two extra esophageal glands supplement

secretions by the dorsal esophageal gland and thus aid in

preoral digestion or may serve to stimulate the "nurse"

cells of the plant.

As stated earlier, there are striking differences

between the second stage larvae and the adult females of

Meloidogyne sp. One of these differences is the position

of the excretory pore. In the second stage larvae, it is

usually found posterior to the metacorpus. In the adult

females it is usually found anterior to the metacorpus

adjacent to the stylet knobs. Christie and Cobb (10)

illustrated the excretory pore opposite the metacorpus.

In M. cruciani the excretory pore is opposite the procorpus,

which agrees with the illustrations of Chitwood (7), Bird









(3) and Triantaphyllou and Hirschmann (61). Siddiqui and

Taylor (52) did not illustrate an excretory system in

M. naasi, and did not mention the location of the

excretory pore. Bird (3) made careful examinations of

the excretory pore and duct of M. javanica throughout the

larval development, but he made no mention of the changes

in position of the excretory pore opening between the

second stage larva and the adult female.

Daily examinations of the developmental stages reveal

the transformation that takes place in the position of the

excretory pore (Figs. 27, 28). In the late second stage

larva (Fig. 26F), the excretory pore is posterior to the

metacorpus with the duct directed posteriorly for some

distance. Immediately after the second molt (Fig. 27B)

the duct is directed anteriorly. Subsequently in the late

third state larva (Fig. 27B) and early fourth stage larva

(Fig. 27C) the duct was observed penetrating the body

opposite the procorpus. In the young adult female (Fig.

28A), the excretory pore on the second stage cuticle was

posterior to the metacorpus with its duct directed anteri-

orly, attaching to the adult female body opposite the pro-

corpus where the new excretory pore formed with the duct

directed posteriorly. After the final molt (Fig. 28B), the

excretory pore and duct became sclerotized and very

prominent, ending in a renette-type cell (Fig. 28C).

Observation of this excretory gland was possible in the

adult female immediately after the last molt and before










enlargement (Figs. 28C, D). In older females the body is

full of fat globules and ovaries making it impossible to

observe.

It is probable that during the molting stages the

excretory system is non-functional, forming a new duct

directed anteriorly, a new excretory pore and a new and

larger excretory duct in the final stage.

The post-infection development of the gonads of

Meloidogyne cruciani agrees in general with the descriptions

and illustrations presented by Triantaphyllou and Hirschmann

(61) and Siddiqui and Taylor (52). The genital primordium

enlarged by cell division and migrated posteriorly. As it

approached the posterior end, the genital primordium

assumed a V-shape, attached itself to the body wall, and

formed two branches which grew anteriorly.

Maggenti and Allen (35) gave a complete account of the

formation of the rectal glands and the origin of the

gelatinous matrix in Meloidogyne sp. They found six rectal

glands present in the early post-infective second stage

larvae before enlargement. My study agrees with Maggenti

and Allen's findings. Since males do not have rectal

glands, it is possible, therefore, to determine the sex of

the developing second stage larvae based on the presence

of the rectal glands. Thus, sex can be determined in the

very early stages of development, when the genital

primordium is still in the six-cell stage (Fig. 26C) and

has not migrated to the posterior end of the body or

assumed a V-shape (Fig. 26D).










Previous studies of the life cycle and development of

the genus Meloidogyne reveal few details on the molting of

the three cuticles by the females. Siddiqui and Taylor

(52) working with M. naasi were the first to mention

shedding of the cuticles. They observed the old cuticles

lying in the cortex in close proximity to the adult female

bodies. Observations during my study indicate that the

second stage larval cuticle is shed (Fig. 28B) while the

third and fourth stage larval cuticles are either

absorbed by the developing adult female or are lysed away.

No feeding takes place during the third and fourth stages

while the female is still enclosed within the second, and

second and third stage larval cuticles, respectively.

Before the female resumes feeding it has to get rid of

the barrier that the second, third, and fourth stage

larval cuticles present. By absorption or lysing action,

the third and fourth stage cuticles are eliminated, while

by force (mechanical action from body movements) the

second stage cuticle is broken in half and molted

(Fig. 28B).















CHAPTER IV
ESOPHAGEAL GLANDS OF ADULT FEMALES
OF Meloidogyne cruciani n.sp.

Introduction


Under the light microscope, the esophageal glands of

Meloidogyne cruciani appear composed of five individual

lobes. This is a deviation from the typical tylenchoid

esophagus which is considered to be composed of only

three esophageal glands. Previous original descriptions

of Meloidogyne spp. illustrate the esophageal region as

one, two or three lobes with three nuclei. Bird (3)

reported that the esophageal region in the developmental

stages of M. javanica was composed of only one gland. This

differs from Chitwood's (7) original description of M.

javanica in which he reported three glands.

The purpose of this study was to develop a fixation

technique that could be used to study internal structures

of nematodes with the scanning electron microscope (SEM)

and to corroborate the existence of five esophageal lobes

that can be seen with the light microscope.



Materials and Methods

Egg laying females were dissected from galled 'Rutgers'

tomato roots (Lycopersicon esculentum Mill.) in 2% formalin









and divided into two groups. One group of females was

placed in a glass cylinder (10 mm long x 6 mm inside

diameter) and both ends covered with a fine mesh nylon

screen (6 vim openings). The cylinder containing the

females was placed in 5 ml of 2.5% glutaraldehyde solution

in pH 7.2 phosphate buffer and fixed for 24 hours.

Subsequently they were transferred to 2% osmium tetroxide

for 24 hours at 8 C. The specimens, still inside the glass

cylinder, were dehydrated for 15 minutes in a series of 10,

20, 30, 40, 50, 75, 95 and 100% ethyl alcohol. They were

dehydrated in two changes of 100% ethyl alcohol for 15

minutes each. Then the specimens were placed in a Pelco

Critical Point Dryer with an ethanol-liquid CO2 system and

dried. The dried specimens were removed from the glass

cylinder, transferred by means of a dental root canal file

onto a stub covered with double-sided adhesive tape. Under

a dissecting microscope the anterior end of the specimens

were cut with an eye knife to expose the internal organs.

They were coated with gold with an Iako Sputter Coater,

viewed and photographed with a Hitachi S-450 scanning

electron microscope operated at 20 KV.

The other group of females was placed in 2% formalin

in a stendor dish, heated to 68-70 C and fixed and stained

in lactophenol-cotton blue (21). The females were

transferred to glycerin and the cuticle was cut and removed

(using an eye knife under a dissecting microscope) exposing

the esophageal region. These sections were placed in a









glass cylinder and fixed, dehydrated, critical point dried,

coated, mounted and examined as for the first group.



Results


The first group of females was not satisfactory for

studying the esophageal region. All the internal structures

had been fixed properly but the osmium tetroxide made the

internal contents of the females very brittle and, when

cut with the eye knife, the internal structures crumbled

(Fig. 29).

The second method was successful. By first staining

the females, the esophageal glands could be seen thus

facilitating cutting and removing the cuticle. Once the

cuticle was punctured, the pseudocoelomic fluids flowed

out, the cuticle was removed, exposing the esophageal glands.

The SEM corroborated the presence of two additional lobes

in the esophageal region of M. cruciani as seen with the

light microscope (Figs. 30-33).



Discussion


The use of the SEM to observe internal organs of

nematodes requires further study. Most of the SEM studies

of Meloidogyne females have been limited to external fea-

tures such as perineal patterns (20, 28, 32, 36, 57, 68) and

to the anterior ends (20, 28). Hogger and Estey (30) used

cryofracturing techniques to observe internal structures









Figure 29

Scanning electron micrograph of Meloidogyne cruciani
female internal structures crumbled.






88









Figure 30

Scanning electron micrograph of Meloidogyne cruciani
female dissected anterior region.




Full Text
64
Discussion
Perineal patterns of Meloidogyne cruciani differ from
perineal patterns of other species of the genus in having
punctations around the anus. Some variations of the
perineal patterns (Figs. 18A, 20-23) resemble perineal
patterns of Meloidogyne javanica Chitwood, 1949 (7) in
their pronounced lateral fields; however, length of larvae
easily separate the two species.
Andrassy (1) stated that as far as the triradial
symmetry of the esophagus is concerned, the 5-gland
condition in nematodes represents a more advanced
evolutionary stage. This condition would certainly aid the
feeding process of highly advanced sedentary parasites such
as members of the genus Meloidogyne. I have found that
females of M^ incognita and M^ arenaria also have 5-lobed
esophageal glands.
Andrassy (1) further stated that the uninucleate gland
(renette-type) excretory system is considered typical of
Torquentia and Penetrantia and does not occur in the
Secernentea (1), but this excretory system which was first
called a renette cell-type by Cobb (13), has been shown in
other genera of the Secernentea (11, 12, 14-16, 41).
The development of five glands in the esophagi of
females of Meloidogyne cruciani and the type of excretory
system are reported in Chapter III.


Figure 31
Enlarged portion of Fig. 30
showing esophageal glands.


Figure 4
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Xiphinema americanum


82
enlargement (Figs. 28C, D). In older females the body is
full of fat globules and ovaries making it impossible to
observe.
It is probable that during the molting stages the
excretory system is non-functional, forming a new duct
directed anteriorly, a new excretory pore and a new and
larger excretory duct in the final stage.
The post-infection development of the gonads of
Meloidogyne cruciani agrees in general with the descriptions
and illustrations presented by Triantaphyllou and Hirschmann
(61) and Siddiqui and Taylor (52). The genital primordium
enlarged by cell division and migrated posteriorly. As it
approached the posterior end, the genital primordium
assumed a V-shape, attached itself to the body wall, and
formed two branches which grew anteriorly.
Maggenti and Allen (35) gave a complete account of the
formation of the rectal glands and the origin of the
gelatinous matrix in Meloidogyne sp. They found six rectal
glands present in the early post-infective second stage
larvae before enlargement. My study agrees with Maggenti
and Allen's findings. Since males do not have rectal
glands, it is possible, therefore, to determine the sex of
the developing second stage larvae based on the presence
of the rectal glands. Thus, sex can be determined in the
very early stages of development, when the genital
primordium is still in the six-cell stage (Fig. 26C) and
has not migrated to the posterior end of the body or
assumed a V-shape (Fig. 26D).


u>


Figure 28
Adult females of Meloidogyne cruciani n.sp.
A) Early adult female still enclosed
in old cuticles of the second, third
and fourth stages.
B) Early adult female in the process of
shedding the second stage cuticle.
C-D) Fully developed females.


