• TABLE OF CONTENTS
HIDE
 Copyright
 Front Cover
 Table of Contents
 Introduction
 Description of h. leuceilyma...
 Pathogenicity of h. leuceilyma...
 Summary, literature cited
 Back Cover






Group Title: Bulletin - University of Florida. Agricultural Experiment Station - no. 687
Title: Heterodera leucilyma n. sp. (Nemata Heteroderidae)
CITATION PAGE IMAGE ZOOMABLE PAGE TEXT
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00027502/00001
 Material Information
Title: Heterodera leucilyma n. sp. (Nemata Heteroderidae) a severe pathogen of St. Augustinegrass in Florida
Series Title: Bulletin University of Florida. Agricultural Experiment Station
Physical Description: 35 p. : ill. ; 23 cm.
Language: English
Creator: Di Edwardo, A. A
Perry, Vernon George, 1921-
Publisher: University of Florida Agricultural Experiment Station
Place of Publication: Gainesville Fla
Publication Date: 1964
 Subjects
Subject: Plant nematodes -- Florida   ( lcsh )
Grasses -- Diseases and pests -- Florida   ( lcsh )
Genre: government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Bibliography: Bibliography: p. 34-35.
Statement of Responsibility: A.A. Di Edwardo and V.G. Perry.
General Note: Cover title.
Funding: Bulletin (University of Florida. Agricultural Experiment Station) ;
 Record Information
Bibliographic ID: UF00027502
Volume ID: VID00001
Source Institution: Marston Science Library, George A. Smathers Libraries, University of Florida
Holding Location: Florida Agricultural Experiment Station, Florida Cooperative Extension Service, Florida Department of Agriculture and Consumer Services, and the Engineering and Industrial Experiment Station; Institute for Food and Agricultural Services (IFAS), University of Florida
Rights Management: All rights reserved, Board of Trustees of the University of Florida
Resource Identifier: aleph - 000929271
oclc - 18361241
notis - AEP0048

Table of Contents
    Copyright
        Copyright
    Front Cover
        Page 3
    Table of Contents
        Page 4
    Introduction
        Page 5
        Page 6
    Description of h. leuceilyma n.sp.
        Page 7
        Materials and methods
            Page 7
        Generic anatomy
            Page 8
        Heterodera leuceilyma n.sp.
            Page 9
        Females
            Page 10
        Males
            Page 10
            Page 11
            Page 12
            Page 13
        Second stage larvae
            Page 14
            Page 15
    Pathogenicity of h. leuceilyma to st. augustinegrass
        Page 16
        Materials and methods
            Page 16
            Page 17
        Results and discussion
            Page 18
            Page 19
            Page 20
            Page 21
            Page 22
            Page 23
            Page 24
            Page 25
            Page 26
            Page 27
            Page 28
            Page 29
            Page 30
            Page 31
            Page 32
            Page 33
    Summary, literature cited
        Page 34
        Page 35
    Back Cover
        Page 36
Full Text





HISTORIC NOTE


The publications in this collection do
not reflect current scientific knowledge
or recommendations. These texts
represent the historic publishing
record of the Institute for Food and
Agricultural Sciences and should be
used only to trace the historic work of
the Institute and its staff. Current IFAS
research may be found on the
Electronic Data Information Source
(EDIS)

site maintained by the Florida
Cooperative Extension Service.






Copyright 2005, Board of Trustees, University
of Florida




-F













CONTENTS


Page

Introduction -...----------5-- -- ------ ------------- ----------------- 5

Description of H. leuceilyma n.sp. ..___. ------------------------ 7
Materials and Methods ------. ------- ----------------- 7
Generic Anatomy .------------- ------------------- 8
Heterodera leuceilyma n.sp ... ---.. ---------------- 9
Females ------------ .. .. ..---------------------- ----- 10
Males .------------------ --- ---------------- ---------- 10
Second Stage Larvae ..------ -----------14

Pathogenicity of H. leuceilyma to St. Augustinegrass ------ 16
Materials and Methods ._....- ---------------------- 16
Results and Discussion .__--- ...-.......... .------------------- 18

Summary -----.....-- -----.------ ------------ 34


Literature Cited










Heterodera leuceilyma n.sp.
(Nemata: Heteroderidae), a Severe

Pathogen of St. Augustinegrass in Florida

A. A. Di Edwardo and V. G. Perry1

INTRODUCTION
Most species of the Genus Heterodera appear to be adapted
to climates as found in the northern portion of the north tem-
perate zone of the world. The soybean cyst nematode, H. gly-
cines, occurs in North Carolina, Tennessee, and certain other
southern states but not south of latitude 300. The golden nema-
tode of potato, H. rostochiensis, occurs in the tropical zone of
Central and South America, but apparently only at high eleva-
tions. This nematode is widespread throughout northern Europe,
but the only known infestations within the United States are on
Long Island, New York. Most of the other species of significant
economic importance are also limited to cooler climates than are
found in Florida.
In Florida, several species of cyst nematodes have been re-
ported, but damage to economic plants has not previously been
considered important. Heterodera fici has been found on figs.
Cysts similar to H. weissi or H. cacti are found quite commonly,
especially in samples of turf. The clover cyst nematode, H. tri-
folii, has been reported from Florida, but the present authors
have never identified this species within the state. Recently
Golden, Rau, and Cobb (5)2 described H. cyperi from nutgrass
at Sanford, Florida. Other members of Heterodera that occur in
Florida have received little attention.
A great deal of difficulty is experienced when specific iden-
tification of cyst nematodes is attempted. Most morphological
studies have been conducted on the dead female body, or cyst,
and its contents including eggs. The first species to be described
was H. schachtii by Schmidt in 1871 (12) from sugar beets in
Germany. Other hosts were recorded later, but in most cases

1 Dr. Di Edwardo was formerly Assistant Nematologist and Dr. Perry
is Nematologist in the Entomology Department, Florida Agricultural
Experiment Stations.
2 Numbers in parentheses refer to Literature Cited.








