Esmenjaud, D.

Minot, J.C.

Salesses, G.

Scotto La Massèse, C.

Voisin, R.

Horizon / Pleins textes

Fundam. appl. Nemalol., 1992, lS (5), 385-389
Method and criteria to evaluate resistance to Meloidogyne arenaria
in Prunus cerasifera Ehr.
Daniel ESMENJAUD *, Claude Scorro LA MAssÈsE *, Georges SALESSES **,
Jean-Claude MINOT * and Roger VOISIN *
* INRA, Laboratoire de Biologie des Invertébrés, B.P. 2078, 06606 Antibes Cedex, France and
** INRA, Station de Recherches d'Arboriculture Fruitière, B.P. 81, 33883 Villenave d'Ornon Cedex, France.
Accepted for publication 18 September 1991.
Summary - The inoculaùon of Prunus cerastfera hardwood cuttings in containers by a population of Me/oidogyne arenaria was
done by transfering roots and soil of a tomato plant grown in pot that was inoculated 2 months before with 500 juveniles (2) of
this nematode. Containers with a non host clone of P. cerasifera were first used to estimate the release of J2 inoculum into the soil
from rotting tomato roots. Mean total inoculum produced in one container was 160 000 J2s + eggs. Juvenile inoculum recovered
monthly in Prunus soil varied between 5000 and 17 000 ail along the 4-month experiment thus providing a high and durable
inoculum pressure. Then eleven other P. cerasifera clones (five hosts and six non hosts) inoculated with the same method were
harvested after 4 months to determine suitability of several criteria of evaluation of resistance. Gall index was highJy significantly
correlated with the 10glO(x + 1) transformed numbers of the different nematode stages in roots of host clones. The best linear
correlaùon was observed with females, followed by eggs and J2s. Presumably because of the extracùon technique, gall index, root
eggs and root J2s resulted the best criteria to differentiate sratistically weakly-galled clones from non host clones. Gall index is
therefore a good crirerion ro evaluare resistance in P. ceras ifera.
Résumé - Méthode et critères d'évaluation de la résistance à Me1oidogyne arenaria chez Prunus cerasifera Ehr. -
L'infestation de boutures ligneuses de Prunus cerasifera en conteneur par une population de Me/oidogyne arenaria est assuree par
le transfert du systeme racinaire et du sol d'un godet de tomate inocule 2 mois auparavant avec 500 juveniles (2) de ce nematode.
Un clone non hôte de P. cerasifera est d'abord uùlise pour estimer l'evolution des effectifs de J2 libéres dans le sol par les racines
de tomate en décomposition. L'inoculum total moyen produir par conteneur est de 160 000 juvéniles et œufs. Les effectifs de J2
retrouvés mensuellement dans le sol des Prunus varient de 5000 à 17400 durant les 4 mois d'experimentaùon et assurent donc une
pression elevée et durable d'inoculum. Ensuite onze autres clones de P. cerastfera (cinq hôtes et six non hôtes) sont inocules avec
la même méthode et analyses 4 mois plus tard pour préciser la valeur des differenrs criteres d'évaluation de la résistance. L'indice
de galles est correlé de façon hautement significaùve avec les effectifs endoradiculaires transformés en loglO(x + 1) des differents
stades du nematode dans les clones hôtes. La meilleure correlation linéaire est obtenue avec les adultes femelles, suivies des œufs
et des J2. Vraisemblablement du fait de la technique d'extraction, l'indice de galles, les effectifs endoradiculaires d'œufs et de J2
s'avèrent les meilleurs critères de differenciation des clones portant peu de galles par rapport aux clones non hôtes. L'indice de galles
est donc un bon critere d'évaluation de la resistance chez P. cerasifera.
Key-words : Me/oidogyne, Prunus, resistance.
Methods ta study resistance of plants to root-knot
nematades (Meloidogyne spp.) are based on inoculation
of a defined number of juveniles (}2) at a determined
development stage of the plant. But screening of strong
sources of resistance needs very high levels of inoculum
and repeated inoculations to apply a high pressure at
different development stages of the plant, particularly
