pathozone dynamics of meloidogyne incognita in the rhizosphere of tomato plants in the presence and...
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Plant Pathology (2008) 57, 354362 Doi: 10.1111/j.1365-3059.2007.01776.x
2007 The Authors
354 Journal compilation 2007 BSPP
BlackwellPublishingLtd
Pathozone dynamics of Meloidogyne incognita
in the
rhizosphere of tomato plants in the presence and absence of
the nematophagous fungus, Pochonia chlamydosporia
D. J. Bailey
a
*, G. L. Biran
a
, B. R. Kerry
b
and C. A. Gilligan
a
a
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA; andb
Rothamsted Research, Harpenden,
AL5 2JQ, UK
Pathozone dynamics were derived for approx. 50 Meloidogyne incognita
juveniles infecting a single root of tomato under
controlled conditions from a point source of inoculum and described using simple, non-linear models. The pathozone
decayed sigmoidally with distance, but increased over time as progressively more nematodes were able to infect the root.
Despite the reported ability ofM. incognita
juveniles to travel up to 50 cm in some conditions, the maximum width of
the pathozone for a single tomato root was estimated as 181 mm. It is conjectured that this was because of (i) diffusionfrom a point source of inoculum, (ii) a small infection court (a single root tip) and (iii) the limited life span of the
nematode. A second experiment was used to assess the effect of the nematophagous fungus Pochonia chlamydosporia
on the pathozone dynamics of the nematode. This fungus is known to produce nematicidal products in vitro
, which may
affect invasion of roots by the free-living nematode. To examine the possibility of a change in the position of the site
of infection, changes in the probability of gall formation along the root length were also examined. In the absence
ofP. chlamydosporia
, the pathozone dynamics ofM. incognita
were very similar to those of the first experiment. It was
shown that P. chlamydosporia
did not significantly affect the pathozone dynamics ofM. incognita
nor the site of gall
formation, which appear to have little importance for the role of the fungus as a biological control agent.
Keywords
: biocontrol, epidemiology, Lycopersicon esculentum
, root-knot nematode, soilborne parasite
Introduction
The nematode Meloidogyne incognita
is a root-infectingparasite that attacks a wide range of cultivated plants andcauses extensive economic damage, worldwide(Whitehead, 1998). The epidemiology of root-knotdisease is well known. Epidemics are initiated by primaryinfection from egg masses that persist in soil followinginfection of a previous crop and from which second-stagejuvenile (J2) nematodes hatch, migrate and infect nearbyroots. Within the roots, further nematode developmentdepends upon the formation of giant multinucleate
feeding cells. Swelling of cortical cells around the giantcells gives rise to galls on the roots of infected plants thatare characteristic of parasitism by root-knot nematodes.Female nematodes obtain nutrients from the plant via thegiant cells and eventually produce egg masses from which
a new generation of J2s hatch and spread to fresh roots,thus initiating the secondary phase of the epidemic (Bridge& Starr, 2007).
For soilborne parasites, the distance over which theycan migrate and infect a susceptible host root is a criticalfactor affecting the outcome of an epidemic. Thesecharacteristics are defined within the concept of thepathozone (Gilligan, 1985). The pathozone is the regionof soil surrounding the root in which inoculum must bepresent if it is to have any chance of infecting the root. Theprobability of infection is not constant when inoculum islocated at any site within the pathozone, but depends on
the distance between inoculum and host. Changes in theprobability of infection with distance can be derivedexperimentally by challenging replicate hosts with singleunits of inoculum placed at different distances fromthe host and then recording the proportion of successfulinfections at each distance after a given period of time.The resulting data can be described by a curve known asthe pathozone profile, which typically decays as thedistance between inoculum and host increases, butevolves over time, as progressively more inoculum issuccessful in causing infection. In combination with
*E-mail: [email protected]
Current address: INRA Agrocampus Rennes, UMR BiO3P
BP 35327, F-35653 Le Rheu Cedex, France.
