Annals of Applied Biology ISSN 0003-4746

RESEARCH ARTICLE

Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites of root-knot and potato cyst nematodes
1 , I. Esteves2 , S.J. Powers3 & B.R. Kerry1 R.H. Manzanilla-Lopez

1 Plant Pathology and Microbiology Department, Rothamsted Research, Harpenden, Hertfordshire, UK 2 Departamento of Life Sciences, Faculty of Sciences and Technology, IMAR-CMA, Coimbra, Portugal 3 Statistics Unit, Biomathematics and Bioinformatics Department, Rothamsted Research, Harpenden, Hertfordshire, UK

Keywords Endophytes; Meloidogyne incognita; Monographella cucumerina; oilseed rape; Paecilomyces lilacinus; rhizodeposits; sugarbeet; wheat. Correspondence R.H. Manzanilla-Lopez, Plant Pathology and Microbiology Department, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK. Email: rosa.manzanilla-lopez@bbsrc.ac.uk Received: 2 November 2010; revised version accepted: 5 April 2011. doi:10.1111/j.1744-7348.2011.00479.x

Abstract
The effects of a host plant on reproduction/abundance of fungal populations in relation to soil nutrients released by plants in the rhizosphere were studied. Abundance in the soil and potato rhizosphere of the fungi Paecilomyces lilacinus, Monographella cucumerina (CABI 380408) and Pochonia chlamydosporia var. chlamydosporia (Pc280, potato cyst nematode biotype) and P. chlamydosporia var. catenulata (Pc392, root-knot nematode biotype) were assessed. The different ability of break crops (oilseed rape, sugarbeet and wheat) in the potato rotation to support Pa. lilacinus, Pochonia isolates Pc280 and Pc392 and abundance of the latter two isolates in soil and rhizosphere of potato plants infected with Meloidogyne incognita were also studied. Potato chits and crop seedlings were planted into boiling tubes containing 5000 chlamydospores or conidia g1 in acid washed sand (pH 6) and kept in a growth chamber at 20 C, and 16 h of light for up to 9 weeks. The abundance of the fungi in sand (fallow) differed signicantly between fungal species, being in general less abundant in the absence than in the presence of the plant, although there was no interaction between plant species and fungal isolate. There was evidence of a different response to Me. incognita for Pc392 than for Pc280 but there was no signicant effect of the presence of the nematode on the rate of increase of the fungus.

Introduction
The fungus P. chlamydosporia (Goddard) Gams & Zare (Clavicipitaceae) occurs saprophytically in soils and the rhizosphere. It is has been reported as a parasite in eggs of various invertebrates such as molluscs (Zare et al., 2001), helminths (Araujo et al., 2009a,b) and both animal and plant-parasitic nematodes (Braga et al., 2010; Frassy et al., 2010). Pochonia is a potential biological control agent of plant endoparasitic nematodes of the genera Meloidogyne spp. [root-knot nematodes (RKNs)], Nacobbus spp. (false RKNs) and Globodera and Heterodera spp. (cyst nematodes). Globodera rostochiensis (Wollenweber) and Globodera pallida Stone, commonly known as potato cyst nematodes (PCN), are important pests in commercial potato production in the UK (Atkins et al., 2003; Tobin et al., 2008). Both PCN species multiply only on
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solanaceous crops and weeds; hence, keeping soil free of them for a number of years leads to a decline in nematode populations (Whitehead & Turner, 1998). Integrated pest management (IPM) for PCN includes the use of resistant cultivars and nematicides in addition to crop rotation, although the latter is not always effective or economically viable. As a result, there is a need for effective novel control strategies that can be included in an IPM framework. The use of biological control agents such as nematophagous fungi is a potential strategy to control these pests (Kerry et al., 1993; Jacobs et al., 2003; Tobin et al., 2008). However, the potential success of such a biological control agent should be based on the careful selection and combination of the fungal isolate biotype (i.e. from the original nematode host) and host plant to be included as break crops in the potato crop rotation.
Ann Appl Biol 159 (2011) 118129 2011 Rothamsted Research Ltd Annals of Applied Biology 2011 Association of Applied Biologists

