Taxonomy and biology of Verutus volvingentis N. Gen. N. Sp. (Tylenchida-Nemata)

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Title:
Taxonomy and biology of Verutus volvingentis N. Gen. N. Sp. (Tylenchida-Nemata)
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xi, 101 leaves : ill. ; 28 cm.
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Esser, Robert Paul, 1924-
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Thesis--University of Florida.
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Includes bibliographical references (leaves 90-99).
Statement of Responsibility:
by Robert Paul Esser.
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Vita.

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TAXONOMY AND BIOLOGY OF Verutus volvingentis
N. GEN. N. SP. (TYLENCHIDA-NEMATA)








BY

ROBERT PAUL ESSER


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


1980















ACKNOWLEDGEMENTS


The author is deeply grateful to the chairmen of his

supervisory committee, Dr. V. G. Perry and Dr. A. C. Tarjan,

who have given considerable time and immeasurable assistance

during the term of this study.

Gratitude is also expressed to Dr. R. A. Dunn and

Dr. D. F. Rothwell for invaluable assistance and encourage-

ment, while serving as members of my supervisory committee.

A special debt of thanks must also go to Dr. G. C.

Smart, Mr. A. L. Taylor, and Dr. K. R. Langdon for sugges-

tions and assistance pertinent to this study.

Thanks are also given to Mr. W. W. Smith who origi-

nally found the new nematode, and provided much data and

material essential to expediting the objectives of this

study.

I am also deeply grateful to Agricultural Commissioner

Doyle Conner, and H. L. Jones, director of the Division of

Plant Industry, for encouragement and very generous vouch-

safement in this endeavor.

Finally, I am very appreciative for considerable en-

couragement and forebearance from my wife Hannelore in this

term of trial.















TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS .. . ii

TABLE OF CONTENTS . ... iii

LIST OF TABLES . .. ... v

LIST OF FIGURES . ... vi

ABSTRACT . . x

INTRODUCTION ... . ... 1

HISTORY . . .. 2

SECTION I
TAXONOMY AND SYSTEMATICS . 3

Taxonomic Position of the New Genus 3

Taxonomy of the New Genus . 17

Anatomy .. . 36

SECTION II
REPRODUCTIVE DEVELOPMENT . .. 45

Early Development . 45

Male Development .. . 47

Female Development . .. 50

SECTION III
HOST-PARASITE RELATIONSHIPS . .. 54

Methods . .. 54

Behavior Studies . .. 56


iii













SECTION IV
HOST PLANT INVESTIGATIONS .

Host Plant . .

Host Habitat . .

Host Testing . .

Host Symptoms . .

Pathogenicity . .

Distribution of Verutus volvingentis
in Florida . ...

SECTION V
BIOLOGICAL CONTROL INTERACTIONS .

LITERATURE CITED . .

BIBLIOGRAPHY . .

BIOGRAPHICAL SKETCH . .


Page


. 70

. 70

. 70

. 72

.. 76

. 78


. 83


87

90

95

100
















LIST OF TABLES


Page


Table


1. Comparative distinguishing characteristics
of females in genera of Heteroderidae .

2. Comparative distinguishing characteristics
of males contained in the Heteroderidae .

3. Male development . .

4. Number of fully developed eggs detected
in 123 mature females . .

5. Survival stage and numbers of nematodes
recovered before and after a longivity test

6. Larval hatch from eggs kept in fallow
soil 3 years . .

7. Phytoparasitic nematodes found in soil
associated with roots of buttonweed .

8. Verutus volvingentis host testing results

9. Effect of Verutus volvingentis on foliage
and seed pod production of inoculated
plants . . .

10. Nematode population density in treated
and untreated soil . .

11. Areas in Florida sampled for Verutus
volvingentis . .

12. Plants examined for the new nematode
in the Florida survey . .


. 49


. 66


. 67


. 68


. 73

. 74



. 81


S. 81


. 84


. 85
















LIST OF FIGURES


Figure

1. Meloidogyne sp. showing spheroid shape, and
terminal vulva of a mature female .

2. Meloidodera floridensis . .

3. Verutus n. gen. female in root tissue with
a single deposited egg . .

4. Cryphodera eucalypti . .

5. Hypsoperine graminus . .

6. Heteroderidae male tail types .

7. A comparison of female appearances in three
subfamilies. A) Verutinae, B) Nacobbinae,
C) Rotylenchulinae . .

8. First-stage larva of Verutus volvingentis
n. gen., n. sp . .

9. A comparison of first and second-stage larva
esophageal glands in the Heteroderidae .

10. Water agar en face method . .

11. Verutus volvingentis n. gen. n. sp.
Mature female . .

12. Verutus volvingentis n. gen. n. sp.,
female body shapes . .

13. Vestigial larval tail tip on posterior
area of a mature female . .

14. En face presentation of a mature female

15. Mature male of Verutus volvingentis
n. gen. n. sp. . .

16. Verutus volvingentis male en face view .

17. Female tail area showing lateral field
irregularities . .

vi


Page


S 5

5


5
. 5


. 5

. 10

. 10

. 11


S. 21


S. 22


. 23

. 23


S. 25

. 28


S. 28











Figure

18. Telorhabdions of a mature female posterior
view . . .

19. Posterior region of mature females .

20. Anus (left) and rectum of a mature female

21. Excreta exuded from the anus of a
mature female . .

22. Vulva and vulva muscles of a mature female

23. Lateral views of an unprolapsed (left) and
prolapsed (right) vagina of mature females

24. Uterus of a mature female .

25. Male tail showing tubus . .

26. Male tail in a ventral view .

27. Larval tail shapes . .

28. Ova . . .

29. Anterior portion of third-stage larval male

30. Rectal musculature . .

31. Anterior nervous system in a first-stage
larva . . .

32. Ventral nerve cord in the area of the
genital primordia . .

33. Nerves in the tail of a first-stage larva

34. Crystalline layer . .

35. Gubernaculum . .

36. Larvae forced from egg by applying
cover slip pressure . .

37. Spicular primordia cells in the cloacal
area of an early second-stage male .

38. Male reproductive system development .

39. Early third-stage female gonad .


Page


. 28

. 28

* 28


. 29

. 29


. 30

. 31

. 31

. 34

. 34

. 37

37

. 40


. 40


. 42

. 42

. 43

. 46


. 46


. 46

. 48

. 51


vii











Figure

40. Vaginal development of a late third-
stage female . .

41. Virgin female with gonad development
complete . . .

42. Final female ecdysis . .

43. Mature female . .

44. Anatomy of the female reproductive system
from vulva to uteri . .

45. A root map . .

46. Mode of root entry by larvae .

47. Larval head in contact with middle lamella

48. A mature female in a shallow, hand-cut
epidermal section . .

49. Tissue discoloration . .

50. Large lesion occupied by 4 larvae .

51. Attack sites . .

52. Spheroid bodies attached to anterior
region of a mature female .

53. Appearance of a single spheroid body .

54. Exudates on the anterior end of
Meloidodera floridensis . .

55. Female containing 7 eggs . .

56. Diodia virginiana in flower .

57. A mat of buttonweed mixed with
herbaceous plants . .

58. Paynes Prairie, Gainesville, Florida .

59. Young female in root, showing large
dark lesion at feeding site .


60. Appearance of inoculated and uninoculated
plants at the conclusion of the
pathogenicity trial . .

viii


Page


. 51


. 51

. 52

. 52


. 52

. 58

. 58

. 59


. 59

. 60

. 62

. 63


. 65

. 65


. 65

. 65

. 71


. 71

. 72


. 77




. 80










Figure Page

61. A comparison of leaves from inoculated
and uninoculated plants . ... 82

62. A comparison of seeds from inoculated
and uninoculated plants . ... 82

63. Biological control interactions ... 88

64. Sporangium of Rhizophidium sp. attached
to an egg . .... 88














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


TAXONOMY AND BIOLOGY OF Verutus volvingentis
N. GEN. N. SP. (TYLENCHIDA:NEMATA)

By

ROBERT PAUL ESSER

JUNE, 1980

Chairman: Armen C. Tarjan
Major Department: Entomology and Nematology

A new subfamily Verutinae is proposed. Females dif-

fer from all other subfamilies in the Heteroderidae in pos-

sessing a sausage shaped body with an uncommonly large sub-

equatorial vulva.

The genus and species named Verutus volvingentis is

described. Larval ecdysis was not noted within the egg.

Eggs are not deposited in a gelatinous matrix. Anatomical

features include a phasmid that appears only on tail of

cast cuticles of the first stage larvae. A previously un-

described muscle, the "median dilator vulvae,"was named.

A description of the rectal musculature and nervous system

is given. A detailed account of male and female develop-

ment is presented. Male development is completed in 6-15

days; female development took 17 days. Larvae entered the

root by pushing through the middle lamella between 2









epidermal cells. Tissue discoloration occurred 3-4 days

after feeding. Nuclei of invaded cells enlarged and exu-

date production by the host was incited. Larvae and eggs

survived at least 3 years in the absence of food. Eggs are

the dominant survival stage.

The nematode is widely distributed in Florida in

moist habitats. Testing of selected economic crops as

hosts proved negative. Host plants inoculated with a

minimal number of nematodes died in 13- months. Control

plants were maintained in a healthy vigorous condition.

Catenaria anguillulae killed males but not larvae or

eggs in biological control tests.














INTRODUCTION


The family Heteroderidae contains a large number of

highly pathogenic species included in 17 genera, 14 of

which have been erected since 1956. Pathogenicity has not

been proved for the following genera: Atalodera Wouts &

Sher, 1971; Meloidoderita Poghossian, 1966; Meloidodorella

Khan, 1972; Meloinema Choi & Geraert, 1973; Punctodera

Mulvey & Stone, 1976; Sarisodera Wouts & Sher, 1971;

Sherodera Wouts, 1973; and Thecavermiculatus Robbins, 1978.

The principal objectives of this research were to

establish the systematic position of the new genus of nema-

todes described in this work and to investigate the patho-

genic potential of the new taxon.

Secondary objectives included: host testing, life

cycle and developmental studies, longevity, host-parasite

relationships and anatomical studies.















HISTORY


In March, 1969, Mr. Wayne W. Smith, "Agricultural

Products Specialist," with the Florida Department of Agri-

culture submitted 14 samples from a field near Apopka,

Florida,for regulatory analysis. Four of the samples were

infested with larvae that resembled Heterodera sp. A

search of the sample material for Heterodera cysts revealed

females that did not fit the generic concept of any known

nematode phytoparasitic genus described at that time.

