Orion, D.

Sharon, E.

Spiegel, Y.

Horizon / Pleins textes

Fundam. appl. NemalOl., 1993, 16 (1), 5-9
Binding of soil microorganisms and red blood cells by the
gelatinous matrix and eggs of Meloidogyne javanica
and Rotylenchulus renifarmis
Edna SHARON, Daniel ORION and Yitzhak SPIEGEL
Deparlrnenl of Nernalology, AfJ'icultural Research Organizalùm,
The Volcani Cenler, Bel Dagan 50 250, Israel.
Accepted for publication 14 March 1992.
Summary - Egg masses of both Meloidogyne javanica and Rotylenchulus reniformis, and the gelarinous matrix (GM) suspension of
M. ]avamca bound various bacteria, as weB as human red blood cells (RBC). Encerobaaer aerogenes and Escherichia coli did not have
the abiliry to be agglutinated by the egg masses or the GM. Four rhizobacteria isolates from tomato roots which did not multiply on
the GM, had the abiliry to agglutinate by the GM. However, four other rhizobacteria isolates which did not agglutinate, were able to
utilize the GM for growth. The agglutination intensiry of RBC by the GM was not inhibited by several carbohydrates, depleted with
preheated GM, but increased after trypsinization. The possibiliry of antibiotic(s) involvement in the GM-bacteria interaction was
ruled out. The authors suggest that the agglurination phenomenon may partly explain the GM abiliry to protect the eggs from
surrounding microorganisms in soil.
Résumé - Agglutination de bactéries du sol et d'hématies hU1naines par la matière gélatineuse et les œufs de
Meloidogyne javaIÙca et de Rotylenchulus reIÙfonnis - Les masses d'œufs de Meloidogyne javanica et de Rotylenchulus
reniformis, de même que des suspensions de matière gélatineuse (MG) provenant de M. javanica, agglutinent différentes bactéries
ainsi que les hématies humaines. Emerobacrer aerogenes et Escherichia coli ne sont agglutinés ni par les masses d'œufs ni par la MG.
Quatre rhizobactéries isolées de racines de tomate, et ne se multipliant pas sur la MG, sont agglurinées par cene dernière.
Cependant, quatre autres isolats de rhizobactéries, non agglutinés, sont capables d'utiliser la MG pour leur croissance. L'agglutina-
tion des hématies humaines par la MG n'est pas inhibée par différents hydrates de carbones; eUe est diminuée si la MG est
préchauffée et augmentée après trypsination. La possibilité de l'intervention de subsrance(s) antibiotique(s) dans l'interaction
GM-bactéries est écartée. Les auteurs suggèrent que le phénoméne d'agglutination peut expliquer la capacité de la MG à protéger
les œufs des microorganismes présents dans le sol.
Key-words: Nematodes, Melozdogyne, Rotylenchulus, soil microorganisms.
Females of several sedenrary nematodes of plants en-
velop their eggs with a gelatinous matrix (GM). In the
root-knot nematode, Meloidogyne, the GM is synthe-
sized in a voluminous quantity by the female anal glands
and secreted through its anus just before and during egg
laying; eggs are deposited into the GM to form the
egg-mass (Maggenti & Allen, 1960). Information about
the chemical composition of the matrix is very limited.
In the root-knot nematode, Meloidogyne, it was found to
be a tanned protein which contains carbohydrate, but no
lipids, and appeared to be morphologicaUy homogene-
ous (Bird, 1958; Bird & Rogers, 1965).
Several studies have been performed on the produc-
tion process of the matrix (Maggenti & Allen, 1960;
Maggenti, 1962). Orion el al. (1987) showed that the
GM dissolved ceUs in the gall cortical parenchyma to
form a canal through which the egg-mass protruded
outside the gall surface. Later, Orion and Franck (1989)
described the cell Iysis process by e1ectron microscopy
observations.
The biological function of the GM is still speculative.
Recently, Orion and Kritzman (1991) found that M. ja-
vanica GM was able to agglutinate two microorganisms,
thus interpreting that this properry of the GM might
enable it to protect the nematode eggs from the micro-
bial fauna in the soil.
In the current paper we explore the agglutination abil-