Accepted 15 July 2007
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Pathozone dynamics ofMeloidogyne incognita 355
simple non-linear models, the dynamics of the pathozoneare defined by a three-dimensional surface for change inthe probability of infection over distance and time (Bailey& Gilligan, 1997; Kleczkowski et al
., 1997).Pathozone dynamics have been carefully quantified for
particulate inoculum of certain soilborne oomycetes andfungi, but not for nematodes. For example, the pathozone
ranges from 5 mm for Phytophthora
spp. (Reynolds et al
.,1985) and Sclerotium rolfsii
on Phaseolus
spp. (Punja &Grogan, 1981) to 50 mm for Rhizoctonia solani
on sugarbeet petioles (Henis & Ben-Yephet, 1970). Nematodes arecapable of moving much faster and further than fungi, andProt (1978) reported Meloidogyne
populations migrating50 cm vertically in 9 days. Thus, one might expect a pro-portional increase in the magnitude and dynamics of thepathozone for nematodes compared to that for fungi.Moreover, the ability of nematodes to detect the presence ofa host from distances of up to 45 mm (Wallace, 1963) furtherincreases the probability of locating a susceptible root.
The potential for reducing the spread of disease canbe quantified using the pathozone bioassay (Bailey &
Gilligan, 1997), where disease control may be associatedwith either the inoculum (a reduction in infectivity) or thehost (increase in resistance via protection of the infectioncourt). For the root-knot nematode, M. incognita
, thesoilborne fungus Pochonia chlamydosporia
(Zare et al
.,2001) is a well-known parasite (Kerry, 2000) andstrategies for its exploitation as a biological control agenthave been developed (Atkins et al
., 2003). The fungus is afacultative parasite and develops saprotrophically inthe rhizosphere of many healthy and nematode-infectedplant species. The egg masses of root-knot nematodesproduced on the surface of infected roots may be colonizedby the fungus and the eggs containing infective juvenile
nematodes destroyed (Segers et al
., 1996; Kerry, 2000).Importantly, P. chlamydosporia
also produces bioactivecompounds in vitro
(Lopez-Llorca & Boag, 1993;Khambay et al
., 2000) that may act to protect the rootfrom infection, thus augmenting the hosts own resistance.Phomalactone was produced by P. chlamydosporia
during in vitro
culture and this weakly nematicidalmetabolite significantly reduced M. incognita
invasion oftomato roots when applied as a soil drench (Khambay
et al
., 2000). However, the importance of this mode ofaction in the regulation of Meloidogyne
populations inthe rhizosphere is not known.
In this paper a simple experimental bioassay combinedwith non-linear modelling was used to quantify the
pathozone dynamics for the infection of tomato plantsby the root-knot nematode, M. incognita
. The bioassaywas then used to test for any nematicidal effect of
P. chlamydosporia
in the rhizosphere of tomato plantscolonized by the fungal antagonist. A further level ofanalysis was also included involving the effect of P.chlamydosporia
on the distribution of galls producedalong a root (defined as the probability of gall formationat a given position). The potential of the methodology foranalysis and interpretation of nematode dynamics andtheir control are briefly discussed.
Materials and methods
Inoculum
Second-stage juveniles (J2) of the nematode Meloidogyneincognita
, race 2, population 1135, supplied as a gift fromNorth Carolina State University, USA and maintained
under quarantine at Rothamsted Research, were hatchedfrom egg masses produced on roots of infected aubergine(
Solanum melongena
) cv. Purple Ruby plants (grown inglasshouses at 27
C with a day length of 16 h) using themethod of de Leij & Kerry (1991) to provide an inoculumsuspension of 500 juveniles mL
1
water.An inoculum ofP. chlamydosporia isolate Vc-10 was
prepared from 21-day-old cultivars on cornmeal agarcontaining 50 mg each of streptomycin sulphate, chlortet-racycline and chloramphenicol (Sigma, USA) L
1
in Petridishes, incubated in the dark at 23
C. Aliquots of 1 mLsterile reverse-osmosis (RO) water were pipetted onto thecentre of each agar plate and the surface of each colonylightly agitated with a surface-sterilized spatula to
dislodge chlamydospores. The spore suspension waspipetted into a sterile universal tube and the concentrationadjusted to 500 chlamydospores mL
1
with sterile RO water.