In May, 1969,the site from which the samples origi-

nated was surveyed in an attempt to isolate and identify

the host plant of the undescribed nematode. The host was

found to be buttonweed (Diodia virginiana L.) and was sub-

sequently infected with the nematode in greenhouse culture.














SECTION I
TAXONOMY AND SYSTEMATICS

Taxonomic Position of the New Genus


Verutinae n. subf.

Diagnosis: Heteroderidae (Filipjev, 1934) Skarbilovitch,

1947.

Female: Mature female saccate, sausage to reniform-shaped

(Fig. 12), vulva uncommonly large, subequatorial in posi-

tion, vulval lips strongly protuberant (Fig. 11), ovaries

reflexed, anus subterminal, cyst stage absent, body striae

present, phasmid obscure, strong sexual dimorphism present.

Male: (Fig. 15) Body vermiform, caudal alae absent, tail

rounded flatly to truncate, body untwisted, one testis

present.

Type genus Verutus n. gen. (from the Latin "armed with a

dart").


Affinities with the Family Heteroderidae

Females. Table 1 shows comparative female distin-

guishing characteristics of genera contained in the sub-

families of Heteroderidae.

Verutus n. gen. differs from all other members of the

Heteroderinae in lacking a cyst stage and a terminal vulva.

It differs from all members of the Ataloderinae, and









Meloidogyninae in lacking a terminal or subterminal vulva,

and a spheroid body (Fig. 1). Verutus is most closely re-

lated to Meloidoderinae, one member of which, Meloidodera

floridensis Chitwood, Hannon, & Esser, 1956, possesses a

subequatorial vulva (Fig. 2) and a spheroid or subspheroid

body shape. Verutus eggs are deposited as they mature

(Fig. 3), and not retained in large numbers within the fe-

male body as in females of Meloidoderinae (Fig. 4). The

body is completely annulated in the subfamilies Meloidoderi-

nae, Meloidogyninae, and Verutinae n. subfam. Members of

Ataloderinae and Heteroderinae possess an irregular body

pattern, or lack body markings. In some members of these

2 subfamilies, annulation may be present on the cervical

area or about the vulva, but not on the body. The vulva

is widely separated from the anus in the Verutinae (Fig. 11)

and in the genus Meloidodera (Fig. 2) in the Meloidoderinae.

In all other subfamilies the vulva is located in the peri-

neal area or near the anus (Fig. 4, Table 1). In some mem-

bers of Ataloderinae, Heteroderinae, and Meloidoderinae the

vulva is situated on a papule (Fig. 5). Neither Meloido-

derinae or Verutinae are so equipped. A labial disc (Fig.

14) is described only in Ataloderinae, Meloidoderinae, and

Verutinae. An attempt was made to utilize the presence of

a gelatinous matrix, or a sub-crystalline layer (Fig. 34)

in the diagnosis. This was not possible due to lack of

data concerning these criteria in many species descriptions.

Genera of the Heteroderidae were also compared to the new







































Figure 3. Verutus n. gen.
female in root tissue with
a single deposited egg.


Figure 1. Meloidogyne sp.,
showing spheroid shape and
terminal vulva of a mature
female.


1.
,~awr ;



-'.


I -

:.. -
......... "


Figure 2. Meloidodera floridensis
showing spheroid shape and equatorial
vulva position.


i.
:-1 c-n."
ri+;u









genus on the basis of the measurement (length/greatest body

width) alpha. This was found to be infeasible due to

omitted data and differences in the measurement criteria

used by some authors. Some measure the total body length,

others exclude the neck and head from the measurement

(Mulvey & Stone, 1976).

A key to the subfamilies of Heteroderidae based on

mature females is as follows.

Key to subfamilies of the Heteroderidae

1. Female forms a cyst---------------------Heteroderinae
Female does not form a cyst-------------------------2

2. Annulation absent or limited only to
cervical or vulva area---------------Ataloderinae
Annulation present on body (may be sparse)----------3

3. Body sausage or reniform-shaped vulva
uncommonly large---------------------Verutinae n. gen.
Body ovoid or pear-shaped, vulva not
uncommonly large--------------------------------4

4. Eggs retained in large numbers in female
body, labial disc present------------Meloidoderinae
Eggs not retained in body in large
numbers (exception Meloidoderita),
labial disc present------------------Meloidogyninae


Males. Table 2 compares selected male characteris-

tics of genera included in the subfamilies of the

Heteroderidae. It can be seen that few definitive differ-

ences exist between males. Only the subfamilies Ataloder-

inae and Verutinae contain genera without a twist in the

male body (Fig. 6-C). Males of both subfamilies also pos-

sess a truncate tail terminus, similar spicules and guber-

naculum, and a tubus (Fig. 6-A,B). Stylet and body length
















Table 1.


Comparative distinguishing characteristics of
females in genera of Heteroderidae.


Vulva
Cyst Cuticle Anus


Vulva
on a


Egg
Lip Reten-


Galls
on


** Vulva


Sub-
family


Genus Stage Markings Gap Papule Disc tion HPR Host Sac C L Position
1/70 no head & very yes yes yes ? ? ? ? terminal
vulva close
striae
1/89 no none .... "
1/121 head & no semi- ? no yes "
vulva endo
striae
2/118 yes pattern close no no no "
2/13 no & yes yes
yes
2/99 pattern no endo yes ?
& punc-
tations
2/102 semi- no "
endo
2/71 lace- very ? ? ? terminal
like close recessed
3/63 no striae not yes semi- no no yes terminal
close endo
3/16 far mid-body
apart
3/95 not ? ? ? ? terminal
close
4/41 very yes no endo yes yes yes "
close
4/56 "V ? ? yes semi- no "
endo
4/17 no no & no no endo "
yes
4/92 striae close no ? ? ? ? ? "
sparse
5/130 striae far yes no semi- rare no yes mid-body
apart endo
*HPR = Host parasite relationship, Semi-endo=semi-endoparasite, Endo=endoparasite.
**CL = Crystalline layer.

Legend Subfamily & Genus
l=Ataloderinae; 70=Atalodera, 89=Sherodera, 121=Thecavermiculatus
2=Heteroderinae; 118=Globodera, 13=Heterodera, 99=Meloidodorella, 102=Punctodera
71=Sarisodera
3=Meloidoderinae; 63=Cryphodera, 16=Meloidodera, 95=Zeylandodera
4=Meloidogyninae; 41=Hypsoperine, 56=Meloidoderita, 17=Meloidogyne, 92=Meloinema
5=Verutinae; 130=Verutus

Egg filled cyst develops. Golden, 1976; Andrews et al, 1977.




















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measurements also overlap in both subfamilies. Except for

the striated gubernaculum of the genus Sherodera it would

be difficult to differentiate between males of Ataloderidae

and Verutinae. The only other genus with a truncate tail

terminus and tubus is Sarisodera in the subfamily Hetero-

derinae. The Sarisodera male can be separated from males

of Ataloderinae and Verutinae by its long stylet (38-46 pm).

Meloidoderita possesses a sharp conoid tail and is the only

male in the Heteroderidae with caudal alae. All other

genera, not included in the aforementioned, have rounded

or bluntly conoid tails (Fig. 6-C) with or without a twist.

The only small males have been described in Meloidoderita

(350-432 pm), and Meloidodera (457-505 pm). Males in the

genus Meloinema stand apart from all other males in posses-

sing a distinct subacutely conoid tail (Fig. 6-D).


Larvae. The larvae of the new genus (Fig. 8) close-

ly resemble larvae in the subfamilies Atalodorinae,

Meloidoderinae, and all of the larvae in the genera of

Heteroderinae (except Meloidodorella, which has a short

stylet [11-16 vm and reduced telorhabdions]). Larvae of the

Meloidogyninae differ from the new genus, for the most part,

in having a small stylet, small telorhabdions, and fine

body striae. The principal character peculiar to first-

stage larvae of the new genus is the absence of a detect-

able phasmid.






10





















Figure 4. Cryphodera eucalypti (after
Colbran), showing egg retention in
the mature female, and separation
of the vulva and anus.
,...;. .. '. *L i

',_ ', .-. ,

































Figure 5. Hypsoperine graminus Sledge
and Golden, 1964. A mature female
showing a vulva situated on a papule.











A B C \D




/ -









Figure 6. Heteroderidae male tail types; A,B Truncate
with tubus. A Sherodera (redrawn from Wouts & Sher),
B Verutus, C Rounded twisted type. Meloidodera (after
Hopper), D Blunt conoid. Meloinema (redrawn from Choi
& Geraert).



Comparisons were made of esophageal gland structures

in the 16 genera of the Heteroderidae to determine if the

glands could be used in generic or subfamily diagnosis.

Esophageal glands with two lobes, the anterior lobe over-

lapping the posterior lobe, were found in the Verutinae,

Meloidoderinae, and Heteroderinae (Fig. 9 A,D,F,H,J).

Considerable variation in esophageal gland structure was

noted in the 27 species descriptions examined in 4 genera

of the Heteroderinae (Fig. 9 F-K). Esophageal glands in

29 species of Meloidogynidae all consisted of a single

lobe. Meloinema differed from all other genera in having

an extremely long esophagus (300 pm). However, in the

description the esophagus length range was listed at 125-

130 pm, which contradicts the illustration. The genera







A


C \




/,' s / im 5-----< "
I\ '(K \





Figure 7. A comparison of female appearances in three
subfamilies. A) Verutinae, B) Nacobbinae, C) Rotylenchulinae.






































30,m


Figure 8. First stage larva of
Verutus volvingentis n. gen.,
n. sp.









Sherodera and Zelandodera, Wouts, 1973 are not included in

Figure 9 since neither genus is represented by an illustra-

tion of the larval esophageal glands in the literature.

The Verutinae also differ from the Ataloderinae and

Meloidogyninae in possessing two esophageal gland lobes.


Affinities with the Subfamily Rotylenchulinae, Husain &
Khan, 1967

The females of Verutus (Fig. 7-A) closely resemble

females in Rotylenchulinae (Fig. 7-C). Verutus females

differ in the absence of a well-defined tail tip, a dorsal

gland orifice that originates less than one stylet length

from the base of the telorhabdions, possession of a large

muscular uterus, and, principally, by the absence of a

vermiform, vulvate juvenile female stage.

Verutinae males differ markedly in general appearance

from Rotylenchulinae males which have a tapering conoid

tail, are usually less than 500 pm long, and assume a C-

shaped body position. Rotylenchulinae males possess an

elongate, truncate cephalic framework in contrast to the

slightly convex, shallow cephalic framework of Verutinae

males.