ity of Meloidogyne javamca and ROlylenchulus reniforrnis
intact egg masses, GM suspensions, and separated eggs
- using identified microorganisms, rhizobacteria, and
human red blood cells (RBC).
Materials and methods
BACTERIA
The following bacteria were utilized in this study:
Aeromonas caviae, Bacillus cereus, B. SUblilis, Enterobacter
aerogenes, Escherichia coli, Micrococcus sp., Pseudomonas
pUlida, Serralia marcescens and several unrecognized to-
mato rhizobacteria. The rhizobacteria were isolated
from tomato roots as follows : one gram oftomato roots,
grown for a month in a natural non-infected sandy soil,
was carefully washed with water and shaken on a rotato-
ry shaker (250 rpm), for 30 min, in 50 ml saline with
50 glass beads (5 mm diameter). The mixture was then
transferred wough a 15 f1.m-pore sieve, centrifuged
ISSN 1164-5571/93/01/05 05 S 2.50/ © Gauthier-Villars - ORSTOM 5
E. Sharon, D. Orion & Y. Spiegel
(10000 g; 10 min) and washed rwice with 0.1 M phos-
phate buffer saline (PBS), pH7.2 which included Ca++
and Mg++.
MEDIA AND CULTIVATION CONDITIONS
For the experiments described herein, the different
bacteria strains were grown in 250 ml Erlenmeyer f1asks,
each containing 50 ml of Difco Bacto Nutrient Broth.
The f1asks were shaken on a rotatory shaker (150 rpm)
at 25 oC, and after 24 h the bacteria were separated from
the medium by centrifugation (10 000 g; 10 min) and
washed rwice with PBS. The cells were resuspended in
sterile PBS. Bacterial concentrations, ca 108 colony-
forming units (cfu)'ml-', were determined spectropho-
tometrically at 560 nm.
EGGS AND GELATINOUS MATRIX
Meloidogyne javanica and Rotylenchulus renijorrnis
were monoxenically cultured on tamato (Lycopersicon
esculenlum Mill) cv. Hosen Eilon and on sunflower (He-
lianlhus annuus L.) excised roots, respectively. Egg-
masses were dissected from 4-week-old cultures. Gela-
tinous matrix suspension was prepared as follows:
150 egg masses, drawn with a sterile Pasteur pipet from
28-day-old cultures, were suspended in 1 ml PBS and
vigorously shaken for 1 min. The suspension was then
centrifuged at 1000 g for 1 min, and the supernatant
fraction was separated from the eggs by decantation.
Eggs were resuspended in PBS.
INTERACTION OF EGGS, EGG-MASSES AND GM OF AI.
JAVANICA WITH RED BLOOD CELLS (RBC)
Binding.
Human red blood cells (RBC) (A, B, and 0), were
obtained from Zerifin Medical Center Blood Bank (Zer-
ifin, Israel). RBC were washed four times with PBS to
remove serum proteins and used without fixation. Bind-
ing to eggs and egg-masses were performed at 37 oC, in
a 24 multi-wel1 plate (Becton Dickinson Labware, CA,
USA). Final volume was 400 fJ..I, containing 200 fJ..I of
0.3 % suspension of RBC and 200 fJ..I of egg suspension
(100 eggs per weil) or three to four egg-masses in PBS.
Binding was observed under a dissecting microscope
after incubation for 60 min.
Agglutznation
Agglutination titers with fresh RBC were recorded
only with M. javanica GM. Assays were carried out at
37 oC, in a 96 multi-well u-shape plate (Becton Dickin-
son Labware, CA, USA), in a final volume of 0.1 ml
containing 50 fJ..I of 0.5 % suspension of RBC and 50 fJ..l
of GM suspension diluted seriaily in rwo-fold incre-
ments up to 1024 tÎmes. Agglutination was observed
after 60 min.
To verify the specificity and to assess the nature of the
observed agglutination and binding phenomena, eggs
and GM suspensions in PBS were incubated for
120 min, at 37 oC, with fucose, galactose, N-acetylga-
6
lactasamine, glucose, N-acetylglucosamine and alpha-
methyl-mannoside at final concentrations of 0.2 M. As a
control, PBS was substituted for the GM suspension.
Eggs were pre-treated with : a) 0.5 % sodium hypo-
ctùorite solution for 1 min and washed three times with
PBS, and b) thoroughly washed with PBS.
Trypsznzzalion
Eggs or GM were incubated in a water-bath shaker for
150 min, at 37 oC, with 0.5 and 0.05 mg/ml trypsin (Sig-
ma), in PBS, respectively.
Each treatment was replicated in four wells, and the