Florida Agricultural Experiment Stations


specimens from these other hosts would not parasitize sugar
beets. Gradually other species, totaling 28, have been described.
Characteristically, species of cyst nematodes have relatively
narrow host ranges, and in some cases the host plant, when
known, will help narrow the list of possible species when identi-
fication is attempted. For example only four species were pre-
viously known to attack members of the grass family (Grami-
nae). The oat cyst nematode, H. avenae, is an important parasite
of cereals in Europe. The grass cyst nematode, H. punctata, was
described as a parasite of wheat in Canada by Thorne (15).
Recently Luc and Merny (9) described H. sacchari from sugar-
cane in Africa, and Luc and Brizuela (8) described H. oryzae
as a parasite of rice in Africa.
Pathogenic effects of cyst nematodes have not been elucidated
on many hosts; however, certain species on certain hosts are
recognized as being among the most highly pathogenic of all
plant nematodes. The sugar beet cyst nematode, the golden
nematode of potato, and the soybean cyst nematode, when pres-
ent in moderate to large numbers, bring about such severe
damage to their respective hosts that crop production is dras-
tically reduced. Eradication, if. possible, is economically infeas-
ible. Fortunately infestations of most cyst nematodes within
the United States are confined to rather small areas. Spread of
the golden nematode of potato and the soybean cyst nematode
is contained by regulatory programs.
Species of Heterodera are quite similar to species of Meloi-
dogyne in their host-parasite relationships and life cycles. In
both genera, infection is by second stage larvae penetrating near
the root tip and moving a short distance before becoming seden-
tary. The larvae come to rest and begin feeding; and the body
begins to swell, becoming sausage-shaped. The host responds
to the feeding by initiating a group of abnormally enlarged
multinucleate cells (giant cells) about the head of the parasite.
The giant cells produced as a result of parasitism by cyst nema-
todes are generally smaller than those produced by root-knot
nematodes. Hyperplasia is also significantly less than that caused
by root-knot nematodes. After coming to rest, larvae of cyst
nematodes do not remain wholly imbedded in the roots, but
rather the major portion of the adult female body is external
to the root. Males regain the eelworm shape and move about
as do those of Meloidogyne. Cyst nematodes produce much smal-








Heterodera leuceilyma n. sp.


ler galls on host roots than do the root-knot nematodes. Endo (4)
has recently determined the life cycle and some host-parasite
relationships of the soybean cyst nematode.
In October 1960, a sample of St. Augustinegrass turf was
received by the authors from Boynton Beach, Florida, for nema-
tode analysis. Plant parasitic nematodes of several species were
found, and the specimens were used to inoculate St. Augustine-
grass growing in soil which had been treated with methyl bro-
mide. The plants were held in a plant growing room at tempera-
tures ranging between 750F and 80F. Within five months the
St. Augustinegrass in these pots exhibited a very pronounced
interveinal chlorosis and stunting. On removal, large numbers
of a Heterodera species were found on the grass roots and in
the soil. The original area was then resampled, and large num-
bers of cysts were found. The St. Augustinegrass within this
area was in an advanced state of decline (Fig. 7). Detailed
studies of the nematodes revealed certain morphological differ-
ences from all reported species of Heterodera. No previous re-
port of a cyst nematode infection of St. Augustinegrass was
found in the literature.
Therefore investigations were undertaken 1) to prepare the
taxonomic description of the St. Augustinegrass species and 2)
to elucidate certain aspects of the host-parasite relationships.

DESCRIPTION OF H. LEUCEILYMA3 n.sp.
Materials and Methods
Cysts for microscopic studies were collected from both
naturally infested soil and experimental colonies established in
plant growing rooms. All specimens were considered to be from
St. Augustinegrass since this is the only known host, although
other plants occurred in the naturally infested areas. The soil
was processed by a routine method utilizing an 80 mesh standard
soil sieve, and the cysts were isolated to distilled water. The
eggs within the cysts were killed by exposure to 50'C tempera-
tures for about 12 minutes. Fixation was accomplished in 5
percent formalin which had also been heated to 50'C.
Slides were prepared by placing individual cysts in a drop
of fixative on a glass microscope slide. The cysts were cut at the
3 The name leuceilyma was suggested by Dr. R. F. Hussey. It is de-
scriptive of the "white cover" afforded the cysts by the extensive sub-
crystalline layer.








Heterodera leuceilyma n. sp.


ler galls on host roots than do the root-knot nematodes. Endo (4)
has recently determined the life cycle and some host-parasite
relationships of the soybean cyst nematode.
In October 1960, a sample of St. Augustinegrass turf was
received by the authors from Boynton Beach, Florida, for nema-
tode analysis. Plant parasitic nematodes of several species were
found, and the specimens were used to inoculate St. Augustine-
grass growing in soil which had been treated with methyl bro-
mide. The plants were held in a plant growing room at tempera-
tures ranging between 750F and 80F. Within five months the
St. Augustinegrass in these pots exhibited a very pronounced
interveinal chlorosis and stunting. On removal, large numbers
of a Heterodera species were found on the grass roots and in
the soil. The original area was then resampled, and large num-
bers of cysts were found. The St. Augustinegrass within this
area was in an advanced state of decline (Fig. 7). Detailed
studies of the nematodes revealed certain morphological differ-
ences from all reported species of Heterodera. No previous re-
port of a cyst nematode infection of St. Augustinegrass was
found in the literature.
Therefore investigations were undertaken 1) to prepare the
taxonomic description of the St. Augustinegrass species and 2)
to elucidate certain aspects of the host-parasite relationships.