Affinities with the Subfamily Nacobbinae, Chitwood &
Chitwood, 1937

Nacobbinae females differ from females of Verutinae

in body shape (Fig. 7-B), position of a posteriorly sit-

uated vulva, and in having a single gonad. Nacobbinae










A
(



I
c/


BCD











o@
(I


E F G H I J K








0 OD
0?'^ ??

o 00^
vs) r
^ @ @


LM







0 )




Figure 9. A comparison of first and second-
stage larval esophageal glands in the
Heteroderidae. A) Verutinae; Verutus. B,C)
Ataloderinae; B) Atalodera, C) Thecavermiculatus.
D,E) Meloidoderinae; D) Meloidodera, E)
Cryphodera Colbran,1966. F,K) Heteroderinae;
F) Sarisodera, G) Meloidodorella, H) Punctodera,
I,J) Globodera or Heterodera. K) Heterodera.
L-O Meloidogynidae; L) Meloidoderita, M)
Meloidogyne, N) Hypsoperine, O) Meloinema.
(Esophageal glands are directly proportional
to the size of the metacorpus shown.)


NO0









females form prominent root galls while Verutinae females

do not.

Nacobbinae males possess conoid tails and caudal

alae, both of which are absent in Verutinae males.

Nacobbinae larvae differ from Verutinae larvae in

having a bluntly rounded tail tip.


Discussion. The new subfamily Verutinae does not fit

within the concepts of the subfamilies in the family

Heteroderinae, or the subfamilies, Nacobbinae or Rotylen-

chulinae. It is therefore proposed as a new subfamily.

Subfamilies proposed by Husain, 1976, but not included in

the analysis are Meloineminae and Meloidoderellinae.

The position of the Verutinae in the Animal Kingdom

is shown in the following scheme:

Kingdom-Animalia

Subkingdom-Metazoa

Branch-Enterozoa

Division-Bilateria

Section-Pseudocoelomata

Phylum-Nemata (Rudolphi,1808), Cobb,1919

Class-Secernentia (von Linstow,1905)Chitwood,1958

Order-Tylenchida Thorne,1949

Suborder-Tylenchina Pearse,1942

Superfamily-Heteroderoidea (Filipjev,1934)Golden,
1971

Family-Heteroderidae (Filipjev,1934)Skarbilovitch,
1947









Subfamily-Verutinae

Genus-Verutus

Species-volvingentis

In the above scheme categories above Phylum are based

on Storer and Usinger, 1957. Classifications below Section

are based on schemes proposed by Golden, 1971, Wouts, 1972,

Andrassy, 1976, Husain, 1976, and Stone, 1977.


Taxonomy of the New Genus

Methods; Measurement Preparation

Specimens to be measured were placed in water within

a "Zut" ring on a glass microscope slide (Esser, 1973-b), and

a cover slip placed on the zut. The nematodes ceased moving

in 3 to 5 min., and measurements were taken according to

the method proposed by Esser, 1971. While the nematodes

are in the quiescent state one has 20 to 30 min. to make

observations and measurements before deterioration, swelling

and/or shrinkage occur.


Permanent Fixation

When specimens on slides are to be fixed permanently

the cover slip is removed and 2 or 3 drops of 2% formalin

are added to the exposed zut well. The specimens are then

transferred to a BPI watch glass for permanent fixation in

lactophenol (Esser, 1973-a).









En face Preparation

A new method was devised to study en face prepara-

tions. Live immobile females or males, or freshly killed

nematodes in 2% formalin were used as subjects.

Procedure. A 12 X 12 X 3 mm square of 1.7% water

agar is cut very evenly with a razor blade (Fig. 10-A) and

placed on a microscope slide. A 3- to 4-mm piece is pre-

cisely cut from the square (Fig. 10-A) and laid with the

outer face down (Fig. 10-B). Nematodes are placed on the

upper side of the cut piece with the longitudinal axis of

the head parallel with the outer edge of the cut piece

(Fig. 10-B). The cut piece is then placed back into the

same position it occupied in Fig. 10-A, then gently pushed

into its original position against the parent block (Fig.

10-C). A small (4-mm) drop of water is applied to a 15-mm

cover slip that is then placed waterside down over the cut

line (Fig. 10-C). A drop of immersion oil is applied to

the center of the cover slip at the junction of the cut

pieces. When the body of the nematode is properly aligned

the en face appears as in Fig. 10-D If the en face is

off-center or below the field of focus, the cover slip is

removed, the cut piece placed backside down, and the speci-

mens reoriented. Water must be added to the cover slip

each time it is placed on the agar block. It takes 10 to

15 min. to prepare an en face ready for viewing using this

technique. Locating the en face is rather easy since it

lies within the cut line.


























































Figure 10. Water agar en face method; A) 12 X 12 X 3 mm
square of water agar with a 3- to 4-mm piece cut off, B)
Specimens aligned on outer edge of the inner face of the
cut piece, C) Re-alignment of the separated agar pieces,
with cover slip in place, D) Closeup of en face in
junction line of re-aligned agar.









External Cuticle Preparation

Lateral lines and phasmids were not well-defined in

live or fixed specimens. Several stains were tested to

bring out the lateral lines including: methyl blue, acid

fuchsin, merthiolate, iodine, chlorazol black-E, and pro-

pionic carmine, none of which enhanced cuticular incisures

or phasmids. Lateral incisures were brought out clearly

by making squash mounts. Cut or uncut females, males, and

larvae were placed in a 4-mm drop of water, and a 18-mm

cover slip applied. A needle point was pressed against the

cover slip until the body contents gushed out. Examination

for the phasmid was made of each squash mount specimen.


Nervous System Preparation

Chlorazol black-E in lactophenol was used with the

4-min. fixation method (Esser, 1973-a).


Verutus n. gen.

Diagnosis: Verutinae, with characters of the subfamily.

Mature female (Fig. 11): Body swollen, reniform or sausage-

shaped (Fig. 12). Cephalic framework moderately sclerotized,

lips striated, set off, amphids obscure, oral disc hexagonal

(Fig. 14). Body striated, lateral lines irregular, some-

times indistinct. Crystalline layer present (Fig. 34).

Stylet tylenchoid, dorsal gland orifice near telorhabdion

base. Uncommonly large protuberant post-equatorial vulva.





























































Figure 11.
Verutus
volvingentis
n. gen. n. sp.
Mature female.

















































200,u


Figure 12. Verutus volvingentis n. gen. n. sp.,
female body shapes.













5Sym









Figure 13. Vestigial larval
tail tip on posterior area
of a mature female.



Figure 14. En face view of
a mature female.



Gonads didelphic and amphidelphic. Ovaries reflexed. Anus

subterminal forming small depression (Fig. 13). Tail

vestigial (Fig. 13) or absent.

Female. (Table 1) Females differ from all other fe-

males in the Heteroderidae in possessing a reniform or

sausage-shaped body, with an uncommonly large vulva in a

post equatorial position with strongly protuberant lips.

Esophagus typically tylenchoid, procorpus moderately expan-

ded, metacorpus moderate in size. Isthmus narrower than

procorpus, esophageal gland a single lobe moderately over-

lapping the intestine. Deirids and phasmids not observed.
sausageshapedbody, wth an ncommo lylrevvaia
post~~~~~~~~~~~~ eqaoil oiinwihsrngypouern is
Esophagus tyial tlnhid rcrusmdrteyepn









Male. (Fig. 15) Body vermiform, monodelphic, lips

striated not set off, oral disc circular (Fig. 16), amphid-

ial openings elliptical on lateral lips. Body untwisted.

Spicules and gubernaculum tylenchoid. Caudal alae absent,

tail terminus angular truncate (Fig. 6-B). Phasmids or

deirids not detected.

.1
Verutus volvingentis n. sp.

Female. (35 specimens) Total length = 662.7 (500-

930) pm; width = 141.4 (94-207) pm; tail = 10.2 (3.9-15.7)

pm; esophagus = 188.3 (150-290) pm; a = 4.7 (3.0-6.5); c =

69.4 (34-155); total stylet length = 26.1 (23.5-29.4) vm;

vulva % = 67.5 (50-75); excretory pore 139.8 (122-183) pm.

Female holotype. Total length = 540 pm; width =

118 pm; tail = 15 pm; esophagus = 148 pm; a = 4.6; b = 3.6;

c = 36; stylet = 27.2 pm; vulva % = 71.2; excretory pore =

114 pm from anterior end.

Female description. (Fig. 11) Body pearly white,

reniform or sausage-shaped (Fig. 12), anterior part of body

sometimes twisted upward lying in a different plane than

the posterior swollen portion. Head and neck occasionally

reflexed across the posterior body. Six equidistant lips

surround a hexagon-shaped oral disc (Fig. 14). Amphid

apertures or lip papillae not observed. Lips set off, com-

prised of 2 annules. Cuticle 9-10 pm thick, evenly stria-

ted, striae about 2.5 pm apart. The occurrence and


1volv = vulva, ingen = remarkable size.




















































Figure 15.
Mature male
of Verutus
volvingentis
n. gen. n. sp.









appearance of lateral lines are variable: lines may proceed

for a short distance beyond anus and fade out, or appear as

midline cuticular interruptions or irregularities extending

slightly past the vulva area. They appear as 1 or 2 lines

of irregular blocks in the cervical area. In the tail

area they appear as a mass of irregularities in the tail

tip area with sometimes a wide separation (5 pm) of the

striae (Fig. 17). In a few females lateral lines were not

observed. Stylet tylenchoid, telorhabdions (Fig. 11, 18),

4-5 wide by 1-2 ym long, directed posteriad. Prorhabdions

14 pm. Two stylets with tips protruding from the body

measured 25.5 and 25.6 pm, respectively. The dorsal gland

orifice appears 7-11 pm posterior to the telorhabdion base.

A moderately swollen procorpus narrows prior to the well-

developed metacorpus. A clearly defined metacorpal valve

is present. A short narrow isthmus leads from the meta-

corpus followed by a single distinct esophageal gland lay-

ing ventrally over the intestine. The intestine extends

from beneath the mid-area of the esophageal gland to the

rectal intestinal valve. The sclerotized portion of the

rectum is 12 pm long in a lateral view. The rectum dilates

anteriorly extending 30 pm beyond the sclerotized portion

(Fig. 19) as a finely sclerotized tube (15 pm long) which

joins the intestine. The oval anus (Fig. 20) lies in a

depression (Fig. 19). Tail is usually absent, occasionally

vestigial (Fig. 19). Differences in orientation of the body









do not permit an accurate tail annule count, or anal body

diameter measurement. One female was noted (Fig. 21) with

a granular mass over the anus, assumed to be excreta. The

excretory pore lies at the level of the esophageal gland

134.4 (113-183) pm from the oral opening. The nerve ring

appears as a mass of tissue surrounding the isthmus.