with ligneous plant material. In a study of host suit-
ability of the genus Prunus to M. arenaria., we used a
technique that was presumed to provide a high and
durable pressure of Meloidogyne J2s during several
months (Scotto La Massèse et al., 1990).
The first objective of this study was to estimate the
progressive release of J2 inoculum in soil by this techni-
que.
Furthermore, resistance in Prunus spp. stocks to
different species of Meloidogyne can be evaluated by
several criteria e.g. visual appearance of galls or gall
index (Sharpe et al., 1981; Kochba & Spiegel-Roy, 1975;
Sherman et al., 1981) coupied with an estimation of final
numbers of nematodes (Kochba & Samish, 1971; SCOttO
La Massèse et al., 1984; Pinochet et al., 1989). U sing
sorne of these criteria, resistance of P. cerasifera Ehr.
(Myrobalan plum) ta Meloidogyne arenaria resulted
very variable (Scotto La Massèse et al., 1990). This
self-incompatible species is used alone or crossed with
ISSN 1164-5571/92/05/38505 $ 2.50/ © Y""loWoI:IOr.l:,l;;;.~~~-~ORSTOM 385
1 1 SEP. 1992
D. Esmenjaud et al.
other plum, peach or almond trees as a rootstock for
stone fruit trees (Bernhard et al., 1979; Salesses et al.,
1988).
The second objective was to de termine the suitability
of the gall index or numbers of nematode to study
resistance in Myrobalan plum especially to determine
" resistant " and" susceptible" classes.
Material and methods
Host suitability of several P. cerasifera clones to the
" Monteux " population of M. arenaria has been
established by Scotto La Massèse et al. (1990). Prunus
clones harbouring females and eggs of M. arenaria were
considered " hosts " and others were considered " non
hosts ". Inoculation method was the following. Rooted
hardwood cuttings of the clones to be tested were in-
dividually planted in a glasshouse in 5 dm) containers in
sterilised sandy soil. Tomato cv. " St Pierre" grown to
the 5-leaf stage in sterilized soil in 250-ml pots were
simultaneously inoculated with 500 J2s of the Meloido-
gyne population. After 2 months, the tOP of each tomato
plant was cut off, and the content of its pot (galled roots
and soil) was transfered into a hole of the same volume
dug with a trowel into the soil of each Prunus container.
Nematodes of each Prunus plant were extracted
4 months later to evaluate resistance of the tested clones.
During our experiments, containers were drip irrigated
with a nutrient solution, and temperature varied from 20
to 28 oc.
RELEASE OF J2 INOCULUM FROM TOMATO ROOTS IN PRu-
NUS SOIL
Twenty four rooted hardwood cuttings of a non host
P. cerasifera clone followed the procedure described
above. Thirty tomato plantS were inoculated with M.
arenaria " Monteux" population. After 2 months, the
tomatoes were transfered into the Prunus container and
the six more tomato containers were analysed on the date
of transfer for an estimation of the mean nematode
numbers they harboured. In this objective the content of
each tomato container was individually and carefully
placed into a bucket and the roots were washed under
tap water above it ta prevent any loss of nematodes. The
six tomato root systems were cut in 2-cm pieces, placed
into a miSt chamber and eggs and J2s counted every
3 day. After 30 days, they were ground and centrifuged
with the grinding-centrifugation technique (Coolen &
D'Herde, 1972) before counting. Mean soil numbers of
J2 were estimated from the six buckets analysed indivi-
dually by three sedimentations each followed by sieving
on a 40 !lm pore sieve and then using a double centrifu-
gation (Jenkins, 1964).
Each month, from 1 month to 4 months after the
transfer of tomato roOtS and soil into Prunus containers,
nematodes of six Prunus containers, taken randomly,
were extracted. The content of each container was
carefully placed into a bucket and (non host clone)
386
Prunus roots taken off. Then the six tomato root systems
(or remaining part of them) were individually placed into
