Pinochet, J.

Verdejo, S.

Horizon / Pleins textes

Reproduction of the nematode otylenchus guevarai 
(Nemata : Pratylenchidae) in monoxenic cultures 
Soledad VERDEJO and Jorge PINOCHET 
Departamento de Patologia Vegetal, Institute de  Recerca i Tecnologia Agroalimentariès, 
IRTA, Crta. de Cabris1 s/n 08348 Cabrils, Barcelona, Spain. 
SUMMARY 
Zygotylenchus guevarai reproduced monoxenically on carrot disc and on Agrobacterium rhizogenes transformed potato root 
cultures. The life  cycle  was  completed  in 45 days  at 25 OC. An initial  inoculum  level of 50 individuals  provided threë times  more 
nematodes than  ten or  twenty  individuals.  In  al1  tests  most  nematodes  were found in the egg  stage  and  only a low  proportion  of 
the  population  reached  the  adult  stages. 
RÉSUME 
Reproduction du nématode Zygotylenchus  guevarai (Nemata : Pratylenchidae) en culture monoxénique 
Zygotylenchus  guevarai se reproduit  en élevage  monoxénique  sur  disques  de  carotte et sur  cultures  de  racines  de  pomme  de  terre 
modifiées  par Agrobacterium rhizogenes. Le  cycle  biologique  est  accompli  en 45 jours, à 25 OC. Un inoculum  de 50 individus produit 
trois  fois  plus  de  nématodes  que  des  inoculums  de 10 ou 20 individus.  Dans tous les  essais  la plupart des  nématodes  sont  rencontrés 
au  stade  d'œuf  et  seule  une  faible  proportion  de  la  population  atteint  l'âge  adulte. 
The migratory  endoparasitic  nematode Zygotylenchus 
guevarui (Tobar Jimenez) Braun & Loof, invades the 
root cortex causing root lesions and the formation of 
large cavities, destroying the cortical  tissues (Varo 
Alcala, Tobar Jiménez &. Mufioz  Medina, 1970; Vovlas, 
Inserra & Lamberti, 1976). Although the nematode is 
widespread in the Mediterranean countries (Siddiqi, 
1975; Lamberti,  198l),  little  information is available on 
its pathogenicity, perhaps due  to diffïculties found  in 
rearing large numbers of individuals needed for such 
studies. Tobar Jiménez,  Guevara  Benitez and  Martinez 
Sierra (1968), found that Z. guevarai increased only 
twelve fold  on  potted violet, Viola tricolor L. after 
10 months  in  a  lath  house. 
Carrot disc cultures have been used to propagate a 
number of root lesion nematodes  providing  large num- 
bers of highly infective nematodes  (O'Bannon & Taylor, 
1968;  Moody,  Lownsbery & Ahmed, 1973; Huettel & 
Rebois, 1985; Lawn & Noel, 1986). Since  contamination 
due  to bacteria associated to  the carrot  tissue  sometimes 
occurs in carrot  disc  cultures, Agrobacterium rhizogeizes 
transformed-potato  root  cultures were considered  as an 
alternative in this  study  to  propagate Z. guevarai. Trans- 
formed root cultures have been successfully used to 
propagate sedentary endoparasitic nematodes (Paul et 
al., 1987; Verdejo, Jaffee & Mankau, 1988). In this paper 
the monoxenic  culture  of, 2. guevarai in  both  carrot  and 
transformed  potato  root is reported. 
Revue  Nématol. 14 (2) : 291-297 (1991) 
Materials  and  methods 
The nematode was isolated from soil around  almond 
trees in Tarragona,  Spain.  Adult  individuals recovered 
from soil were hand  picked and surface  disinfected in a 
100 ppm mercuric  chloride  and  1 O00 ppm streptomycin 
sulphate  solution for 5 min.  Nematodes were then 
pipetted to carrot  disc  cultures  prepared as indicated by 
O'Bannon  and  Taylor (1968). Such  cultures were used 
as the  source of inoculum  for  the reproduction  studies. 
Transformed potato roots were grown on solid Gam- 
borg's B5 medium plus vitamins (Verdejo, Jaffee & 
Mankau, 1988) and  incubated  for 3 days at 25 "C before 
nematode  inoculation. 
To  compare  nematode  reproduction  on  differentiated 
and non-differentiated tissues, 15 & 1 surface disin- 
fected  females were added  to  each of 20 cultures  con- 
taining  either  tfansformed-potato  roots  or  carrot discs 
(one disc  per  culture). The  number of Z. guevarui added 
per culture was determined  after  nematode  inoculations. 
Cultures were maintainded at 25 "C in the dark for 
68 days. Nematodes were recovered from carrot disc 
cultures by adding 5 ml distilled water per culture. 
Nematodes that migrated from the disc to the clear 
water were collected 24 h later. The carrot disc was then 
weighted and  blended in a  blender  for  a  total of 30 s 
given as 10 second  periods.  Both  suspensions were 
mixed and nematodes  counted  determining the  number 
3 17 
S. Verdejo & J. Pinochet 
of eggs, juvenile stages (J2, J3 and J4), males and 
females. To recover Z.  guevarai from  root  cultures,  the 
agar was melted in a microwave oven  for 1-2 min, roots 
blotted  dry  and  weighted.  Nematodes  remaining  in  the 
