Beane, J.

Perry, R.N.

Horizon / Pleins textes

The effect of brief exposures to potato root difisate 
on the hatching of Globodera rostochiensis . 
Roland N. PERRY and Jack BEANE 
Nematology  Department,  Rothamsted  Experimental  Station,  Harpenden,  Herts.,   England. 
L’effet d’immersions brèves dans l’exsudat radiculaire de pomne de terre sur  l’éclosion de Globodera rostochiensis 
L’éclosion d’œufs  libres de Globodera  rostochiensis est  stimulée  par  une immersion limitée à 5 minutes  par  semaine 
pendant 5 semaines dans  un  exsudat  radiculaire  de  pomme  de  terre ; l’allongement de la  durée d’immersion jusqu’à 
24 heures  n’entraîne  aucune  augmentation significative du  taux d’éclosion. Une immersion répétée de même que 
l’emploi  d’exsudat  frais  améliorent significativement l’éclosion, comparativement à celle résultant  d’une immersion 
unique ou à celle obtenue en employant le même exsudat  pendant 5 semaines  d’expérience. 
The  hatching  mechanisms of nematodes  have 
been reviewed  (Perry & Clarke,  1981)  and  one  aspect 
that  was  apparent  was  the  rapid  action of root 
diffusate  in  initiating  the  hatching of Globodera 
rostochiensis (Woll.)  and G.  pa l l ida  Stone.  During 
the first 24  h  after  the  hatching  stimulus  physiological 
changes in the unhatched G. rostochiensis juvenile, 
such as an increase  in  the  oxygen  uptalte  (Atltinson & 
Ballantyne,  1977a), a decrease  in the  adenylate 
energy charge (Atkinson & Ballantyne, 1977b) and 
an  increase  in  water  content  (Ellenby & Perry,  1976) 
, occur  3-4  days  prior t o  movement  and  hatching 
(Doncaster & Shepherd,  1967). A 5 mn  exposure 
each week is sufficient t o  induce a 43% hatch of 
juveniles  from  free eggs of G.  pa l l ida  (Forrest & 
Perry, 1980). Similar data are laclting for the hatch 
of G. rostochiensis so we investigated the response 
of G. rostochielzsis t o  brief exposures of potato 
root  diffusate t o  learn  more  about  he  hatching 
mechanism of this species. 
Revue   Némato l .  5 (2): 221-224 (1982) 
Materials  and Methods 
Cysts of G. rostochiensis R o l ,  grown  on  Rotato  cv. 
Arran Banner, in pot cultures were from a single 
generation harvested in ‘1979 and had been subse- 
quently  stored a t  room  temperature (200). They  were 
used in batches of 25 and soalted in artificial tap 
water  (Greenaway,  1970)  for  four  days a t  200. 
They were then  cut,   the eggs were  freed  from  the 
cyst  and  any  free  juveniles  were  removed.  The 
suspension of eggs and cyst debris from each batch 
was added to 6 mm diameter glass cylinders with 
45 Pm mesh  nylon  etting fixed over  the  nds 
(Forrest & Perry,  1980). Soi1 leachate  and  potato 
root  diffusate  were  obtained  by  the  method of 
Fenwick  (1949)  and  the  latter  was  diluted wiLh 
artificial tap  water 1 in  4  by  volume.  Four  different 
experimental  protocols  were followed, each  involving 
exposure of the eggs t o  root diffusate  for  seven 
different periods from 5 mn  to  24 h (Fig. 1) a t  200. 
221 
R.N. Perry h J. Beane 
Tests were 'also carried out to determine the per- 
centage  hatch  over  a five weelr period  from  batches 
of eggs kept continuously in potato root diffusate 
and soil leachate. Counts of hatched juveniles were 
made  weekly  when  diffusate  and  leachate  were 
replenished  from  stock  solutions  kept a t  50. 
EXPERIMENT 1 : REPEATED STIMULUS USING FRESH 
DIFFUSATE EACH WEEK 
Each cylinder was placed in a 9 ml  capacity  vial 
containing 3 ml of diffusate, for the  appropriate 
time.  After  treatment  he  cylinders were  rinsed 
under running tap water for 30 mn  then placed in 
a  vial  containing 3 ml of artificial tap  water.  Previous 
tests khowed that washing for 30 mn completely 
removed the  hatching  stimulus.  The  above procedure 
was  repeated  with  fresh  diffusate  from  the  stock 
solution  each  week  for 5 weeks  and  counts of hatched 
juveniles were taken weekly. Each  test  was  repeated 
three  times a t  t he  end of which the  number of 
unhatched eggs was  counted  and  the  percentage 
hatch was determined over the test period. 
EXPERIMENT 2 : REPEATED STIMULUS USING THE 
SAME DIFFUSATE EACH WEEK 
The above procedure was followed except that, 
for each period of exposure,  the  same  root diffusate 
was  retained  throughout  the 5 weeks. 
EXPERIMENT 3 : SINGLE STIMULUS 