a mist chamber and eggs and J2s counted every 3 day.
When counting had become null, tomato roots were
ground and centrifuged in the same conditions as
described above. J2 mean soil numbers per container
were also estimated from the 6 buckets as previously
described.
EVALUATION OF PRUNUS RESISTANCE TO MELOIDOGYNE
Five hOSt and six non host clones of P. cerasifera from
the collection of the" Station de Recherches Fruitières"
of INRA at Bordeaux (France) were seleeted for this
study. Host clones (S 1 tO S5) except " 1090 " (S 1) were
intraspecific hybrids : " (2032 x 26 466) Il " (S2);
" (2032 x 2646) 13 " (S3); " (2175 x 18) 32 " (S4)·
" '(2175 x 18) 27 " (55). Non hoSt clones (R 1 to R6)
were ail intraspecific hybrids : " (2175 x 1079) 16 "
(RI); " (2175 x 1079) 29 " (R2); " (1079 x 18) 37 "
(R3); " (1079 x 18) 32 " (R4); " (2175 x 2032) 39 "
(R5); " (2175 x 18) 2 " (R6).
Eight rooted hardwood cuttings of each Prunus clone
were individually inoculated as described at the begin-
ning of this chapter. Four months after inoculation,
Prunus plantS were removed from soil and their roOtS
carefully rinsed under tap water to eliminate any remain-
ing tomato root. Gall index of each cutting was noted on
a 0-5 scale (0 = no galls; 5 = fully galled roots)
modified from Barker (1985) with steps of 0.5. Nema-
todes were then extracted from a 10 g representative
sample of roots, and 1 kg of soil per container respect-
ively by grinding-centrifugation (Coolen & D'Herde,
1972) and flotation-centrifugation (Jenkins, 1964) tech-
niques.
Females, males, third and fourth stage juveniles
(]3-J4), J2s and eggs were estimated from rwo I-ml
aliquots from 50-ml suspensions. Nematode numbers
were log,o(x + 1) transformed, subjected to a single
classification analysis of variance (factor" clone ") and
means compared by Newman-Keuls multiple range test
at p <;;; 0.05. Gall index was submitted without trans-
formation to the same statistical procedure.
Results
RELEASE OF J2 INOCULUM FROM TOMATO ROOTS IN PRU-
NUS SOIL
Table 1 gives the evolution of numbers of J2s and
eggs per container. On the date of transplanting, the
initial total inoculum given by tomato roOts and soil was
about 160000 J2s + eggs. After 1 month, rotting roots
yielded very few J2s and soil J2s were more numerous
than on transplanting date. After 2 and 3 months,
tomato roots were rotten and after 4 months there were
no longer available roots for nematode extraction while
J2s recovered from soil were berween 5000 and 7000 per
container.
Fundam. appl. NemalOl.
Resislance co Meloidogyne arenaria
4
3
2
o
1 a
b
H GALL INDEX t--
f-e-
L- e
il; d
.r::JI
1..- ..- ..-..- .. -,,"""""7
51 52 53 54 55 R1 R2 R3 R4 R5 R6
3
2
o
1,0
0,8
0,8
0,4
0,2
0,0
a
b
FEMALES 1ROOTS ~
e
Z -d
~
e
fj.e-e-e-e-e-e7
ka a
U J3-J4 1ROOTS l-
I--b
~n e~e-e-e-e-e-e--:
1,5
1,0
0,5
0,0
4
3
2
o
a
a
Ir
~ MALES 1ROOTS l-
bber.
n e~e-e-e-e-e-e7
a
b /"
Ir
e- J J2/ROOTS L
L dd L/ e-
el ~
1-1-fJ-I-f~l
51 52 53 54 55 RI R2 R3 R4 R5 R6
a
b /
e- -J EGGS 1ROOTS l-
L f-d d
I.;j /"
e-e-e-e-e-e"""'
4
3
2
o
HOST NON HOST
3
2
o
1 J2/S01L r
ab a_ab-a-ab ab-
Ir abcabc/ F t. F c..
r--abc,bc-
c/:c..
ri":
51 52 53 54 55 RI R2 R3 A4 A5 A6
HOST NON HOST
Fig. l. Gall index, numbers of nematodes at different development stages in roOts (10 g), and numbers of second stage ;uveniles
in soil (J2/100 g) in host and non host clones of P, ceraslfera, Nematode numbers reported on the figure are loglo(x + 1) transfonned.
Bars with the same lener do not differ at p ~ 0.05 using Newman-Keuls test.
Vol. 15, nO 5 . 1992 387
D. Esmenjaud et al.
, Estimated in the 250-ml tomato pot on the date of transplanting.
Discussion
Initial total inoculum brought by tomato roots trans-
Table 1. Evolution of numbers of Meloidogyne arenaria re-
covered from 5 dm) containers inoculared by transplanting the
root system and soil of a tomato plant infested 2 months before
with 500 J2s. Values are means and standard deviation (in
brackets) of six replications.
EVALUATION OF PRUNUS RESISTANCE TO MELOIDOGYNE
