Duponnois, Robin

Mateille, Thierry

Netscher, Caspar

Horizon / Pleins textes

I -  - 
EFFECT OF THE RHIZOSPHERE MICROFLORA. ON PASTEURXA PENÆTmNS 
PARASITIZING MELOIDOGYNE GRAMINICOLA 
1 
Summary. The effect of the rhizosphere microflora associated with spores of Pastèu?-ia peizstrmzs on the at- 
tachment of the spores on juveniles of Meloidogyne grnmiizicola has been studied. The microflora stirnulated 
the attachment of the spores on the juveniles and consequently reduced the invasion of the roots of tomato 
plants by the nematodes. The role of a helper rhizosphere niicroflora for biocontrol of root-knot nematodes by 
P. peizetruns is suggested. 
Pasteuria peneti-ai?s is a n  obligate endopara- 
site of nematodes which has potential as a bio- 
logical control agent (Oostendorp et al., 1991; 
Zaki a n d  Maqbool, 1992). The relationship 
between this actinoniycete and root-knot nema- 
todes is well documented (Davies et al., 1992; 
Davies and Danks, 1992). However, the ways in 
which soil factors influence the P. peizeti-aizs - 
nematode interaction are virtually unknown, 
particularly that between the nematodes, P. 
penetram and the microflora. The aim of this 
paper is to examine the influence of the rhizo- 
sphere microflora on  the parasitic relationship 
between P. penetrans Sayre et Starr and Meloi- 
dogyize graminicola Golden et Birchfield. 
Materials and methods 
A population of A4eloidogyize graininicola 
was cultured for two months on tomato (Ljco- 
persicon esculeiztum Mill.) cv. Roma. Then the 
tomato roots were harvested, cut into short 
lengths and placed in a mist chamber for one 
week to allow second stage juveniles (T2) to 
hatch from the eggs (Seinhorst, 1950). 
Seeds of tomato cv. Roma were planted in 
0;3 dm3 pots previously filled with a sandy soil 
(sand 84.7%; silt 4.1%; clay 11.2%; pH (H2O) 
7.6) sampled in a vegetable field from the ex- 
perimental s ta t ion of the  École Nationale 
Supérieure d'Agriculture (ENSA, Thiès, Sénégal) 
heavily infested with juveniles of M. javanica 
parasitized by P. penetram (20,000 J2 per dm3, 
SO% of infected juveniles). Two months after 
sowing, the plants were uprooted, the root 
systems were gently washed, cut into short 
lengths and blended in 100 ml sterile water us- 
ing an Ultraturax blender. The suspension was 
sieved through 3 45 pm sieve and the filtrate 
was divided in two parts, each of which were 
centrifuged (3000 g, 20 min); the supernatants 
were discarded. The pellet: of the first sample 
was resuspended in alcohol (97%) for 12 h at 
25 "C to obtain P. penetram spores free of con- 
taminant microflora, and centrifuged (3000g, 20 
min) three tinies to replace the alcohol with 
sterile sodiuni-phosphate buffer (50ni1, pH 7). 
'I'he pellet of the second sample was directly re- 
suspended in the same sterile bu f fe r  without 
sterilization by alcohol. The two suspensions of 
spores disinfected or not were incubated for 
two weeks in the dark at 25 O C .  
Each week, attachment tests were performed 
in glass tubes (1Ss2 cm) with the spores of P. 
peizti-mzs disinfected or not, adding 5 p1 of the 
suspensions (lo4 spores) to one ml of distilled 
water containing 100 juveniles of M. g~í"ii7ico- 
la. There were five replicates per condition. 
The suspensions -ïvere incubated for 12 hours at 
2 j OC. The number of juveniles encoumbered 
with P. peizetraizs spores and the number of 
spores per juvenile were determined with the 
aid of a dissecting microscope (magnification s 
400). All the data were statistically analysed ac- 
cording to the Mann Whitney U test (P < 0.0s). 
At the same time, the populations of bacteria 
were determined in the- two suspensions of P. 
peizeti-aizs spores disinfected or not by spread- 
plate counts on  0.3% tryptic soy agar (h?artin, 
1975). 
Two week-old seedlings of tomato cv. Roma 
were transplanted in 1 dm3 pots filled with the 
same sandy soil that was used for the produc- 
tion of spores of P. peizeti-ans but which had 
been previously autoclaved (140 OC, 40 min). 
Two weeks after transplanting, the plants were 
inoculated either with 100 juveniles of M. gm-  