Gonad amphidelphic, anterior branch 136-147 pm long,

posterior branch 117-130 pm long. The vulva appears as a

transverse slit (Fig. 22) about 62 m wide. In some fe-

males the vulva lips protrude markedly. The vulva striae

do not form a distinctive pattern, but surround the vulva

rather uniformly (Fig. 22). In some females prolapse of

the vaginal walls causes the vulva to widen, and the vaginal

lining prolapses externally (Fig. 23). Wide muscle bands,

the dilator vulvae appear at either end of the vulva under-

lying the cuticle (Fig. 22). Vulva epitygma were not ob-

served. The vagina extends 42 to 70.5 pm into the body

where it joins the well-developed vagina uterina (58-70 pm;

Fig. 11). A constriction appears at the junction of the

vagina uterina and the uterus. The uterus is a large mus-

cular sac (70 X 35 pm) that joins directly with the sper-

matheca (Fig. 24). It comprises 4 to 5 rows of large cells

with a furrow in the center for expansion. The spermatheca

is a roughly circular thick walled chamber 35 to 40 pm in

diameter. The oviduct, a thickened area comprised of small

cells,lies between the spermatheca and the maturation zone

of the ovaries. The ovary is reflexed at the spermatheca,



























Figure 16. Verutus
volvingentis
male en face view.


Figure 18. Telorhabdions of a
mature female posterior view.


Figure 17. Female tail area
showing lateral field
irregularities.












Figure 20. Anus (left) and
rectum of a mature female.


Figure 19. Posterior region of mature
females, showing vestigial larval tail
tips (T), and rectum (R).






29




V. /
I"' ":k' """^."" II



















Figure 21. Excreta exuded Figure 22. Vulva and vulva
......tt ..



1/ \ \ \

/: / '/ ,'










Muscles, M-median dilator

vulvae muscles (striae cutaway
to show underlying muscles).
-- -,
/ I















and or 2 times in the maturation zone area. The cap cell





and germinal zone cells are rarely delineated in live or

fixed specimens.

Males. (24 specimens, Fig. 15) Body length = 830.8
l -.( specimens, Fig."15)_.ody // lnt 330.8


























(650-1020) pm; width = 28.9 (25.5-35.5) pm; tail = 9.5

(5-12.7) pm; esophagus = 153.5 (122-188) pm; a = 28.7

(24.3-32.8) 1jm; b = 5.4 (4.5-6.6) pm; c = 99.2 (59.1-178.6)

pm; total stylet length = 25.3 (21.5-27.4) pim; dorsal gland

orifice = 4.6 (2-6.8) pm behind the base of the telorhabdions;
orifice = 4.6 (2-6.8) Dm behind the base of the telorhabdions;















I
I
I
/
I
I


I
I
I
/

I
~\~A.WLLULAJWUtLLJ


Qi


Figure 23. Lateral views of an unprolapsed (left)
and prolapsed (right) vagina of mature females
P=prolapsed vaginal tissue.



spicules = 40.4 (36.2-46.6) pm; gubernaculum = 16.2 (14.7-

18.6) pm.

Allotype. Total body length 790 pm; width = 25 pm;

tail = 6 im; esophagus = 140 pm; a = 31.6; b = 5.6; c =

131.7; total stylet length = 22 pm; dorsal gland orifice =

6 pm; spicules = 40 pm; gubernaculum = 15 pm.

Male description. Body vermiform, untwisted (Fig.

15), 6 equidistant lips (Fig. 16) surround a circular oral

disc that stands out clearly in profile. Crescent-shaped

amphids appear indistinctly on posterior margin of lateral


























Figure 24. Uterus of a mature female:
1) spermatheca, 2) uterus, 3) egg.


Figure 25. Male tail showing tubus
(arrow).









lips. Labium moderately sclerotized, comprising 4 to 7

labial annules, counting from first reduced annule at onset

of cephalic sclerotization. Labium rounded, not set off,

papillae not observed. Body striae about 2 vm wide, some-

times ending irregularly at the terminus (Fig. 15). Four

unareolated lateral fields present, extending from region

of corpus to cloacal area. Phasmid not observed. Excre-

tory pore lying in posterior esophageal gland area, 103-

146 pm from oral disc (mean = 124 pm). Hemizonid 4 pm long,

located just posterior to excretory pore. Stylet typi-

cally tylenchoid. Telorhabdions sloping posteriorly.

Cheilorhabdions extending through lip annules 2 to 4.

Esophagus comprising a moderately swollen procorpus that

dilates just prior to oval, distinct metacorpus containing

a valve slightly smaller than that of female. A narrow

isthmus precedes a single ventral esophageal gland with

single nucleus. Cardia not observed. Nerve ring appear-

ing as an irregular band of tissue overlapping isthmus,

and extending past the esophageal gland about 1/3 of its

length (Fig. 15). Intestine overlapping about 1/2 of

esophageal gland and extending uniformly to cloaca. Tail

bluntly hemispherical to truncate; tail terminus annu-

lated. Anal lips in form of tubus (Fig. 25). Caudal

alae absent. Spicules equal and slightly arcuate when

seen in lateral view. Capitulum moderately swollen,

followed by slight constriction, and moderately swollen

calomus. Lamina wide at the center tapering at both









extremities. Sclerotized piece arising at junction of

lamina and calomus and projecting along ventral wall of

calomus. The gubernaculum with teeth on lateral sides of

cuneus seen in ventral.view when cuneus is situated between

spicules (Fig. 26). Male gonaduct originating from ventral

face of the cloaca. Narrow vas deferens (Fig. 15) about

90 pm long is followed by rather long seminal vesicle, usu-

ally filled with sperm. Germinal and growth zones sometimes

indistinguishable. Testes have been observed in which en-

tire tube was filled with sperm and an observable germinal

and growth zones were not present. Cephalids and deirids

not observed.

Larval description. (49 first-stage larvae; Fig. 8)

Length = 492 (430-540) pm; width = 18.5 (16-20.2) pm; tail =

53.6 (46-64) m; esophagus = 163 (132-190) pm; a = 26.5 (22-

30); b = 3.2 (2.6-3.7); c = 9.1 (6.7-10.5); anal body diam-

eter = 4.5 (4.0-5.2) pm; stylet = 23.1 (21.5-24.5) pm; dorsal

gland orifice 6.4 (4-8.9) pm posterior to telorhabdions;

excretory pore 93.8 (79-103) pm from oral disc.

Body vermiform, labium rounded, cephalic framework

consisting of 16, C-shaped sclerotized pieces lying 4 pm

below oral disc. Head bearing 6 annules. Four lateral

incisures beginning as single line, 45 pm posterior to

oral disc forming 4 lines at median procorpus. The 4

lines resolve into a single line just posterior to anus

(30 pm from the tail tip). Width of lateral incisures at

mid-body is 5-7 pm. Excretory pore located in mid-isthmus































Figure 26. Male tail in a
ventral view, showing
toothed cuneus of
gubernaculum (arrow)


Figure 27. Larval tail


shapes.









area. Hemizonid located 1 annule anterior to excretory

pore. Stylet well-developed, prorhabdion 10.8-12 pm long.

Rounded telorhabdions usually laying in an even plane, occa-

sionally sloping posteriorly. Procorpus (38 pm long by 6-7

pm wide), moderately swollen, narrowing just prior to the

well-developed metacorpus (15 pm long by 12 pm wide).

Metacorpus valve a wide oval shape. Isthmus narrow, 25 pm

long. Esophageal glands about 35 pm long, lying on ventral

side of body. Posterior lobe sometimes filled with coarse

granules (digestive fluid). Coarse granules also appearing

in anterior end of anterior lobe, in isthmus, and in a

large vesicle in posterior part of metacorpus (Fig. 8). A

single large nucleus present in the posterior esophageal

gland lobe. Anterior esophageal gland lobe not strongly

set-off. It is delineated by a weak line of demarcation on

posterior lobe, and has a very large nucleus surrounded by

a large clear area (Fig. 8). Esophageal glands overlapping

intestine by about 1/2 of their length, extending as a

straight tube to undilated rectum. Anus oval, 1 to 2 pm

wide. Nerve ring appearing either as group of nerve cells

(Fig. 31), or as fine band of tissue surrounding isthmus

(Fig. 8). Genital primordia appearing about 160 pm ante-

rior to tail tip (Fig. 8). Tail conoid (Fig. 27), with

26-29 annules. Tail tip usually awl-shaped. Hyaline area

of tail 25.4 (23.5-31.3) pm long. Deirids, phasmids and

cardia not observed.









Ova. (Fig. 28) Eggs broadly oval 50 X 100 pm. No

markings observed on shell. An en-utero egg was 52 X 103

pm.

Third-stage larvae. (10 specimens) Length = 572

(500-677) pm; width = 31.3 (27.4-34.3) pm; tail = 11.1

(9.8-13.7) pm; a = 19.5 (17.3-22.6); c = 54.5 (46.7-63.2);

dorsal gland orifice = 3.9 (2.9-6.8) pm; excretory pore =

115.9 (109-122) pm.

Body slightly swollen, tail rounded, head and esoph-

agus similar to that of first-stage larvae (Fig. 29).

Type specimens. Holotype collected May, 1969 by

Wayne W. Smith. Collection number B-5018; Allotype same

data as holotype. Type slides in Bureau of Nematology nema-

tode collection, Division of Plant Industry, Florida De-

partment of Agriculture.

Type habitat. Soil about roots, and roots of Diodia

virginiana growing near bodies of water.

Type locality. Irrigation ditch bank bordering Hwy

50, 4 miles west of Hwy 27 near Clermont, Florida. (Orig-

inal site now commercially developed.


Anatomy


Lateral Incisures

These structures are very difficult to see in live or

fixed specimens even when various stains were used. It was

possible to see them, however, by squeezing out the body

contents and examining the lateral sides of the integument










1 "1


'; .. -. ..' ^



*,,, _.r-. > ...




-: .. ;... *' .r -, ^ .-- _. .* ,
i,- -' 1.




a:. '-, s '-a.S .'



