Des juvéniles et des mâles du nématode des citrus, #Tylenchulus semipenetrans$, infestés par #Pasteuria$ sp. ont été détectés dans des échantillons de sol de vergers de citrus de la partie centrale de la Floride. Au cours d'essais au laboratoire, il a été observé que trois à seize endospores s'attachent à la cuticule du nématode. Des endospores mûres sont présentes à l'intérieur du nématode 18 jours après l'attache des endospores. Le développement du parasite est asynchrone, dépendant du spécimen du nématode. Chez les mâles, la région du corps contenant les spermatocytes n'est pas colonisée. Les juvéniles de deuxième stade et les mâles infestés contiennent de 320 à 400 endospores (18 jours après l'attache des endospores, à 25 °C). Les femelles ne sont pas parasitées et aucune endospore n'a été détectée sur leur cuticule. Les endospores de #Pasteuria$ sp. ne s'attachent pas à la cuticule de #Meloidogyne incognita, M. javanica, Radopholus citrophilus$ ou #R. similis$. (Résumé d'auteur

Kaplan, D.T.

Horizon / Pleins textes

Fundam. appl. NemalOl., 1994,17 (6),509-512
Partial characterization of a Pasteuria sp. attacking
the citrus nematode, Tylenchulus semipenetrans, in Florida (1)
David T. KAPLAN
U.S. Department oJ Agriculture, Agricultural Research Service, 2120 Camden Road, Orlando, FL 32803, USA.
Accepted for publication 7 January 1994.
Summary - Juveniles and males of the citrus nematode, Tyleruhulus semiperzelrans, infected with a Pasleuria sp. were detected in soil
samples collected from a citrus grove in Central Florida. In laboratory studies, three to sixteen endospores attached to cuticles of
individual nematodes. Mature endospores were observed within the nematode body 18 days after endospore attachment. Parasite
development was asynchronous within individual nematodes. In males, the region of the body filled with spermatocytes was not
colonized. Infected second stage juveniles and males each contained 320-400 endospores 18 days after endospore attachment at
25°C. Adult females infected by Pasteuria sp. were not detected nor were endospores observed attached to the cuticles of aduJt
females. Endospores of the Pasleuria sp. did not attach to the cuticles of Meloidogyne iruognita, M. javanica, Radopholus cilrophilus, or
R. similis.
Resume - Caracterisation partieUe de Pasteuria sp. attaquant k nematode des citrus, Tylenchulus semipenetrans,
en Floride. - Des juveniles et des males du nematode des citrus, Tylenchulus semipenelrans, infestes par Pasleuria sp. ont ete detectes
dans des echantiIlons de sol de vergers de citrus de la partie centraJe de la Floride. Au cours d'essais au laboratoire, il a ete observe
que trois aseize endospores s'attachent ilia cuticuJe du nematode. Des endospores mures sont presentes al'interieur du nematode
18 jours apres \'attache des endospores. Le developpement du parasite est asyncillone, dependant du specimen du nematode. Chez
les males, la region du corps contenant les spermatocytes n'est pas colonisee. Les juveniles de deuxieme stade et les males infestes
contiennent de 320 iI 400 endospores (18 jours apres l'attache des endospores, a 25 QC). Les femelles ne sont pas parasitees et
aucune endospore n'a ete detectee sur leur cuticule. Les endospores de Pasteuria sp. ne s'attachent pas ilIa cuticuIe de Meloidogyne
incognita, M. javanica, Radopholus cilrophilus ou R. similis.
Key-words: bacteria, biological control, Citrus, parasitism, Pasteuria, Tylenchulus, nematodes.
Past.euria penetrans (Thome) Sayre & Starr have been
associated with Tylenchulus semipenetrans Cobb in
France (Corsica), Florida, Iraq, and Samoa (Sturhan,
1985, 1989; Fattah et al., 1989; Walter & Kaplan,
1990). To date, the only published detailed report of a
PaSleuria sp. attacking T. semipenetrans characterized
the endospores \vithin the nematode's pseudocoelom
and illustrated endospore attachment to the nematode
body wall of second-stage juveniles in Iraq (Fattah et al.,
1989). A Pasteuria sp. was among a diverse group of
predators, parasites, and antagonists of the cirrus nema-
tode identified in a survey of Florida cirrus groves (Wai-
ter & Kaplan, 1990). The purpose of this paper is to
further characterize the Pasteuria sp. detected in that
survey.
Materials and methods
LIFE CYCLE AND DEVELOPMENT
Second-stage cirrus nematode juveniles 02) and adult
males were retrieved from 50 cm3 soil samples collected
from a cirrus grove using modified Baermann funnels.
Approximately 250 females were collected by either me-
chanically removing them from untreated roots or by
enzymatically treating roots to facilitate release of intact
females prior to dissection of females from the roots
(Kaplan & Davis, 1990). The grove was located in Ona,
Florida, where a Pasteuria penetrans-like organism at-
tacking cirrus nematodes was previously detected (Wai-
ter & Kaplan, 1990).
Citrus nematodes were examined using an inverted
light-microscope (200 x) and those with Pastel/ria sp.
endospores attached to their cuticles were transferred to
individual drops of sterile distilled water on microscope
slides and observed at 400 x with a light microscope.