DESCRIPTION OF H. LEUCEILYMA3 n.sp.
Materials and Methods
Cysts for microscopic studies were collected from both
naturally infested soil and experimental colonies established in
plant growing rooms. All specimens were considered to be from
St. Augustinegrass since this is the only known host, although
other plants occurred in the naturally infested areas. The soil
was processed by a routine method utilizing an 80 mesh standard
soil sieve, and the cysts were isolated to distilled water. The
eggs within the cysts were killed by exposure to 50'C tempera-
tures for about 12 minutes. Fixation was accomplished in 5
percent formalin which had also been heated to 50'C.
Slides were prepared by placing individual cysts in a drop
of fixative on a glass microscope slide. The cysts were cut at the
3 The name leuceilyma was suggested by Dr. R. F. Hussey. It is de-
scriptive of the "white cover" afforded the cysts by the extensive sub-
crystalline layer.








Florida Agricultural Experiment Stations


mid-portion of the body, and the contents were removed. The
cyst walls were then mounted in dessicated glycerine between
two coverslips, using a Cobb aluminum slide holder. The re-
maining eggs and larvae were then mounted for temporary
studies in the manner usual for this genus. In order to observe
vulvar cone tops, posterior portions of some cysts were cut so
that about one-fifth of the posterior body remained. A small
globule of hard glycerine jelly was placed on a glass slide, melted,
and spread. After the glycerine cooled, a warm needle was used
to melt a small portion near the center. The vulvar cone was
inserted into the melted area and manipulated to stand upright
as the media hardened. A cover slip was then applied and sealed
with paraffin wax.
Males and second stage larvae were removed from the soil
through the use of a modified Seinhorst elutriator. They were
killed with exposure to temperatures of 50C for 12 minutes
and fixed in 2 percent formalin, which had also been heated to
50'C. The males, mature cysts, larvae from the soil, larvae
mechanically removed from eggs, and eggs were processed to
dehydrated glycerine using the technique outlined by Seinhorst
(14). They were then mounted in dessicated glycerine between
two cover slips held in Cobb aluminum slide holders. Measure-
ments of each life cycle stage given above were obtained, and
the data are presented elsewhere in this paper. Measurements of
second stage larvae obtained from the soil only are herein
recorded, but little difference was found between these and the
artificially hatched larvae.

Generic Anatomy
Species of Heterodera, especially members of the so-called
H. schachtii group, are similar in most morphological details.
Unfortunately most attention has been directed to the cysts
and their contents. The cysts constitute a persistent stage in the
life cycle, since eggs contained within them may survive several
years in an absence of suitable hosts and favorable environment.
Oostenbrink and den Ouden (11), Cooper (3), Mulvey (10), and
Thorne (16) have contributed recently to knowledge of cyst
characteristics. Such factors as cyst wall patterns, punctation,
bullae position and development, fenestra, length of vulva,
structure of the underbridge, and structure of the vagina do
vary somewhat between species. Punctations, for example, occur








Heterodera leuceilyma n. sp.


in the group but are usually difficult to observe, being tiny,
porelike markings of the body wall. However, the punctations
on H. avenue and the species herein described are relatively easy
to observe because of the large size and abundance. Cysts of
H. avenue and the species herein described are normally covered
by a thick sub-crystalline layer. Actually it seems logical to
postulate that the sub-crystalline layer is laid down by the hypo-
dermis through pores of the cuticle which are called punctations.
A great deal of information is lacking concerning reproduc-
tion of the species of the nematode family Heteroderidae. Some
species of Meloidogyne and Heterodera will reproduce in an ab-
sence of males, apparently parthenogenetically. However, sperm
have been observed in females, but the process of copulation re-
mains undescribed for the family. Males are commonly found
within the egg masses of Meloidogyne, indicating the possibil-
ity that copulation does occur. The males are non-bursate and,
most if not all species, have a peculiar twist of the body, usually
900. Males of the species herein described apparently occur spo-
radically, indicating that females possibly reproduce without
mating.
The male reproductive opening in the phylum Nemata is
common with the anal opening. This opening, a cloaca, is usually
a pore with no protrusions or lips, but Hirschmann (6) illus-
trates a protuberance about the cloacal opening of H. glycines.
Luc and Merny (9) illustrate a marked protuberance about the
cloacal opening of H. sacchari. The protuberance of the St.
Augustinegrass species is so distinct that a name seems desir-
able. Although copulation has not been observed, the spicules are
obviously exerted through this tube and into the female vagina.
It appears logical to assume that the tube is inserted through
the female vulva during copulation and that spermatozoa are
transferred between the spicula through the tubular apparatus.
However, there appears little reason to consider this a penis-
like organ, so it is proposed that the structure be called a "tubus".

Heterodera leuceilyma n.sp.
Measurements: Cysts (10)-Length=650 930 (830) p; Width
=420 -570 (480)p.
Males (10) Length = 950 1450 (1230) p; a = 25.0 41.9
(34.8); b=5.0 -8.6 (6.7); c=? (Anus near terminus) ; t=54.9-
67.6 (61) %; stylet=32-40 (35) /; spicules=30 -37 (35.5) /.







Florida Agricultural Experiment Stations


Second stage larvae (10)-Length=520 580 (550) /; a--
26.3 31.5 (28.3) ; b = 4.0 4.9 (4.4) ; c=7.9 9.3 (8.6) ; stylet
=23 28 (26) p; clear portion of tail = 31 41 (38) p.

