Plant Pathology (2008) 57, 354362

Doi: 10.1111/j.1365-3059.2007.01776.x

Pathozone dynamics of Meloidogyne incognita in the rhizosphere of tomato plants in the presence and absence of the nematophagous fungus, Pochonia chlamydosporia
Blackwell Publishing Ltd

D. J. Baileya*, G. L. Birana, B. R. Kerryb and C. A. Gilligana

a Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA; and bRothamsted 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 of M. 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) diffusion from 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 of P. chlamydosporia, the pathozone dynamics of M. incognita were very similar to those of the rst experiment. It was shown that P. chlamydosporia did not signicantly affect the pathozone dynamics of M. 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-infecting parasite that attacks a wide range of cultivated plants and causes extensive economic damage, worldwide (Whitehead, 1998). The epidemiology of root-knot disease is well known. Epidemics are initiated by primary infection from egg masses that persist in soil following infection of a previous crop and from which second-stage juvenile (J2) nematodes hatch, migrate and infect nearby roots. Within the roots, further nematode development depends upon the formation of giant multinucleate feeding cells. Swelling of cortical cells around the giant cells gives rise to galls on the roots of infected plants that are characteristic of parasitism by root-knot nematodes. Female nematodes obtain nutrients from the plant via the giant cells and eventually produce egg masses from which
*E-mail: djb21@cam.ac.uk Current address: INRA Agrocampus Rennes, UMR BiO3P BP 35327, F-35653 Le Rheu Cedex, France. Accepted 15 July 2007

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 they can migrate and infect a susceptible host root is a critical factor affecting the outcome of an epidemic. These characteristics are dened within the concept of the pathozone (Gilligan, 1985). The pathozone is the region of soil surrounding the root in which inoculum must be present if it is to have any chance of infecting the root. The probability of infection is not constant when inoculum is located at any site within the pathozone, but depends on the distance between inoculum and host. Changes in the probability of infection with distance can be derived experimentally by challenging replicate hosts with single units of inoculum placed at different distances from the host and then recording the proportion of successful infections at each distance after a given period of time. The resulting data can be described by a curve known as the pathozone prole, which typically decays as the distance between inoculum and host increases, but evolves over time, as progressively more inoculum is successful in causing infection. In combination with
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simple non-linear models, the dynamics of the pathozone are dened by a three-dimensional surface for change in the probability of infection over distance and time (Bailey & Gilligan, 1997; Kleczkowski et al., 1997). Pathozone dynamics have been carefully quantied for particulate inoculum of certain soilborne oomycetes and fungi, 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 sugar beet petioles (Henis & Ben-Yephet, 1970). Nematodes are capable of moving much faster and further than fungi, and Prot (1978) reported Meloidogyne populations migrating 50 cm vertically in 9 days. Thus, one might expect a proportional increase in the magnitude and dynamics of the pathozone for nematodes compared to that for fungi. Moreover, the ability of nematodes to detect the presence of a host from distances of up to 45 mm (Wallace, 1963) further increases the probability of locating a susceptible root. The potential for reducing the spread of disease can be quantied using the pathozone bioassay (Bailey & Gilligan, 1997), where disease control may be associated with either the inoculum (a reduction in infectivity) or the host (increase in resistance via protection of the infection court). For the root-knot nematode, M. incognita, the soilborne fungus Pochonia chlamydosporia (Zare et al., 2001) is a well-known parasite (Kerry, 2000) and strategies for its exploitation as a biological control agent have been developed (Atkins et al., 2003). The fungus is a facultative parasite and develops saprotrophically in the rhizosphere of many healthy and nematode-infected plant species. The egg masses of root-knot nematodes produced on the surface of infected roots may be colonized by the fungus and the eggs containing infective juvenile nematodes destroyed (Segers et al., 1996; Kerry, 2000). Importantly, P. chlamydosporia also produces bioactive compounds in vitro (Lopez-Llorca & Boag, 1993; Khambay et al., 2000) that may act to protect the root from infection, thus augmenting the hosts own resistance. Phomalactone was produced by P. chlamydosporia during in vitro culture and this weakly nematicidal metabolite signicantly reduced M. incognita invasion of tomato roots when applied as a soil drench (Khambay et al., 2000). However, the importance of this mode of action in the regulation of Meloidogyne populations in the rhizosphere is not known. In this paper a simple experimental bioassay combined with non-linear modelling was used to quantify the pathozone dynamics for the infection of tomato plants by the root-knot nematode, M. incognita. The bioassay was then used to test for any nematicidal effect of P. chlamydosporia in the rhizosphere of tomato plants colonized by the fungal antagonist. A further level of analysis was also included involving the effect of P. chlamydosporia on the distribution of galls produced along a root (dened as the probability of gall formation at a given position). The potential of the methodology for analysis and interpretation of nematode dynamics and their control are briey discussed.
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Materials and methods