67
The experiment was carried out at a constant temper
ature of 28 + 1 C in a temperature controlled growth
chamber. The seedlings were fertilized twice a week with
2 ml of a 390 ppm NutrisolR (12-10-20) nutrient solution.
Every 24 hours, five seedlings were removed and the roots
washed and fixed using De Guiran's (29) method.
After fixation, the developmental stages were recovered
by dissecting them from roots. The nematodes then were
mounted in glycerin on glass slides, a cover glass applied
and sealed with Zut.
Morphological observations and drawings were made with
the aid of a camera lucida attached to an Olympus Vanox
compound microscope equipped with a Nomarski reflected
light differential interference contrast attachment.
Results
For the first 7-8 days, the post-infective second
stage larva underwent very few changes but generally
decreased in length when compared with the pre-infective
stage and increased in diameter (Fig. 26A, B). After 7-8
days, the genital primordium in the four-cell stage began
to migrate posteriorly, the esophageal glands became
shorter in length but larger in diameter and volume, and
there was a slight increase in the width of the metacorpus
and an increase in the body width around the esophageal
region (Fig. 26B). Inside the nuclear envelopes of the


PLANT PARASITIC NEMATODES OF THE U.S. VIRGIN ISLANDS
WITH THE DESCRIPTION, LIFE CYCLE AND MORPHOLOGY
OF Meloidogyne cruciani n.sp. (NEMATODA:
MELOIDOGYNIDAE) AND ITS INTERACTION WITH
Rotylenchulus reniformis
BY
ROBERTO GARCIA MARTINEZ
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1981


y n
fsj
-J


46
(Nicotiana tabacum L. cv 'NC 95') watermelon [Citrullus
lanatus (Thunb.) Matsum and Nakai cv 'Charleston Gray'],
and tomato (Lycopersicon esculentum Mill. cv 'Rutgers').
Each treatment was replicated five times. Inoculum was
prepared by shaking egg masses for two minutes in a 1%
NaOCl solution to obtain a suspension of eggs and larvae
(31). Sixty days after inoculation at a greenhouse
temperature of 22-26 C, roots were removed from soil,
washed and examined for galls and egg masses.
Results
Meloidogyne cruciani n.sp.
Females, (21): Length: 426.0-1121.8 ym (mean 787.5
ym, 95% confidence interval + 86.2); body width: 315.7-770.0
ym (505.1 ym + 61.5); a: 1.2-2.1 (1.5 + 0.1); stylet length:
11.4-16.2 ym (14.2 ym + 0.6); stylet knob height: 2.1-2.9
ym (2.4 m + 0.1); stylet knob width: 3.8-5.1 ym (4.5 ym +
0.2); dorsal gland orifice to base of stylet knobs: 3.2-5.1
ym (3.9 ym + 0.2); excretory pore to anterior end: 25.7-45.1
ym (32.2 ym + 2.2); center of median bulb to anterior end:
65.4-93.3 ym (78.3 ym + 3.4); vulva slit length: 20.0-25.7
ym (23.2 ym + 0.7); vulva slit to anus: 15.9-20.3 ym (18.1
ym + 0.6); interphasmidial distance: 25.7-36.8 ym (30.3
ym + 1.2).
Holotype. (female): Length: 949.9 ym; body width
536.4 ym; a: 1.8; stylet length: 12.7 ym; stylet knob


107
Discussion
Rotylenchulus reniformis populations increased over
two-fold more in the clay soils than in sandy soils while
Meloidogyne cruciani increased over three-fold more in sand
than in clay. Other researchers (38, 39, 42, 44, 46, 66)
have found that Meloidogyne spp. develop better in sandy
soils than in fine textured soils. This study corroborated
their findings. The lack of population increase of M.
cruciani in the clay soil indicates the presence of an
abiotic factor that restricts their reproductive capacity
under these conditions. Their inability to reproduce and
increase in numbers in clay soils as compared to sandy soils
will restrict their distribution. R^ reniformis, on the
other hand, had a large increase in numbers in the clay
soils. This indicates that R^ reniformis is more adapted
to and develops better in fine texture soils than does M.
cruciani. This advantage that R_^ reniformis has over M.
cruciani answers the question of why IL_ reniformis was
distributed throughout the U.S. Virgin Islands, while M.
cruciani was found only in isolated areas. Since the U.S.
Virgin Islands are composed mainly of clay-type soils, R.
reniformis will be able to reproduce and increase their
numbers at a faster rate than M^ cruciani. While the former
has the ability to spread throughout, the latter is
restricted to areas where the soil conditions are more
suitable to their development.


Figure 10
St. Thomas, U.S. Virgin Islands.
Shading indicates total area sampled.
Rotylenchulus spp. were found in all of the above areas.


6
cruciani n.sp. and Hemicriconemoides cocophillus (Loos,
1949) Chitwood and Birchfield, 1957 (8) were found on both
St. Croix and St. Thomas (Tables 2 and 3). Hoplolaimus
Columbus Sher, 1963 (49) were recovered only on St. Croix
from the three golf courses on the island (Table 2).
Rotylenchulus reniformis were present in every sample
taken from both islands while R^ parvus was present in 8 and
5% respectively, of the samples taken from St. Croix and St.
Thomas (Tables 2 and 3, Figs. 1 and 10). Helicotylenchus
dihystera, H. multicinctus, Tylenchorhynchus mashhoodi,
Xiphinema americanum and Pratylenchus pratensis were
recovered from 70, 4, 65, 58 and 58%, respectively, of
the samples taken from St. Croix (Table 2, Figs. 2-5), and
from 74, 3, 45, 16 and 13%, respectively, of the samples
taken from St. Thomas (Table 3, Figs. 11-14).
Meloidogyne, an economically important nematode genus
which is widespread in most tropical regions (9), was found
in only 11 samples from St. Croix (Table 2, Fig. 7) and five
samples from St. Thomas (Table 3, Fig. 15). It was found
only in home gardens, but not in all home gardens sampled.
Criconemoides citri and Hemicriconemoides cocophillus
were recovered in relatively low numbers from both St. Croix
and St. Thomas. Criconemoides was found in 15% of the
samples from St. Croix (Table 2, Fig. 6) and only in 8% of
the samples from St. Thomas (Table 3, Fig. 17).
Hemicriconemoides was recovered from 9% of the samples from
St. Croix (Table 2, Fig. 8) and in 11% of the samples from
St. Thomas (Table 3, Fig. 16).


Meloidogyne cruciani n.sp. differs from other species
of the genus by having punctations around the anus of the
female and by the larvae possessing extremely long tri-
lobed esophageal glands. Females, males and larvae possess
a uninucleate gland excretory system. Post-infection
developmental stages of females of It cruciani were dissected
from tomato roots, killed and fixed in lactophenol-cotton
blue and mounted in glycerin. Eleven days after inoculation,
the procorpus, metacorpus and esophageal glands of the
second stage larva were enlarged and prominent. Two small
lobes were present just posterior to the metacorpus. The
excretory duct of the second stage larva was directed
anteriorly and seemingly connected to the cuticle of the
third stage larva opposite the procorpus. The esophagus of
the adult female appeared typical for the species, having
a prominent procorpus, metacorpus and five nucleated lobes.
The excretory pore was opposite the procorpus with the
excretory duct directed posteriorly and terminating in
a uninucleate gland.
Rotylenchulus reniformis was distributed throughout
the two islands while M^_ cruciani was restricted in
occurrence. The interaction between these two species
was investigated in a clay vs. a sandy soil and at two
temperatures. While reniformis populations developed
better in clay soils, cruciani developed better in sandy
soils. When cruciani was present, R^ reniformis
populations did not increase as much as when the latter
x


57


Figure 14
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Pratylenchus pratensis


60


70
esophageal glands, the nucleoli and the chromocenters
enlarged. At this point, the rectal glands were not seen,
but six irregularly arranged nuclei were present near the
anal region (Fig. 26B).
Eleven days after inoculation the second stage larva
had enlarged considerably in size (Fig. 26C). At this
time, the esophagus had decreased in length, but increased
in width and volume; the procorpus and metacorpus were
enlarged and more prominent. For the first time, two small
lobes were seen connected to the esophagus just posterior
to the metacorpus; chromocenters of the esophageal glands
were very prominent. The dorsal gland had enlarged more
than the subventral esophageal glands. The genital
primordium was in the six-cell stage and had enlarged and
migrated further posteriorly. The rectal glands were
visible in this stage. Triantaphyllou and Hirschmann
(61) and Siddiqui and Taylor (52) refer to this stage as
the "developed but sexually undifferentiated" second stage
larva based on the genital primordium. The genital
primordium had not reached the V-shape characteristic
of the developing female gonad, but the presence of
rectal glands indicated the sex as a developing female.
The "developed and sexually differentiated" second
stage larva continued to enlarge. By fourteen days after
inoculation (Fig. 26D) the procorpus, metacorpus and
esophageal glands decreased still further in length, but


85
and divided into two groups. One group of females was
placed in a glass cylinder (10 mm long x 6 mm inside
diameter) and both ends covered with a fine mesh nylon
screen (6 ym openings). The cylinder containing the
females was placed in 5 ml of 2.5% glutaraldehyde solution
in pH 7.2 phosphate buffer and fixed for 24 hours.
Subsequently they were transferred to 2% osmium tetroxide
for 24 hours at 8 C. The specimens, still inside the glass
cylinder, were dehydrated for 15 minutes in a series of 10,
20, 30, 40, 50, 75, 95 and 100% ethyl alcohol. They were
dehydrated in two changes of 100% ethyl alcohol for 15
minutes each. Then the specimens were placed in a Peleo
Critical Point Dryer with an ethanol-liquid C02 system and
dried. The dried specimens were removed from the glass
cylinder, transferred by means of a dental root canal file
onto a stub covered with double-sided adhesive tape. Under
a dissecting microscope the anterior end of the specimens
were cut with an eye knife to expose the internal organs.
They were coated with gold with an Iako Sputter Coater,
viewed and photographed with a Hitachi S-450 scanning
electron microscope operated at 20 KV.
The other group of females was placed in 2% formalin
in a stendor dish, heated to 68-70 C and fixed and stained
in lactophenol-cotton blue (21). The females were
transferred to glycerin and the cuticle was cut and removed
(using an eye knife under a dissecting microscope) exposing
the esophageal region. These sections were placed in a


5
Table 1continued
Host plants
Nematodes
Okra IAbelmoschus esculentus (L.) Moench]
Onion (Allium cepa L.)
Pangla grass (Digitaria decumbens Stent)
Papaya ( Carica papaya L.)
Pepper ( Capsicum anmaon L.)
Pineapple [Ananas comosus (L.) Merrill]
Spanish bayonet ( Yucca aloifolia L.)
Sorghum [Sorghum bicolor (L.) Moench]
Squash ( Cucrbita pepo L.)
Sugar apple (Annona squamosa L.)
Sugar cane ( Saccharum officinarum L.)
Sour sop (Annona muricata L.)
Sweet potato [Ipomoea batatas (L.) Lam.]
Tomato (Lycopersicon esculentum Mill.)
Watermelon [ Citrullus lanatus (Thunb.)
Matsum. and Nakai]
Yam (Dioscorea alata L.)
Yuca CManihot esculenta Crantz)
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+ + + + + +
+ + + + +
+ + + + + + + +
+ + + + +
+ + + + + + + +
+ + ++ + +
1Hoplolaimus columbus recovered from St. Croix only.


110
Temperature had no significant effect on cruciani
in the sandy soil when alone or with reniformis, but in
clay, populations increased more at 30 C than at 22 C. The
temperature ranges for cruciani have not been determined;
therefore, it is possible that the lower temperatures in
the growth room (20-24 C) might have slowed the development
of these nematodes.
The soil texture conditions of the U.S. Virgin Islands
plus the faster reproduction rates of R_^ reniformis may be
the reasons why reniform nematodes have adapted themselves
throughout these islands. Those same conditions may have
kept cruciani from further spreading. The former were
found in every sample examined, while the latter were
recovered only in specific sitesintensively worked
vegetable gardens.
Experiments of longer duration (more than three months)
are needed to determine whether R_^ reniformis is capable of
displacing Meloidogyne spp. under field conditions in sand
or clay soils. Bird et al. (4) reported that when field
populations of Hoplolaimus columbus and incognita were
together on cotton, columbus populations increased in
number, suppressing populations of incognita and
eventually replacing them.