minicola, or with 5 . 9 ~ 1 0 ~  spores of F. penetram 
disinfected o r  not with alcohol following the 
technique described above, or with 100 juve- 
niles of M. gi-anziuicola and with 5 . 9 ~ 1 0 ~  spores 
of P. penetram disinfected o r  not, together. 
There were ten replicates for each inoculum 
condition placed in 3 randomisecl block design 
in a glasshouse (25 "C day, 20 OC night). One 
month later, each root system was gently 
washed free of soil and the g:i11s with egg n i x s -  
es were counted. The roots were then cut into 
short lengths and placed in a mist chamber for 
two weeks to collect the juveniles as described 
above. The oven dried weights of shoot and 
root (1 week at 65 OC) were measured and  
compared to non inoculated plants (control). 
'l'lien the root systems wcre Sround to a pow- 
der and resuspended in  distilled water. The 
spores of 1'. peizcti-ans were recovered as de- 
scribed before and counted using a Mallassez 
counting cell. The data were analysed with the 
Mann Whitney U test (P < 0.05). 
Results 
In the attachment tests 100?/0 of the juveniles 
of At. gi-niiaiizicoln were parasitized by 1'. pei7e- 
trcms whatever the time of incubation of the 
spores in the buffer and there were no differenc- 
es between the disinfected and the non disinfect- 
ed spores. Only the number of spores per juve- 
nile differed (Fig. 1). Twenty to twenty-four 
spores attached to the juveniles in the first test 
with no difference between the disinfected and 
the non-disinfected spores. However, after one 
and two weeks of incubation, the number of 
spores attached per juvenile decreased, more 
with the spores previously disinfected with alco- 
hol than with the non-disinfected spores. In the 
suspension containing the non-disinfected spores 
of P. penetram, the population of bacteria re- 
mained constant during the two weeks of incu- 
bation in the sodium-phosphate buffer. No bac- 
teria were detected in the suspension containing 
the spores previously disinfected with alcohol. 
One month after the inoculation of tomato 
plants with juveniles of M. gmmiizicola, 87.8% 
of the juveniles were able to invade the roots 
and to mature in gravid females (Table I). When 
the nematode inoculum was supplemented with 
the spores of P. perzetmizs. the de\.elopmenr 
rate of the juveniles w3s strongly reduced, sig- 
nificantly more when the spores were not disin- 
fected with alcohol than when they were. Coni- 
paring with the non-inoculated plants (control), 
the juveniles of M. gi-anziizïcola alone reduced 
the development of the roots without any effect 
on  the shoots. When the spores of P. perietizrizs 
were added to the juveniles, the root growth 
- 100 - 
Q sporcs not disinfcctcd 
bacteria in  tlic non-disinfcclcd spor 
2 suspcnsion 
1 2 
Time (wech) 
Fig. 1 - Evolution of the number of spores of Pasteru-iape- 
netruiu disinfected or not with 97% alcohol on the juveni- 
les of MeZoidogJuze grarninicoZa &ars represent standard 
errors) and evolution of the total .population of bacteria in 
the suspension of non disinfected spores (columns with 
the'same letter are not significandy dif5rer.t: P 2 C.05). 
ci 
was restored. Plant growth was greater when 
the spores, especially not disinfected, were in- 
oculated alone. Most of the next generation of 
juveniles which were exqracted from the roots 
in the mist chamber were encumbered with 
spores of P. penetram (66 to 74% of the juve- 
niles), but without any significant effect of the 
alcohol disinfection previous to the inoculation 
of the plants. After the roots were dried and 
ground to powder, the number of spores of P. 
penetram extracted was the same whether the 
parent spores were disinfected or not. 
Discussion 
The disinfection of the spores of P.peizet~nn.s 
with alcohol eliminated the associated microflo- 
ra as no bacteria were detected later in the sus- 
pensions, but it did not affect the attachment of 
the spores to the juveniles of hl. g7.nmiizicoln. 
'The spores of P. peizchzciu wliicli were incu- 
Iiated in the sodium-phosphate buffer became 