experiments were repeated three times.
INTERACTION OF EGG-MASSES AND GM WITH DIF-
FERENT BACTERIA
Binding assays
Egg-mass (one per volume), suspended in 25 fJ..1
PBS, was mixed on a microscope slide with the same
volume (25 fJ..l) of bacterial suspension in PBS. The
slide was maintained at 25 oC, in a covered Petri plate to
prevent evaporation. The same tests were performed
with 25 fJ..1 of GM suspension. Observations were made
under a microscope, after incubation for 30 min.
Effect of CM on bacteria growlh and viability
The experiments described herein were accomplished
with M. javanzca. Two growth media were used : Nutri-
ent Agar (Difco) (Na) and Mueiler Hinton (MH) (Di-
agnostics Pasteur), which was used to determine bacte-
rial antibiotic sensitivity. Various experimental
combinations were conducted to determine the qual-
itative effect of GM suspension on bacteria growth and
viability: Ma) different bacterial suspensions (25 fJ..1)
were spread onto agar plates and rwo or three egg mass-
es were then positioned in the center 15 mm apart each
other; Mb) three filter paper discs (5 mm diameter)
were absorbed with 25 fJ..I GM suspension, ailowed ta
dry, and then placed in each agar plate with MH, which
had already contained the bacteria; Mc) GM suspension
(10 fJ..I) was pipetted on the agar (NA) which had al-
ready contained the bacteria; Md) GM suspension
(10 fJ..I) was pipetted on the agar (NA) and ailowed ta
dry before the different bacterial suspensions were
spread onto the agarj Me) equal volumes of GM and
bacteria suspension were mixed and 10 fJ..1 were pipe-
tted on the agar (NA) plates; Mf) GM and bacteria
suspensions (50 fJ..1, each) were mixed, and incubated at
25 oC for 60 min; 10 fJ..l of the mixture was then pipe-
tted on agar (NA) plates.
Within ail treatments, plates were incubated at 25 oC
for 24 h before the observations were recorded.
In order to evaluate quantitatively the effect of GM
suspension on the viability of B. SUblilis, Micrococcus sp.,
P. putida, and the tamato rhizobacteria, 90 fJ..1 of the
GM suspension and 10 fJ..I of the bacteria suspension
were mixed in rwo ml u-shape tubes and incubated at
28 oC, for 48 h. Vigorously mixed suspensions were di-
Fundam. appl. Nemal.Ol.
Binding of microorganisms 10 geLalinous malrix
luted ten-fold and 10 fLI were pipetted on agar (NA)
plates and cfu were counted.
Results
INTERACTION OF EGGS, EGG-MASSES AND GM sus-
PENSION WITH RED BLOOD CELLS (RBC)
Eggs and egg-masses of M. ja-vamca (Fig. 1A, B) and
egg-masses of R. remformis bound RBC. The binding
was not blood-group-specific. Binding of RBC de-
creased when M. javanica eggs were pre-exposed ta so-
dium hypochlorite or pre-washed extensively wim PBS
(Table 1). The intensity of RBC binding tO M. javanica
eggs decreased after pre-incubation wim fucose, glu-
cose, N-acetyl-glucosamine, alpha-memyl-mannoside
and trypsin (0.5 mg/ml) (Table 1). Galactose and N-
acetyl-galactosamine increased me binding intensity
(Table 1).
GM suspension of M. javanica agglutinated RBC.
The agglutination was not blood-group-specific. Ag-
glutination occurred up to me x 128 dilution. None of
me sugars mentioned above was found to inhibit me
agglutination. Trypsinization, however, caused a partial
increment of me agglutination intensity (IWO times in-
crement), while boiling me GM (15 min in boiling wa-
ter) completely inhibited me agglutination.
INTERACTION OF EGGS, EGG-MASSES AND GM WITH
DIFFERENT BACTERlA
Binding ability of several bacteria ta M. jàvanica
(Fig. 1 C) and R. remformis egg masses is summarized
in Table 2. A. caviae, B. cereus, B. subtilis, Micrococcus
Table 1. Agglutination and binding of human red blood cells
(RBC) ta MeLoidogyne javanica gelatinous matrix (GM) and
eggs.
sp., P. putida and S. marcescens bound to A1. javanica
egg-mass, while E. aeorogenes and E. coli did not.
The agglutination capabiliry of M. jàvanica GM sus-
pension was identical to me bacterial binding pattern
recorded wim me egg mass.