: .: -. .- .- .. .. .. .., .,.. .
'A.'
; ^^ \
''I' ,- "- '. -'? :" l







'. .. .-.: .. -- -; ,.- .,,:; A W
;" ." "I^

-I ."". ..' -
...~ ~~U '..:.<.-. :.'





.. ... : .- .-- _* .- ,
C '. f.


;' :':s










A- ^ r4-?' '
i z-. I. ,.:": :..". : : '
Li -.
















*; -, ~:-. ","* -*, ,-"-"" ," "-a ,'* 3 -. -

Figure 23. Ova. Top to bottom:
=__- ......











2-cell, 3.cell, 4-c-], 5.cell,

tadpole stage, first-stage
,larva' "- ,
i.,- : ...-'*" '
Figure ~ '= 23 Ova.Top o botom
2-cll 3-el 4cl, 5ce
tadpol stage firsct-stag
la v.


Figure 29. Anterior
portion of third-
stage larval male.









using an oil immersion objective. This procedure was

not necessary for males and females.

Phasmid. Over a hundred each of males, females, and

first-stage larvae were examined for phasmids with negative

results in both ventral and lateral views. The phasmid and

its lining were only detected on the cast integument of

first-stage larvae early in the first molt. The phasmid

was located between the 14th and 18th annule from the tail

tip. When its location was known, fixed and living first-

stage larvae were examined to see if the phasmid was detect-

able; in no case was it observed.


Muscles

Vulva. The dilator vulvae musculature are well devel-

oped in broad bands in mature females, extending from vagin-

al epithelium to a ventrolateral insertion in the hydodermis

(Fig. 22). A band of muscle also attaches to vagina on

either side of median part of vulva, herein called "median

dilator vulvae" (Fig. 22).

Rectal muscles. Rectal musculature was observed in a

third-stage female (Fig. 30). The H-shaped muscle surrounds

the rectum or rectal intestinal valve. The depressor ani

extends into the dorsal hypodermis, and the dilator ani is

inserted in ventral hypodermis. A sarcoplasm band nucleus

as described by Chitwood & Chitwood, 1937 was not observed.









Procorpus and Metacorpus


In third-stage larvae the procorpus is short and

stout while metacorpus is a well-developed, wide oval. The

esophagus of mature females is very similar to that of

third-stage larvae. Geraert, 1978, found that the metacor-

pus enlarges in saccate females (Heterodera carotae Jones,

1950), as was the case in V. volvingentis.

In males the esophagus is shorter, more slender and

the metacorpus is smaller and more elongate.


Nervous System

In males and females stained with chlorazol black-E

the circum-esophageal commissure appears as a flat band of

tissue that surrounds the posterior part of isthmus (Fig. 11,

15) and proceeds posteriorly a short distance past the ante-

rior part of the basal bulb as 2 ventral ganglion. Anterior

and posterior nerve cords were not seen. In first-stage

larvae stained with chlorazol black-E, the circum-esophageal

commissure appears looped around the isthmus, either as a

flat band of tissue (Fig. 8) or as an accumulation of nerve

cells (Fig. 31). The ventral ganglion proceeds posteriorly

a short distance, branching dorsally and ventrally. The

dorsal nerve arises from the dorsal portion of the ventral

ganglion, and becomes indistinguishable a short distance

posterior to the esophageal gland. The ventral nerve arises

from the ventral portion of the ventral gnaglion, and pro-

ceeds as a chain of ganglia (92 in 1 specimen) in the












A


























Figure 30. Rectal musculature.
A) Rectal-intestinal valve
area: (1) sarcoplasm, (2)
depressor ani, (3) dilator ani, /'i
(4) rectal intestinal valve. .
B) Rectal area: (5) mid-rectum.
D) Dorsal side.
V) Ventral side.

Figure 31. Anterior nervous
system in a first-stage larva.
A) Anterior ventral nerve
cord; b) Circum-oral
commissure; C) Ventral nerve;
D) Dorsal nerve; E) Hemizonad.









hypodermis. The ganglial chain forms rectal commissures

that surround the rectum with 3 dorsal and 5 ventral ganglia

(Fig. 33). A dorsal rectal ganglion (Fig. 33) is present

where rectal commissures rejoin post-rectally in the dorsal

position. Three ganglia are present in the medial caudal

nerve (Fig. 33). Anteriorly, a large ganglion arises from

the dorsal portion of the nerve ring, and one from the ven-

tral side (Fig. 31). The 2 nerve cords extend around either

side of the metacorpus forming a small mass of nerve cells

just anterior to the metacorpus. Dorsal and ventral nerves

proceed from this ganglion to sclerotized area of the labium.

Cephalic nerves appear as elongate, spindle-shaped proc-

esses. Lateral and papillary nerves were not detected.


Crystalline Layer

Brown et al, 1971, reported the subcrystalline layer

is a complex of long-chain fatty acids. It was hypothesized

that sugar exudates from the integument of Heterodera spp.

are converted to long chain fatty acids by soil fungi there-

by producing the crystalline layer. A crystalline layer was

observed on the integument of about 10% of the females of

the new genus. This layer assumes the form of the striae

and other designs and modifications present in the parent

integument (Fig. 34-B,C). The subcrystalline layer is usu-

ally fragmented and sloughs off the female body (Fig. 34-A,


D).
























Figure 32. Ventral nerve cord (arrow) in the area of the
genital primordia.


L D


Figure 33. Nerves in the tail of a first-stage larva. A)
ventral ganglion; b) rectal commissure; C) dorsal rectal
ganglion; D) dorsal view; E) medial caudal nerve; L)
lateral view.






43







.







r L2
-, .






4








lrr
"- ,. ..
'e5rfk


gue 34 Crytaline ayer A)Seprto fth ae
from ih ancrren;BC aera ,-od;D ae
frgmnt~cjfrm ta~ area.i";


B


y
p






44


Gubernaculum

A gubernaculum was isolated from the surrounding tissue for

observation of the dorsal and ventral faces (Fig. 35). The

dorsal face is longer, measuring 16 pm, and shows serrated

margins on the cuneus (Fig. 35-D). The ventral face is

shorter (11 im), ventrally grooved, and serrations were not

observed in the focal plane (Fig. 35-V).














SECTION II
REPRODUCTIVE DEVELOPMENT

Early Development


The female deposits a naked undivided egg in the

environment (gelatinous matrix absent).

Four-hundred eggs in lots of 50 were examined under

the oil immersion lens to determine if a molt occurred in

the egg as described for Heterodera rostochiensis Wollen-

weber by Hagemeyer, 1951, and in Meloidogyne sp. by

Christie and Cobb,1941. In no case was evidence of ecdysis

present. After examination, the larvae were expelled from

the eggs (Fig. 36) by exerting a gentle pressure with a

fine needle tip on the cover slip. None of the larvae ex-

pelled from the 400 eggs showed evidence of ecdysis. It

is concluded based on these data that a molt does not occur

in the egg.

First-stage larvae possess binucleate genital primor-

dia with posterior and anterior cap cells (Fig. 32, 38-A).

Shortly after the first molt, determination of sex is pos-

sible by examination of the rectal area. If spicular pri-

mordia cells are present (Fig. 37), a male is developing.

Absence of spicular primordia cells indicate a female is

developing.





























Figure 35. Gubernaculum (top
is anterior). V) ventral face
(4 pm wide by 11 pm long);
D) dorsal face (5 pm wide by
16 m long); l=cuneus, 2=
corpus, 3=crura.


"A





o..:3 I



Figure 36. Larvae forced
from egg by applying
cover slip pressure.







:IIII-zI


Figure 37. Spicular primordia
cells in the cloacal area of
an early second-stage male.
Phasmid is shown (arrow) on
first-stage exuviae.









Male Development


Shortly after the first molt, and before the first-

stage integument is cast off, the genital primordium begins

to divide and proliferate posteriorly (Fig. 38 B,C). The

body widens, and the testes join the spicular primordia

shortly before, and after, the first molted integument is

lost (Fig. 38-D). Sperm cells are large and angular at

this stage. Following the second molt, little development

is evident in the testes and spicular primordia. The esoph-

agus is not clearly differentiated at this stage of devel-

opment. The gubernaculum is the anlage of sclerotization,

followed by the lamina of the spicules. The calomus and

capitulum are the last to become sclerotized. Development

proceeds to completion after the second exuviae is cast.

In 2 cases observed, the male left the third-stage exuviae

embedded in the root. Empty exuviae are not uncommon in in-

fected roots. Table 3 shows the length of time required for

development of males. Male development from penetration of

the first-stage larva until a fully developed male was ob-

served took place in a minimum of 6 days and a maximum of

15 days in roots growing in water agar. Three first-stage

larvae that entered a root about the same time all molted to

the second-stage in 48 hours. Twenty-four hours later, all

3 molted to the third-stage. Three days later the final
1
molt occurred for all 3 males within 92 hours. Total aver-

age time required was 10 days and 20 hours. Other periods

















C


A I
I1O,_ oo 0
0 o

;oo 0 00 .





Figure 38. Male reproduc-
tive system development:
A) genital primordium; B)
Genital primordium, 4-cell
stage; C) Genital primor-
dium, early third-stage
male; D) Reproductive
system in late third-
stage male. l=spicular
primordia. 2=vas
deferens.










of male development observed were: a 15-day cycle; a 10-day,

4-hour cycle (Table 3); and a 6-day cycle.


Time(hours)


Table 3. Male Development

Activity


Width (ym)


0
24
32
48
72
81
96
105
122
145
168
176-
2
1922
195

197

202

212
236
244

Total time


penetration
feeding
11 1

ecdysis (first)
1 I1 1
It II
feeding

shedding integument
feeding
It It
ecdysis (second)
Third-stage larva emerged from
exuviae which remained in root.
Migration along root (length
700 im), stylet 24 im.
Migration to a new root, no
physical change.
no change
ecdysis (third & fourth)
development complete

= 0 days, 4 hours.


It is shown in Table 3 that the width of the feeding larva
1
increases with time until 176- hours have elapsed when a

maximum width of 51.7 pm is attained. After the second

ecdysis the width decreases until the male is fully devel-

oped with a width of 33.3 pm. Feeding has ceased at this

time and it is postulated that the decrease in width is due

to energy expended during ecdysis and migration.


28.2
32.9
32.9
42.3
37.6
37.6
37.6
42.3
49.3
49.3
47.0
51.7
42.3
42.3

37.6

38.6

38.6
38.6
33.3









Female Development


Shortly after the first molt the body swells and the

genital primordium proliferates anteriorly, and posteriorly

(Fig. 38-A,B). The cells in the center bulge toward the

body wall forming the vaginal primordium, after which the

anterior and posterior branches elongate and develop (Fig.

