Kritzman, G.

Orion, D.

Horizon / Pleins textes

Antimicrobial activity of Meloidogyne javanica 
gelatinous matrix (l) 
Daniel ORION and Giora KRITZMAN 
Department of Nematology and Department of Plant Pathology, Institute of Plant Protection, Agricultural Research Organization, 
Volcani Cemer, Bet Dagan, Israël. 
SUMMARY 
Young egg masses obtained from monoxenic cultures of the root-hot nematode Meloidogyne javanica were placed in a 
suspension  of Pseudomonas syringae pv. towlato (PS), Bacillus  subtilis (BS) or Saccharomyces sp. (SS). The population  growth  rates 
of BS and SS were  significantly  reduced in the  presence  of the egg  masses,  while PS was not  affected at all.  Microscope  observations 
showed  strong  agglutination of both BS and SS cells in  the vicinity  of  the  gelatinous  mat& It  is  concluded  that  the  gelatinous  matrix 
has  antimicrobial  a tivity proba lyfor  the  protection of the nematode  ggs. I ', 
i 
N S U M E  
Activité antimicrobienne de la sécrétion  gel'atineuse  de Meloidogyne  javanica 
De jeunes  masses  d'œufs  provenant  d'une  culture  monoxénique  de Meloidogyne javanica sont  transférées  dans  des  suspensions 
de Pseudomonas syritzgae pv. tonzato (PS), de Bacillus  subtilis (BS) ou de Saccharomyces sp. (SS). En présence  des  masses  d'œufs, 
la  croissance  des  populations  de BS et SS est  réduite de façon  significative,  alors  que  celle de PS n'est  pas  affectée.  L'observation 
microscopique  révèle  une  forte  agglutination  des  cellules BS et SS au voisinage  de  la  sécrétion  gélatineuse. Il en est  déduit que la 
sécrétion  gélatineuse  a  une  activité  antimicrobienne  jouant  probablement un rôle dans la  protection  des œufs du nématode. 
The root-knot  nematodes (Meloidogyne spp.) gelati- 
nous matrix (GM) is a substance synthesized by six 
rectal  gland cells and  secreted  through the  anus (Mag- 
genti & Allen,  1960). The GM dissolves the  host cells, 
forming a cavity or a canal  leading  from the  nematode 
posterior  end to  the gall surface  (Orion,  Loots & Orion, 
1987; Orion & Franck, 1990) and  into which the female 
deposits its eggs to form the egg mass. The latter, 
protruding  from  the gall,  is exposed to  the soil environ- 
ment. In spite of the rich variety of soil microorganisms 
which apparently  could  consume  the  nutritious eggs, the 
egg  mass  remains  intact in  the soil. 
One could  speculate,  therefore, that  the GM serves as 
an  agent protecting the root-knot  nematode eggs against 
microflora. With this idea in mind, the antimicrobial 
properties of the GM were explored in  the present  study. 
Materials and methods 
The root-knot  nematode Meloidogyne  javanica was 
monoxenically cultured  on excised tomato (Lycopersicon 
esculentunz cv. E-203) roots as described  previously 
(Orion, Weren & Endo, 1980). Four weeks after in- 
oculation egg masses were removed  from the cultures by 
means of a scalpel under a stereoscopic  microscope and 
placed separately in a suspension of three  microorgan- 
isms  cultured,  obtained and assessed as described below. 
ORGANISMS  AND  GROWTH  MEDIA 