Females
Body usually lemon shaped with protruding neck and vulvar
cone. Size and shape variable. Living female white in appear-
ance changing to yellow, light brown, dark brown, and glossy
black, respectively, with increasing age of cysts. Extensive sub-
crystalline layer formed prior to death, thoroughly covering
cysts to a thickness of 50 / or more (Figs. 1F and 6). Cuticle
thick, exhibiting a zigzag pattern externally (Fig. 2) and ex-
tensive and randomly arranged punctations (Fig. 3) internally.
Excretory pore not observed. Anus obscure with cuticular pat-
tern as shown in Figure 1B.
Lip region distinctly set off with 2 prominent disc-shaped
annules, the second of which is larger. Labial sclerotization
weak. Dorsal gland orifice about 5 p from base of stylet. Pro-
corpus enlarged in basal portion. Metacorpus large, well de-
veloped, with conspicuous valvular apparatus. Basal lobe of
esophagus variable in shape, which is affected by enlargement
of intestine and ovaries. Basal lobe overlaps intestine.
Vulva a transverse slit at terminus of protruding cone, length
about 55 j,. Fenestra obscure externally on cone, apparently
thick cuticle extends to vulva (Fig. 4). Vagina as shown in
Figures 1E and 1F. Underbridge of cysts similar to that shown
in Figure 5. Bullae present and similar to those of H. trifolii.
Ovaries convoluted at maturity, occupying much of the swollen
body.

Males
Specimens sporadic in occurence. Body vermiform, tapering
toward both extremities. Body twisted 900 in posterior third.
Lip region set off, marked by 4 to 5 annules. Cuticle marked by
distinct transverse striae which are interrupted on the lateral
fields by 3 lines. Excretory pore located near anterior portion
of basal lobe of esophagus. Hemizonid 6 annules anterior to
excretory pore. Phasmids subterminal (Fig. 1G).
Cephalic framework conspicuous with anterior extensions
from the basal plate. Stylet well-developed with rounded basal
knobs but concave anteriorly when viewed laterally. Dorsal







Florida Agricultural Experiment Stations


Second stage larvae (10)-Length=520 580 (550) /; a--
26.3 31.5 (28.3) ; b = 4.0 4.9 (4.4) ; c=7.9 9.3 (8.6) ; stylet
=23 28 (26) p; clear portion of tail = 31 41 (38) p.

Females
Body usually lemon shaped with protruding neck and vulvar
cone. Size and shape variable. Living female white in appear-
ance changing to yellow, light brown, dark brown, and glossy
black, respectively, with increasing age of cysts. Extensive sub-
crystalline layer formed prior to death, thoroughly covering
cysts to a thickness of 50 / or more (Figs. 1F and 6). Cuticle
thick, exhibiting a zigzag pattern externally (Fig. 2) and ex-
tensive and randomly arranged punctations (Fig. 3) internally.
Excretory pore not observed. Anus obscure with cuticular pat-
tern as shown in Figure 1B.
Lip region distinctly set off with 2 prominent disc-shaped
annules, the second of which is larger. Labial sclerotization
weak. Dorsal gland orifice about 5 p from base of stylet. Pro-
corpus enlarged in basal portion. Metacorpus large, well de-
veloped, with conspicuous valvular apparatus. Basal lobe of
esophagus variable in shape, which is affected by enlargement
of intestine and ovaries. Basal lobe overlaps intestine.
Vulva a transverse slit at terminus of protruding cone, length
about 55 j,. Fenestra obscure externally on cone, apparently
thick cuticle extends to vulva (Fig. 4). Vagina as shown in
Figures 1E and 1F. Underbridge of cysts similar to that shown
in Figure 5. Bullae present and similar to those of H. trifolii.
Ovaries convoluted at maturity, occupying much of the swollen
body.

Males
Specimens sporadic in occurence. Body vermiform, tapering
toward both extremities. Body twisted 900 in posterior third.
Lip region set off, marked by 4 to 5 annules. Cuticle marked by
distinct transverse striae which are interrupted on the lateral
fields by 3 lines. Excretory pore located near anterior portion
of basal lobe of esophagus. Hemizonid 6 annules anterior to
excretory pore. Phasmids subterminal (Fig. 1G).
Cephalic framework conspicuous with anterior extensions
from the basal plate. Stylet well-developed with rounded basal
knobs but concave anteriorly when viewed laterally. Dorsal







1Ar


K I ^ -/ v^
I(J


fl$\.i'/ j~':
r
r:i~- :
-4 .-1
V


Fig. 1-Heterodera leuceilyma. A-Anterior portion of female. B-Cuti-
cular pattern surrounding anus. C-Head and tail of second stage larva.
D-Head of male. E-Dorso-ventral view of vulvar cone. F-Lateral view
of vulvar cone. G-Tail of male.


6::









Florida Agricultural Experiment Stations


Ci. et*t


... '9.. %






A> .ma$ \*


Fig. 2-Photomicrograph of H. leuceilyma n.sp. body wall showing zig-
zag patterns. (1200x)


t 9 '
',* 4"* -*
**'
-* S,

9. 4


-9


C-
*


I a t
p *^ *9 *? '<.
4 vt -
*k 0' *

Ib IV A- ,

y b.. *V



p sear'.
'^- +,r:;E.^

^^^


4 4, *
Ca *.'a1
*. I p 0"' I -







Fg V



Fig. 3-Photomicrograph showing punctation in the body wall of female
H. leuceilyma, n.sp. (1200X)


S -i
Cs


*








Heterodera leuceilyma n. sp.


Fig. 4-Photomicrograph showing the cuticle in the vicinity of the vulva.


-~7p
Fig. 5-Photomicrograph taken through the vulvar cone showing the
underbridge and bullae.