39). The vagina first appears as a large opening with very

large vaginal primordia cells on either side (Fig. 40).

After the second molt the body swells and the gonad com-

pletes its development (Fig. 41). The reproductive system

is complete when the third-stage exuviae is cast (Fig. 42).

Mature females (Fig. 11,43) are usually swollen more than

virgin females, possess convoluted ovaries, and contain

sperm in the spermatheca (Fig. 43). The vagina uterina was

very narrow in a few females (Fig. 44-C). In older females

the vagina uterina is well-developed, with thick folds capa-

ble of containing several eggs. In several females a

severely prolapsed vagina was noted (Fig. 23-right).


Female Life History

Only 1 female developed to maturity in life history

tests. The onset of ecdysis was never observed. Vulva

development was seen 4 days after root penetration by the

first-stage larva. The ovaries were defined 7 days after

penetration, a fully developed female was evident 17 days

after penetration. Seventeen eggs were deposited on the



























Figure 39. Early third-stage female gonad.


Figure 40. Vaginal
development of a
late third-stage
female.


Figure 41. Virgin female with
gonad development complete.


_ZZ~
~2_






















Figure 44. Anatomy of the female reproductive
system from vulva to uteri: A=Egg in uterus;
B=uterus cells; C=Narrow vagina uterina; D=
Vagina; and E=Vaginal muscles.












^ A' ,. .* "f.".' '.

\ " 0


Figure 42. Final
female ecdysis.


Figure 43. Mature female.









same day development was considered complete. Males were

not observed near the female prior to oviposition.


Conclusions

Critical examination failed to reveal ecdysis in the

egg. Early development proceeded as known in most phyto-

parasitic nematodes. The distinct spicular primordia that

appeared early in male development was not noted in other

similar studies (Chitwood & Buhrer, 1946; Christie and Cobb,

1941; Hirschmann & Triantaphyllou, 1971; and Raski, 1950).

Another unique feature of male development occurred

when the male abandoned the third-stage larval integument,

leaving it embedded in the root following final ecdysis.

Male growth measured by body widths during develop-

ment has not been reported in other developmental reports

examined by the author. A loss in width of 18 im was shown

from a maximum of 52 pm. Time for male development varied

from 6 to 15 days.

The unique feature of female development was the

occurrence of huge vaginal primordia cells.














SECTION III
HOST-PARASITE RELATIONSHIPS

Methods


A variety of devices and ideas were tested to study

the host-parasite relationships and life history of the

new nematode species, all but one of which were unsuccess-

ful.


Macro-Observation Boxes

Wood chambers were patterned after rearing chambers

used by Dean, 1929,and by Minton, 1962. The box is 23-cm

square with a 2.3-cm chamber enclosed by glass and removable

wood sides. Buttonweed plants, well established in white

sand in the chamber, were inoculated with groups of 100

larvae at the site of healthy root flushes under the glass.

Development of the nematode either failed to occur or took

place in areas away from visible root sites. This method

was abandoned after a number of failures. In the next

trial plastic boxes,17.5 cm long by 9.3 cm wide by 3 cm

deep, were filled with either white, or with black volcanic

sand, planted with buttonweed and then inoculated with 100

larvae of the new species. In this system the activities of

the nematodes were obscured by the substrate. The macro-

observation boxes were used to observe nematodes on roots

growing in soil. Limitations of magnification and depth










of focus severely handicapped close observation by this

method.


Micro-Observation Units

Trials were conducted utilizing small plastic boxes

of various sizes, and plastic petri dishes containing a

poured 4-mm layer of water agar. Success was assured using

the following procedure: A 4-mm layer of 1% sterile water

agar is poured into a 9.2-cm plastic petri dish lid. A

5-mm ring of water agar is removed from the outside peri-

meter of the agar ring after hardening. A 5-mm glass rod

is heated over a glass flame until slightly red,then used

to burn a hole into the side of the closed petri dish. The

burn area should be sanded so the dish can be easily sepa-

rated. A stem cutting of buttonweed with 1 or 2 small

leaves is inserted through the hole and into the agar with

the leaves external to the dish. When primary roots emerge

and grow into the agar, a 5-mm well is cut into the agar

1-cm lateral to a primary root. Twenty-five first-stage

larvae, and 5 mature males in a small drop of sterile water

were inoculated into the agar well. A root map was drawn

(Fig. 45) when larvae made contact with the root. Each

larvae that situated itself at a particular site on the root

was assigned an alphabetical letter which was placed at the

approximate site on the root map. For each observation, the

date, time, dish number, and larva letter was recorded. Ob-

servations and measurements were made using the oil









immersion lens by placing a small drop of water on a cover

slip, which was inverted and placed over the root site

where larvae were attached. Basic data taken when possible

included: time elapsed from the inoculation to the time

larvae penetrated the root, time elapsed between penetration

of the larva to the appearance of root discoloration, if and

when a larva left its feeding site, body width measurements,

and ecdysis observations.


Behavior Studies


Eighteen plates containing plants in agar were inocu-

lated. Life history activities were observed in 5 plates;

the remainder were abandoned due to plant death, visibility

problems, or severe bacterial contamination.

As soon as the water in the inoculation well dried

the males and larvae migrated into the agar.


Male Behavior

Males migrated at random in the agar. A proclivity

to the root by males was not noted. Several males were

seen with lips in contact with the epidermis of a primary

root. In no case was stylet movement or metacorpus valve

pulsation noted in such contacts. One male lay quiescent

very close to a primary root the duration of the trial.

Most males migrated slowly through the agar after which

they became quiescent.









Larval Behavior

A total of 49 larvae of 450 inoculated was observed

penetrating roots. Data were taken until they departed,

ceased activity, or completed development.

Root penetration. Larvae migrated to the root follow-

ing pathways peculiar to most nematodes in agar (Wallace,

1964). One group of 4 larvae reached the root in 19, 23,

30, and 36 min., respectively,,following inoculation. Sty-

let movement was initiated about 4 min. after lip contact

with the root. Stylet thrusts were recorded at 92 per min.,

and 112 per min. by 2 larvae shortly after contact with the

root. Klinkenberg, 1963, recorded 69 thrusts per min. for

Pratylenchus crenatus Loof, 1960. Pressure was exerted on

the epidermal surface by thrusts of the nematode head and

stylet. The head slid over the cell surface as it thrust

until the stylet tip was over the middle lamella between 2

epidermal cells (Fig. 46-A). At this point the metacorpus

valve moved intermittently indicating digestive enzymes

were extruded into the attack site (Fig. 47). The lamella

between the 2 cells separated and the nematode slipped

laterally into the opening (Fig. 46-B). After penetration,

the larvae migrated obliquely 1-3 cells and 1-3 cells deep

(Fig. 46-C). One root with a 100 pm diameter was penetrated

61 pm laterally and 50 pm deep.

Feeding. Once the larvae were situated in the root,

feeding began immediately. In very small roots, feeding























































Figure 45. A root map
charting the progress
of each nematode that
occupied a feeding
site in one of the
inoculated petri
dishes. Each letter
represents a larva
at a feeding site.


Figure 46. Mode of root entry by
larvae. A=larva with head cen-
tered on middle lamella between
two cells.
B=Penetration. C=Feeding site.












i7

SJ' i' I r,*



._ ',

: el i. -











Figure 47. Larval head in Figure 48. A mature female
contact with middle in a shallow, hand-cut
lamella. Note small area epidermal section.
of discoloration in front
of oral aperture.


occurred in the cortex or pericycle. One larva fed in a

cortical cell occupied by another larva feeding in the peri-

cycle. Feeding sites were rather shallow (1-4 epidermal

cells deep) in mature roots. A very shallow hand cut longi-

tudinal section (Fig. 48) underneath a feeding female rare-

ly cuts the female.

Tissue discoloration. Yellowing of the tissue (Fig.
1
49-A) appeared initially 3, 4, and 4- hours following pene-

tration. In some cases (Fig. 49-B) the discoloration was

confined to the cell wall. Larvae were also noted with the

stylet inserted in the cell wall (Fig. 49-B). Nuclei of
























































Figure 49. Tissue discoloration. A) Heavy stippled
area indicates yellow discoloration in cells 4
hours after larval entry. B) Stippled area indicates
yellowing in cell walls. Note difference in nuclei
size between healthy and attacked cell.









cells with discolored walls were consistently enlarged

(11-13 pm) in comparison to healthy cells containing nuclei

(5-7 pm, Fig. 49-B).

Feeding migration. A few larvae touched the root and

departed without entry. Eleven larvae entered the root,

fed, and after a few hours or several days departed. Some

of the departing nematodes took up a new feeding position

on the same root or entered a different root and resumed

feeding. Some larvae were never seen again after leaving a

feeding site. Entry of an epidermal site predisposes the

site for entry of searching larvae. Nine larvae were seen

feeding together in a single, large longitudinal lesion

(Fig. 50). Such lesions are usually abandoned by feeding

larvae. One assumes the excess of enzymes and metabolites

in such a large open lesion renders the site unfavorable

for the development of the nematode.

Attack sites. Larvae have been detected feeding at

root tips (Fig. 51-A), root scales (Fig. 51-B), along

feeder roots, and rhizomes, singly, or in groups (Fig. 51-

C,D). In mature rhizomes, females are commonly seen either

singly or in groups (Fig. 51-E). Females can almost always

be found at the junction of secondary roots emerging from

the rhizome (Fig. 51-F). Larvae and third-stage males

have also been observed on chlorophyll-bearing stem tissue

at the soil line. Larvae and mature females have been de-

tected in root leaf scales just below chlorophyll-bearing

aerial leaf scales (Fig. 51-B). Many females resembling




















I I ill


li I yI







.09'.0










P Ij) )






Figure 50. Large lesion occupied
by 4 larvae.





63


Srr
A
,. < ;',. Hl


































Figure 51. Attack sites. A) Larvae feeding at root tip;
B) Larvae feeding on root-leaf scale; C) Larvae feeding on
feeder root; D) Large group of larvae in rhizome; E) Group
of mature females in mature rhizome; F) Females at secon-
dary root juncture.









small white sausages lie appressed to large rhizome pieces

(Fig. 51-E).

Host exudates. About 5% of the females examined pos-

sessed an accumulation of spheroid objects around the cervi-

cal region (Fig. 52). The exudates were closely associated

with the integument but did not adhere as do the cement

bodies adhering to the integument of cyst nematodes de-

scribed by Shepherd and Clark, 1978. The cement bodies are

depicted as brown hardened exudates originating from the

integument and grossly resemble the spheroid bodies of

V. volvingentis. Exudates of the new species differ in ap-

pearing to have a crystalline composition (Fig. 53).