Inoculum
Second-stage juveniles (J2) of the nematode Meloidogyne incognita, race 2, population 1135, supplied as a gift from North Carolina State University, USA and maintained under quarantine at Rothamsted Research, were hatched from egg masses produced on roots of infected aubergine (Solanum melongena) cv. Purple Ruby plants (grown in glasshouses at 27C with a day length of 16 h) using the method of de Leij & Kerry (1991) to provide an inoculum suspension of 500 juveniles mL1 water. An inoculum of P. chlamydosporia isolate Vc-10 was prepared from 21-day-old cultivars on cornmeal agar containing 50 mg each of streptomycin sulphate, chlortetracycline and chloramphenicol (Sigma, USA) L1 in Petri dishes, incubated in the dark at 23C. Aliquots of 1 mL sterile reverse-osmosis (RO) water were pipetted onto the centre of each agar plate and the surface of each colony lightly agitated with a surface-sterilized spatula to dislodge chlamydospores. The spore suspension was pipetted into a sterile universal tube and the concentration adjusted to 500 chlamydospores mL1 with sterile RO water.

Sand microcosms
Sand packs were prepared by adding approximately 330 g of sand (acid-washed, quartz sand grade 16/30; Hepworth Minerals & Chemicals), moistened (10% v/w) with Hoaglands solution (Hoagland & Arnon, 1950), to 20-cm lengths of 10-cm-wide Layat tubing (1000gauge). Each end of the tubing was partially sealed with staples to allow for drainage. A small hole was cut 10 mm from the top of each sand pack and a tomato seed (Lycopersicon esculentum cv. Tiny Tim; E. W. King & Co. Ltd) planted beneath the sand surface at a depth of approximately 3 mm. The sand packs were incubated in humid chambers at 23C with 16 h light (1828 moles m2 s1) on a slope (30 from vertical) to force the roots to grow towards the rear of the packs.

Experiments
Two experiments were performed. Experiment 1 was used to examine the pathozone dynamics of M. incognita and to identify the form of non-linear model with which to describe changes in the probability of gall formation with distance and time. The second experiment, whilst demonstrating reproducibility of the pathozone bioassay, examined the effect of root colonization by P. chlamydosporia on the pathozone dynamics of M. incognita inoculum and on the precise location at which galls developed along the root. This latter experiment was designed to demonstrate whether nematicidal metabolites produced by the fungus in vitro were important for the interactions between the nematode and the fungus in the rhizosphere. Additional tests (results not shown) were

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performed to show that the fungus had no effect on the growth of tomato roots and that it could be re-isolated after the experiment had nished. Experiment 1: pathozone dynamics of M. incognita Tomato plants grown in sand packs were inoculated slowly with 100 L of nematode inoculum (approx. 50 J2s) injected 0, 5, 10, 15, 20 or 25 mm from the tip of a root (the target root) to prevent spread of inoculum during the injection process. The packs were completely randomized and incubated for 8 days in humid chambers at 23C with 16 h light per day. The experiment included 16 replicates for all inoculation distances except 20 mm, for which there were 15 replicates. Roots were examined for the presence of galls with the aid of a dissecting microscope (20 magnication) each day for 8 days following inoculation. Experiment 2: effect of root colonization by P. chlamydosporia on the pathozone dynamics of M. incognita Tomato plants grown in sand packs were inoculated with 100 L nematode inoculum injected 0, 5, 10, 15, 20 or 25 mm from the tip of the target root. Half of the packs for each distance were also inoculated with P. chlamydosporia on the target root tip (+Pc). The remaining packs were inoculated with 100 L tap water on the target root tip (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; and 16 for 25 mm + Pc. The packs were completely randomized and incubated in humid chambers for 8 days at 23C with 16 h light. Roots were examined for the presence and precise location (with respect to the site of inoculation with nematodes) of galls with the aid of a dissecting microscope (20 magnication) each day for 8 days after inoculation.

Eqns 2 and 3 into Eqn 1, the probability of infection over distance and time was described by the surface Pg (r, t) = 1(1 exp( 2(t 3))) exp (1 + ( 2 exp( 3t 4 ))r 2) (4)

The furthest extent of the pathozone over time was estimated from Pg 005 as t approached innity. Experiment 2 Equation 4 was used as the basis for a statistical comparison of parameters estimated from data describing change in the probability of gall formation with distance and over time (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 obtained from data (pooled for all distances between inoculum and root) describing the position of galls along the root 8 days after incubation. Data were divided into 10 classes corresponding to contiguous 5-mm lengths of root extending 45 mm below the point of inoculation and described by a gamma function. Distributions for gall production in the presence and absence of P. chlamydosporia were compared using Kolmogorov-Smirnov tests (Kanji, 1995). All curves were tted using genstat (V42, VSN International Ltd) assuming a binomial distribution of errors (Anonymous, 1993).