14 Florida Agricultural Experiment Stations

gland orifice about 2 to 3 p posterior to base of stylet knobs.
Procorpus relatively long. Metacorpus relatively smaller than
in female, ovoid in shape; the valvular apparatus relatively
small. Basal lobe of esophagus containing at least 3 gland nuclei,
with long overlap of intestine. Tail very short, rounded. Body
wall extruded at cloacal opening to form a tube or tubus. Spi-
cules paired, arcuate. Gubernaculum, spicules, and tubus as
shown in Fig. 1G. Testis 1, well developed, outstretched.

Second Stage Larvae
Body vermiform, tapering from midportion towards both
extremities but more markedly posteriorly. Lip region set-off,
marked by 4 to 5 annules. Cuticle marked by distinct striae
which are interrupted on the lateral fields by 3 lines. Excretory
pore near anterior portion of basal esophageal bulb. Hemizonid
6 annules anterior to excretory pore. Phasmids pore-like, pos-
terior to the latitude of anal opening. Tail long, with hyaline
portion about 11/2 times stylet length. Cephalic framework rela-
tively heavy. Stylet well-developed with prominent knobs, as
shown in Fig. 1C. Dorsal gland orifice about 5 p posterior to
stylet knobs. Metacorpus normal for the genus. Nerve ring about
middle of isthmus. Basal lobe of esophagus overlapping intestine
and 3 gland nuclei were observed. Genital primordium usually
in 2-cell stage.
Holotype Cyst containing eggs and larvae collected from
St. Augustinegrass by the authors at West Palm Beach, Florida,
slide No. 6, Nematode Collection, Department of Entomology,
University of Florida, Gainesville, Florida, U.S.A. Slide No. 6A
containing anterior portion of female; slide No. 6B with larvae;
slide No. 6C posterior portion of cyst, and slide No. 6D posterior
view of vulva cone all collected same date from the same loca-
tion as the holotype.
Paratypes 5 cysts, 6 males, and 6 larvae on slides No. 6E,
6F, and 6G respectively, Nematode Collection, Department of
Entomology, University of Florida, Gainesville, Florida, U.S.A.
Also 7 cysts, 6 males, and 6 larvae on slides No. T295P, T296P,
and T297P respectively USDA Nematode Collection, Nematology
Investigations, Agricultural Research Service, Beltsville, Mary-
land. Also 5 cysts, 6 males, and 6 larvae on slides No. 18, 19,
and 20 respectively in cabinet C-2724, tray 2, Nematode Collec-
tion, Citrus Experiment Station, Lake Alfred, Florida, U.S.A.








Heterodera leuceilyma n. sp.


Diagnosis Heterodera with the above measurements and
general description. H. leuceilyma is similar in certain charac-
teristics to H. trifolii and H. glycines. Cysts are distinguished
from these two by the distinctive cuticular markings, puncta-
tions, and extensive sub-crystalline layer. Larvae differ in their
very long size and ratio of stylet to clear portion of the tail.
Males are distinctive from all other species except H. sacchari
by the well developed tubus and from H. sacchari by the 3 lines
of the lateral field on adult males. H. leuceilyma is also somewhat
similar to H. avenue but differs by the cysts having bullae in a
more anterior location, by the 3 lines of the lateral field on males
and larvae, and by the well developed tubus of males. H. leu-
ceilyma is the only species of Heterodera known to parasitize
St. Augustinegrass.
Type host St. Augustinegrass, Stenotaphrum secundatum
(Walt.) Kuntze.
Type location 151 Gregory Place, West Palm Beach, Flo-
rida. Specimens of this species also have been collected from
Boynton Beach, Miami, Ft. Lauderdale, Boca Raton, Lake Worth,
and Lake Park, all on the lower east coast of Florida. St. Augus-
tinegrass was the predominant plant growing in each of the
above locations from which samples were obtained.


Fig. 6-Cysts of H. leuceilyma. Note heavy sub-crystalline layer.







Florida Agricultural Experiment Stations


PATHOGENICITY OF HETERODERA LEUCEILYMA
TO ST. AUGUSTINEGRASS

Materials and Methods
Isolation of the nematode. For inoculation purposes, several
methods were used to extract nematodes from the soil. Initially
specimens were extracted through the use of a modification of
the centrifugal-flotation technique (1). Nematodes obtained
with this method were used to start colonies which were main-
tained in a plant-growing room or in a greenhouse during the
course of the investigations.
Inoculum for subsequent tests was obtained from those
colonies by removing mature cysts from the host roots or di-
rectly from the soil after screening onto a 30 and 100 mesh
sieve combination. Debris collected on the 100 mesh sieve was
examined with a dissecting microscope, and the cysts were re-
moved. The Seinhorst elutriator technique (13) and a modifi-
cation of the Christie-Perry technique (2) were used to obtain
males and larvae.
Inoculation Experiments. Experiment 1. Eight 6-inch stand-
ard clay pots were filled with autoclaved soil and sprigged with
St. Augustinegrass. After the sprigs rooted, the soil in each of
four pots was inoculated with 30 cysts collected from an infected
lawn in Boynton Beach, Florida. Each cyst was cut open to re-
lease the eggs just prior to inoculation. The remaining four
pots received no nematodes. The pots were randomly arranged
in a plant-growing room and maintained at a temperature of
approximately 80F.
Experiment 2. Eighteen 6-inch standard clay pots were filled
with autoclaved soil and sprigged with St. Augustinegrass. After
the sprigs rooted, each of 12 pots was inoculated with 10 cysts.
Six were inoculated with cysts obtained from West Palm Beach,
Florida, and the other six were inoculated with cysts from North
Miami. The remaining six pots served as checks. The pots were
arranged in a randomized block design and maintained in the
plant growing room at a temperature of approximately 800F.
Experiment 3. Four 6-inch standard clay pots were filled
with autoclaved soil and sprigged with St. Augustinegrass. Each
of the four pots was inoculated with a single cyst at the time of
planting. The cysts were treated in the following manner prior
to inoculation. Each cyst was surface disinfested by immersion