Bacterial growm was not affected by whole egg
masses, or GM suspensions when tested by me paper
disc (Mb) or memod Md (see above). However, when
GM suspension drops were applied onto me agar plates
(Mc), or by omer memods described in me " Materials
and memods » section (Me, Mf), me bacteria which had
me ability to bind to egg masses (Table 2), showed a
different growth pattern wimin me GM drop zone - as
compared ta me non-agglutinating bacteria (Fig. 1 D).
Pre-heated GM, however, did not exhibit mis effect on
bacterial growth.
The viability of B. subtilis and P. pullda slightly de-
creased in me presence of me GM suspension: cfu
decreased by one order of magnitude, after incubation
for 48 h, compared wim me bacteria viability in me
presence of PBS (Table 3). MzCrococcus sp. growth was
Table 2. Bacteria binding to MeLoidogyne Jàvanica and Roly-
LenchuLus remformis egg masses.
Bacteria A1.. javanica R reniformis
Aeromonas caviae + +
BaàLLus cereus + +
BaciLLus subtiLis + n.t.
EnterobaCler aerogenes n.t.
Eschen'chia coLi
Micrococcus sp. + +
Pseudornonas plaida + n.t.
Treatrnent Agglutination
to the GM
Binding
to eggs + = positive binding; - = negative binding; n.t. = not tested.
Tests were performed at 37 oc. Binding was recorded 1 h after
incubation with RBC - (+ = detectable agglutinationlbinding.
++, +++, and ++++ =increasing intensities of agglutination!
binding. -- = No agglutinationlbinding ta GM or eggs, respec-
tively).
+++ ++
+++ +++.,.
+++ +
+++ ++++
+++ ++
+++ ++
++++ +++
++
" Counts were recorded in colony-forming units (cfu) per ml-
".' PBS (0.1 M phosphate buffer saline, pH 7.2) was used as a
control.
Table 3. Viability of different bacteria populations after expo-
sure to MeLoidogyne javamca gelatinous matrix (GM) suspen-
sion.
Counts Counts
at time 0 after 48 h
(log) (log)
GM PBS"~
7.34 572 6.38
693 28 4.95
738 5.2 6.76
655 769 655
Bacteria
BaciLLus SubliLis
MicTOCOCCUS sp.
Pseudornonas putida
Tomato rhizobacteria
++++++Non-treated (control)
Eggs pre-exposed to NaOCI
Eggs thoroughly washed with PBS
GM suspension heated up ta
100 oC
Fucose (0.2 M, 2 h)
Galactose (0.2 M, 2 h)
Glucose (0.2 M, 2 h)
N-acetyl-galactosamine
N-acetyl-glucosamine
-methyl-mannoside (0.2 lv1, 2 h)
Trypsin (005 mg/ml, 2.5 h)
Trypsin (0.5 mg/ml, 2.5 h)
Vol. 16, n° 1 - 1992 7
E. Sharon et al.
Fig. 1. Red blood cells binding, A : To the gelatinous matrix of Meloidogyne javanua egg-mass; B : To separated eggs; C : Bacillus
cereus binding to the gelatinous matrix of M. javanica egg-mass; D : Colonies of B. cereus exposed to M. javanica gelatinous matrix
suspension (area signed by star) compared with non-exposed colonies.
tremendously inhibited, while the tomato rhizobacteria
growth increased (Table 3).
Eight morphologicaUy different colonies isolated from
the rhizobacteria population, were further tested for
their agglutinarion ability by the GM. Four isolates
which were not able to multiply on the GM suspension
were able to agglutinate, while the bacteria which did not
have the agglurination ability, were able to utilize the
GM for growth.
Discussion
Egg masses of both M. javanica and R. renifonnis had
the ability to bind various bacteria and RBC to their
surfaces (Table 2, Fig. 1). These results confirm pre-
8
liminary observations reported by Orion and Kritzman
(1991).
The resemblance between the binding reactions to
M. javanica and R. remfonnis egg-masses may hint a
presence of a similar agglutination factor in the GM of
both nematode species, in spite of their distant phylo-
genic relations.
Erythrocytes have been used as a tool to trace carbo-
hydrate recognition domains (CRD; Jungrey & Weath-
eraU, 1986) since the preponderance of their membrane
carbohydrate consists of oligosaccharides linked to three
types of glycophorine molecules, also known as sialogly-
coproteins (Mirelman, 1986). RBC agglutination by the
Fundam. appl. NemalOl.
GM was not inhibited by several carbohydrates, which,
\\-ithin our experimental conditions, excluding the possi-