Sand microcosms
Sand packs were prepared by adding approximately330 g of sand (acid-washed, quartz sand grade 16/30;Hepworth Minerals & Chemicals), moistened (10% v/w)with Hoaglands solution (Hoagland & Arnon, 1950), to20-cm lengths of 10-cm-wide Layflat tubing (1000-gauge). Each end of the tubing was partially sealed withstaples to allow for drainage.
A small hole was cut 10 mm from the top of each sandpack and a tomato seed (
Lycopersicon esculentum
cv.Tiny Tim; E. W. King & Co. Ltd) planted beneath the sandsurface at a depth of approximately 3 mm. The sandpacks were incubated in humid chambers at 23
C with16 h light (1828
moles m
2
s
1
) on a slope (30
fromvertical) to force the roots to grow towards the rear of thepacks.
Experiments
Two experiments were performed. Experiment 1 was usedto examine the pathozone dynamics ofM. incognita
andto identify the form of non-linear model with which to
describe changes in the probability of gall formation withdistance and time. The second experiment, whilstdemonstrating reproducibility of the pathozone bioassay,examined the effect of root colonization by P. chlamy-dosporia
on the pathozone dynamics of M. incognita
inoculum and on the precise location at which gallsdeveloped along the root. This latter experiment wasdesigned to demonstrate whether nematicidal metabolitesproduced by the fungus in vitro
were important for theinteractions between the nematode and the fungus in therhizosphere. Additional tests (results not shown) were
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D. J. Bailey et al.
performed to show that the fungus had no effect on thegrowth of tomato roots and that it could be re-isolatedafter the experiment had finished.
Experiment 1: pathozone dynamics ofM. incognitaTomato plants grown in sand packs were inoculatedslowly with 100
L of nematode inoculum (approx. 50
J2s) injected 0, 5, 10, 15, 20 or 25 mm from the tip of aroot (the target root) to prevent spread of inoculumduring the injection process. The packs were completelyrandomized and incubated for 8 days in humid chambersat 23
C with 16 h light per day. The experiment included16 replicates for all inoculation distances except 20 mm,for which there were 15 replicates. Roots were examinedfor the presence of galls with the aid of a dissectingmicroscope (
20 magnification) each day for 8 daysfollowing inoculation.
Experiment 2: effect of root colonization byP. chlamydosporia
on the pathozone dynamics ofM. incognita
Tomato plants grown in sand packs were inoculated with100
L nematode inoculum injected 0, 5, 10, 15, 20 or25 mm from the tip of the target root. Half of the packsfor each distance were also inoculated with P. chlamydosporia
on the target root tip (
+
Pc). The remaining packs wereinoculated with 100
L tap water on the target roottip (Pc). The numbers of replicates per treatment were:16 for 0, 5 and 20 mm Pc; 17 for 10, 15 and 25 mm Pc;17 for 0, 5, 10 and 20 mm +
Pc; 15 for 15 mm +
Pc; and16 for 25 mm +
Pc. The packs were completely randomizedand incubated in humid chambers for 8 days at 23
C with16 h light.
Roots were examined for the presence and precise
location (with respect to the site of inoculation withnematodes) of galls with the aid of a dissecting microscope(
20 magnification) each day for 8 days after inoculation.
Modelling and statistical analyses
Experiment 1
Data for the probability of infection (gall formation), P
g
with distance, r
, were described by a sigmoidal curve:
(1)
at each time of observation, where
was the maximumprobability of infection when inoculum was placed at thesurface of the root and
was a measure of the rate at
which the probability of infection decayed as the distancebetween inoculum and the root increased. Changes in theparameters and
over time, t
, were described bymonomolecular and sigmoidal functions, respectively:
(2)
and
(3)
Note that the power on t
,
4
, was introduced to avoid verylarge values for
as t approached
3
. Incorporating
Eqns 2 and 3 into Eqn 1, the probability of infection overdistance and time was described by the surface
(4)
The furthest extent of the pathozone over time was
estimated from P
g
005 as t
approached infinity.