agar plate and in  the roots were counted as indicated 
above. 
The reproduction  rate of the nematode was studied on 
carrot disc cultures inoculated with 10 & 1 surface 
disinfected females. Nematode  reproduction was asses- 
sed, as decribed previously, in  ten cultures at 45,85 and 
125  days, respectively. 
The effect of the initial  inoculum level on nematode 
multiplication was determined  after 125 days by adding 
10,20 and 50 surface  disinfected  nematodes (a mixture 
of al1 life cycle stages) to 250 ml  flasks  containing five 
carrot discs. Each inoculum level was replicated four 
times. 
Results  and  discussion 
Zygotylenchus guevarai was established in monoxenic 
cultures on both carrot discs and A. rhizogenes trans- 
formed  roots  (Table 1). The population  increased 
104 times  on  carrot discs in  about  2  months.  Clusters of 
nematodes were observed in  the plate  outside the  carrot 
disc (Fig. 1 A). Although the nematode was able to 
complete  its  life cycle on transformed  potato  roots,  as 
indicated by the presence of males, these  cultures  were 
apparently a poor host for the nematode. Nematodes 
were found  in  the agar but no nematode was observed 
into the root tissue. The nematode completed its life 
cycle in 45 days which seems to be the  minimun  time 
required to  undergo  development from  adult  to  adult at 
25 "C (Table 2). The reproduction rate of Z. guevarai 
was 106 and 1504  fold  after 85 and 125 days, respect- 
Table 1 
Reproduction  of Zygotylenchus  guevarai on  carrot  disc  and Agrobacterium rhizogenes 
transformed  potato  roots  cultures  inoculated  with  15 f 1 females  after  68  days at 25 OC. 
Substrate Tissue No eggs/ No juveniles/ N o  fernales/ N o  males/ Total nem.  R production 
weight culture culture culture culture culture rate 
Carrot  discs 3.3* 808 440 92 78 1417 104 
Potato  roots 1.1 1 21 9 2 33 2 
* Mean of ten  replications. 
Fig. 1. Monoxenic culture of Zygotylenchus  guevarai. A : Clusters of Z. guevarai observed  in  carrot  disc  cultures  inoculated with 
15 k 1  females  after  68  days  at  25 'Cc; B : Z. guevarai female  containing  eggs  from  both  ovaries  found  in  carrot  disc  cultures. 
Monoxenic reproduction of Zygotylenchus  guevarai 
ively. On occasion  females  with eggs from  both ovaries 
were found  but  most of the observed females  contained 
one or no eggs (Fig. 1 B). An initial  inoculum level of ten 
or  twenty  individuals  provided similar numbers of 
nematodes  per  culture  after  107 days (Table 3). How- 
ever, cultures inoculated with 50 individuals yielded 
three times more nematodes. The egg stage was pre- 
dominant  in carrot  disc  cultures (40-60 O/O of the popula- 
tion) in  the  three experiments  except  when  the  repro- 
duction rate was assessed at 45 days. At this time, the 
population was composed mainly by females (52 "0). 
The high  proportion of females  could  be due  to  the lack 
of nematode reproduction in five of the  ten  cultures 
checked at 45 days, only females  used as inoculum were 
recovered  from  those  cultures.  Carrot  disc  cultures of 
Radopholus similis and Pratylenchus brachyurus yielded 
large  numbers of eggs (O'Bannon & Taylor, 1968). High 
numbers of eggs were also found in cultures of P. 
brachyurus on carrot  callus tissue; however, those  cul- 
tures of Pratylenchus agilis, P. scribneri, Radopholus 
citrophilus and R. similis, yielded low numbers of eggs 
(Reise, Huettel & Sayre,  1987). Only a low percentage of 
the population  reached the  adult  stage,  the  proportion of 
males to females never being  higher than 8 and 11 O/O of 
the population, respectively. 
Table 2 
Reproduction  of Zygotylenchus  guevarai on  carrot  discs  inoculated  with 10 f 1 females  after 45, 85 and 125 days at 25 OC. 
Days after Carrot tissue N o  eggs/ No juveniles/ No fenzales/ No males/ Total  nem. Reproduction 
inoculation weight (g) culture culture culture culture culture rate 
45  3.4* 5 16 24 1 46 5 
85  3.0 400 412 106 85 1 003 106 
125  2.6 7  148 5 818 1 270 852 15  O88 1 504 
* Mean of ten  replications. 
Table 3 
Reproduction  of Zygotylenchus  guevarai on carrot  discs  inoculated  with 10, 20 and 50 nematodes  after 107 days at 25 OC. 
Inoculation No eggs/ No juveniles No fernales/ N o  ritales/ Total  nem. Reproduction 
level culture culture culture culture culture rate 
10  5  827* 4  715 441 186 11 169 1 117 
20  7  786 4  582 379 234  12 981 649 
50 22 230 13 264 1 499 949  37  942 759 
* Mean of four  replications. 
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Reproduction of the nematode Zygotylenchus guevarai (Nemata : Pratylenchidae) in monoxenic cultures

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