Plant parasitic nematodes of the U.S. Virgin Islands with the description, life cycle and morphology of Meloidogyne cruc...

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Material Information

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
Physical Description:
xi, 117 leaves : ill. ; 28 cm.
Language:
English
Creator:
Garcia Martinez, Roberto, 1946-
Publication Date:

Subjects

Subjects / Keywords:
Plant nematodes -- Virgin Islands of the United States   ( lcsh )
Nematode diseases of plants -- Virgin Islands of the United States   ( lcsh )
Meloidogyne cruciani   ( lcsh )
Rotylenchulus reniformis
Entomology and Nematology thesis Ph. D
Dissertations, Academic -- Entomology and Nematology -- UF
Genre:
bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 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
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 020730768
oclc - 07518991
System ID:
AA00022195:00001

Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
        Page vii
        Page viii
    Abstract
        Page ix
        Page x
        Page xi
    Chapter 1. Survey of the plant parasitic nematodes of the U.S. Virgin Islands
        Page 1
        Page 2
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        Page 42
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    Chapter 2. Meloidogyne cruciani n.sp., a root-knot nematode from St. Croix, U.S. Virgin Islands
        Page 44
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        Page 46
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    Chapter 3. Post-infection development of females of Meloidogyne cruciani n.sp.
        Page 65
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        Page 68
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        Page 70
        Page 71
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        Page 82
        Page 83
    Chapter 4. Esophageal glands of adult females of Meloidogyne cruciani n.sp.
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
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        Page 91
        Page 92
        Page 93
        Page 94
        Page 95
        Page 96
        Page 97
    Chapter 5. Interaction of Rotylenchulus reniformis and Meloidogyne cruciani on tomato
        Page 98
        Page 99
        Page 100
        Page 101
        Page 102
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    Literature cited
        Page 111
        Page 112
        Page 113
        Page 114
        Page 115
        Page 116
    Biographical sketch
        Page 117
        Page 118
        Page 119
Full Text












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


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7*) **













I



4

~74~



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





'7,















( 461.







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/









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.