62
Paratypes. Females (whole mounts, perineal patterns),
males and larvae. Same data as allotype. USDANC,
Beltsville, Maryland; Laboratorie voor Nematologie,
Binnehaven, Wageningen, The Netherlands; Nematology
Department, Rothamsted Experimental Station, Harpenden,
Herts., England; Canadian National Collection of Nematodes,
Ottawa, Canada; Division of Plant Industry, Florida
Department of Agriculture and Consumer Services, Gainesville,
Florida; and Entomology and Nematology Department,
University of Florida, Gainesville, Florida.
Diagnosis. Meloidogyne cruciani differs from other
published descriptions of species of the genus by its
perineal pattern with punctations around the anus. The only
other species with punctations in the perineal area is M.
hapla Chitwood, 1949 (8) but the punctations of M^ hapla are
around the tail terminus. The larvae of M_;_ cruciani differ
from most other species of the genus in possessing extremely
long, distinctly tri-lobed esophageal glands.
Other morphological characters found in this species
and not reported for other members of the genus are: 1)
The presence of a guiding ring around the stylet shaft of
males and larvae (Figs. 24C, 25A). (These rings are often
associated with the order Dorylaimida, class Adenophorea
(1), but have not been reported in the class Secernentea.)
2) Three distinct lobes of the esophagi of males and larvae
of this species (Figs. 24A, 25A). Original descriptions
and illustrations of 29 other species of this genus examined


Intermittent itru _
1
Jersey Bay
Deck
) 4 lilM
20000 fMt


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.
Dr. G. C. Smart, Jr^:
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.
A), k/.
Dr. D. W. Dickson
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.
Dr. S. fiy. Locascio
Professor of Horticultural
Science
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 egpej o^poci^o^ off Philosophy.
D. E. Stokes
Assistant Professor of
Entomology and Nematology


CHAPTER III
POST-INFECTION DEVELOPMENT OF FEMALES
OF Meloidogyne cruciani n.sp.
Introduction
In most genera of nematodes, the adult females
resemble the larval stages in many of their morphological
characters. In the genus Meloidogyne, the developing
larvae undergo a series of morphological changes and the
mature females appear very different than the second stage
larvae. The former have a saccate pear-shaped body, a
very large robust stylet, very prominent esophageal glands
and an enlarged metacorpus. The larvae are filiform with
a small stylet and small esophageal glands.
Before Chitwood (7) placed what was known as
Heterodera marioni into the genus Meloidogyne and created
four species and one subspecies, the life cycle and
morphological studies of unknown species of Meloidogyne
(Heterodera marioni) were carried out by Nagakura (37) and
Christie and Cobb (10) Nagakura described three molts
taking place within the plant roots and the existence of
third and fourth larval stages. Christie and Cobb disagreed
with Nagakura, stating there is no third larval stage
and that the fourth is just theoretical.
65


CHAPTER II
Meloidogyne cruciani n.sp., A ROOT-KNOT NEMATODE
FROM ST. CROIX, U.S. VIRGIN ISLANDS
Introduction
During a survey to determine the plant-parasitic
nematode complement in the U.S. Virgin Islands, an
undescribed species of Meloidogyne was recovered from
tomato roots. Specimens of the nematode were brought to
the University of Florida and populations established on
'Rutgers' tomato plants. Single egg mass isolations were
made to establish a population originating from a single
female. One isolate was selected and used for the
taxonomic and morphologic studies reported herein.
Materials and Methods
Egg masses dislodged from 'Rutgers' tomato (Lycopersicon
esculentum Mill.) roots were teased apart to expose the eggs.
These eggs were placed in water and left overnight at 28-29
C. Newly-hatched second stage larvae were mounted in 2%
formalin on slides. The specimens were measured and drawn
immediately using a camera lucida (23). Males were dissected
from large root galls and from egg masses and prepared as
above. Females were dissected from roots, placed in 2% for
malin and their posterior ends were excised. These sections
44


Figure 5
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Pratylenchus pratenses.


113
24. Estores, R.A., and T.A. Chen. 1972. Interactions of
Pratylenchus penetrans and Meloidogyne incognita
as coinhabitants in tomato. J. Nematol7 4:
170-174.
25. Filipjev, I.N. 1936. On the classification of the
Tylenchinae. Proc. Helminthol. Soc. Wash. 3:
80-82.
26. Gay, C.M., and G.W. Bird. 1973. Influence of
concomitant Pratylenchus brachyurus and Meloidogyne
spp. on root penetration and population dynamics.
J. Nematol. 5: 212-217.
27. Golden, A.M. 1956. Taxonomy of the spiral nematodes
(Rotylenchus and Helicotylenchus), and the
developmental stages and host-parasite relation
ships of R^ buxophilus, n.sp., attacking boxwood.
Bull. Md. Agrie. Exp. Stn. A-85, 1-28.
28. Golden, A.M. 1979. Descriptions of Meloidogyne
camelliae n.sp. and M^ querciana n.sp. (Nematoda:
Meloidogynidae), with SEM and host-range
observations. J. Nematol. 11: 175-189.
29. Guiran, G. De. 1966. Coloration des nematodes dans
les tissue ve'ge'taux par le bleu coton a froid.
Nematologica 12: 646-647.
30. Hogger, C.H., and R.H. Estey. 1977. Cryofracturing
for scanning electron microscope observations of
internal structures of nematodes. J. Nematol. 9:
334-337.
31. Hussey, R.S., and K.R. Barker. 1973. A comparison of
methods of collecting inocula of Meloidogyne spp.,
including a new technique. Plant Dis. Reptr. 57:
1025-1028.
32. Khan, Z.N., and W.H. Thames. 1978. SEM study of
perineal patterns of four species of Meloidogyne.
J. Nematol. 10: 292-293 (Abstr.).
33. Kheir, A.M., and A.A. Osman. 1977. Interaction of
Meloidogyne incognita and Rotylenchulus reniformis
on tomato Trematologa Mediterrnea 5: 113-6 .
34. Linford, M.B., and J.M. Oliveira. 1940.
Rotylenchulus reniformis, nov.gen., n.sp., a
nematode parasite of roots. Proc. Helminthol
Soc. Wash. 7: 35-42.
35. Maggenti, A.R., and M.W. Allen. 1960. The origin of
the gelatinous matrix of Meloidogyne. Proc.
Helminthol. Soc. Wash. 277 4-10.


Kriu* Ft
VJqtha*


109
(53, 54) had lower reproduction, less galling, and lower
root penetration when in the presence of other genera of
plant parasitic nematodes.
When comparing the population densities of R^_ reniformis
and cruciani together in sand and clay, there was no
significant difference in their numbers between either soil
type. Soil type had no effect on either nematode when they
were together.
There was a significant increase in population
development with both nematodes alone and combined in both
soil types from one month to three months. Sampling at one
month after inoculation is too short a period of time to
detect any significant effect soil type might have on the
nematodes. After three months, reniformis increased
greater in clay than in sand while cruciani showed the
opposite effect. Regardless of soil type, the increases
shown by R^ reniformis were much greater than the increases
shown by cruciani. This could be due to R_^ reniformis
having a shorter life cycle than RL_ cruciani. Rao and
Prasad (43) found that R^ reniformis increased in numbers
faster than did javanica; and they attributed this to
R. reniformis having a shorter life cycle.
Populations of R^ reniformis alone or with concomitant
populations of cruciani increased with no significant
difference between 22 or 30 C. The experiments showed
that the temperature conditions used had no significant
effect on R^ reniformis growth.


Figure 26
Second stage larvae of Meloidogyne cruciani n.sp.
A) Infective second stage larva.
B) Post-infective sexually
undifferentiated second stage
larva.
C-F) Sexually differentiated, female
second stage larvae.


100
reniformis were obtained by processing infested soil using
a sieving and sugar flotation-centrifugation technique
(5). As for cruciani, these nematodes were trans
ferred in lots of 100 to vials containing distilled water.
One month and three months after inoculation, nine
pots of each treatment were sampled as follows: the soil
in each pot was placed in a container and water added to
bring the total volume to 10 liters. The soil in the
container was mixed thoroughly with the water and an
aliquot of 500 ml was poured through a 500 mesh sieve
(pore size 30 ym). The residue on the sieve was washed
into 50 ml centrifuge tubes and the nematodes extracted
using a modification of the sugar flotation-centrifugation
technique described by Caveness and Jensen (5). The
extracted nematodes were placed in Syracuse dishes and
counted.
A computerized statistical analysis of variance
procedure (ANOVA) was run on all the different treatments
and two way tables used to compare means followed by a
Tukey's HSD (honestly significant difference) test of
those comparison of means that were found significant with
the tables (6) .
Results
Statistical analyses were used to interpret the
following variables:


71
became wider and more voluminous. The two esophageal lobes
seen first at day 11 were larger and nuclei could be seen
for the first time. The nucleoli and chromocenters inside
the nuclei of the other three esophageal glands were
prominent. The genital primordium in this stage had
assumed the V-shape characteristic of the developing female
gonad. The genital primordium was associated closely with
the now visible and prominent rectal glands but had not
attached to the body wall.
Sixteen days after inoculation the early second stage
female larva (Figs. 26E, F) showed further enlargement of
the esophagus. The genital primordium now assumed a
definite V-shape with two branches directed anteriad.
The branches grew in length as the gonad moved towards the
anal region and attached to the body wall (Fig. 26F); the
female second stage larva possessed six well-developed
rectal glands. At this point, a new cuticle was evident
posteriorly as the second stage cuticle began to separate
from it.
The third stage larva (Figs. 27A, B) could be recognized
enclosed in the second stage larval cuticle; it did not
possess a stylet and the posterior end was round. The
second stage larval stylet remained attached to the old
cuticle. During the molt the cone and shaft of the stylet
and the lumen of the stylet knobs were shed with the
second stage cuticle, the stylet knobs disappeared and
there was a void at the anterior part of the body where the
stylet and stylet knobs normally would be.


4
Table 1
Plants from which soil samples were taken through the root
zones and nematodes recovered on St. Croix and
St. Thomas, U.S. Virgin Islands
Host plants
Nematodes
Bahia grass (Paspalum notatum Flgge)
<15
to .
, 3>+>
K .
a
El, to to
3 3 rC
to ^
3 15 15
c e
o> 05 3;
i-O 1^5 S|
O O 15
15 15 C
3i 3iM -rl tS5
(1 -WnIM M
££££
to
<15
Si
to
3
r-1
3 3
s §
05 05
r-l
+ + + + + + + +
Banana (Musa acuminata Colla)
+ + + + + + + + + +
Bean iphaseolus vulgaris L.) + +
Bermuda grass [Cynodon dactylon (L.) Pers.] + +
Citrus [Citrus aurantiifolia (Christm.) Swingle] + +
Corn (Zea mays L.) + + +
Grapes (Vitis rotundigolia Michx.) +
Guayaba (Psidium guajada L.) + +
Guinea grass (Panicum maximum Jacq.) + +
Hairy crabgrass [ Digitaria sanguinalis (L.)
Scop.] + +
Hibiscus (Hibiscus rosa-sinensis L.) + +
Hurricane grass [ Sporobolus cryptandrus (Torr.)
Gray] +
Mango (Mangifera indica L.) + +
+ + + +
+ + + + + + +
++++++
+ + + + +
+ + +
+ + + +
+ + + + +
+ + + +
+ +
+ + +
+ + +


LITERATURE CITED
1. Andrassy, I. 1976. Evolution as a basis for the
systematization of nematodes. Pitman Publishing
Ltd. 288 pp.
2. Baldwin, J.G., and J.M. Sasser. 1979. Meloidogyne
megatyla n.sp., a root-knot nematode from loblolly
pine. J. Nematol. 11: 47-56.
3. Bird, A.F. 1959. Development of the root-knot
nematodes Meloidogyne javanica (Treub) and
Meloidogyne hapla Chitwood in the tomato.
Nematologica 4: 31-42.
4. Bird, G.W., O.L. Books, and C.E. Perry. 1974.
Dynamics of concomitant field populations of
Hoplolaimus columbus and Meloidogyne incognita.
J. Nematol. 6: 190-194.
5. Caveness, F.E., and H.S. Jensen. 1955. Modification
of the centrifuge-flotation technique for the
isolation and concentration of nematodes and their
eggs from soil and plant tissue. Proc. Helminthol.
Soc. Wash. 22: 87-89.
6. Chew, V. 1976. Comparing treatment means: a
compendium. HortScience 11: 348-357.
7. Chitwood, B.G. 1949. Root-knot nematodes. Part 1.
A revision of the genus Meloidogyne Goeldi, 1887.
Proc. Helminthol. Soc. Wash. 16: 9"CT-104.
8. Chitwood, B.G., and W. Birchfield. 1957. A new
genus, Hemicriconemoides (Criconematidae:
Tylenchina). Proc. Helminthol. Soc. Wash. 24:
80-86.
9. Christie, J.R. 1959. Plant nematodes, their bionomics
and control. Florida Agricultural Experiment
Station. 256 pp.
10.Christie, J.R., and G.S. Cobb. 1941. Notes on the
life history of the root-knot nematode Heterodera
marioni. Proc. Helminthol. Soc. Wash. 8: 23-26.
Ill


Figure 18
A) Drawings of perineal patterns o
Meloidogyne cruciani n.sp.
B) Outlines of females in varying
sizes and shapes.