less aggressive to the juveniles of Ag. gi-niniizico- 
lu over one and two weeks. The attachment is 
between t u 7 0  orgmisms whose external surfac- 
es are electronegatively charged and liydropho- 
bic (Himmelhoch et al., 1979; Afolabi et al., 
1995; Davies et nl., 1996). Thus, the decrease of 
agressiveness could be due to a change of hy- 
drophobic characteristics or to a progressive 
saturation of the neg:itive sircs of the spore sur- 
face by the ai'+ cations of the buffer at neutral 
pH. For example, these characteristics of the 
Na2+ cations are commonly used in soil parti- 
cule dispersion (Bartoli et al., 1990; Feller et al., 
199&). Nevertheless, the spores of P. peizetraizs 
which were incubated together with the micro- 
flora over two weeks, were more aggressive 
against the nematodes than the spores whose 
associated microflora was eliminated with alco- 
hol. Even if the microflora did not develop in 
the buffer, bacterical metabolites would have 
been excreted in the suspension. They could 
act enzymatically on the external tissues of the 
spores thereby exposing the parasporal fibres 
and enabling their attachment to the cuticle of 
the nematodes (Davies and Danks, 1993). They 
might also modify the chemical environment of 
the spores and  facilitate their attachment by 
producing or excreting directly (Rozycki, 1987) 
simple sugars and oligosaccharides which could 
involve lectin-carbohydrate interaction (Bird et 
al., 1989; Ibrahim, 1991). 
The same helper potential of the microflora 
may act efficiently in the soil. Despite steriliza- 
tion of the soil, the microflora associated with 
the spores of P peizetmizs induces the best at- 
tachment of the spores to the cuticle of the 
juveniles, unless compounds toxic to nema- 
todes (Hoffmann-Hergarten and Sikora, 1995) 
are produced in addition. Consequently, the 
motility of the juveniles (Stirling et nl., 1990) 
and the invasion of the plant roots (Brown and 
Smart, 1985; Davies et al., 1988) are reduced. 
The subsequent production of spores detected 
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TAULE I - EfJecCl oJ'lbe e¡isiirfcclioir of lhe spoiz~ of l'asieuria penetram oii tbc cieueIopitIeIzL of Akloiciogyize gi-aitzi,zicola 
oii toinalo plaiils niici c o i r s ~ c ~ ~ ~ i u e ~ ~ l c i ~ ~ ~  groru b. 
Inoculum No o f  galls Shoot Iiioinass Ibot Iiiom:iss NO of spores 
per pl:rnt (111.q dry \vcigilL) (111~ dry weight) per plant 
Control 1880 b 818 b 
M. gi'anziizicola 87.8 a 1700 b 372 d - 
P. peizetizizs disinfected - 2070 ab 1117 a - 
P. penetinns not disinfected - 2400 a 1112 a - 
M. gi-utniizicola + 
P. peizetiwizs disinfected 43.9 b 2200 ab 540 bc 4 . 6 ~ 1 0 ~  a 
M. grnn?iizicoln + 
P. peizermizs not disinfected 20.7 c 1900 b 424 c 3.7~10~ a 
In each column, data followed by the same letter x e  not significantly different according to the kkinn Wiitney U test 
(1' < 0.05) 
in the powdered roots are not different, certain- 
ly because most of the spores were spread dur- 
ing nematode extraction. in the water mist. 
Thus, the rhizosphere microflora can enhance 
the biocontrol fo the root-knot nematodes by P. 
pe7zetlwizs by increasin3 the attachment of the 
spores to the nematodes. Further research is re- 
quired to study the bacteria which are concerned 
and the helper mechanisms they induce, and to 
study and manage the abiotic factors which in- 
volve such favorable biocenotic relationships. 
Acknowledgements The study was support- 
ed by a grant from the EC Project STD 3 no TS3 
* CT92-0098: Biocontrol of damaging root-knot 
nematode (Meloidogyze spp.) pests of staple 
food and cash crops by including suppressive 
soils with the bacterial parasite Pusteuna pene- 
t7z77s. The authors are grateful to Dr D.L. Trud- 
gill (SCRI, Dundee, Scotland) for reviewins the 
manuscript. 
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Accepted for public:ition on  70 February 1996 
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Effect of the rhizosphere microflora on Pasteuria penetrans parasitizing Meloidogyne graminicola

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