bllity of CRD involvement in the agglutination phenom-
enon.
M. javanica separated eggs also bound RBC
(Fig. 1 B); however, the binding could be easily eliminat-
ed by washing thoroughly the eggs either with PBS or a
hypochlorite solution. Agglutination of RBC by GM
increased after trypsinization but was not affected by
carbohydrates; RBC binding to eggs, however, was a
trypsin- and carbohydrate-dependent process (Ta-
ble 1), much like the RBC binding to several plant-para-
sitic nematades described elsewhere (Spiegel el al.,
1991). This suggests that the tested egg surface con-
tained a binding factor different from that in the GM.
Bacterial growth inhibition by antibiosis is generally
expressed by the appearance of a halo araund the source
of the antibiotic material. Gelatinous component de-
rived from the egg-mass or the GM suspension which
had been placed on the agar plate - already containing
the different bacteria - did not react in this manner.
Since a halo was not observed in our experiments be-
yond the egg-mass or the GM drop zone, the authors
concluded that the results did not support the possibility
of the involvement of antibiotic activity in the GM-
bacteria interaction. The agglutination phenomena ex-
pressed by the ability of the GM suspension ta agglut-
inate bacteria (and RBC), and thus ta reduce their
growth or viability, may explain part of the GM capa-
bility to protect the eggs from surraunding microorgan-
isms in soil. The highly viscous GM, which forms a hard
layer around the egg-mass surface, could support a fur-
ther physical (or mechanical) barrier.
Out of the different rhizobacteria isolated fram toma-
to raots, several populations were able to utilize the GM
suspension as a sole nutrient source, and multiply. Ag-
glutination tests with populations which either multiply
on the GM suspension or did not, revealed a good corre-
lation bw.veen the ability of growth on the GM and
agglutination. This observation further supports the raie
Vol. 16, n° 1 - 1992
Binding of microorganisms co gelalinous malTlX
of the agglutination phenomenon in the GM protection
process.
Additional chemical characterization of the GM sus-
pension is underway to further understanding of the
agglutination phenomenon described in this paper.
References
B1RD, A. F. (1958). The adult female cuticle and egg sac of the
genus Meloidogyne Goeldi, 1887. NemacologlCQ, 3 : 205-212.
BIRD, A. F. & ROGERS, G. E. (1965). Ulrrasrructural and
histochemical stucties of the cells producing the gelatinous
matrix in Meloidogyne. Nemacologica, 11 : 231-238.
JUNGREY, M. & WEATHERALL, D. J. (1986). Carbohydrate
recognitions mediate attachmenr of Plasmodium falâpantm
malaria to erythrocytes. ln.' Mirelman, D. (Ed.). Microbial
ieclins and agglltlinins, New York, John Wiley & Sons: 335-
357
MAGGENTI, A. R. (1962). The production of the gelatinous
marrix and its taxonomic significance in Tyienchulus (Nema-
toda : Tylenchulina). Proc. helmimh. Soc. Wash., 29: 139-
144.
MAGGENTI, A. R. & ALLEN, M. W. (1960). The origin of the
gelatinous marrix in Meloidogyne. Proc. helminlh. Soc. Wash.,
27: 4-10.
MlRELMAN, D. (1986). lvlicrobialleClins and agghllinins. New
York, John Wiley & Sons, 443 p.
ORlON, D. & FRANCK, A. (1989). An elecrron rnicroscopy
study of cell wall lysis by Meloidogyne javanica gelatinous
marrix. Revue NémaLOI., 13: 105-107.
ORlON, D. & KR1TZMAN, G. (1991). Antimicrobial activity of
,Weloidogynejavanica gelatinous marrix. Revue Némacol., 14 :
481-483.
ORlON, D., LOOTS, G. C. & ORlON, T. (1987). Cell lysis
activity of Meloidogyne gelatinous marrix. Revue Némacol.,
10 : 463-465.
SPIEGEL, Y, SH1\RON, E., KAHANE, I. & MCCLURE, M. A.
(1991), Surface binding of red blood cells to differenr nema-
todes. J. Nemacol., 23 : 551 [Absrr.].
9


Binding of soil microorganisms and red blood cells by the gelatinous matrix and eggs of Meloidogyne javanica and Rotylenchulus reniformis

Abstract

Similar works

Full text

Available Versions

Horizon / Pleins textes