Strains Pseudo?nonas syringae pv. tmnato (PS), Bacil- 
lus subtilis (BS) and Saccharomyces sp. (SS) were Ob- 
tained from Our culture collection. The bacteria were 
maintained at 26 "C in  the  dark  on slants of nutrient  agar 
(Difco) and  the  fungus  on  potato dextrose  agar  (PDA; 
Difco). 
PROCEDURES WITH INOCULA 
Cultures  used as inoculum were incubated  for 24 h on 
slants : PS on  King's medium B agar  (agar F, Difco), BS 
on  nutrient agar (NA, Difco) with glycerol (1 '10 wlv), 
and SS on PDA. The cells were suspended in sterile 
water. After  centrifugation at 12 000 g for 10 min, the 
pellet was resuspended in saline (0.85 O/O W/V NaCl)  to 
give 0.65 absorbance unit at 480 nm in a Beckman 
Mode1 25  spectrophotometer. This corresponded to 
(1) Contribution No. 2700-E, 1989 series,  from  the  Agricultural  Research  Organization, The Volcani  Center,  Bet  Dagan,  Israël. 
Revue Nématol. 14 (4) : 481-483 (1991) 48 1 
l 
D. Orion & G. Kritzrnan 
Table 1 
The effect of Meloidogyne javanica gelatinous  matrix,  on the growth  rate  during 24  h at 28 "C of Pseudomonas syringae pv. tornato 
(PS), Bacillus subtilis (BS) and Saccharomyces sp. (SS) in  comparison with various  compounds  (expressed in CFU per  ml). 
Cornpounds PS BS ss 
~ 
Control 5.06 x 107 f 1.1 x 103 1.27 x 108 f 1.7 X 104 8.12 x 108  1.1 x 104 
Gelatinous  matrix 5.06 x 107 k 1.1 x 103 1.30 x 107 k 0.9 x 104 1.73 x 107 t- 5.0 x 103 
Gelatin (5 pg) 5.07 x 107 k 1.1 x 103 1.28 x 108 k 1.7 x IO4 8.11 x 108 k 1.1 x 104 
Pectin (5 pg) 5.50 x 107 -+ 1.2 x 103 1.27 x 108 k 1.6 x 104 8.12 x 108 f 1.1 x 104 
Albumin (5 pg) 5.11 x 107 f 1.2 x 103 1.32 x 108 f 1.8 x 104 8.10 x 108 t- 1.1 x 104 
Carboxy-methyl-cellulose (5  pg) 6.06 x 107 C 1.1 x 103 1.26 x 1S8 t- 1.7 x 104 8.13 x 108 k 1.2 x 104 
Penicillin (500 pg/ml) 5.06 x 107 2 1.1 x 103 O 8.13 x 108 f 1.3 x 10' 
Penicillin  (45  pg/ml) 5.07 x 107 e 1.1 x 103 9.34 x 107 k 1.6 x 104 8.11 x 108 f 1.1 x 104 
Cycloheximide (500 pg/ml) 5.06 x 107 k 1.1 x 107 1.27 x 108 k 1.7 x 104 O 
Cycloheximide  (45  pg/ml) 5.07 x 107 k 1.3 x 103 1.27 x 108 f 1.7 X 104 5.73 x 108 k 9.2 x 103 
Polymixin (50 pg/ml) O 1.25 x 108 C 1.6 x 104 7.94 x 108 f 9.6 x 103 
Polymixin (4.5 pg/ml) 3.80 x 107 f 1.9 x 102 1.29 x 108 +. 1.7 x 104 8.03 x 108 t- 1.0 x 104 
Chloramphenicol  (250  pg/ml) O O 8.14 x IO8 k 1.3 x 104 
Chloramphenicol  (23  pg/ml) 4.12 x 107 k 2.0 x 102 1.06 x 108 f 1.5 X 104 8.13 x 108 t- 1.1 x 104 
approximately 10' colony-forming  units  (CFU)  per ml 
of PS, 5 x 107 CFU of BS, and 6 x 105 CFU of SS. 
ASSAY PROCEDURES 
The effect of various  substances (see Table 1) on  the 
growth rates of the three test micro-organisms was 
studied in microcentrifuge  tubes (1.5 ml) with attached 
lids. 
Each of the  test  micro-organisms was diluted in 
Nutrient  Broth (Difco)  supplemented with 1 O/o glucose 
(W/V), to a final concentration of ca 500 CFU/ml. 
Aliquots of 0.1 ml from each diluted inoculum were 
delivered to  sterile  microcentrifuge  tubes in five repli- 
cations. 
The microcentrifuge tubes contain al1 the supple- 
ments as described above and the controls, were in- 
cubated for 24 h at  28 OC. After the incubation, the 