81
(3) and Triantaphyllou and Hirschmann (61). Siddiqui and
Taylor (52) did not illustrate an excretory system in
M. naasi, and did not mention the location of the
excretory pore. Bird (3) made careful examinations of
the excretory pore and duct of javanica throughout the
larval development, but he made no mention of the changes
in position of the excretory pore opening between the
second stage larva and the adult female.
Daily examinations of the developmental stages reveal
the transformation that takes place in the position of the
excretory pore (Figs. 27, 28). In the late second stage
larva (Fig. 26F), the excretory pore is posterior to the
metacorpus with the duct directed posteriorly for some
distance. Immediately after the second molt (Fig. 27B)
the duct is directed anteriorly. Subsequently in the late
third state larva (Fig. 27B) and early fourth stage larva
(Fig. 27C) the duct was observed penetrating the body
opposite the procorpus. In the young adult female (Fig.
28A), the excretory pore on the second stage cuticle was
posterior to the metacorpus with its duct directed anteri
orly, attaching to the adult female body opposite the pro
corpus where the new excretory pore formed with the duct
directed posteriorly. After the final molt (Fig. 28B), the
excretory pore and duct became sclerotized and very
prominent, ending in a renette-type cell (Fig. 28C).
Observation of this excretory gland was possible in the
adult female immediately after the last molt and before


53


Figure 19
A) Anterior region of female.
B) Face view showing cephalic
framework.
C) Variations in size and shape
of esophageal glands.


Picara Pt
Sturdy Pt
Botany Pt
Intermittent ttrcam
1
Com Pt
20000 ?Mt
LO
vo


Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
PLANT PARASITIC NEMATODES OF THE U.S. VIRGIN ISLANDS
WITH THE DESCRIPTION, LIFE CYCLE AND MORPHOLOGY
OF Meloidogyne cruciani n.sp. (NEMATODA:
MELOIDOGYNIDAE) AND ITS INTERACTION WITH
Rotylenchulus reniformis
By
Roberto Garcia Martinez
March, 1981
Chairman: G. C. Smart, Jr.
Major Department: Entomology and Nematology
A nematode survey was conducted on St. Croix and St.
Thomas, U.S. Virgin Islands. Soil samples were collected
and processed from all agricultural and some non-agricul-
tural lands. The nematodes recovered were preserved and
brought to the University of Florida for identification.
Eleven species in nine genera of plant parasitic nematodes
were recovered: Rotylenchulus reniformis, R, parvus,
Helicotylenchus dihystera, H. multicinctus,
Tylenchorhynchus mashhoodi, Xiphinema americanum,
Pratylenchus pratensis, Criconemoides citri, Meloidogyne
cruciani n.sp. and Hemicriconemoides cocophillus on both
islands, and Hoplolaimus columbus only on St. Croix.
ix


24
Male of Meloidogyne cruciani n.sp. A)
Entire specimen (curvature of specimen for
convenience in illustrating). B) Face view
showing cephalic framework. C) Anterior
portion. D) Tail (lateral view). E)
Lateral field
25 Larvae of Meloidogyne cruciani n.sp.
A) Entire specimen (curvature of specimen
for convenience of illustrating). B) Face
view showing cephalic framework. C) Tails
(lateral view). D) Lateral field at tail
region. E) Tail (ventral view)
26 Second stage larvae of Meloidogyne cruciani
n.sp. A) Infective second stage larva. B)
Post-infective sexually undifferentiated
second stage larva. C-F) Sexually
differentiated, female second stage larvae. .
27 Third and fourth stage larvae of Meloidogyne
cruciani n.sp. A-B) Third stage larvae
encased in the second stage cuticle. C)
Fourth stage larva encased in the second
and third stage cuticles
28 Adult females of Meloidogyne cruciani n.sp.
A) Early adult female still enclosed in old
cuticles of the second, third and fourth
stages. B) Early adult female in the
process of shedding the second stage
cuticle. C-D) Fully developed females. . .
29 Scanning electron micrograph of Meloidogyne
cruciani female internal structures crumbled.
30 Scanning electron micrograph of Meloidogyne
cruciani female dissected anterior region .
31 Enlarged portion of Fig. 30 showing
esophageal glands
32 Scanning electron micrograph of Meloidogyne
cruciani female dissected anterior region .
33 Enlarged portion of Fig. 32 showing
esophageal glands and metacorpus
57
60
69
73
77
88
90
92
94
96
Vlll


74
The esophagus compressed, with the procorpus and
metacorpus enlarging. The esophageal lumen and metacorpus
valve were visible but very faint. The esophageal glands
lost their chromocenters but the nucleoli remained very
prominent. In the early third stage (Fig. 27A) the
excretory pore was located opposite the esophageal glands
with the duct pointing anteriorly. In the late third stage
larva (Fig. 27B) the excretory pore was located opposite the
esophageal glands with the anteriorly directed duct
penetrating the body opposite the procorpus. The ovaries
of the third stage larva continued to elongate and the
uterus and vagina began to develop.
With the onset of the third molt, the body of the
fourth stage larva formed and separated from the third
stage cuticle but was still enveloped in the third and
second stage cuticles. The early fourth stage larva (Fig.
27C) differed from the late third stage larva (Fig. 27B) in
being enclosed in the old second and third larval cuticles.
There was no stylet visible, and a void was seen at the
anterior end where the stylet would have been. The
esophageal lumen and metacorpus valve were very faint; the
valve was now located in the posterior portion of the
metacorpus. The excretory duct was similar to that of the
third stage larva; it ran anteriorly and penetrated the body
opposite the procorpus. At the posterior end, the gonads
continued to elongate, the uterus and vagina formed
completely.


92


Figure 2
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Helicotylenchus spp.


97
of Xiphinema americanum and Caenorhabditis briggsae. The
method they used presents the problem of not being specific
as to where the cuts are going to occur. Furthermore, the
observation plane is a cross section of the nematode, not
a tridimensional view of the organs exposed during
fracturing. More recently, Eisenback et al. (20) used the
SEM and light microscopes to study the excised stylet, lumen
and metacorpus valve of Meloidogyne hapla, M. arenaria, M.
incognita, and M^ javanica females. These are the first
accounts of using the SEM to study excised internal organs
of nematodes. Their results show that it is possible to
excise sclerotized internal structures of nematodes and
view them with the SEM. My results show that with adequate
techniques, non-sclerotized internal structures also can be
studied (Figs. 30-33).
Light microscope studies of Meloidogyne cruciani
revealed five-lobed esophageal glands; one large dorsal
gland, two small dorsal glands and two subventral glands.
SEM micrographs confirm the presence of the two small dorsal
glands (Figs. 30-33). The external surface of the meta
corpus appears ridged (Figs. 32, 33).


ACKNOWLEDGMENTS
I extend my sincere appreciation to Dr. G.C. Smart,
Jr., my committee chairman, Dr. D.W. Dickson, Dr. D.E.
Stokes and Dr. S.J. Locascio, my committee members for
their patience, understanding and valuable assistance.
I also express my appreciation to Dr. R.P. Esser,
Dr. J.R. Rich and Mr. A.L. Taylor for all their help and
encouragement during this study.
11


90


112
11. Cobb, N.A. 1891. Strawberry-bunch (A new disease
caused by nematodes). Agr. Gaz. N.S.W. 2: 390-
400.
12. Cobb, N.A. 1893. Nematodes, mostly Australian and
Fijian. Macleay Memorial Volume, Linn. Soc. N.S.W.
(Sidney) 252-308.
13. Cobb, N.A. 1913. In The Helminthological Society
of Washington meeting. Terms amphid and renette
introduced. Science, N.S., 37: 498-499.
14.
Cobb, N.A. 1914.
Res. 2: 217-230.
Citrus-root nematode.
J. Agr.
15.
Cobb, N.A. 1915.
root disease of
Res. 4: 561-568.
Tylenchus similis, the
sugar cane and bananas.
cause of a
J. Agr.
16.
Cobb, N.A. 1917.
infesting cotton
27-33.
A new parasitic nematode found
and potatoes. J. Agr. Res. 11:
17.
Dasgupta, D.R., D.
J. Raski, and S.A. Sher.
1968.
A revision of the genus Rotylenchulus Linford and
Oliveira, 1940 (Nematoda: Tylenchidae). Froc.
Helminthol. Soc. Wash. 35: 169-192.
18. De Grisse, A.T.L., and P.H.A. Loof. 1965. Revision
of the genus Criconemoides (Nematoda). Meded.
Landb. Opzoekstns. Staat Gent. 30: 577-603.
19. Eisenback, J.D., H. Hirschmann, and A.C. Triantaphyllou
1980. Morphological comparison of Meloidogyne
female head structures, perineal patterns, and
stylets. J. Nematol. 12: 300-313.
20. Esser, R.P. 1973. A four minute lactophenol fixation
method for nematodes. Plant Dis. Reptr. 57:
1045-1046.
21. Esser, R.P. 1980. Taxonomy and biology of Verutus
volvingentis n.gen., n.sp. (Tylenchida: Nemata).
Diss. Abstracts. In press.
22. Esser, R.P., J.B. MacGowan, and H.M. Van Pelt. 1965.
Two new nematode subsampling tools. Plant Dis. Reptr
49: 265-267.
23. Esser, R.P., V.G. Perry, and A.L. Taylor, 1976. A
diagnostic compendium of the genus Meloidogyne
(Nematoda: Heteroderidae). Proc. Helminthol.
Soc. Wash. 43: 138-150.


CHAPTER V
INTERACTION OF Rotylenchulus reniformis AND
Meloidogyne cruciani ON TOMATO
Introduction
During a survey of two of the U.S. Virgin Islands, as
described in Chapter I, Rotylenchulus reniformis Linford
and Oliveira, 1940 (34) was recovered from all samples
while Meloidogyne cruciani n.sp. was recovered from only 14%
of the samples. Since both islands surveyed, St. Croix and
St. Thomas, are tropical and would appear to have conditions,
including ample host plants, suitable for both nematode
species, the question arises as to why the low incidence of
Experiments were initiated to determine the influence
of soil type and temperature on each nematode. Since the
islands have predominently clay-type soils, a clay soil
and a sandy soil were compared as to their influence on
population development at two different temperatures.
Materials and Methods
Seeds of tomato, Lycopersicon esculentum Mill, cv
'Rutgers', which is susceptible to both R_^ reniformis and
M. cruciani, were germinated in moist sterile vermiculite
in a temperature controlled water bath set at 29 C. When
98


79
be determined in the second stage larva of M^ cruciani as
early as 11 days after inoculation and before the genital
primordium had assumed the V-shape typical of developing
females (Fig. 26C). This determination was based on the
presence of rectal glands in second stage immature females
and the absence of rectal glands in males (61).
I found that the stylet cone, shaft and lining of the
lumen through the stylet knobs are molted. This differs
from previous reports on other species of Meloidogyne.
According to Christie and Cobb (10), Bird (3) and
Siddiqui and Taylor (52) only the anterior conical portion
of the stylet is shed, with the basal portion of the stylet
and stylet knobs disappearing. However, my findings agree
with the illustrations of incognita presented by
Triantaphyllou and Hirschmann (61).
The formation of two extra esophageal lobes in females
but not in males indicates that females have different
digestive requirements than do males or larvae. (There is
no evidence that adult males feed.) Original descriptions
of other Meloidogyne females illustrate the esophagi as
tri-lobed glands or as an amorphous mass below the
metacorpus. Bird (3) made careful examination of the
esophageal region of M^ javanica throughout larval
development, and reported only one lobe in the esophagus.
Chitwood's (7) illustrations showed three lobes.
Andrssy (1) stated that the five-gland condition
represents a more advanced evolutionary state. The


was alone. cruciani populations also were suppressed
when R_^ reniformis was present. This indicates compe
tition for available feeding sites. The soil texture of
the U.S. Virgin Islands and the faster reproduction rate
of R^_ reniformis may be reasons why reniformis was
found throughout those islands, while cruciani was
restricted in distribution.
xi


*<>,-. d?>
U)
MIm
o
OOOO
20000 fMt


fropwcc
Stuwp/ Pi
Intermittent etr;**
Coki K
Lk)
Ln
o
10000
20000 Tmmt


106
Table 7
Effects of soil type and temperature on populations of
Meloidogyne cruciani when inoculated alone and
simultaneously with an equal number of
Rotylenchulus
reniformis.
Soil
type
Sand
Clay
Temperature
Temperature
Inoculum1
density
22 C
30 C
22 C
30 C
(No./pot)
(No.
/pot)
0/200
12332a2
12396a
2455c
5418b
200/200
1316c
2573c
1283c
2710c
inoculum density of Rotylenchulus reniformis/Meloidogyne
cruciani, respectively.
2Means followed by the same letter are not significantly
different at 5% level according to Tukey's HSD comparison
of means. Significance applies to vertical and
horizontal columns.