Three J2 or males containing Pasteuria sp. endospores
were individually transferred to watch glasses (BPI)
containing 200 I-LI of sterile distilled water. A dissecting
needle was used to break open and crush the parasitized
nematodes in order to release the Pasteuria sp. endos-
pores.
After endospore release, 200 I-LI of water containing
100 J2 and males were added to each of the BPI watch
glasses containing Pasteuria sp. endospores. These were
(1) Mention of a trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the V.S. Department of
AgricuJture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
lSSN 1164-5571/94/06 S 4.00/ © GaUlhier-Villars - ORSTOM 509
D. T. Kaplan
subsequently placed on a shaker in moist chambers in
the dark at 25 QC. The citrus nematodes added to the
endospore suspension were hatched from eggs isolated
from a citrus grove located in Ocoee, Florida, where
Pasteuria sp. had not been detected. To anain adequate
numbers of freshly hatched citrus nematodes, infested
fibrous roots of sour orange (Citrus auranlium L.) were
cut into small pieces (ca 5 mm) and 250 ml of water was
added to create a slurry of root pieces. This was vigor-
ously shaken for 5 min and then passed trough a series
of sieves (2000-, 354-) 90-, 63-, and 30-!-lm opening) to
separate the eggs from root pieces, soil particles, and
debris. Nematode eggs and fine soil debris were collect-
ed on a 20-!-lm opening nylon-mesh sieve. Citrus nema-
tode eggs were then isolated by adding 10.0 ml of 1.103
specific gravity MgS04 - 7H20 beneath 20 ml of the
egg-debris suspension and centrifuged for 10 min
(2000 g). Eggs were collected again on the 20-!-lm open-
ing sieve. The eggs were then rinsed with distilled water.
The egg suspension was placed in a vial fined with
20-!-lm nylon mesh over the open end. The vial was
inverted for 20 min into a watch glass filled with water to
allow males and J2 that had previously hatched to mi-
grate from the eggs. Nematodes that migrated through
the mesh-fined end of the vials were discarded. The
vials were then turned upright, sealed, and placed on a
shaker in a dark incubator at 25 QC. Citrus nematode J2
were subsequently isolated from the egg suspensions
after 48 h by collecting nematodes that migrated
through the 20-!-lm nylon mesh as before. These nema-
todes were visually inspected at 400 x with a light micro-
scope to ensure that neither Pasteuria sp. nor any other
organisms were present prior to their addition to the
aqueous endospore suspension.
Three watch glasses were removed from the moist
chamber at 3-day intervals over a 30-day period and the
nematodes fixed in T AF (Courtney et al.) 1955). Nema-
todes in each of the watch glasses were then examined
using an inverted light-microscope (200 x). Nematodes
examined for the presence of endospores or other Pas-
teuria sp. life cycle stages on/in the nematodes at 1000-
1600 x with a Zeiss Photomicroscope II using Normar-
sky optics fined with aDAGE MTI CCD72 video cam-
era and viewed on aDAGE MTI-HR 1000 high
resolution monitor. Developmental stages of the
parasite, morphometrics (diameter of endospore and
central core) of endospores anached to the nematode
cuticle and within the nematode host, the location of
endospores within the nematode body, and the number
of endospores within each of 20 J2 and males were
determined with a Hamamatsu Argus-l0 Image
processor and recorded with a Panasonic Time Lapse
Video Cassene Recorder AG-6750A. Images recorded
for publication were either photographed off the
screen or produced with a Sony Color Video Printer
UP 3000.
510
HOST SPECIFICITY
An experiment was conducted to determine if the
Pasteuria sp. would anach and/or parasitize Meloidogyne
incognita) M. javanica and Tylenchulus semipenetrans col-
lected from Pasteuria sp.-free greenhouse pot cultures.
The burrowing nematodes, Radopholus citrophilus and
R. similis) retrieved from carrot disk cultures (Kaplan &
Davis, 1990) were also evaluated. Then, 200 cm3 soil
samples from the Ona site were placed in beakers. A
5.0-ml aqueous suspension containing 1000 nematodes
was added to each soil sample. The samples were in-
cubated at 25 QC for 5 days and then placed on Baer-
mann funnels. Nematodes retrieved from each of five
samples per nematode species were examined using an
inverted light-microscope (200 x) for the presence of
anached endospores. Individuals with endospores at-
tached to their bodies were transferred to BPI watch
glasses containing distilled water and incubated in a dark
moist chamber at 25 QC. Nematodes were examined for
the presence of endospore development 20 days after
placem.::nt in incubators.
Results and discussion
Males and J2 of T. semipenetrans infected with Pas!eu-
ria sp. were detected in soil samples collected from a
citrus grove in Central Florida (Fig. 1 A). Three to six-
teen endospores were anached to the cuticles of J2 and
males six days after the Pasteuria sp-free citrus nema-
todes were added to the Pasteuria sp-endospore suspen-
sion. The diameter of endospores anached to the nema-
tode cuticles were 2.7 ± 0.3 !-lm and their central cores