contents of each  microcentrifuge tube was diluted  ten- 
fold in sterile saline solution and the population was 
estimated using the most probable number (MPN) 
method, of ten  tubes of inoculum  from  each of three 
successive tenfold  dilutions  (Meynell & Meynell, 1970; 
Kritzman, 1989) : King's medium B for PS;  NA  for  BS; 
and  PDA  for SS.  For microscope  observations,  a drop of 
either BS or SS suspension was placed  on  a  microscope 
slide, and a young egg mass was added and observed 
with  a compound microscope over a  period of 20 min. 
Results and discussion 
Table 1 summarizes the effect of the various sub- 
stances on  the growth  rates of the tested  organisms. PS 
was not affected by the presence of the egg masses 
482 
whereas BS and SS showed significantly lower growth 
rates as compared  with  the  controls. 
In  the presence of the egg masses BS reached only 
10.2 Yo and SS only 2.1 O/O of the expected  population 
after  24  h at 28 O C .  Under  the  experiment  conditions 
gelatin, pectin, albumin and CMC had no significant 
effects on any of the tested  microorganisms. The  anti- 
biotics, penicillin, cycloheximide, polymixin and chlor- 
amphenlcol,  inhibited the growth of bacteria  or  fungus. 
In microscope  observations  clear  agglutination of 
both ES and SS by the G M  after 5-10 min of exposure 
(Fig.  1) was evident. The findings of the  present  study 
indicate that  the GM provided  protection to  the root- 
h o t  nematode eggs against at least two microorganisms. 
It is possible, however, that this activity is efficient 
against a broader spectrum of microorganisms, thus 
explaining how root-hot nematode eggs can  survive in 
the soi1 for  long  periods. 
REFERENCES 
KRITZMAN, G. (1989).  Detection,  quantification  and  classifi- 
cation of soft rot Erwinias associated with potato  tubers. 
Phytoparasitica, 17 : 205-219. 
WGGENTI, A. R. & ALLEN, M. W. (1960). The origin of the 
gelatinous  matrix  in Meloidogyne. Proceedings of the helrnin- 
thological Society of Washington, 21 : 4-10. 
MEYNELL, G. G. & MEYNELL, E. (1970). n e o y  and Practice 
in Experimental Bacteriology. 2nd ed. Cambridge  University 
Press,  346  p. 
ORION,  D. & FRANCK, A.  (1990). An electron  microscopy  study 
of  ce11  Wall  lysis  by Meloidogyne javanica gelatinous  matrix. 
Revue de Nématologie, 13 : 105-107. 
Revue Nématol. 14 (4) : 481-483  (1991) 
Anti?nicrobial  activity of Meloidogyne  javanica gelatinous nzatrix 
Fig. 1. Agglutination  of Bacillus  subtilis (A)  and Saccharomyces sp. (B) in  the  presence of a Meloidogyne javanica egg mass. 
ORION,  D., LOOTS, G. C. & ORION, T. (1987). Ce11  lysis activir, ORION, D., WERGIN,  W. P. & ENDO, B. Y. (1980).  Inhibition  of 
of Meloidogyne gelatinous matrix. Revue de Nématologie, syncytia  formation  and root-hot nematode  development in 
10 : 463-465.  cultures  of excised  tomato ro ts. Journal of Nenzatology, 12 : 
196-203. 
Accepté pour publication le 23 juillet 1990. 
Revue  Nématol. 14 14) : 481-483  (1991) 483 


Antimicrobial activity of Meloidogyne javanica gelatinous matrix

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