Florida Agricultural Experiment Stations


PATHOGENICITY OF HETERODERA LEUCEILYMA
TO ST. AUGUSTINEGRASS

Materials and Methods
Isolation of the nematode. For inoculation purposes, several
methods were used to extract nematodes from the soil. Initially
specimens were extracted through the use of a modification of
the centrifugal-flotation technique (1). Nematodes obtained
with this method were used to start colonies which were main-
tained in a plant-growing room or in a greenhouse during the
course of the investigations.
Inoculum for subsequent tests was obtained from those
colonies by removing mature cysts from the host roots or di-
rectly from the soil after screening onto a 30 and 100 mesh
sieve combination. Debris collected on the 100 mesh sieve was
examined with a dissecting microscope, and the cysts were re-
moved. The Seinhorst elutriator technique (13) and a modifi-
cation of the Christie-Perry technique (2) were used to obtain
males and larvae.
Inoculation Experiments. Experiment 1. Eight 6-inch stand-
ard clay pots were filled with autoclaved soil and sprigged with
St. Augustinegrass. After the sprigs rooted, the soil in each of
four pots was inoculated with 30 cysts collected from an infected
lawn in Boynton Beach, Florida. Each cyst was cut open to re-
lease the eggs just prior to inoculation. The remaining four
pots received no nematodes. The pots were randomly arranged
in a plant-growing room and maintained at a temperature of
approximately 80F.
Experiment 2. Eighteen 6-inch standard clay pots were filled
with autoclaved soil and sprigged with St. Augustinegrass. After
the sprigs rooted, each of 12 pots was inoculated with 10 cysts.
Six were inoculated with cysts obtained from West Palm Beach,
Florida, and the other six were inoculated with cysts from North
Miami. The remaining six pots served as checks. The pots were
arranged in a randomized block design and maintained in the
plant growing room at a temperature of approximately 800F.
Experiment 3. Four 6-inch standard clay pots were filled
with autoclaved soil and sprigged with St. Augustinegrass. Each
of the four pots was inoculated with a single cyst at the time of
planting. The cysts were treated in the following manner prior
to inoculation. Each cyst was surface disinfested by immersion








Heterodera leuceilyma n. sp.


Fig. 7-The site in Boynton Beach, Florida, from which H. leuceilyma
specimens were first recovered. Late stages of damage are quite similar to
chinch bug damage.

in a mercuric chloride solution (1:1000) for 30 seconds, washed
in three changes of sterile water, placed on a microscope slide in
a small quantity of water, and covered with a sterile coverslip.
While observing under low magnification of a compound micro-
scope, enough pressure was applied to the cover slip, with a dis-
secting needle, to rupture the cyst wall and expell the body con-
tents. The cyst contents including unhatched eggs were examined
carefully, under high magnification, to determine whether the
eggs, larvae, or their contents contained a parasitic fungus
which had been observed in cysts from some locations. In in-
stances where no fungus was detected, the coverslip was removed
carefully, and the eggs were washed into the pot being inocu-
lated. Each of the four pots was inoculated with the contents
of a single fungus-free cyst, and placed in the greenhouse.
Experiment 4. Progenies of single cysts obtained in Experi-
ment 3 provided fungus-free inoculum for this experiment.
Fifteen 6-inch standard clay pots were filled with autoclaved







Florida Agricultural Experiment Stations


soil and sprigged with St. Augustinegrass. Each of 10 pots was
inoculated with 10 mature cysts. The remaining pots served as
checks. The pots were placed in a greenhouse and arranged in
a randomized block design with each block containing two inocu-
lated and one check pot. All pots received the same treatment
insofar as watering and fertilization were concerned.
Fertilization. Fertilizer was applied to each pot in all experi-
ments at the following rate: K2S04 1.6 g per liter of water;
50 ml were added per pot every four weeks. (NH4) 2S04 8 g per
liter of water; 50 ml were added per pot every two weeks.
Histopathology. Portions of parasitized roots were killed and
fixed in Randolph's modified Navashin fluid, dehydrated by a
tertiary butyl alcohol method, and imbedded in Fisher's "Tissue-
mat". Transverse sections were cut at 10 p, fixed to slides with
Haupt's adhesive, stained with Johansen's quadruple stain (7),
and mounted in Fisher's "Permount".

Results and Discussion
Inoculation Experiments. Experiment 1. A distinct inter-
veinal chlorosis (Fig. 8) similar to an iron deficiency appeared
on the foliage in three of the four pots containing H. leuceilyma
approximately four and one-half months after inoculation. There
was also a very marked reduction in top growth (Fig. 9). Root
systems were greatly abbreviated (Fig. 10). Clusters of H.
leuceilyma were observed on parasitized roots (Fig. 11). In one
of the inoculated pots, injury by the nematode was not observed.
Examination of soil and roots failed to reveal the presence of
the parasite, and its failure to become established could not be
explained.
Experiment 2. Symptoms of chlorosis began to appear on
the grass within five months in five of the six pots inoculated
with cysts obtained from the West Palm Beach location, while
only one of the pots inoculated with cysts obtained from Miami
was affected. Careful examination of cysts obtained from the
Miami location revealed the presence of a parasitic fungus with-
in the cysts. The fungus parasitized the eggs and larvae within
the cysts. In many cases the eggs and larvae were completely
filled with fungus hyphae. Microscopic examination of numerous
cysts obtained from the Miami location indicated that approxi-
mately 95 percent were parasitized. Apparently, the presence








Heterodera leuceilyma n. sp.