In t2hese tests, for  each  time  period,  root  diffusate 
was used for the initial stimulus only. Counts were 
taken  at  weekly  intervals  when  the  tap  water  in  the 
vials  was  changed ; the  eggs  were neither re-exposed 
to  diffusate  nor  rinsed  for 30 mn. 
EXPERIMENT 4 : SINGLE STIMULUS WITH RINSING 
To  check  whether  the  extra  mechanical  stimulation 
and oxygenation associated with rinsing enhanced 
hatch, the single stimulus procedure was followed 
but  each week the cylinders were flushed with tap 
water for 30 mn before  being  placed  in 3 ml of 
fresh artificial tap  water. 
Results 
The mean number of free eggs recovered from a 
batch .- of 25 cyst? !as 4 945 & 247, The-total  hatch 
222 
for  eggs kept  continuously  in  root  diffusate  and 
soil leachate for 5 weeks was 69% and 2y0, ,respec- 
tively. The total percentage hatch of juveniles for 
each exposure to root diffusate is given in Fig. 1. 
Results  were  analysed  using  two  way  analysis of 
variance  after  arcsin  t>ransformation of percentages. 
A 5 mn  exposure  to fresh  root  diffusate  each  week 
induced a 51% hatch and, there was no significant 
increase or decrease in hatch with increase in the 
period of stimulation (P > 0.05). Using the same 
diffusate  ach  week for re-stimulation  gave less 
consistent  results  with  the  greatest  hatch (36%) 
after 15 mn exposure  each week and  the  least (14%) 
after 1 h  exposure  ach weelr. The  variation  in 
hatch with period of exposure was not st,atist>ically 
significant (P > 0.05). However, for each period of 
exposure  the  percentage  hatch  was  much  greater 
when fresh diffusate was used each week and the 
difference between results for the repeated stimula- 
tion  tests  using  the  same diffusate and  fresh  diffusate 
is highly significant (P < 0.01). Even weekly expo- 
sures of up to  24 h to the same diffusate did not 
elicit as great a hatch as 5 mn per week of fresh 
diffusate (28% and 51% respectively) even though 
the  total  time exposed to diffusate in  the  latter  test 
was  only  25  mn.  Thus,  the use of fresh  diffusate  from 
stock  solutions for re-stimulation  is of greater  import- 
ance  than  the  duration of st<imulation. 
Using  root  diffusate for the  initial  stimulation 
without,  any subsequent.  rinsings  elicited  hatches 
which  did  not exceed 15% (Fig. 1). The  smallest 
hatch  was  after  24  h  exposure  (9%)  and  the  largest 
after 30 mn (15%). Including weekly rinsing, with 
the  concomitant mechanical  stimulation,  did  not 
significantly increase , hatach (P > 0.05). For both 
sets of data an increase in the period of exposure 
did  not significantly  affect the  total  percentage 
hatch (P > 0.05). For each time period the  hatches 
from both tests using single stimulation was lower 
than  hatches  from  experiments  on  repeated  exposure 
to  diffusate  and  this difference was  tatistically 
significant (P < 0.01).  Thus,  repetition of the 
stimulus,  even  with  the  same  diffusate  and  for 
short  periods,  stimulated  greater  hatch. 
Discussion 
Al1 treatments  with  potato  root  diffusate  increased 
hatch from free eggs when compared to the hatch 
induced  by soil leachate.  However,  a  single  stimula- 
'tion,  whether or not   i t  was  accompanied  by  weekly 
rinsing,  enhanced  hatch  only  slightly. A much 
greater hatch was obtained with repeated stimula- 
tions  especially if fresh  diffusate  was  used  each  time. 
Duration of stimulation - is of . only .~ marginal . -  - import- 
Revue Nématol. 5 ( 2 ) :  221-224 (1982) 
Hatching of Globodera  rostochiensis 
70 
50 
x 
O 
I I I 1 1 I I ’ I  I 
O “2 1 2. 6 ‘-4 I I  
Time, hr, exDosed to PRD 
Fig. 1. The  total  percentage  hatch of free eggs of Globodera rostochiensis exposed 
to potato root diffusate (PRD) for various periods : black circles : repeated 
weekly exposure to fresh diffusate ; white circles : repeated weekly exposure to 
the  same  diffusate;  black  squares : single exposure  with  subsequent weekly 
rinsings; white  squares : single exposure  with  no rinsings. 
ance in hatching ; this work shows that repetition 
of the  stimulation  and  fresh  diffusate are of overrid- 
ing importance in stimulating hatch from free eggs 
of G. rostochierzsis. 
Clarke,  Perry  and  Hennessy (1978) proposed tha t  
a  change  in  permeability of the G. rostochierzsis 