Figure 9
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Hoplolaimus columbios .


83
Previous studies of the life cycle and development of
the genus Meloidogyne reveal few details on the molting of
the three cuticles by the females. Siddiqui and Taylor
(52) working with ft naasi were the first to mention
shedding of the cuticles. They observed the old cuticles
lying in the cortex in close proximity to the adult female
bodies. Observations during my study indicate that the
second stage larval cuticle is shed (Fig. 28B) while the
third and fourth stage larval cuticles are either
absorbed by the developing adult female or are lysed away.
No feeding takes place during the third and fourth stages
while the female is still enclosed within the second, and
second and third stage larval cuticles, respectively.
Before the female resumes feeding it has to get rid of
the barrier that the second, third, and fourth stage
larval cuticles present. By absorption or lysing action,
the third and fourth stage cuticles are eliminated, while
by force (mechanical action from body movements) the
second stage cuticle is broken in half and molted
(Fig. 28B).


Figure 8
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Hemicriconemoides cocophillus.


TABLE OF CONTENTS
ACKNOWLEDGMENTS
LIST OF TABLES V
LIST OF FIGURES vi
ABSTRACT ix
CHAPTER I
SURVEY OF THE PLANT PARASITIC NEMATODES OF THE U.S.
VIRGIN ISLANDS 1
Introduction 1
Materials and Methods 2
Results 3
Discussion 7
CHAPTER II
Meloidogyne cruciani n.sp., A ROOT-KNOT NEMATODE FROM
ST. CROIX, U.S. VIRGIN ISLANDS 44
Introduction 44
Materials and Methods 44
Results 46
Meloidogyne cruciani n.sp 46
Females 46
Holotype 46
Description 47
Males 54
Allotype 54
Description 55
Second stage larvae 55
Description 58
Holotype 61
Allotype 61
Paratypes 62
Diagnosis 62
Type host and type habitat 63
Type locality 63
Discussion 64
iii


51


2
in the survey because most of the island is a national park
with very little agriculture on the remainder.
Materials and Methods
Large maps of St. Croix and St. Thomas Islands were
constructed with aerial photographs and the areas under
cultivation and those areas that could be cultivated were
marked on the maps for easy reference and location. The
survey extended over a period of four months covering
agricultural fields, home gardens, golf courses, nurseries,
lawns and non-cultivated areas that had a potential for
agricultural development. Soil subsamples were taken with
a cone-type soil sampler (22) and combined in the field to
form a composite sample that was used for the extraction of
nematodes. The number of subsamples comprising a composite
sample was determined by the size of the area and diversity
of crops sampled, but ranged from three from about 3 m^
garden plots to 10 from 2 hectare fields. The subsamples
were mixed thoroughly and approximately 500 cm of soil were
placed in plastic bags, numbered, and the number recorded on
the map of each island. The crop or plants from which a
sample was taken also was recorded. A total of 80 composite
soil samples were taken from the root zone of 30 different
plants on St. Croix and 26 samples from the root zone of 16
different plants on St. Thomas. Samples were processed on
St. Croix using a modification of the centrifugation-


Figure 7
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Meloidogyne cruciani n.sp.


Figure 1
St. Croix, U.S. Virgin Islands.
Shading indicates total area sampled.
Rotylenchulus spp. were found in all of the above areas.


54
Males. (25): Length: 1160-1620 ym (mean 1378.8 ym,
95% confidence interval + 52.3); body width: 23.2-46.3 ym
(33.8 ym + 2.4); stylet length: 19.4-24.1 ym (22.0 ym +
0.5); stylet base to anterior end: 21.2-26.3 ym (24.4 ym
+ 0.5); stylet knob height: 2.7-3.8 ym (3.3 ym + 0.1);
stylet knob width: 4.1-6.0 ym (5.2 ym + 0.2); dorsal
esophageal gland orifice to base of stylet knobs: 3.2-7.9
ym (4.9 ym + 0.4); center of metacorpus valve to anterior
end: 66.7-118.1 ym (88.9 ym + 4.1); excretory pore to head
end: 127.3-189.5 ym (149.2 ym + 5.8); anterior end of
testis to posterior end: 489.4-1127.0 ym (823.4 ym +
60.1); spicule length: 28.7-38.0 ym (31.3 ym + 0.9);
gubernaculum: 6.7-11.1 ym (8.8 ym + 0.5); phasmid to
posterior end: 8.3-22.9 ym (16.7 ym + 1.4); a: 31.9-
71.2 (43.2 + 3.7); c: 89.2-238.2 (132.6 + 13.1); c':
0.3-0.7 (0.5 + 0.1); 0 (distance from dorsal esophageal
gland orifice to base of stylet knobs, expressed as % of
stylet length): 14.3-36.8 (22.6 + 1.9); T% (distance from
anterior end of testis to posterior end, expressed as % of
body length): 39.8-79.7 (60.1 + 4.7).
Allotype. (male): Length: 1440 ym; body width:
29.9 ym; stylet length: 22.8 ym; stylet base to anterior
end: 24.8 ym; stylet knob height: 3.5 ym; stylet knob
width: 4.8 ym; dorsal esophageal gland orifice to base of
stylet knobs: 4.9 ym; center of metacorpus valve to anterior
end: 97.4 ym; excretory pore to anterior end: 139.2 ym;
anterior end of testis to posterior end: 933.8 ym; spicule


Figure 33
Enlarged portion of Fig. 32
showing esophogeal glands and metacorpus.


40 urn


108
When M;_ cruciani was present, R^ reniformis did not
increase as much as when the latter was alone. This
influence that cruciani had on R^_ reniformis was
detected in both sand and clay soils. Singh (55) and
Thomas and Clark (60) found that R^ reniformis populations
are suppressed in the presence of M^ incognita.
M, cruciani numbers were lower under both soil types
when R^_ reniformis was present than when cruciani was
alone. The lack of reproduction of M_^ cruciani in sand was
greater proportionally than for R^ reniformis. Other
studies with R^_ reniformis and Meloidogyne spp. have shown
that in the presence of FL_ reniformis, populations of
Meloidogyne spp. are inhibited, their growth retarded,
or penetration and developmental rates are slower (33, 40,
43, 67).
The mutual antagonistic effect found in this study
between R^ reniformis and M^ cruciani indicates a compe
tition between both nematodes for available feeding sites.
This agrees with the work reported by Estores and Chen (24).
They found that when Pratylenchus penetrans and M.
incognita were together on tomato, populations of both
species were lower than when alone. Turner and Chapman (62)
also found that when penetrans and M^ incognita were
together on alfalfa or red clover, root invasion by M.
incognita was reduced because of fewer suitable feeding sites.
Other researchers also have reported that M. incognita (26,
47, 56, 64), M^ hapla (45), M. javanica (65), and M. naasi


to
u>
IntwtitlMt tr


114
36. Mulvey, R.H., P.W. Johnson, J.L. Townsend, and J.W.
Potter. 1975. Morphology of the perineal pattern
of the root-knot nematode Meloidogyne hapla and
M. incognita. Can. J. ZooT^ 53~: 370-373.
37. Nagakura, K. 1930. Ueber den Bau and die
Lebensgeschichte der Heterodera radiciola (Greeff)
Miiller. Jap. J. Zool~ 3l 95-160.
38. Nardacci, J.F., and K.R. Barker. 1978. Influence of
temperature and soil type on Meloidogyne incognita
on soybean. J. Nematol. 10: 294-295.
39. O'Bannon, J.H., and H.W. Reynolds. 1961 Root-knot
nematode damage to cotton yields in relation to
certain soil properties. Soil Sci. 92: 384-386.
40. Oteifa, B.A., and A.A. Osman. Host-parasitic relations
to Rotylenchulus reniformis on Lycopersicon
esculentum. In Simposio International (XII) de
Nematologia, Sociedad Europea de Nematologos, 78-79.
41. Perry, V.G., H.M. Darling, and G. Thorne. 1959.
Anatomy, taxonomy and control of certain spiral
nematodes attacking blue grass in Wisconsin.
University of Wisconsin, Research Bulletin 207,
24 pp.
42. Prot, J.C., and S.D. Van Gundy. 1979. Influence of
soil type and temperature on the migration of
M. incognita juveniles towards tomato roots.
Nematropica 9: 104-105.
43. Rao, B.H.K., and S.K. Prasad, 1971. Population
studies on Meloidogyne javanica and Rotylenchulus
reniformis occurring together and separately, and
their effect on the host. Indian J. Entomol. 32:
194-200.
44. Santo, G.S., and W.J. Bolander. 1976. Effects of soil
temperature and soil type on the reproduction of
Heterodera schachtii and Meloidigyne hapla on
sugarbeets. J. Nematol. 8: 301-302.
45. Santo, G.S., and W.J. Bolander. 1977. Separate and
concomitant effects of Macroposthonia xenoplax and
Meloidogyne hapla on Concord grapes. J. Nematol.
9: 282-283.
Santo, G.S., and W.J. Bolander. 1979. Interacting
effects of soil temperature and type on reproduction
and pathogenicity of Heterodera schachtii and
Meloidogyne hapla on sugarbeets. J. Nematol. 11:
289-291.
46.


Figure 32
Scanning electron micrograph of Meloidogyne
female dissected anterior region.
cruciani


7
Table 2
Incidence and percentage frequency of occurrence
of plant parasitic nematodes in St. Croix
Nematode genera
Incidence
%
of
Frequency
occurrence
Rotylenchulus reniformis
80
100
Rotylenchulus parvus
6
8
Heliootylenohus dihystera
56
70
Helicotylenchus multicinctus
3
4
Tylenchorhynchus mashhoodi
52
65
Xiphinema americanum
46
58
Pratylenohus pratensis
46
58
Criconemoides citri
12
15
Meloidogyne cruciani n.sp.
11
14
Hemicriconemoides cocophillus
7
9
Hoplolaimus columbus
4
5
Table 3
Incidence and percentage frequency of occurrence
of plant parasitic nematodes in St. Thomas
Nematode genera
Incidence
%
of
Frequency
occurrence
Roty1enahulus reniformis
38
100
Rotylenchulus parvus
2
5
Heliaotylenchus dihystera
28
74
Helicotylenchus multicinctus
1
3
Tylenchorhynchus mashhoodi
17
45
Xiphinema americanum
6
16
Pratylenohus pratensis
5
13
Meloidog-yne cruciani n.sp.
5
13
Hemicriconemoides cocophillus
4
11
Criconemoides citri
3
8


CHAPTER I
SURVEY OF THE PLANT PARASITIC NEMATODES
OF THE U.S. VIRGIN ISLANDS
Introduction
The U.S. Virgin Islands consists of three islands, St.
Croix, St. Thomas and St. John. They are located between
1740' and 1824' latitude north, 6430' and 6504'
longitude west and have an area of 207,199; 77,699; and
51,799 square kilometers, respectively. These islands are
of volcanic origin with elevations up to 366 meters; they
have a tropical trade wind climate, with an average
temperature of 27 C and an average annual rainfall of
965 millimeters.
The tropical and subtropical regions of the world have
climates that will permit year round production of a great
variety of agricultural crops. These continuous crop
productions favor nematode pests, since they are able to
reproduce continuously, increasing their numbers and their
damage. In 1978, I conducted a survey on St. Croix and St.
Thomas Islands to determine the genera of plant parasitic
nematodes present, their relative abundance and geographic
distribution, and the crops with which they were associated.
All cultivated areas and some non-cultivated areas were
sampled on each island. St. John Island was not included
1