Results
Experiment 1: pathozone dynamics of M. incognita on tomato
After 2 days incubation, the probability of infection decayed from 025 when inoculum was placed at the root surface to zero when inoculum was placed 10 mm from the root surface (Fig. 1). The probability of infection increased for a further 3 days so that, after 5 days incubation, the probability of infection for inoculum placed at the root surface was 075 and decayed to zero when inoculum was placed at a distance of 25 mm from the root surface. Equation 1 provided a good description of the pathozone data at each time of observation. The parameters and both changed signicantly over time (Fig. 2). Parameter , describing change in the probability of infection when inoculum was placed at the surface of a root, increased monotonically and was described by a monomolecular curve rising from zero after 175 days to 082 after 8 days (Fig. 2a, Eqn 2). Parameter was described by an exponential decay over time (Fig. 2b, Eqn 3). Using Eqns 2 and 3, a single surface: Pg (r, t) = 079 (1 exp(129(t 169))) exp (0009 + (641 exp(341t 0 61))r 2) (5)

Modelling and statistical analyses

Experiment 1 Data for the probability of infection (gall formation), Pg with distance, r, were described by a sigmoidal curve: Pg (r) = exp(r 2) (1) at each time of observation, where was the maximum probability of infection when inoculum was placed at the surface of the root and was a measure of the rate at which the probability of infection decayed as the distance between inoculum and the root increased. Changes in the parameters and over time, t, were described by monomolecular and sigmoidal functions, respectively: (t) = 1(1 exp ( 2(t 3))), and (t) = 1 + ( 2 exp( 3t )).
4

(2)

(3)

Note that the power on t, 4, was introduced to avoid very large values for as t approached 3. Incorporating

was used to describe the evolution of the pathozone over time (Fig. 3, Table 1). Change in the furthest
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Figure 1 Pathozone proles 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. Proles were described by Eqn 1: exp(r 2).

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(3t 4)).

Table 1 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): Pg (r , t ) = 1(1 exp(2( t 3))) exp (1 + (2 exp( 3t 4 ))r 2). Parameter 1 2 3 1 2 3 4 Value ( SE) 079 005 129 073 169 024 0009 0002 641 (xed) 341 125 061 035

extent of the pathozone was estimated at Pg = 005. Pathozone width increased monotonically from 62 mm after 2 days to a maximum of 1811 mm after 8 days (Fig. 4).

Experiment 2: effect of root colonization by P. chlamydosporia on pathozone dynamics of M. incognita

In the absence of P. chlamydosporia, data describing change in the probability of gall formation with distance between inoculum and root were similar to those

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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 tted with the model Pg (r , t ) = 1(1 exp(2(t 3))) exp (1 + (2 exp( 3t 4 ))r 2).

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): Pg (r , t ) = 1(1 exp(2(t 3))) exp (1 + (2 exp( 3t 4 ))r 2). in the presence (+Pc) or absence (Pc) of Pochonia chlamydosporia Treatment Parameter 1 2 3 1 2 3 4 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. Pc ( SE) 05769 011 05020 045 12350 085 00061 0001 641 (Fixed) 17100 122 11100 062 +Pc ( SE) 06123 005 22100 248 18540 018 00068 0001 641 (Fixed) 27900 115 06780 034

obtained from experiment 1. After 2 days incubation, the probability of gall formation when inoculum was positioned at the root surface was 02 and decayed to zero when inoculum was placed at a distance of 5 mm. However, in contrast to the rst experiment, after 7 days incubation the maximum probability of gall formation Pg = 068 occurred from inoculum placed 5 mm from the root surface and declined to zero at a distance of 20 mm. In the presence of P. chlamydosporia, the probability of gall formation was marginally increased during the rst 5 days when inoculum was positioned at or 5 mm from the root surface and marginally decreased when placed

10 mm from the root surface. After 8 days, the probability of gall formation in the presence of P. chlamydosporia was slightly higher when inoculum was placed 15 mm from the root surface, but lower when it was placed at a distance of 5 or 10 mm. Using the pathozone model (Eqn 4) to describe surfaces for change in the pathozone over time (Table 2, Fig. 5), no signicant effect of P. chlamydosporia was detected on the pathozone dynamics of M. incognita. The pathozone increased monotonically from 28 mm after 2 days to a maximum of 20 mm after 8 days in the absence of the fungal antagonist and from 395 mm after 2 days to a maximum of 192 mm in the presence of the fungal antagonist.
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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 tted with the model Pg (r , t ) = 1(1 exp(2(t 3))) exp (1 + (2 exp( 3t 4 ))r 2).