The spheroid bodies appear to be exudates originating

from the host in response to feeding activities of the new

genus. Exudates have also been noted in Meloidodera flori-

densis (Fig. 54).

Oviposition and fecundity. Eggs are deposited naked

in the substrate. Five to 25 eggs usually lie inside the

ventral space formed by the body coil, or they are scattered

about the female body near the vulva. One large egg mass

contained 185 eggs in various states of development. Stan-

dard soil washing procedures usually wash the eggs from the

female so the number of eggs deposited is difficult to as-

certain. To determine the number of eggs contained in ma-

ture females, 123 female specimens were examined (Table 4).

















1 .
/






Figure 52. Spheroid bodies
attached to anterior region
of a mature female.


I


-. .E *-


Figure 53. Appearance
of a single spheroid
body.


Figure 55. Female
containing 7 eggs.


Figure 54. Exudates on the
anterior end of Meloidodera
floridensis.


r-'t









Table 4.


Number of fully developed eggs detected
in 123 mature females.


Egg number Females

0 47
1 28
2 22
3 8
4 12
5 5
6 0
7 1


Most females contained 1 or 2 eggs with a maximum of

7 eggs in 1 female (Fig. 55). The largest egg measured was

128 X 70.5 pm, inside a female.

Longevity. All plants which had been inoculated with

V. volvingentis for pathogenicity trials had died by

September, 1973. Five 1.9 X 20.3 cm soil plugs were re-

moved from each of 7 pots and the nematode population

counted (Table 5). Most pots contained large numbers of

eggs. The pots were maintained in the greenhouse in a fal-

low condition until October, 1976,when a sample similar to

the soil sample in September, 1973,was taken and the nema-

tode population counted (Table 5).

No females survived the longevity test while male and

larval survival was minimal. It appears feasible that eggs

are the prime survival stage in this species.

Egg viability. A 15-cm clay pot in which buttonweed

was root-bound, but nematode free,was saturated with sterile

water. The pot was then placed in a beaker and an addition-

al 100 ml of sterile water added. The effluent was














Table 5. Survival stage and numbers of nematodes recovered
before and after a longevity test.

Pot Females Males Larvae Eggs

Sept. Oct. Sept. Oct. Sept. Oct. Sept. Oct.
1973 1976 1973 1976 1973 1976 1973 1976

1 2 0 0 0 18 6 552 257

2 0 0 18 0 8 0 1282 5

3 1 0 0 0 0 0 198 6

4 0 0 1 0 3 0 41 287

5 0 0 0 0 0 0 1 2

6 1 0 1 0 6 0 811 2

7 0 0 0 2 8 6 336 129


Total 4 0 20 2 43 12 3221 688

Mean .57 0 2.85 .28 6.1 1.7 460 98.2









collected, and added back to the pot. The collection and

addition procedure was repeated 9 times, after which the

final effluent was filtered and the leachate placed in an

Erlenmeyer flask in the refrigerator.

To test egg viability, 10 eggs from 3-year-old fal-

lowed soil were placed in a drop of sterile water in each

of 4 dishes. Ten drops of stock leachate was added to the

water containing eggs in each of the 4 dishes. Four simi-

lar dishes containing eggs in sterile water but without

leachate served as controls. Results of the test are

shown in Table 6.


Table 6. Larval hatch from eggs kept in fallow
soil 3 years.
Larvae emerged
Examination Sterile water Sterile water
date and leachate only

Dish no. Dish no.
1-2-3-4 1-2-3-4
10/26/76 0-0-0-0 0-0-0-0
10/27/76 0-0-0-0 0-0-0-0
10/28/76 1-0-2-0 0-0-0-0
10/29/76 0-0-1-0 0-0-0-0
11/ 1/76 1-0-0-0 0-0-0-0
11/ 4/76 0-0-1-0 0-0-0-0
11/ 8/76 1-3-4-2 0-0-0-0
Total 3-3-8-2 0-0-0-0



Results. Larvae emerged from eggs only to which

leachate was added. Eggs placed in sterile water failed to

hatch. The test demonstrated that eggs can survive 3 years

in fallow soil. Attempts to inoculate buttonweed with lar-

vae hatched from 3-year-old eggs from fallow soil were









unsuccessful. Failure is attributed to low inoculum levels

and reduced nematode viability.


Conclusions


Larvae entered roots by penetrating the middle lamel-

la between 2 epidermal cells. Tissue discoloration became

evident 3-4 hours after entry of the nematode into the root.

A number of larvae abandoned the site after actively feed-

ing. Nuclei in invaded cells were distinctly larger than

nuclei in cells not entered by the nematode.

Host exudates were noticeably extruded at attack

sites and these exudates adhered to the cervical area of

female feeding at the site.

In longevity tests, eggs and larvae survived 3 years

in the absence of a host. Results indicate that ova are

the survival stage of this nematode.















SECTION IV
HOST PLANT INVESTIGATIONS

Host Plant


Diodia virginiana L. (buttonweed) in the family Rubi-

aceae is a perennial of no known economic importance.

Buttonweed (Fig. 56) is comprised of smooth, weedy stems

bearing lanceolate leaves. The plant creeps across the

soil as it grows forming a dense mat when abundant (Fig.

57). It usually is found in a mixture of herbaceous plants

peculiar to vegetation growing near bodies of water. The

vegetative form is found from Florida West to Texas, and

North to New England and Missouri (Small, 1933; Rickett,

1967).

Host Habitat


Buttonweed is found growing on moist soil adjacent to

bodies of water such as lakes, ponds, water-bearing ditches,

swamps, and prairies (Fig. 58). Maximum growth appears from

1 to 40 meters from the water's edge. As soil becomes less

moist and elevation increases, buttonweed decreases until

none are found. When collecting buttonweed a body of water

will almost always be in sight.

Norton, 1978,lists 15 genera of phytoparasitic nema-

todes comprising 30 species in aquatic habitats. Eight

genera and 2 species listed by Norton were found associated

70
































Figure 56. Diodia virginiana in flower. (A Susan
Anthony dollar is in the background for comparison.)


Figure 57. A mat of buttonweed mixed with
herbaceous plants.
































Figure 58. Paynes Prairie, Gainesville, Florida. A
prime habitat for buttonweed and Verutus volvingentis.

with buttonweed in an aquatic habitat. Ten genera and 23

species of phytoparasitic nematodes not included in Norton's

list were detected (Table 7).


Host Testing


A primary consideration, when a previously undescribed

phytoparasite is encountered, is the determination of its

potential as a parasite of economic crop plants. In the

original site where the new genus was collected corn and

soybean plantings were established adjacent to buttonweed

plants. The 2 aforementioned plants, in addition to 19

other plants, were placed in soil infested with the new

genus. Results of the host testing are shown in Table 8.













Table 7. Phytoparasitic nematodes found in soil
with roots of buttonweed.


associated


Phvtouarasite


Occurrence


Belonolaimus sp.
Cacopaurus sp.
Criconema sp.
Criconemoides curvatum Raski, 1952
mutabile Taylor, 1936
xenoplax Raski, 1952
Dolichodorus heterocephalus Cobb, 1914
Helicotylenchus crenicauda Sher, 1966
iI" dihystera (Cobb, 1893)
Sher, 1961
erythrinae (Zimmermann,
1904) Golden,
1961
longicaudatus Sher, 1966
I" paxilli Yuen, 1964
Hemicriconemoides wessoni Chitwood &
Birchfield, 1957
Hemicycliophora zuckermani Brzeski, 1965
Heterodera sp.
Meloidogyne arenaria (Neal, 1889)
Chitwood, 1949
Pratylenchus brachyurus (Gocfrey, 1929)
Filipjev and
Schuurmans Stek-
hoven, 1941
Trichodorus christ= e Allen, 1957
roxims "
Trophonema arenariam (Raski, 1956) Raski,
1957
Tylenchorhynchus irregularis Wu, 1969
Xiphinema americanvum Cobb, 1913


infrequent
moderate
infrequent
frequent
infrequent

moderate
frequent
infrequent




11 II











11 11
II 11
11 II






frequent
infrequent
moderate


infrequent


Phvtnarsit



















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Methods

Plants to be tested were seeded into or propagated

by cuttings in 20-cm clay pots filled with steamed soil.

When a healthy state of growth was evident, the pot was in-

oculated with 50 cc of soil and roots taken from a soil box

heavily infested with V. volvingentis. Plants were har-

vested in a minimum of 4 weeks. Roots were removed, washed,

and examined using a dissecting microscope to see if any

stage of the new nematode was in the root system. Five

1.9 X 20.3 cm soil plugs were removed from each inoculated

plant and processed for the nematode (Table 8). A few

plants were placed directly in a population maintainence

box.


Results

Females of the new taxon developed only in roots of

Ludwigia peruviana (Onagraceae), a weed of no economic im-

portance. Larvae were not detected in any of the other

host plants examined.

Results of this host test and because this nematode

has not been reported in numerous surveys of economic crop

plants indicate the nematode is doubtful as a threat to

economic crops, and very likely has a limited host range.

In the original site, the infested buttonweed was found

growing into plantings of both corn and soybeans, neither

of which proved to be hosts in field samples or host

tests. Since the nematode habitat includes irrigation









ditch banks serving as borders for numerous crops, the nema-

tode has ample opportunity to infest a variety of crops,

should they prove susceptible.


Host Symptoms


Aboveground symptoms of nematode injury were not

noted in any site examined. Symptoms were noted only in

inoculated population maintainence boxes, inoculated pots,

and in plants grown in water agar.

Generalized aboveground symptoms include stunting,

chlorosis, seed pod reduction, and death. Healthy green

stolons and leaves gradually turn chlorotic until the entire

plant is a pale yellow-green colo:. Plants begin to decline

and die 6 months after inoculation until no living plants

remain. In the final stages of decline, germinating seeds

produce small plants with 2-4 leaves that rarely assume a

healthy green color. Such plants rarely survive very long,

and their roots are almost always infected with females and

larvae of V. volvingentis.

Root symptoms are expressed as shallow epidermal

lesions of a pale yellow to brown or dark brown color.