45
were transferred to 45% lactic acid, cleaned of debris,
trimmed to the perineal region and mounted in glycerin for
observation. Anterior ends of females were prepared by
fixing and staining whole females in lactophenol-cotton
blue (21), after which the anterior ends were excised and
mounted in glycerin. All type material was prepared using
the same technique as for the female anterior ends.
Photomicrographs of perineal patterns were made with
an automatic 35-mm camera using an interference contrast
system (Nomarski) attached to a compound microscope. In
making the scanning electron microscope (SEM) micrographs,
females were killed and fixed in 2.5% glutaraldehyde
solution with phosphate buffer for 12 hours, transferred to
2% aariium tetroxide for 24 hours at 8 C, dehydrated in an
ethanol series (10-100%) for 15 minutes in each
concentration, dried to critical point, coated with gold,
and examined with a SEM.
A differential host test (59) was conducted by trans
planting seedlings of the following plants into steam-
sterilized, sandy soil contained in 10-cm clay pots and
inoculating the plants with 5000 eggs per pot: sweet corn
(Zea mays L. var. rugosa Bonaf. cv 'Silver Queen'), cotton
Gossypium hirsutum L. cv 'Delta pine'), peanut (Arachis
hypogaea L. cv 'Florunner'), pepper (Capsicum annuum L. cv
'California Wonder'), strawberry (Fragaria ananassa Duch.
cv 'Albritton' and 'Florida 90'), sweet potato [Ipomoea
batatas (L.) Poir. cv 'Allgold' and 'Porto Rico'], tobacco


86
glass cylinder and fixed, dehydrated, critical point dried,
coated, mounted and examined as for the first group.
Results
The first group of females was not satisfactory for
studying the esophageal region. All the internal structures
had been fixed properly but the osmium tetroxide made the
internal contents of the females very brittle and, when
cut with the eye knife, the internal structures crumbled
(Fig. 29).
The second method was successful. By first staining
the females, the esophageal glands could be seen thus
facilitating cutting and removing the cuticle. Once the
cuticle was punctured, the pseudocoelomic fluids flowed
out, the cuticle was removed, exposing the esophageal glands.
The SEM corroborated the presence of two additional lobes
in the esophageal region of M^_ cruciani as seen with the
light microscope (Figs. 30-33).
Discussion
The use of the SEM to observe internal organs of
nematodes requires further study. Most of the SEM studies
of Meloidogyne females have been limited to external fea
tures such as perineal patterns (20, 28, 32, 36, 57, 68) and
to the anterior ends (20, 28). Hogger and Estey (30) used
cryofracturing techniques to observe internal structures


3
flotation technique described by Caveness and Jensen (5).
The nematodes recovered from 100 cm^ of soil were killed in
hot water, fixed and preserved in 4% formalin-2% glycerin,
placed in vials and brought back to the Nematology
Laboratory, University of Florida, Gainesville, Florida,
where the plant parasitic nematodes were identified to genus
and the number per sample determined.
Twenty adult nematodes of each genus were mounted in
2% formalin on glass slides and using an Olympus Vanox
compound microscope with a Nomarski reflected light
differential interference contrast attachment, measurements
and other morphological characters of the nematodes were
recorded and the species determined.
Results
There were nine genera and 11 species of plant
parasitic nematodes recovered from soil associated with 30
different host plants (Table 1). Rotylenchulus reniformis
Linford and Oliveira, 1940 (34), R_;_ parvus (Williams, 1960)
Sher, 1961 (48), Helicotylenchus dihystera (Cobb, 1893)
Sher, 1961 (48) multicinctus (Cobb, 1893) Golden, 1956
(27), Tylenchorhynchus mashhoodi Siddiqui and Basir, 1959
(51), Xiphinema americanum Cobb, 1913 (14), Pratylenchus
pratensis (de Man, 1880) Filipjev, 1936 (25) Criconemoides
citri Steiner, 1949 (58) = Macroposthonia sphaerocephala
(Taylor, 1936) De Grisse and Loof, 1965 (18), Meloidogyne


104
following interactions statistically significant:
Soil by time by treatment; and
Soil by treatment by temperature.
Sampling time had a significant effect on both
treatments, with both soil types (Table 6). Treatment
effect was not significant one month after inoculation
regardless of soil type, but it was significant at three
months in both soil types. Soil type had a significant
effect three months after inoculation with cruciani
alone, but its effect was not significant for any of the
other treatments or sampling times.
Temperature effect was significant with cruciani
alone in the clay soil; it was not significant with the
other treatments (Table 7). Treatment effect was not
significant in the clay soils at the variable temperature
conditions, but was significant in the other interactions.
Regardless of temperature conditions, soil type was
significant with cruciani alone but not significant
when both genera of nematodes were together. Soil type has
a significant effect on M;_ cruciani reproduction, while
temperature does not.


This dissertation was submitted to the Graduate
Faculty of the College of Agriculture and to the
Graduate Council, and was accepted as partial fulfillment
of the requirements for the degree of Doctor of Philosophy.
March 1981
Dean
Dean for Graduate Studies and
Research


Figure 12
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Tylenchorhynchus mashhoodi.


96



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PAGE 129

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PAGE 130

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Figure 15
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Meloidogyne cruciani n.sp.


Figures 20-23
Perineal patterns of Meloidogyne
cruciana n.sp.
20-22) Photomicrographs showing
subcuticular punctations.
23) Scanning electron micrograph.


Picera Pt
Hot any Ft
Intermittent itrtM
1
Cabrita ft
(jJ
U)
0
10000
10000 fwt


80
sedentary parasite feeding habit of Meloidogyne sp. can be
interpreted as one of the most advanced evolutionary states
of parasitism. This high degree of specialization in
feeding habit of these nematodes would undoubtedly require
also a highly specialized digestive system. A five-gland
esophageal condition should aid in the digestive process by
increasing the quantity of digestive enzymes secreted by
those glands. The two extra glands described in this study
have always been found in close association with the dorsal
esophageal gland. Baldwin and Sasser (2) and Eisenback et
al.(19) found that the dorsal gland orifice of various
species of Meloidogyne branched into three channels. That
indicates that species other than M^_ cruciani may have five
glands and that the two extra esophageal glands supplement
secretions by the dorsal esophageal gland and thus aid in
preoral digestion or may serve to stimulate the "nurse"
cells of the plant.
As stated earlier, there are striking differences
between the second stage larvae and the adult females of
Meloidogyne sp. One of these differences is the position
of the excretory pore. In the second stage larvae, it is
usually found posterior to the metacorpus. In the adult
females it is usually found anterior to the metacorpus
adjacent to the stylet knobs. Christie and Cobb (10)
illustrated the excretory pore opposite the metacorpus.
In M_;_ cruciani the excretory pore is opposite the procorpus,
which agrees with the illustrations of Chitwood (7), Bird


105
Table 6
Effects of soil type and sampling time on populations of
Meloidogyne cruciani when inoculated alone and
simultaneously with an equal number of
Rotylenchulus reniformis.
Inoculum1
density
Soil
Sand
type
Clay
Sampling time
Sampling time
1 mo.
3 mo.
1 mo.
3 mo.
(No./pot)
(No./pot)
0/200
261d2
24467a
369d
7505b
200/200
282d
3607c
562d
3431c
'inoculum density of Rotylenchulus reniformis/Meloidogyne
cruciani, respectively.
^Means followed by the same letter are not significantly
different at 5% level according to Tukey's HSD comparison
of means. Significance applies to vertical and horizontal
columns.


61
tip of head to posterior extremity of glands averaging 46.4%
of the total body length. Metacorpus well developed with
well sclerotized valve. Esophageal glands contained in
three distinct nucleated lobes, each with a smaller
satellite nuclear body. Excretory pore position variable;
always posterior to esophago-intestinal valve. Hemizonid
2-4 annules anterior to excretory pore. Excretory duct
long, terminating in a sac-like gland. Lateral fields
originate as two lines one stylet length posterior to base
of knobs, becoming four near metacorpus. Two inner lateral
lines terminate at phasmids and outer two terminate
posteriorly. Genital primordium in the two-cell stage,
seen easily. Rectum dilated. Phasmids small and difficult
to see; one anal body width posterior to level of anus.
Tail gradually tapering, with annules disappearing near
hyaline area. Tail terminus notched, with smooth, bluntly
conoid tip.
Holotype. (whole female): Originally recovered in
tomato roots from the Agricultural Community Gardens, St.
Croix, U.S. Virgin Islands in September 1977. It was grown
subsequently on 'Rutgers' tomato in an isolated green
house. [Slide T-333t, USDA Nematode Collection, (USDANC)),
Beltsville, Maryland, USA.
Allotype. (male): Isolated from 'Rutgers' tomato
roots cultured in a greenhouse and established from type
locality. Slide T-334t, USDANC, Beltsville, Maryland, USA.


102
While R^ reniformis alone had a significant increase within
soil types at three months sampling time, in the presence
of cruciani, it was not significant.
Table 4
Effects of soil type and time of sampling
on populations of Rotylenchulus reniformis when inoculated
alone and simultaneously with an equal number
of Meloidogyne cruciani.
Soil type
Sand
Clay
Sampling time Sampling time
Inoculum1
density 1 mo. 3 mo. 1 mo. 3 mo.
(No./pot) (No./pot)
200/0
4021 2
a
94297,
b
4845d
226000_
u
200/200
4677d
51201c
4608d
40483c
inoculum density of Rotylenchulus reniformis/Meloiodgyne
cruciani, respectively.
2Means followed by the same letter are not significantly
different at 5% level according to Tukey's HSD comparison
of means. Significance applies to vertical and horizontal
columns.
Treatment effect was significant in both soil types
regardless of temperature conditions (Table 5). Temperature
conditions had no significant effect on the treatments in


116
59. Taylor, A.L., and J.N. Sasser. 1978. Biology,
identification and control of root-knot nematodes
(Meloidogyne species). North Carolina State Uni-
versity Graphics. Pages 101-103.
60. Thomas, R.J., and C.A. Clark. 1980. Interactions
between Meloidogyne incognita and Rotylenchulus
reniformis on sweetpotato. J. Nematol. 12: 239.
61. Triantaphyllou, A.C., and H. Hirschmann. 1960.
Post-infection development of Meloidogyne incognita
Chitwood, 1949 (Nematoda: Heteroderidae). Ann.
Inst. Phytophathol. Benaki, N.S. 3: 3-11.
62. Turner, D.T., and R.A. Chapman. 1972. Infection of
seedlings of alfalfa and red clover by
concomitant populations of Meloidogyne incognita
and Pratylenchus penetrans. J. Nematol" 31
280-286.
63. U.S. Virgin Islands and U.S. Dept, of Agrie. 1973.
U.S. Virgin Islands project plan. Resource
community and development project prepared by the
Government of the Virgin Islands of the U.S. and
the U.S. Dept, of Agrie, and other cooperating
agencies. 74 pp.
64. Vaishnav, M.U., and C.L. Sethi. 1979. Pathogenicity
of Meloidogyne incognita and Tylenchorhynchus
vulgaris on ba~jra and their interrelationship.
Indian J. Nematol. 8: 1-8.
65. Van Gundy, S.D., and J.D. Kirkpatrick. 1975.
Nematode-nematode interactions on tomato. J.
Nematol. 7: 330-331.
66. Wallace, H.R. 1966. Factors influencing the
infectivity of plant parasitic nematodes. Proc.
R. Soc., Series B, 164: 592-614.
67. Winoto, S.R., and T.K. Lim. 1972. Interaction of
Meloidogyne incognita and Rotylenchulus reniformis
on tomato. Malaysian AgricT Res. H fT^13.
Yik, C., and W. Birchfield. 1978. Scanning electron
microscope of perineal patterns of three species
of Meloidogyne. J. Nematol. 10: 118-122.
68.