Fig. 8-Individual leaves of St. Augustinegrass from cyst nematode
infected plants, left, and nematode free plants, right.


of large amounts of the parasitic fungus in the soil and its
active parasitism of the grass cyst nematode played a major
role in controlling the damage normally caused by this nematode.
Symptoms of the type normally caused by this cyst nematode
were limited to only a very small area in the Miami location,
and did not spread to any great extent over a two-year period.
Examination of an approximately equal number of cysts ob-
tained from the West Palm Beach location indicated that only
15 to 20 percent of the cysts contained the parasitic fungus.
In this instance the lawn declined rapidly and was considered
a total loss within one and one-half years after symptoms were
first observed. Although the parasitic fungus was present in
the West Palm Beach location, certain unknown factors pre-
vented it from increasing and providing effective control against
this cyst nematode.








Florida Agricultural Experiment Stations


Fig. 9-Heterodera leuceilyma inoculated plants, center and right, are
compared to a check pot 41/2 months after inoculation.


Fig. 10-H. leuceilyma infected plant, left; healthy plant, right.








Heterodera leuceilyma n. sp.


Experiment 3. Again, symptoms began to appear on the in-
oculated St. Augustinegrass within four and one-half months
after inoculation. The grass in all inoculated pots developed
an interveinal chlorosis closely resembling the symptoms of
iron deficiency. Small soil and root samples were taken from each
pot, and the nematodes were extracted. Microscopic examination
of 50 cysts indicated that they were free of the fungus. The
colonies thus obtained were used to provide inoculum for Experi-
ment 4.
Experiment 4. This experiment was terminated six months
following inoculation. The parasitism resulted in a 45 to 50 per-
cent reduction in top growth and 60 to 75 percent reduction in
root growth. Roots of infected plants exhibited a light to dark
brown discoloration in the vicinity of points of attachment of
the nematodes to the roots. The roots also were distorted and
contained many abnormal swellings (Fig. 12). In some instances
roots exhibited varying degrees of proliferation beyond the
point where nematodes were feeding (Fig. 13). The foliage
exhibited an interveinal chlorosis closely resembling the symp-


Fig. 11-Cluster of adult H. leuceilyma females in feeding position on
St. Augustinegrass roots. (30x)








Florida Agricultural Experiment Stations


Fig. 12-Development stages of H. leuceilyma in feeding position on
St. Augustinegrass roots. Note abnormal swellings of roots. (30x)


Fig. 13-Single H. leuceilyma female feeding on St. Augustinegrass
root. Note proliferation of roots beyond point of feeding. (30x)








Heterodera leuceilyma n. sp. 23















"W ^ "- ::: : ---- *. "
t.d
,., -.




Fig. 14-Longitudinal section of a St. Augustingrass root showing
immature H. leuceilyma and giant cells.

rb -


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rk


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o r 71
Figf 15--Enlargement of a portion of Fig 14.








Florida Agricultural Experiment Stations


toms of iron deficiency. Application of iron chelate to the foliage
of infected plants temporarily corrected these symptoms. Ap-
parently the damage to roots by H. leuceilyma interferes with
the host plant's ability to take up certain nutrients even though
the mineral nutrients are present in the soil.
Histopathology. Studies of sectioned St. Augustinegrass roots
revealed the presence of giant cells in the vicinity of the female
head (Figs. 14, 15, and 16). The giant cells induced by the feed-
ing of this cyst nematode resemble those caused by species of
Meloidogyne and species of Heterodera on other hosts. Serial
sections of one group of giant cells brought about by the feeding
of one specimen of H. leuceilyma are shown in Fig. 22. Endo (4),
reporting on penetration and development of Heterodera glycines
in soybean roots, referred to these abnormal cells as syncytia
rather than giant cells.
In most instances giant cell formation resulting from para-
sitism by H. leuceilyma involved cells of the central cylinder and
extended into the cortex (Figs. 17 and 18). In some cases the


Fig. 16-Giant cells produced in St. Augustinegrass roots as a result
of feeding by H. leuceilyma. Note multinucleate condition and thick cell
walls.



































Fig. 17-Cross-section of St. Augustinegrass root. Giant cells can be
seen extending from the central cylinder into the cortical region. Note
portion of female head in proximity to giant cells. (470x)


Fig. 18-Cross-section of St. Augustinegrass root showing massive giant
cell formation extending from the central cylinder into the cortex. This is
the result of feeding by two specimens. (470x)








26 Florida Agricultural Experiment Stations

giant cells occupied space in the area of the pericycle and endo-
dermis as well as the cortex (Fig. 19). In cases where more than
one parasite was feeding in a given area, clusters of giant cells
were present in the vicinity of the head region of each individual
(Fig. 20), and often resulted in a compression of conductive
elements in the central cylinder (Fig. 21). In instances where
conducting tissue is involved in giant cell formation it seems
reasonable to postulate that giant cells in this region were initi-
ated prior to differentiation, since shortly after differentiation
the cells either die, as in the case of xylem cells, or lose their
nuclei, as in the case of phloem cells. Relative position of indivi-
dual parasites apparently determines to some extent which cells
in the root will become involved in the production of giant cells.
If penetration occurs in the young root near the actively grow-
ing tip, then undifferentiated cells of the central cylinder may
become involved in the production of giant cells; whereas if
infection occurs in an older root or in an area where differentia-
tion of xylem and phloem cells has already occurred, then cells
of the endodermis, pericycle, and cortex are more likely to


Fig. 19-Cross-section view of St. Augustinegrass root showing giant
cells within the area of the cortex, endodermis and pericycle.