Lesions comprise 3-15 cells and are present at feeding

sites. Lesions enlarge and darken with time (Fig. 59). In

a few cases, epidermal swelling in the form of a small

rounded protuberance appeared at the feeding site. In very

small roots lesions sometimes encircle the root resulting

in a necrotic constriction that causes blocking of





























2A


















Figure 59. Young female in root, showing large
dark lesion at feeding site.









conductive elements in the root and subsequent detachment

of the root at the lesion site.

Feeder roots of buttonweed are very succulent and de-

tach quite easily. Thus, it is not practical to compare

root systems of infected and nematode-free plants under ex-

perimental conditions.


Pathogenicity


Twenty cuttings of buttonweed were taken from plants

grown in steamed soil and transplanted to steamed soil in

20-cm clay pots. Four months later all foliage extending

outside the pot perimeters were clipped off, and 25 larvae

of V. volvingentis were inoculated into each of 10 pots.

Ten pots were left as uninoculated checks. Pots were ar-

ranged in a randomized block design using "Tippets Random

Number Table" (Le Clerg, Leonard & Clark, 1966).

Pathogenicity was evaluated by clipping foliage ex-

tending beyond the pot perimeter, and calculating the dry

weights. Clippings were dried in a heat chamber for 3 weeks

at 570C. The total number of seed pods produced were coun-

ted on each plant just prior to taking perimeter clippings.

Eight 20 cm deep by 2 cm wide soil plugs were removed

from each pot to evaluate the nematode population. Soil

from the 8 plugs was thoroughly mixed and 150 cc subsamples









were removed from each of the mixtures. Samples were proc-

essed using the sugar-centrifuge technique.


Results


Table 9 shows the severe effect of the nematodes on

the plant after 1 year. Thirteen days after final evalua-

tion data were taken, 4 chlorotic, severely stunted plants

still survived the initial inoculation (Fig. 60-A). Leaves

on the surviving plants were very chlorotic (Fig. 61) and

seed production was reduced considerably (Fig. 62). Inocu-

lated plants yielded 2.9 grams of seed pods, while check

plants yielded 15.7 grams. Seeds sprouting in infested

soil in pots inoculated with the nematode were attacked at

a very early stage of development and rarely produced more

than 1 or 2 yellowed leaves before dying.

Within 60 days of the final leaf evaluation, the 4

surviving inoculated plants were dead. All control plants

were in a healthystate of growth.

Table 10 shows the developmental stage and numbers of

individuals in each stage in the 150 cc of soil examined

from each replication. This table indicated that the egg

is the survival stage of the nematode.


Discussion


The severe parasitism by the nematode resulted in

eventual death of all inoculated plants. This occurred

under greenhouse conditions when the plants were maintained









80











'Aft


Figure 60. Appearance of inoculated (A) and

uninoculated (B) plants at the conclusion of

the pathogenicity trial.


.A


i




-~
r,
-
;













Table 9. Effect of Verutus volvingentis on foliage and
seed pod production of inoculated plants.


Days after
inoculation


Mean dry weight
of pot clippings
per plant in grams


Mean number of seed
pods per plant


untreated treated untreated treated


95 11.54 11.40 320.2 324.0

217 4.9 2.6* 0 0

398 24.0 7.2** 512.0 168.3


2 inoculated plants died.
2 inoculated plants died.
** 5 inoculated plants died.








Table 10. Nematode population density in treated and
untreated soil.

Days after Mean numbers of eggs or nema- Uninoculated
inoculation todes from inoculated plants plants

eggs larvae males females

406 1144 26 7 3.0 0

504 728 9 2 0.4 0












i
i
r



i




I






i
Irscj~; -r.
I-*.


Figure 62. A comparison of
seeds from inoculated (left)
and uninoculated (right).


Figure 61. Comparison of leaves
from inoculated (top), and
uninoculated plants (bottom).



several months past their normal annual cycle. Symptoms

produced in the greenhouse were never observed in the natu-

ral habitat. It is believed that the nematode population is

in ecological balance with the environment and is not large

enough to severely damage its host when buttonweed dies out

after about 6 months of growth, following the first frost.

During the next 6 months enough nematode eggs and seeds of

buttonweed survive to establish a well balanced association

between the host and its parasite. In the greenhouse there

is no intervening period to retard nematode population


B
I.
!tN

__









growth. As a consequence the parasite increases unchecked,

severely damaging or killing its host. Factors in the nat-

ural habitat such as biological control agents and

population-limiting physical changes are also absent from

the controlled environment of the greenhouse.


Distribution of V. volvinqentis in Florida


A total of 266 soil samples, and an almost correspon-

ding number of root samples, were examined from collection

sites in 13 Florida counties (Table 11).

Ninety-eight samples were positive and 168 samples

were negative. Plants examined in the survey are shown in

Table 12. The nematode was detected in soil from 13 hosts

in the survey, but in roots of buttonweed only. It is

doubtful if the 12 plants surveyed are true hosts of the

new nematode since most of them were growing close to

buttonweed.

The nematode was detected in Alachua, Lake, Orange,

and Sumter counties. The largest population of the nema-

tode occurred in Alachua county within Paynes prairie (Fig.

57), which is a relatively undisturbed, natural, ecological

unit in Florida. The prairie supports large plantings of

buttonweed. Samples taken at the waterline or from high

dry sites in the prairie are almost always free of the nem-

atode and buttonweed. About 90% of the negative sample

results originate from plants other than buttonweed in













Areas in Florida samples for Verutus volvingentis.


Sample results


Positive Negative


Alachua



Baker
Bradford
Broward
Gadsden
Lake




Marion
Orange

Putnam
Seminole
Sumter


Union
Volusia


Gainesville-Terwilliger school
Newnans lake
Paynes prairie
Earleton
roadside

Pompano Beach
Chattahoochee
Clermont
Grand Island
Howey-in-the-Hills
Leesburg
Minneola
Ocala
P lymouth*
Zellwood
Florahome*
Sanford
Oxford
Rutland
Tarrytown
roadside
Barberville


Buttonweed was not sampled at this site.


County


Site


Table 11.
















Table 12.


Plants examined for Verutus volvingentis in
the Florida Survey.


Scientific name


Vernacular name


Result


Alternanthera sp.
Andropoaon glomeratus (Wa]t)B.S.P,
Baccaris halinifolia L.
Bacopa caroliniana (Walt) G.L. Robinson
Bidens laevis (L.)B.S.P.
Brassica oleraceae L. (acephala group)
Cassia obtusifolia L.
Cephalanthus oc-identalis L.
Circium sp.
Cuphea carthagensis (Jacq) Macbride
Cyperus odoratus L.
Diodia teres Walt
." virginiana L.
Eupatorium sp.
Geranium carolinianum L.
Clottidium vesicarium (Jacq) Mohr
Glvcines max (L.) Merr
Hyp~L cun r:iutilum L.

Juncus effusus L.
Lachnanthes caroliniana (Lam.) Dandy
Linaria sp.
Ludwigia arcuata Walt
leptocarpa (Nutt.) Hara
Mikania scandens (L.) Willd.
Nelumbo lutea (Willd.) Pers.
Paron.chia balwinii Fenzl
- ______ rlel- Steud.
Polygonum hydropiperoides Michx.
persicaria L.
punctatum Ell.
Ptilimnium capi1l1ceum (Michx.) Raf.
Rnexia mariana L.
Sabal palmeti o (Walt.) Todd ex. Schult
& Schult f.
Salix sp.
Scirpus sp.
Scoparla dulcis L.
Stenota hrum secundatum (Walt.)
0. Kuntze
Typha sp.
Zantedeschia sp.
Zea mays L.


bushy beardgrass
saltbush
water hyssop
beggars tick
collards
coffee-weed
buttonbush
thistle


poor Joe
buttonweed

cranebill
bladderpod
soybean
dwarf St. Johns
wort
soft rush
redroot
toadflax
false loosestrife

climbing hempweed
American lotus

vasey grass
smartweed
heartweed
water smartweed
mock bishop weed
meadow beauty
cabbage palr:ttto

willow
bulrush
sweet broom
St. Augustine grass

cattail
calla lily
corn


negative

positive
negative
positive
negative



positive
negative

positive
positive
negative
positive

negative

positive
negative
positive

negative






positive
negative

positive

negative

Sposi
positive









higher, less moist areas, or from areas in water or at the

water's edge.


Conclusions


V. volvingentis occurs in Florida in moist habitats.

Paynes Prairie contains large populations of the nematode

and its host. The host range appears to be very limited.

None of the economic host plants tested were susceptible to

the nematode. Three months following a low level inocula-

tion of the nematode on its host, little or no effect was

evident. Severe symptoms occurred 7 months after inocula-

tion. Fourteen months after inoculation all inoculated

plants were dead. All uninoculated plants in the test were

in a healthy vigorous condition. It is believed that death

of the inoculated plants was a result of growing an annual

plant in the greehouse, devoid of natural inimical ecologi-

cal factors present in its natural habitat, and past its

time of normal growth.















SECTION V
BIOLOGICAL CONTROL INTERACTIONS


Males and larvae were exposed to zoospores of Cate-

naria anguillulae Sorokin. Within 10 min., zoospores were

attached to the cephalic region of larvae (Fig. 63-A), and

males (Fig. 63-B), and to the male cloacal area (Fig. 63-C).

Development of the fungus was only completed in males.

Zoospores were released from an infected male 78 hours after

the initial infection.

Two-hundred eggs were exposed to a culture of zoo-

spores to test the susceptibility of ova to the fungus.

Zoospores became attached to 25% of the eggs in culture but

no thalli developed subsequently.

Eggs were also exposed to a culture of an aquatic

Phycomycete, Rhizophidium sp., a member of the Chytridiales,

known to attack eggs of invertebrates. Sporangia of the

fungus was observed attached to a number of eggs (Fig. 64);

however, further development of the fungus was not noted.

A natural population of the nematode was found infes-

ted with endospores of Pasteuria ramosa Metchnikoff (Fig.

63-D). A single larvae hatching from an egg in the popula-

tion was also noted with endospores attached.











A








\

A'-
A"i .'

\ '\9r


ri* l


*1 1
:-I iit :rli ;




11 t rI %
,-. (r*J


.~a

h i'
I ~ C'

U. 1


Figure 63. Biological control
interactions. Catenaria
anguillulae zoospores attached
to the cephalic region of a
larva (A), a male (B), male
cloacal region (C). Endospores
of Pasteuria ramosa on a
larva (D).


7
-- ". -






Figure 64. Sporangium of
Rhizophidium sp.
attached to an egg.


., 1


no% -
ts






89


Femaleswere not noted in biological control inter-

actions. Biological control agents attached to ova but

failed to penetrate the shell and complete development.