Experiment 2
Equation 4 was used as the basis for a statistical compar-ison of parameters estimated from data describing changein the probability of gall formation with distance and overtime (Gilligan, 1990) in the presence and absence of
P. chlamydosporia
.A distribution of the probability of gall formation with
distance below the point of inoculation was obtainedfrom data (pooled for all distances between inoculum androot) describing the position of galls along the root 8 daysafter incubation. Data were divided into 10 classescorresponding to contiguous 5-mm lengths of root
extending 45 mm below the point of inoculation anddescribed by a gamma function. Distributions for gallproduction in the presence and absence ofP. chlamydosporia
were compared using Kolmogorov-Smirnov tests (Kanji,1995). All curves were fitted using genstat
(V42, VSNInternational Ltd) assuming a binomial distribution oferrors (Anonymous, 1993).
Results
Experiment 1: pathozone dynamics ofM. incognita
on tomato
After 2 days incubation, the probability of infectiondecayed from 025 when inoculum was placed at the rootsurface to zero when inoculum was placed 10 mm fromthe root surface (Fig. 1). The probability of infectionincreased for a further 3 days so that, after 5 daysincubation, the probability of infection for inoculumplaced at the root surface was 075 and decayed to zerowhen inoculum was placed at a distance of 25 mm fromthe root surface.
Equation 1 provided a good description of thepathozone data at each time of observation. The param-eters and both changed significantly over time (Fig. 2).Parameter , describing change in the probability ofinfection when inoculum was placed at the surface of a
root, increased monotonically and was described by amonomolecular curve rising from zero after 175 days to082 after 8 days (Fig. 2a, Eqn 2). Parameterwas describedby an exponential decay over time (Fig. 2b, Eqn 3).
Using Eqns 2 and 3, a single surface:
(5)
was used to describe the evolution of the pathozoneover time (Fig. 3, Table 1). Change in the furthest
P r rg( ) exp( )= 2
( ) ( exp( ( ))),t t= 1 2 31
( ) ( exp( )).t t= + 1 2 34
P r t t
t rg( , ) ( exp( ( )))
exp ( ( exp( )) )
=
+
1 2 3
1 2 32
14
P r t t
t rg( , ) ( exp( ( )))
exp ( ( exp( )) )
=
+
0 79 1 1 29 1 69
0 009 6 41 3 41 0 61 2
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Pathozone dynamics ofMeloidogyne incognita 357
extent of the pathozone was estimated at Pg= 005.
Pathozone width increased monotonically from 62 mmafter 2 days to a maximum of 1811 mm after 8 days(Fig. 4).
Experiment 2: effect of root colonization byP. chlamydosporia on pathozone dynamics ofM. incognita
In the absence of P. chlamydosporia, data describingchange in the probability of gall formation with distancebetween inoculum and root were similar to those
Figure 1 Pathozone profiles describing change in the probability of infection (gall formation) by the nematode Meloidogyne incognita with distance
from roots of tomato after 2, 3, 4, 5, 6 and 8 days of incubation. Profiles were described by Eqn 1: exp(r2).
Figure 2 (a) Change in the probability of gall formation for Meloidogyne incognita inoculum placed at the surface of the root () over time (t)
described by the monomolecular function 1(1 exp(2(t 3))); and (b) change in the rate of decay () with time (t) described by an exponential
decay: 1+ (2 exp(3t4)).
Table 1 Parameter estimates for the pathozone dynamics (change in
the probability of gall formation with distance and over time) ofMeloidogyne
incognita on tomato described using the pathozone model (Eqn 4):
Parameter Value ( SE)
1 079 005
2 129 073
3 169 024
1 0009 0002
2 641 (fixed)
3 341 125
4 061 035
P r t t t r g( , ) ( exp( ))) exp ( ( exp( )) )( .= +
1 2 3 1 2 3
4 21
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obtained from experiment 1. After 2 days incubation, the
probability of gall formation when inoculum waspositioned at the root surface was 02 and decayed to zerowhen inoculum was placed at a distance of 5 mm.However, in contrast to the first experiment, after 7 daysincubation the maximum probability of gall formationPg= 068 occurred from inoculum placed 5 mm from theroot surface and declined to zero at a distance of 20 mm.
In the presence ofP. chlamydosporia, the probability ofgall formation was marginally increased during the first5 days when inoculum was positioned at or 5 mm fromthe root surface and marginally decreased when placed
10 mm from the root surface. After 8 days, the probabilityof gall formation in the presence of P. chlamydosporia
was slightly higher when inoculum was placed 15 mmfrom the root surface, but lower when it was placed at adistance of 5 or 10 mm.