NemalOde densities in the soil
Nematodes recovered from soil were almost exclus-
ively J2s, as the extraction method was not suitable for
obtaining eggs or eggmasses (Fig. 1). A maximum of two
males per 100 g were counted. Variation in host Prunus
clones was lower than in non host clones. Although
significant differences appeared, no major variation was
observed between densities in the soil with host and non
host clones.
Gall index and nemalOde densities in the roolS
Mean gall index varied from 0.5 to 3.5 in host clones
and was zero in non hosts (Fig. 1). In host clones, eggs
and J2s were the most numerous followed by females,
males and lastly J3-J4. Female, egg and J2 numbers were
ranged into the same order as gal1 index. J2s were the
only stage deteeted in the roots of non host clones (R3
and R6) but densities were very low.
Female numbers were ranged into the same order as
males and J3-J4, except in clones SI and S3. Sex ratio
varied from 0.08 to 0.21, except in clone S3 where it
reached 0.43. Linear correlation coefficients were calcu-
lated for all host clones (5 clones x 8 replicates =
40 couples of data) between gall index and loglo(x + 1)
transformed densities of females (r = 0.78), males
(0.66), J3-J4 (0.60), J2s (0.68) and eggs (0.71). Ali the r
values are highly significant.
Aknowledgrnents
We thank Mr. A. Bonnet from the Station de Recherches
Fruitières of INRA at Bordeaux for supplying P. cerasifera
clones.
References
BARKER, K. R. (1985). Design of greenhouse and microplot
experiments for evaluation of plant resistance ra nemarades.
In : Zuckerman, B. M.) Mai, W. F. & Harrison, M. B. (Eds).
PianI nemalology laboralOry manual. Amherst, Univ. Mas-
sachusetts Agric. Exp. Statn : 103-113.
fer was probably under-estimated because only J2s were
obtained from soil. Moreover survival of entire root
system in soil is longer than that of root pieces in the
mist chamber and consequently advanced J4 and young
females might have a higher chance of achieving their
development and laying eggs. Most inoculum was cer-
tainly eggs in eggmasses whose diapause is removed
progressively (de Guiran, 1979). Consequently the re-
lease of J2 is progressive and keeps relatively high and
stable from 2 te 4 months.
Numbers of J2s in the soil at harvest were almost
similar under host and non host clones and thus it is
concluded that J2s in soil came mainly from eggmasses
that developped on tomato. Consequently the release of
J2 from tomate continued to be higher than that of
Prunus 4 months after inoculation, confirrning our
previous results.
The best linear correlation between gall index and
nematode stages in roots was observed with females
followed by eggs and J2s. While host suitability of the
clones was very variable, the proportion of different
nematode stages in roots was very similar. Moreover the
five clones can be ranged into three genetically differem
groups : S2 and S3, S4 and S5 (brother clones), SI.
Despite the high level of initial inoculum, even S4 roots
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multiplication did not occur to an extent on the best
hosts; in another experiment not reported here, we
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These experiments demonstrate that a clear separa-
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The method reported here indicates that a durable
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ligneous plant material. It can also be adapted to study
telerance.
20522
(4455)
5088
(1083)
5707
(1970)
6984
(1877)
161863
(29502)
Total
inoculum
Roots Soil
J2 Eggs J2
127576 24070 10217
(5365)
2588 542 17392
(4630)
5 20 5063
(1087)
0 5706
(1970)
no longer roots 6984
(1877)
Initial'
inoculum
4
3
2
Months
after
trans-
plantation
388 Fundam. appl. Nemalol.
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389


Method and criteria to evaluate resistance to Meloidogyne arenaria in Prunus cerasifera Ehr.

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