Eight days after inoculation with the nematode, a total of 55 galls had been produced on roots in the presence and 52 galls in the absence of P. chlamydosporia. The probability of gall formation rose from zero, at the point of inoculation with the nematode, to a maximum of 033, 1620 mm below the height of inoculation. The probability then decreased again to zero, 45 mm below the height
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of inoculation (Fig. 6). A gamma distribution described the change in the probability of gall formation with distance, x, from the height of inoculation with the nematode. No signicant difference was detected for distributions describing change in the probability of gall formation with distance from the point of inoculation.

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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).

Discussion
The pathozone dynamics of M. incognita on single roots of tomato plants were quantied. Despite the potential of nematodes to spread by several cm each day, the furthest extent of the pathozone in the absence of P. chlamydosporia was estimated to be between 181 mm (experiment 1) and 200 mm (experiment 2). Others estimates of the furthest 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 a root system by individuals from a large population of nematodes. The measurements in the present study estimated the probability of infection of a single root and showed that, although infection from distance is possible, the probability of infection decays very rapidly as the distance between inoculum and root increases. The reason for this may result from a combination of three factors: (i) the concentration of nematodes decreases rapidly as they move away from the site of inoculation, a process which

is most likely related to simple diffusion or a random walk (Feltham et al., 2002); (ii) the life expectancy of nematodes in this system was less than 10 days (results not shown); and (iii) the susceptible portion of the root does not include the entire root, but may well be restricted to the zone of elongation just behind the root tip. This means that, whilst the nematodes in this system may be capable of travelling signicantly greater distances than denoted by 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 susceptible portion of a target root during the course of their migration. Observations were not made on other roots to determine if they had been invaded elsewhere. The variability between reported estimates of pathozone width undoubtedly results from differences in the inoculum source, the host target and the soil environment through which the nematodes migrate. For example, some studies involved inoculum composed of over 300 juvenile nematodes (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 included larger or multiple targets for infection (Bird, 1959; Johnson & McKeen, 1973; Prot & Netscher, 1979; Stephan & Estey, 1982; Pinkerton et al., 1987), thereby increasing the probability of infection. Moreover, for studies involving incubation periods of over 50 days (e.g. Wallace, 1963; Johnson & McKeen, 1973; Stephan & Estey, 1982) resulting in estimates for infection over large distances, secondary, root-to-root, infection cannot be discounted. 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 the pathozone used sand-based soil packs combined with a highly controlled environment providing relatively homogeneous and repeatable growth conditions for the plant, nematode and fungus. This had the advantage of reproducibility within (between replicates) and between experiments, and forms the mechanistic platform from which variability in other factors (inoculum size, host number, soil type, the presence of an antagonist, etc.) can be investigated. Equation 4 accurately described the pathozone dynamics for M. incognita. Changes in the probability of infection for inoculum placed at the root surface, , increased monotonically over time and the rate at which the probability of infection decreased with distance, , decreased exponentially over time. This is consistent with a prole (probability of infection with distance) that increases disproportionately more over time for inoculum placed at the root surface than for inoculum placed away from the root surface. It suggests that nematodes located on the root surface remain more efcient at infecting the root and forming galls and may be related to a more favourable balance between energies invested in foraging versus infection.
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By inoculating P. chlamydosporia directly onto the root surface, and despite the presence of actively growing fungus for the duration of the experiment, no signicant effects of fungal presence on pathozone dynamics (parameters 1, 2, 3, 1 and 2 of Eqn 4) of M. incognita were detected. Either the fungus had no direct effect on infection of the nematode or the nematode was able to avoid the fungus by infecting at another location. Yet, distributions describing change in the probability of infection along the root in the presence and absence of P. chlamydosporia were identical and there was no signicant effect of the fungus on the site of gall formation. Since P. chlamydosporia is a poor saprotroph with a slow rate of mycelial growth (Kerry, 1989), the nematode probably enters the root ahead of extensive colonization by the fungus and the fungus is incapable of providing control during this phase of the nematode infection cycle. Therefore, the role of P. chlamydosporia in the biological control of M. incognita presumably depends on the reduction of secondary inoculum through the parasitism of 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 descriptive models for the complex interactions between pest species and their microbial natural enemies. Scaling up from small experimental plots to eld crops will increase the variation in the system and require such knowledge as presented here to identify the key factors that affect the efcacy of biological control and so help provide consistent and practical levels of nematode management. This paper begins to address this need.

Acknowledgements
This work was funded by the award of a Research Studentship from the Biotechnology and Biological Sciences Research Council (BBSRC), which we gratefully acknowledge. Rothamsted Research receives grant-aided support from the BBSRC and CAG also acknowledges BBSRC support. We are also grateful to Dr D. Hall for technical assistance during the experiments. The work was conducted in accordance with Defra Plant Health Licence No. 174A/4485.

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