Using the pathozone model (Eqn 4) to describe surfacesfor change in the pathozone over time (Table 2, Fig. 5), nosignificant effect ofP. chlamydosporia was detected on thepathozone dynamics of M. incognita. The pathozoneincreased monotonically from 28 mm after 2 days to amaximum of 20 mm after 8 days in the absence of the fungalantagonist and from 395 mm after 2 days to a maximumof 192 mm in the presence of the fungal antagonist.
Figure 3 Pathozone dynamics describing change
in the probability of gall formation over distance
and time for the infection of tomato by Meloidogyne
incognita. Data were fitted with the model
P r t t
t rg( , ) ( exp( ( ))) exp (
( exp( )) )
.
= +
1 2 3 1
2 3
4 2
1
Figure 4 Change in width of the Meloidogyne incognita pathozone
over time, estimated from pathozone dynamics (Eqn 4), where
pathozone width was estimated at Pg= 005.
Table 2 Parameter estimates for the pathozone dynamics (change in
the probability of gall formation with distance and over time) of Meloidogyne
incognita on tomato described using the pathozone model (Eqn 4):
in the
presence (+Pc) or absence (Pc) of Pochonia chlamydosporia
Parameter
Treatment
Pc ( SE) +Pc ( SE)
1 05769 011 06123 005
2 05020 045 22100 248
3 12350 085 18540 018
1 00061 0001 00068 0001
2 641 (Fixed) 641 (Fixed)
3 17100 122 27900 115
4 11100 062 06780 034
P r t t t r g( , ) ( exp( ( ))) exp ( ( exp( )) ).= +
1 2 3 1 2 3
4 21
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Pathozone dynamics ofMeloidogyne incognita 359
Eight days after inoculation with the nematode, a totalof 55 galls had been produced on roots in the presence and52 galls in the absence ofP. chlamydosporia. The probabilityof gall formation rose from zero, at the point of inoculationwith the nematode, to a maximum of 033, 1620 mmbelow the height of inoculation. The probabilitythen decreased again to zero, 45 mm below the height
of inoculation (Fig. 6). A gamma distribution describedthe change in the probability of gall formation withdistance, x, from the height of inoculation with thenematode. No significant difference was detectedfor distributions describing change in the probabilityof gall formation with distance from the point ofinoculation.
Figure 5 Pathozone dynamics describing
change in the probability of gall formation over
distance and time for the infection of tomato by
Meloidogyne incognita in (a) the absence
and (b) the presence of Pochonia
chlamydosporia. Data were fitted with the model
P r t t t r
g( , ) ( exp( ( ))) exp (( exp( )) )
.
= +
1 2 3 1
2 3
4 21
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Discussion
The pathozone dynamics ofM. incognita on single rootsof tomato plants were quantified. Despite the potential ofnematodes to spread by several cm each day, the furthestextent of the pathozone in the absence ofP. chlamydosporiawas estimated to be between 181 mm (experiment 1) and200 mm (experiment 2). Others estimates of the furth-est extent from which nematodes can infect a host
ranged from 5 mm (Wieser, 1955) to 100 cm (Johnson &McKeen, 1973) and were based on the infection of aroot system by individuals from a large population ofnematodes. The measurements in the present studyestimated the probability of infection of a single root andshowed that, although infection from distance is possible,the probability of infection decays very rapidly as thedistance between inoculum and root increases. The reasonfor this may result from a combination of three factors: (i)the concentration of nematodes decreases rapidly as theymove away from the site of inoculation, a process which
is most likely related to simple diffusion or a randomwalk (Feltham et al., 2002); (ii) the life expectancy ofnematodes in this system was less than 10 days (results notshown); and (iii) the susceptible portion of the root doesnot include the entire root, but may well be restricted tothe zone of elongation just behind the root tip. This meansthat, whilst the nematodes in this system may be capable
of travelling significantly greater distances than denotedby the furthest extent of the pathozone detected here,when placed only a small distance from the root surface,few will actually make contact with the susceptibleportion of a target root during the course of theirmigration. Observations were not made on other roots todetermine if they had been invaded elsewhere.