egg shell allowed the diffusion of egg fluid solutes 
out of the egg, which removed the osmotic stress 
on the  juvenile  with  concomitant  increase  in  juvenile 
water content. This increase allows the juvenile to 
become fully  mobile and  start  the  behavioural 
sequence  leading to eclosion. The  change  in  permeabi- 
lity has yet to be established although it is lrnown 
to occur in Ascar i s  suum, a species whose hatching 
mechanism  (Clarke & Perry,  1980)  shows  some simi- 
larities to   that  of G. rostochierzsis. Permeability 
change  could  be  caused  directly or indirectly  by 
the hatching agents (Clarke & Perry, 1977 ; Perry, 
1978) and i t  is possible tha t  the  juvenile could be 
involved in the hatching process at an early stage. 
Revue Nèmatol. 5 (2): 221-224 (1982) 
Fisher  (1966)  found  that  exposing  fourth  stage 
juveniles of Paratylerzchus nanus to  root  diffusate 
induced moulting 8-13 days later but i t  was not 
necessary to expose them to diffusate al1 the time. 
Exposure  for  24 h “ triggered ” moulting  and  Rogers 
and Sommerville (1968) suggested that  root  diffusate 
could stimulate a receptor which in turn restarted 
internal  mechanisms  governing  moulting or could 
replace a missing link in the sequence of internal 
secretions.  The  present  work on G. rostochierzsis 
shows that very brief exposures to root diffusate 
are suffkient to “ trigger ” the sequence of events 
leading  to  hatch.  The  enhancement of hatch  by 
repeated  stimulation  may  indicate a receptor 
threshold  level  being  reached  whereas  an  equivaleni; 
period of single  stimulation does not  reach  the 
threshold  level  and  the  receptor,  therefore,  does  not 
“fire”.  Ellenby  and  Perry  (1976)  suggested  that  the 
G. rostochiensis juvenile could be involved in the 
initial  stages of the  hatching  sequence,  as the 
223 
R. N .  Perry 6 J .  Beane 
hatching  factors  may  initiate  a  n urosecretory 
response  from  the  juvenile. 
Although of importance,  repetjtjon of the  stimulus 
is not  the  only  significant  factor ; using fresh diffusate 
each week, even for periods as short as 5 mn, sig- 
nificantly  increased  hatch  almost  to  the  level  obtained 
with eggs continuously in diffusate. The reasons for 
this  are  unknown  but it is unlikely that  the  diffusate 
activity had been reduced when the same diffusate 
had been used weekly €or 5 weeks because after- 
wards the diffusate used for al1 seven time periods 
was pooled and 3 ml of this diffusate for 3 weeks 
elicited  a  63%  hatch. 
In soil conditions  there  is  a 35-40% " carry  over " 
of viable cyst contents from one year to the next 
(Jones & Parrott, 1969). This wauld seem unlikely 
if hatching is elicited by very short exposures to 
root  diffusate  which  may  persist in the soil for  some 
time  (Tsutsumi,  1976 ; Perry,  Hodges & Beane, 
1982). However, under field conditions not al1 cysts 
would be reached by root diffusate ; the cyst as a 
hatching unit (Ellenby, 1956) is obviously a signifi- 
cant  factor  and,  as  pointed  out elsewhere (Clarke & 
Perry,  1977), tes ts  on  free eggs may  ignore  important 
factors  such  as  inhibition of hatch or distribution of 
eggs within cysts. Changes in the physiology of the 
unhatched juvenile (associated with dormancy, for 
example)  may  block  the  sequence of events leading 
to  eclosion. 
Soaking  for 5 mn in  dif€usat.e is also very effective 
in stimulating hatch of juveniles in whole cysts of 
G. pallida IForrest in Forrest & Perry, 1980) and 
G. rostochiensis (Perry,  unpubl.)  but  with  these 
experiments i t  was  not  certain  that  rinsing  after 
the  xposure  period effectively removes al1 the 
diffusate from wjthin the cyst. 
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ATRINSON,  H.J. & BALLANTYNE,  A.J.  (1977~). Changes 
, in the oxygen consurnption of Globodera  rostochiensis 
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ATKINSON,  H.J. & BALLANTYNE,  A.J.  (1977b). Changes 
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~ ~~~~ ~~ ~ 
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Accepté pour publ icat ion le 10 décembre 1981. 
224 
.~ - - .  - -  . 
Revue   Némato l .  5 ( 2 ) :  221-224 (1982) 


The effects of brief exposures to potato root diffusate on the hatching of Globodera rostochiensisec

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