58
(10.6 ym + 0.2); stylet base to anterior end: 14.3-17.6 ym
(15.2 ym + 0.4); stylet knob height: 1.1-1.6 ym (1.4 ym +
0.1); stylet knob width: 2.1-2.7 ym (2.3 ym + 0.1); dorsal
esophageal gland orifice to base of stylet knobs: 3.2-3.9
ym (3.5 ym + 0.1); center of metacorpus valve to anterior
end: 51.7-61.9 ym (57.8 ym + 1.4); distance from cardia to
anterior end: 69.5-86.7 ym (76.3 ym + 2.1); distance from
posterior end of glands to anterior end: 190.4-250.4 ym
(202.0 ym + 6.4); excretory pore to anterior end: 74.6-
103.2 ym (88.1 ym + 3.4); genital primordium to posterior
end: 148.2-175.5 ym (163.5 ym + 4.3); phasmid to posterior
end: 34.9-43.8 ym (39.2 ym + 1.2); tail length (anus to
posterior end): 41.3-51.7 ym (46.6 ym + 1.3); tail width
(at anus): 9.8-13.0 ym (11.2 ym + 0.4); a: 22.9-29.8
(25.4 + 0.8); b: 5.0-7.1 (5.8 + 0.2); b': 1.8-2.4 (2.2 +
0.1); c: 8.6-10.5 (9.4 + 0.3); c': 3.7-4.6 (4.2 + 0.1);
0 (distance from dorsal esophageal gland orifice to base
of stylet knobs, expressed as % of stylet length): 28.9-
37.9 (33.1 + 0.9) .
Description. Body vermiform, tapering slightly
anteriorly and much more posteriorly (Fig. 25A). Head
offset slightly with one annule; head cap with weakly
visible cephalic framework with lateral sectors larger than
ventral or dorsal sectors. Labial or cephalic sensillae not
observed. Stylet robust, rounded knobs slanting poste
riorly. Cephalids not observed. Amphidial glands prominent,
posterior to stylet knobs. Esophagus extremely long, from


1
2
J
4 n
10000
20000 Tmmt


94


Figure 11
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Helicotylenchus spp.


99
seedlings were one month old, they were transplanted to
15-cm clay pots containing either sterile clay-loam (Esto
series) soil or sandy (Arrendondo fine sand) soil. Each
soil type was inoculated as follows: 200 reniformis/
0 cruciani, 0 reniformis/200 M, cruciani, and 200
R. reniformis/200 M. cruciani. The larval stages were
introduced at random in holes about 5 cm from the base of
each seedling. The inoculated seedlings were placed at
two different temperature conditions: at 30 + 1 C in a
constant temperature chamber or at 22 + 2 C in a growth
room. The plants were watered daily and fertilized once a
week with 100 ml of a solution containing 390 ppm of
£
Nutrisol (12-10-20), a commercially obtained fertilizer
solution. Nine replicates of each treatment were sampled
at one and three months after inoculation.
Inoculum was obtained as follows: Roots of tomato
plants heavily infested with cruciani were washed free
of soil under a gentle stream of water. The roots were
blotted dry, placed in a petri dish in water, set on the
stage of a dissecting microscope and egg masses dislodged
from the roots with a dissecting needle. Individual egg
masses were transferred to a BPI dish containing distilled
water, teased apart with a dissecting needle to expose the
eggs and left overnight at 30 C. The next morning freshly
hatched second stage larvae were transferred, using a
capillary pipette apparatus, in lots of 100 to vials
containing distilled water. Immature females of R.


BIOGRAPHICAL SKETCH
Roberto Garcia Martinez was born in Quepos, Costa
Rica, on 18 May 1946. He graduated from Boca Vieja
Elementary School, and, in 1966, from the Instituto de
Alajuela Miguel Obregon High School. In 1969, he
completed a three year program at the Escuela Agricola
Panamericana in Tegucigalpa, Honduras, receiving his
"Agronomo" degree. In 1972 and 1976, he received the
degrees of Bachelor of Science in soils and Master of
Science in nematology, respectively, from the University
of Florida. In the Winter of 1977, he began studies
toward the degree of Doctor of Philosophy in nematology
at this same University. He is a member of the Society
of Nematologists and the Organization of Tropical American
Nematologists.
After being awarded the Bachelor of Science degree,
he returned to his native country, Costa Rica, to work at
the Escuela Technica Agricola as a teacher and field work
supervisor. While employed at that school, he taught
courses in soil science, plant propagation, organic and
inorganic chemistry and his duties as a field work
supervisor consisted of instructing students in the
production of agronomic, fruit and vegetable crops.
117




49


c
I
-J




8
Discussion
Rotylenchulus reniformis was found to be more
widely distributed than parvus. Dasgupta et al. (17)
gave an extensive list of plants with which R^_ reniformis
has been found associated and the localities from which they
were reported. They found R^ reniformis associated with
banana, citrus, corn, papaya, sugarcane, sweet potato and
tomato. These crops also were found to be common hosts of
R. reniformis in St. Croix and St. Thomas (Table 1).
Helicotylenchus dihystera was found to be the most
widely distributed species of the genus. Sher (50) gave an
extensive list of plants with which dihystera has been
found associated and the localities from which they were
reported. He found dihystera associated with banana,
citrus, corn, mango, onion, papaya, pineapple and sugarcane.
These crops also were found to be common hosts of H.
dihystera in St. Croix and St. Thomas (Table 1).
The occurrence of Hoplolaimus columbus on golf courses
associated with bermuda grass indicates that these genera
of nematodes might have been introduced to St. Croix with
the turfgrass.
During this survey, a new species of the genus
Meloidogyne was discovered. The complete description and
a host differential test of the new species, named
Meloidogyne cruciani, are reported in Chapter II.


Figure 16
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Hemicriconemoides cocophillus.


LIST OF TABLES
Table Page
1 Plants from which soil samples were
taken through the root zones and
nematodes recovered on St. Croix and
St. Thomas, U.S. Virgin Islands 4
2 Incidence and percentage frequency of
occurrence of plant parasitic nematodes
in St. Croix 6
3 Incidence and percentage frequency of
occurrence of plant parasitic nematodes
in St. Thomas 6
4 Effects of soil type and time of sampling
on populations of Rotylenchulus renifopnis
when inoculated alone and simultaneously
with an equal number of Meloidogyne
cruciani 102
5 Effects of soil type and temperature on
populations of Rotylenchulus reniformis when
inoculated alone and simultaneously with an
equal number of Meloidogyne cruciani .... 103
6 Effects of soil type and sampling on
populations of Meloidogyne cruciani when
inoculated alone and simultaneously with an
equal number of Rotylenchulus reniformis . 105
7 Effects of soil type and temperature on
populations of Meloidogyne cruciani when
inoculated alone and simultaneously with an
equal number of Rotylenchulus reniformis . 106
v


103
either soil type. Soil type was significant with R,
reniformis alone but not significant when both R^_ reniformis
and M^ cruciani were together.
Table 5
Effects of soil type and temperature on populations
of Rotylenchulus reniformis when inoculated alone and
simultaneously with an equal number of Meloidogyne cruciani.
Inoculum1
density
Soil
Sand
type
Clay
Temperature
Temperature
22 C
30 C
22 C
30 C
(No./pot)
(No./pot)
200/0
54413b2
43906,
b
120364
a
110481
cl
200/200
30065c
25 812c
20870c
24221c
inoculum density of Rotylenchulus reniformis/Meloidogyne
cruciani, respectively.
2Means followed by the same letter are not significantly
different at 5% level according to Tukey's HSD comparison
of means. Significance applies to vertical and horizontal
columns.
Meloidogyne cruciani
The results of the analysis of variance of the two
treatments containing M^ cruciani (0/200 and 200/200
Rotylenchulus reniformis/Meloidogyne cruciani) showed the


4 HUM
O
OOOO
20000 fMt


Figure 3
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Tylenchorhynchus mashhoodi.


Figure 27
Third and fourth stage larvae
of Meloidogyne cruciani n.sp.
A-B) Third stage larvae encased
in the second stage cuticle.
C) Fourth stage larva encased
in the second and third stage
cuticles.


Figure 24
Male of Meloidogyne cruciani n.sp.
A) Entire specimen (curvature of
specimen for convenience in
illustrating).
B) Face view showing cephalic
framework.
C) Anterior portion.
D) Tail (lateral view).
E) Lateral field.


88


55
length: 28.7 yin; gubernaculum: 8.1 ym; phasmid to
posterior end: 18.4 ym; a: 48.0; c: 151.3; c': 0.4;
0: 21.5; T% 64.9%.
Description. Body long, vermiform, tapering at both
ends (Fig. 24A). Head offset with two annules and distinct
head cap. Labial or cephalic sensillae not observed.
Cephalic framework with lateral sectors larger than ventral
or dorsal sectors; ends of framework slightly forked when
viewed laterally. Stylet robust, with rounded knobs.
Amphidial glands prominent posterior to stylet knobs.
Cephalids not observed. Metacorpus poorly developed,
slightly larger than procorpus with well sclerotized valve.
Esophageal glands consisting of three distinct nucleated
lobes. Excretory pore prominent (139.1 ym from anterior
end). Hemizonid 3.5 annules anterior to excretory pore.
Excretory duct long, terminating in a sac-like gland.
Lateral fields begin anteriorly as two lateral lines
opposite stylet knobs and become four near metacorpus.
There is anastamosis of the lateral lines in the posterior
end of the body. Testis predominantly one, two occasionally.
Spicules slightly arcuate, their tips rounded (Fig. 24D).
Gubernaculum with fine serrations on the cuneus. Phasmids
5.9 ym anterior to cloaca.
Second stage larvae. (20): Length: 418.6-479.8 ym
(mean 435.3 ym, 95% confidence interval + 8.7 ym); width:
14.6-18.7 ym (17.2 ym + 0.5); stylet length: 9.8-12.1 ym


LIST OF FIGURES
Figure Page
1 St. Croix, U.S. Virgin Islands. Shading
indicates total area sampled.
Rotylenchulus spp. were found in all of
the above areas 11
2 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Helicotylenchus spp 13
3 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Tylenchorhynchus mashhoodi 15
4 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Xiphinema
americanum 17
5 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Pratylenchus pratensis 19
6 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Criconemoides citri 21
7 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Meloidogyne
cruciani n.sp 23
8 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by
Hemicriconemoides cocophillus 25
9 St. Croix, U.S. Virgin Islands. Shaded
areas were found infested by Hoplolaimus
columbus 27
10 St. Thomas, U.S. Virgin Islands. Shading
indicates total area sampled.
Rotylenchulus spp. were found in all of the
above areas 29
vi


19


Figure 30
Scanning electron micrograph of Meloidogyne
female dissected anterior region.
cruciani


9
The extensive distribution of R^_ reniformis throughout
these islands indicates favorable environmental conditions
for the growth, development and reproduction of these
nematodes. The widespread distribution of this nematode
may be due to the extensive and intensive production of
sugarcane on both islands in the past (63). Contrary to
this, the low recovery rate of RL_ cruciani indicates the
presence of biotic or abiotic factors that are restricting
further establishment of these nematodes. With this in
mind, experiments were initiated to determine whether
certain factors favored Rotylenchulus reniformis and were
detrimental to Meloidogyne cruciani. These experiments
are reported in Chapter V.


Figure 17
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Criconemoides citri.


47
height: 2.7 ym; stylet knob width: 4.4 ym; dorsal gland
orifice to base of stylet knobs: 4.1 ym; excretory pore to
anterial end: 31.7 ym; center of median bulb to anterior
end: 93.3 ym; vulva slit length: 23.8 ym; vulva slit to
anus: 20.0 ym; interphasmidial distance: 31.7 ym.
Description. Females white, pear-shaped to globular,
without prominent posterior protuberance (Fig. 18B). Neck
tapers, curving gently (Figs. 18B, 19A). Head offset
slightly with labial cap and one or two cephalic annules.
Labial or cephalic sensillae not observed. Amphidial
openings oval, inconspicuous, obscured by labial cap.
Cephalic framework with lateral sectors larger than ventral
or dorsal sectors. Stylet robust, with rounded knobs.
Excretory pore about one stylet length from base of stylet
knobs, variable in exact position; excretory duct seen
easily throughout anterior region terminating in a
uninucleate gland (Fig. 28C). Esophageal lumen between base
of stylet knobs and valve of median bulb well sclerotized
with an average width of 2.4 ym. Prominent metacorpus with
strongly sclerotized valve. Esophageal glands consisting
of five distinct nucleated lobes (Fig. 19A, C). One lobe
always larger than the other four. Perineal pattern (Figs.
18A, 20-23) with subcuticular punctations (stippling) almost
surrounding the anus on lateral and posterior sides (Figs.
18A, 20-23). Striae deep, wavy, sometimes broken. Lateral
field fairly deep with distinct phasmids. Phasmidial ducts
often visible. Vulva lips faintly serrated, margins with
very fine striae.