Heterodera leuceilyma n. sp.


* iq4"


rXI-


--..>



ff


C "-A
'i ikY "'; ", ,
-I'- ft
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Fig. 20--Cross-section views of different St. Augustinegrass roots
wherein more than one specimen of H. leuceilyma was present in each root.


P8~P








Florida Agricultural Experiment Stations


become involved in the formation of giant cells. In instances
where multiple infection occurs, the roots are almost completely
destroyed and unable to function normally.








-- ) 1*

















Fig. 21-Cross-section of a St. Augustinegrass root showing compres-
sion of vascular elements within the central cylinder due to multiple infec-
tion by H. leuceilyma.










Fig. 22-Serial sections of a St. Augustinegrass root portion parasitized
by a single specimen of H. leuceilyma. A female partially embedded within
root tissues was chosen for this purpose, since others would be lost during
the staining and sectioning processes. Giant cells extending from the cortex
into the central cylinder are evident beginning with photograph 1 (which
is the older portion of the root) and are clearly evident through the section
shown in photograph 18. Note also that hyperplasia is associated with the
hypertrophy, but the root shows little evidence of gall formation. Anterior
portions of the parasite become evident in photograph 7, and the parasite's
neck is visible through photograph 18. The remaining photographs (19-50)
show the sections through the body of the nematode and the return to a
normal root condition in photograph 50.









Heterodera leuceilyma n. sp. 29



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30 Florida Agricultural Experiment Stations


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Heterodera leuceilyma n. sp. 31

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32 Florida Agricultural Experiment Stations
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Heterodera leuceilyma n. sp. 33






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Florida Agricultural Experiment Stations


SUMMARY

During the process of examining routine turf samples to
identify populations of plant parasitic nematodes, a species of
Heterodera was found infecting St. Augustinegrass in Boynton
Beach, Florida. Subsequently, the same species was found at
various other locations on the lower east coast of Florida, always
infecting St. Augustinegrass. Larval, female, and male charac-
teristics differed sufficiently from known species so that the para-
site is described as a new species and given the name Heterodera
leuceilyma. The growth of St. Augustinegrass was noted to de-
cline and become chlorotic within infested areas. Root systems
were reduced and necrotic. A series of pathogenicity tests veri-
fied that these symptoms are produced as a result of parasitism
by H. leuceilyma. Microscopic examinations of infected roots
revealed that giant cells are produced wherever the parasite
feeds. These giant cells are similar to those associated with
feeding of other members of Heteroderidae.


LITERATURE CITED
1. Caveness, F. E., and H. J. Jensen. Modification of the centrifugal flo-
tation technique for the isolation and concentration of nematodes
and their eggs from soil and plant tissue. Proc. Helm. Soc. Wash.
22:87-89. 1955.
2. Christie, J. R., and V. G. Perry. Removing nematodes from soil. Proc.
Helm. Soc. Wash. 18:106-108. 1951.
3. Cooper, B. A. A preliminary key to British species of Heterodera for
use in soil examination. Proc. Univ. Nottingham Second Easter
School Agri. Sci., Soil Zool., pp. 269-280. 1955.
4. Endo, B. Y. Penetration and development of Heterodera glycines in
soybean roots and related anatomical changes. Phytopathology.
54:79-88. 1964.
5. Golden, A. M., G. J. Rau, and G. S. Cobb. Heterodera cyperi (Hetero-
deridae), A new species of cyst-forming nematode. Proc. Helm.
Soc. Wash. 29:168-173. 1962.
6. Hirschmann, H. Comparative morphological studies on the soybean cyst
nematode, Heterodera glycines and the clover cyst nematode, H.
trifolii (Nematoda: Heteroderidae). Proc. Helm. Soc. Wash. 23:140-
151. 1956.
7. Johansen, D. A. Plant microtechnique. McGraw-Hill, New York, N. Y.
523 pp. 1940.
8. Luc, M., and R. Berdon Brizuela. Heterodera oryzae n.sp. (Nematoda-
Tylenchoidea) parasite du riz en Cote d'Ivoire. Nematologica.
6:272-279. 1961.








Heterodera leuceilyma, n. sp. 35

9. Luc, M., and G. Merny. Heterodera sacchari n.sp. (Nematoda: Tylen-
choidea) parasite de la canne a sucre au Congo-Brazzaville. Nema-
tologica 9:31-37. 1963.
10. Mulvey, R. H. Taxonomic value of the cone top and the underbridge in
the cyst-forming nematodes Heterodera schachtii, H. schachtii var.
trifolii, and H. avenae (Nematoda: Heteroderidae). Canad. Zool.
35:421-423. 1957.
11. Oostenbrink, M., and H. den Ouden. De structuur van den Kegeltop
als taxonomisch Kenmerk bij Heterodera-soorten met citroenvor-
mige cysten. Tijds. Plantenziekten. 60:146-151. 1954.
12. Schmidt, A. Ueber den Riiben-Nematoden (Heterodera schachtii A.S.).
Zeitschr. ver. Riibenzucker-Ind. Zollver. 21:1-19. 1871.
13. Seinhorst, J. W. The quantitative extraction of nematodes from soil.
Nematologica 1:249-267. 1956.
14. Seinhorst, J. W. A rapid method for the transfer of nematodes from
fixative to anhydrous glycerin. Nematologica 4:67-69. 1959.
15. Thorne, G. Heterodera punctata n.sp., a nematode parasitic on wheat
roots from Saskatchewan. Sci. Agric. 8:707-711. 1928.
16. Thorne, Gerald. Principles of Nematology. McGraw-Hill, New York,
N.Y. 553 pp. 1961.













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