The variability between reported estimates of pathozonewidth undoubtedly results from differences in the inoculumsource, the host target and the soil environment throughwhich the nematodes migrate. For example, some studiesinvolved inoculum composed of over 300 juvenilenematodes (Wieser, 1955; Prot & Netscher, 1979;Stephan & Estey, 1982; Pinkerton et al., 1987; Pline &
Dusenbery, 1987; Diez & Dusenbery, 1989). In contrast,the present study used only 50. Others have includedlarger or multiple targets for infection (Bird, 1959;
Johnson & McKeen, 1973; Prot & Netscher, 1979;Stephan & Estey, 1982; Pinkerton et al., 1987), therebyincreasing the probability of infection. Moreover, forstudies involving incubation periods of over 50 days (e.g.Wallace, 1963; Johnson & McKeen, 1973; Stephan &Estey, 1982) resulting in estimates for infection over largedistances, secondary, root-to-root, infection cannot bediscounted. A wide range of abiotic and biotic factors,such as pH, temperature, moisture, redox potential,presence of a mate and root diffusates, affect nematode
movement in soil or in sand columns in controlled conditions(Prot, 1980). The present experiments to measure thepathozone used sand-based soil packs combined with ahighly controlled environment providing relativelyhomogeneous and repeatable growth conditions for theplant, nematode and fungus. This had the advantage ofreproducibility within (between replicates) and betweenexperiments, and forms the mechanistic platform fromwhich variability in other factors (inoculum size, hostnumber, soil type, the presence of an antagonist, etc.) canbe investigated.
Equation 4 accurately described the pathozone dynamicsfor M. incognita. Changes in the probability of infectionfor inoculum placed at the root surface, , increased
monotonically over time and the rate at which the prob-ability of infection decreased with distance, , decreasedexponentially over time. This is consistent with a profile(probability of infection with distance) that increasesdisproportionately more over time for inoculum placed atthe root surface than for inoculum placed away from theroot surface. It suggests that nematodes located on theroot surface remain more efficient at infecting the root andforming galls and may be related to a more favourablebalance between energies invested in foraging versusinfection.
Figure 6 Probability distributions of galls below the point of inoculation
along a Meloidogyne incognita-infested tomato root in (a) the absence
or (b) the presence of Pochonia chlamydosporia 8 days after
inoculation with M. incognita. Data were summarized using a gamma
distribution (solid lines).
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Pathozone dynamics ofMeloidogyne incognita 361
By inoculating P. chlamydosporia directly onto the rootsurface, and despite the presence of actively growingfungus for the duration of the experiment, no significanteffects of fungal presence on pathozone dynamics(parameters 1, 2, 3, 1 and 2 of Eqn 4) ofM. incognitawere detected. Either the fungus had no direct effect oninfection of the nematode or the nematode was able to
avoid the fungus by infecting at another location. Yet,distributions describing change in the probability ofinfection along the root in the presence and absence ofP. chlamydosporia were identical and there was no significanteffect of the fungus on the site of gall formation. SinceP. chlamydosporia is a poor saprotroph with a slow rateof mycelial growth (Kerry, 1989), the nematode probablyenters the root ahead of extensive colonization by thefungus and the fungus is incapable of providing controlduring this phase of the nematode infection cycle.Therefore, the role ofP. chlamydosporia in the biologicalcontrol of M. incognita presumably depends on thereduction of secondary inoculum through the parasitismof nematode eggs and not the protection of the root from
nematode attack.To date, the development of biological control strategies
for nematodes is hampered by the lack of general descriptivemodels for the complex interactions between pest speciesand their microbial natural enemies. Scaling up fromsmall experimental plots to field crops will increase thevariation in the system and require such knowledge aspresented here to identify the key factors that affect theefficacy of biological control and so help provide con-sistent and practical levels of nematode management. Thispaper begins to address this need.
Acknowledgements
This work was funded by the award of a ResearchStudentship from the Biotechnology and BiologicalSciences Research Council (BBSRC), which we gratefullyacknowledge. Rothamsted Research receives grant-aidedsupport from the BBSRC and CAG also acknowledgesBBSRC support. We are also grateful to Dr D. Hall fortechnical assistance during the experiments. The workwas conducted in accordance with Defra Plant HealthLicence No. 174A/4485.
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