101
1) two types of soil (sand and clay);
2) two sampling times (one and three months after
inoculation);
3) two different temperature conditions (22 and 30 C);
and
4) three different inoculum levels (0/200, 200/0, and
200/200Rotylenchulus reniformis/Meloidogyne
cruciani).
Rotylenchulus reniformis
The results of the analysis of variance of the two
treatments containing R^ reniformis (200/0 and 200/200)
showed the following interactions statistically
significant:
Interaction:
Soil by time by treatment; and
Soil by treatment by temperature.
At one month sampling time, there was no significant
effect of the treatments in either sand or clay, while at
three months sampling time, the treatment effect was
significant in both sand and clay (Table 4). The treatments
were significant in time in both sand and clay. At one
month sampling time, soil type had no significant effect on
the population development of R^_ reniformis alone or with
concomitant populations of cruciani. There was a
significant increase in population growth at three months
sampling time in both soil types with both treatments.


Figure 29
Scanning electron micrograph of Meloidogyne cruciani
female internal structures crumbled.


11
12
13
14
15
16
17
18
19
20
21
22
23
31
33
35
37
39
41
43
49
51
53
53
53
53
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Helicotylenchus spp
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Tylenchorhynchus mashhoodi
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by Xiphinema
americanum
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Pratylenchus pratensis
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by Meloidogyne
cruciani n.sp
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Hemicriconemoides cocophillus
St. Thomas, U.S. Virgin Islands. Shaded
areas were found infested by
Criconemoides citri
A) Drawings of perineal patterns of
Meloidogyne cruciani n.sp. B) Outlines of
females in varying sizes and shapes . .
A) Anterior region of female. B) Face view
of showing cephalic framework. C) Vari
ations in size and shape of esophageal
glands
Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograph showing sub
cuticular punctations
Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograpn snowing sub
cuticular punctations
Perineal patterns of Meloidogyne cruciani
n.sp. Photomicrograph showing sub
cuticular punctations
Perineal patterns of Meloidogyne cruciani
n.sp. Scanning electron micrograph . .
vii


66
More recently, Bird (3), Triantaphyllou and Hirschmann
(61) and Siddiqui and Taylor (52) studied the morphology
and developmental stages of females of M^_ javanica, M.
incognita and M_^ nassi, respectively. They all agree with
the early studies of Nagakura and report the presence of
third and fourth larval stages.
The work reported herein is an attempt to determine
the initiation, development and formation of morphological
structures in the developmental stages of Meloidogyne
cruciani n.sp. with emphasis on the esophageal region,
excretory system and genital region.
Materials and Methods
Seeds of 'Rutgers' tomato (Lycopersicon esculentum
Mill.) which is susceptible to M^ cruciani, were germinated
in sterile vermiculite at 30 C in a water bath. When
seedlings were two weeks old, they were removed from the
vermiculite, their roots washed and trimmed to 1 cm in
length and the seedlings placed in sterile water for two
days. These seedlings were exposed to freshly hatched
second stage larvae for 24 hours. (Larvae were obtained by
placing egg masses in distilled water at 30 C overnight.)
After the 24 hour exposure period, roots of the seedlings
were washed to remove any larvae that had not penetrated
and the seedlings were transplanted into sterile white sand
3
m 33 cm plastic cups with a small hole punched in the
bottom for drainage.


115
47. Sharma, N.K., and C.L. Sethi. 1978. Interaction
between Meloidogyne incognita and Heterodera
ca jani on cowpea. Indian J. Nematol. G~: 1-12.
48. Sher, S.A. 1961. Revision of the Hoplolaiminae
(Nematoda) I. Classification of nominal genera and
nominal species. Nematologica 6: 155-169.
49. Sher, S.A. 1963. Revision of the Hoplolaiminae
(Nematoda) II. Hoplolaimus Daday, 1905 and
Aorolaimus n.gen. Nematologica 9: 267-295.
50. Sher, S.A. 1966. Revision of the Hoplolaiminae
(Nematoda) VI. Helicotylenchus Steiner, 1945.
Nematologica 12: 1-56.
51. Siddiqui, M.R., and M.A. Basir. 1959. On some plant
parasitic nematodes occurring in South India, with
the description of two new species of the genus
Tylenchorhynchus Cobb, 1913. Proc. 46th Meet.
Indian Sci. Congr. Part IV (Abstr.), p. 35.
52. Siddiqui, I.A., and D.P. Taylor. 1970. The biology
of Meloidogyne naasi. Nematologica 16: 133-143.
53. Sikora, R.A., R.B. Malek, D.P. Taylor, and D.I.
Edwards. 1979. Reduction of Meloidogyne naasi
infection of creeping bentgrass by
Tylenchorhynchus agri and Paratrichodorus minor.
Nematologica 25: 179-183.
54. Sikora, R.A., D.P. Taylor, R.B. Malek, and D.I.
Edwards. 1972. Interaction of Meloidogyne naasi,
Pratylenchus penetrans, and Tylenchorhynchus agrT
on creeping bentgrass. J. Nematol. 4:162-165.
55. Singh, N.D. 1976. Interaction of Meloidogyne
incognita and Rotylenchulus reniformis on soybean.
Nematropica 6: 76-81.
56. Slabaugh, W.R., and R.E. Adams. 1973. Interactions
of Meloidogyne incognita, Helicotylenchus nannus,
and Pratylenchus penetrans with breeding lines of
Lycopersicon esculentum in agar culture.
Phytopathology 63: 805.
57. Spaull, V.W. 1977. Meloidogyne propara n.sp.
(Nematoda: Meloidogynidae) from Aldabra Atoll,
western Indian Ocean, with a note on M^_ javanica
(Treub). Nematologica 23: 177-186.
Steiner, G. 1949. Plant nematodes the grower should
know. Proc. Soil Crop Sci. Soc. Florida 4-B: 72-117.
58.


Figure 25
Larvae of Meloidogyne cruciani n.sp.
A) Entire specimen (curvature of specimen
for convenience in illustrating).
B) Face view showing cephalic framework.
C) Tails (lateral view).
D) Lateral field at tail region.
E) Tail (ventral view).


1
Dck Pt
20000 rt


25


CHAPTER IV
ESOPHAGEAL GLANDS OF ADULT FEMALES
OF Meloidogyne cruciani n.sp.
Introduction
Under the light microscope, the esophageal glands of
Meloidogyne cruciani appear composed of five individual
lobes. This is a deviation from the typical tylenchoid
esophagus which is considered to be composed of only
three esophageal glands. Previous original descriptions
of Meloidogyne spp. illustrate the esophageal region as
one, two or three lobes with three nuclei. Bird (3)
reported that the esophageal region in the developmental
stages of M^ javanica was composed of only one gland. This
differs from Chitwood's (7) original description of M.
javanica in which he reported three glands.
The purpose of this study was to develop a fixation
technique that could be used to study internal structures
of nematodes with the scanning electron microscope (SEM)
and to corroborate the existence of five esophageal lobes
that can be seen with the light microscope.
Materials and Methods
Egg laying females were dissected from galled 'Rutgers'
tomato roots (Lycopersicon esculentum Mill.) in 2% formalin
84


CHAPTER III
POST-INFECTION DEVELOPMENT OF FEMALES OF Meloidogyne
cruciani n.sp 65
Introduction 65
Materials and Methods 66
Results 67
Discussion 78
CHAPTER IV
ESOPHAGEAL GLANDS OF ADULT FEMALES OF Meloidogyne
cruciani n.sp 84
Introduction 84
Materials and Methods 84
Results 86
Discussion 86
CHAPTER V
INTERACTION OF Rotylenchulus reniformis AND
Meloidogyne cruciani ON TOMATO 98
Introduction 98
Materials and Methods 98
Results 100
Ro tylenchulus reniformis 101
Meloidogyne cruciani 103
Disucssion 107
LITERATURE CITED Ill
BIOGRAPHICAL SKETCH 117
IV


Figure 6
St. Croix, U.S. Virgin Islands.
Shaded areas were found infested
by Criconemoides citri.


rrvtor ikatad
20000 rmmt


75
In the early stages of the adult female, shortly
after the fourth molt (Fig. 28A) and while still enclosed
in the second, third and fourth larval cuticles, the
stylet could be seen. The esophagus appeared typical of
adult females with the procorpus and metacorpus enlarged
and prominent. The lumen of the esophagus and valve of the
metacorpus were reformed and appeared faintly at first.
Inside the nuclear envelope, the chromocenters had
disappeared but the nucleoli remained prominent. On the
old cuticle of the second stage larva the excretory pore
could be seen below the metacorpus with its duct leading
anteriorly where it appeared to penetrate the female body
opposite the procorpus. From here the excretory duct was
seen leading posteriorly, as normally found in adult
females. At the posterior end, the gonads continued to
elongate. The uterus, vagina and vulva were prominent,
and the perineal pattern could be detected. Nineteen days
after inoculation all organs of the adult female were
developed, and molting of the second, third and fourth
cuticles occurred simultaneously (Fig. 28B).
Immediately after molting, feeding was resumed and the
female enlarged from a sausage-shape (Fig. 28C) to the pear-
shape typical of the genus (Fig. 28D). The stylet was
robust and well-developed. The enlarged procorpus had a
prominent lumen. The massive metacorpus had a strong well-
sclerotized valve. The esophageal glands consisted of five
distinct lobes with prominent nuclei and nucleoli. The


78
excretory pore was located opposite the procorpus with the
duct leading posteriorly to a unicellular gland. The
gonads elongated with one branch extending anteriorly close
to the esophageal region. The rectal glands were large,
with prominent nuclei and nucleoli.
Discussion
The post-infection development of Meloidogyne cruciani
agrees in general with the studies done by Bird (3), Tri-
antaphyllou and Hirschmann (61) and Siddiqui and Taylor (52).
The first noticeable changes that the post-infection
larvae underwent were in body length and in the esophageal
region (Fig. 26B). The esophagus increased in volume and the
body around the esophageal region had a noticeable increase
in width. These changes in the esophageal region may have
been due to the intense feeding activity of the second
stage larvae. The post-infection stage had a slight decrease
in body length when compared to the pre-infective stage.
Bird (3) also found a decrease in size of the infective
second stage larvae after root penetration; he attributed
the decrease in size to the depletion of food reserves
used during penetration and migration into the roots.
Triantaphyllou and Hirschmann (61) reported the shape
of the genital primordium (V-shape for females; straight
cylindrical shape for males) could be used to differentiate
sex in the early second stage larva. I found that sex could


Figure 13
St. Thomas, U.S. Virgin Islands.
Shaded areas were found infested
by Xiphinema americanum.


63
report one single esophageal lobe with three nuclei (23),
but post-infection studies of Meloidogyne naasi (52) and
M. incognita (61) illustrate the parasitic second stage
larvae as having 3-lobed esophageal glands. 3) Inside
each nucleus, a small chromocenter is present beside the
nucleolus in each lobe of the esophagi of the second stage
larvae (Figs. 25A). This has not been reported for other
species of this genus. 4) The esophageal glands of the
females (Fig. 19A, C) consist of five separate and distinct
lobes. 6) A uninucleate gland (renette-type) excretory
system. 7) The gubernaculum of the males (Fig. 24D) has
fine serrations on the cuneus; this condition is also
present in males of Verutus volvingentis (23).
In the host-differential test, peanut, strawberry, and
cotton were not hosts. Tomato, watermelon, sweet potato,
tobacco, corn and pepper were hosts. Based on these
results, Meloidogyne cruciani seems to have a similar host
range as that of M^_ incognita Race 2. One other plant,
cabbage (Brassica olercea L. cv 'Greenback') also was found
to be a suitable host.
Type host and type habitat. tomato, Lycopersicon
esculentum Mill., roots
Type locality. Agricultural Community Gardens, College
of the U.S. Virgin Islands, St. Croix, U.S. Virgin Islands