were 1.4 ± 0.2 !-lm. The morphology of the Pastewia sp.
detected in the Florida citrus grove was not significantly
different from that reported from Iraq (Fanah et al.)
1989) where the endospore diameter was 2.6 !-lm. From
published photographs, the central core diameter of that
isolate was estimated to be 1.6 !-lm (± 0.1).
Mature endospores (" fried-egg" shaped) appeared
within the bodies of T. semipenetrans 18 days after en-
dospore anachment (Fig. 1 B, C). In contrast to P. pene-
trans which developed synchronously in Meloidogyne
(Sayre & Starr, 1985), development of Pastewia sp. was
asynchronous within a single citrus nematode
(Fig. 1 D). In contrast to the Pasteuria sp. isolated from
Iraq (Fanah et al.) 1989), the Florida Pasteuria sp. isolate
produced sporangia and endospores within males
(Fig. 1 E). The endospores probably anached to males
after they had reached the adult stage (Fig. 1 F). In-
fected second-stage juveniles and males contained 320-
400 endospores 18 days after anachment.
Adult female specimens of many sedentary and sever-
al migratory species of plant parasitic nematodes have
also been found filled with endospores of members of
the P. penetrans group (Davies et al.) 1990; Giblin-Da-
vis, 1990). However, endospores were not detected on
the cuticles or within mature citrus nematode females.
Fundam. appl. Nematol.
Pasteuria sp. all£lching Tylenchulus sermipenetrans
Fig. 1. Second-swge juveniles a2) and males ofTylenchulus semipenetrans parasitized by Pasteuria sp. A : J2 with malUre endospore
(ME) allached to wil and conwining mature endospores and sporangia; B : Pasteuria sp.-filledJ2 broken to release mature endospores (ME);
C: Dewil of mature endospores released from broken J2; D : Asynchronous development of Pasteuria sp. within the J2 as evidenced by
sporangia (S) and mature endospores (ME); E: Mature endospore (ME) allached la head region of male T. semipenetrans with germ tube
(GT); F: Mature endospore (ME) allached to male cuticle in vicinity of spermatocytes.
Examination of citrus nematode females is complicated
by the parasitic habit of the citrus nematode; intracellu-
lar invasion of the root accompanied by the enlargement
of the anterior half of the female makes it difficult to
attain entire females without breaking them at the point
Vo!. 17, n° 6 - 1994
where they emerged from roots (semi-endoparasite).
That is, the anterior end of the nematode generally re-
mains in the root when the posterior portion and body
contents are lost. However, it is likely that if present,
endospores would have been observed when females
511
D. T. Kaplart
were broken away from the root surface. In an attempt
to circumvent this, tissue-softening enzymes were used
to release females from root tissue, but none of the fe-
males retrieved had endospores attached to their cuticles
or within their bodies.
Citrus nematode J2 and males remain in soils for ex-
tended periods of time. This, in conjunction with the
relatively short life cycle of Pasteuria sp. (18 days),
would be conducive to the development of mature en-
dospores in the migratory stage of the nematode. Iso-
lates of other Pasteun'a sp. that complete their life cycles
in vermiform stages of some other sedentary plant para-
sitic nematodes have also been noted to have a rapid life
cycle (Sayre & Starr, 1988; Giblin-Davis, 1990).
Pasteun'a sp. did not attach to cuticles of Meloidogyne
incogniw, M. javanica, Radopholus citrophilus, or R. simi-
lis. However, Pasleuria sp. did become attached to
T. semipenetrans J2 and males that migrated through the
soil. After 20 days incubation, endospore development
was observed in several T. semipenetrans J2 and males.
This suggests that Pasleuria sp. is specific for the citrus
nematode.
Pot cultures of the Florida isolate of Pasteun'a sp. have
been established using T. semipenetrans on citrus trees
growing in Pasleuria sp.-infested soil and by direct in-
festation with Pasteuria sp.-infected J2 and males. How-
ever, it has been difficult to recover enough Pasleuria
sp.-colonized nematodes to determine the effect of Pas-
leuria sp. on citrus nematode population dynamics. Fail-
ure to detect females filled with endospores suggests that
previously described tedmiques for P. penelrans culture
(Stirling & Wachtel, 1980) are not useful for this citrus
nematode parasite since Pasleuria sp.-infected stages oc-
cur in the soil. Although the objective of this study was
not directed toward development of a culture method
for Pasleuria sp. on citrus nematode, the ability of Pas-
leuria sp. to attach, penetrate, and produce endospores
under laboratory conditions suggests that this may pro-
vide a means of Pasleuria sp. population increase. We
are exploring this possibility.
Acknoweledgments
I wish to thank D. M. Johnson, Biological Laboratory Tech-
nician, and Dr. D. E. Walter for technical advice and assist-
ance.
512
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Fundam. appl. NemaLOl.


Partial characterization of a Pasteuria sp. attacking the citrus nematode, Tylenchulus semipenetrans, in Florida

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