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Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites

Pochonia spp. are facultative parasites of nematode eggs. Recent studies have shown that some Pochonia species have an endophytic behaviour, which is a more intimate relationship with the plant than just the saprophytic behaviour so far attributed to the fungus in the rhizosphere, and that this may be benecial to the host plants defence against other soil-borne pathogens (Bordallo et al., 2002; Lopez-Llorca et al., 2002; Macia-Vicente et al., 2009). It has been hypothesised that a change or switch from the saprophytic to the parasitic phase of the fungus may be related to nutrients released by the plant into the rhizosphere. Plant root exudation, or rhizodeposition, inuences plant growth, resistance to pests, benecial symbioses, pathogen infection and soil ecology in the rhizosphere via organic inputs and depletion of large supplies of inorganic compounds. Rhizodeposits are primarily composed of carbon-containing compounds derived from photosynthetic products such as small molecules (e.g. organic acids, amino acids, sugars), secretions (enzymes), lysates, mucilage and quantities of NO3 and NH4 + (Bertin et al., 2003; Singh et al., 2004; Wichern et al., 2008). Plant species differ in their root exudates and rhizodeposits, as well as in their ability to support P. chlamydosporia growth in their rhizosphere and in their susceptibility to infection by RKN (Kerry, 2000). A potential bio-management strategy for nematode control incorporates the use of P. chlamydosporia in combination with selected cultivars of host plants (e.g. break crops), which are less susceptible or resistant to the nematode and that support extensive growth of the fungus in their rhizosphere (Bourne et al., 1996; Bourne & Kerry, 1999). The colonization of the root surface is closely linked to egg mass production and changes in root exudation induced by the nematodes (Bourne & Kerry, 1999). Hypothetically, the fungi should translocate nutrients (including carbon and nitrogen) across the mycelial network as far as efciency allows, and low numbers of nematode eggs will maintain the fungi in the parasitic, rather than the saprophytic, phase. Pochonia chlamydosporia (= Verticillium chlamydosporium) is one of the most important parasites responsible for the natural control of both cereal and beet cyst nematodes with precropping applications of the fungus surviving long enough to kill nematode eggs and females that develop on roots of spring-sown crops (Kerry et al., 1993). Other PCN nematophagous fungi include Paecilomyces lilacinus (Thom) Samson, 1974 and Monographella cucumerina (Lindf.) Arx, 1984 (= Plectosphaerella cucumerina). The impact of plant root exudates and rhizodeposition on the parasitic activity of these two species is unknown. Pa. lilacinus has been routinely isolated from infected plant-parasitic nematode eggs and is one of the most
Ann Appl Biol 159 (2011) 118129 2011 Rothamsted Research Ltd Annals of Applied Biology 2011 Association of Applied Biologists

widely tested fungi for the control of root-knot and cyst nematodes (Atkins et al., 2005). M. cucumerina has been isolated from RKN and PCN nematodes (Atkins et al., 2003) and the efcacy of the three fungi has been tested for controlling PCN as part of an IPM regime by Jacobs et al. (2003). Therefore, the objectives of the present study were: (a) to assess if nutrients released in the potato rhizosphere will increase abundance of the three PCN nematophagous species: P. chlamydosporia, including isolates of two Pochonia varieties, viz. P. chlamydosporia var. chlamydosporia (Pc280, PCN biotype) and P. chlamydosporia var. catenulata (Pc392, RKN biotype), Pa. lilacinus and M. cucumerina (isolate CABI 380408), (b) to ascertain if break crops in the potato rotation (oilseed rape, sugarbeet and wheat) differ in their ability to support selected fungal isolates and if P. chlamydosporia occurs as an endophyte within the roots of these crops; and (c) to assess if nematode infection by Me. incognita (Kofoid & White, 1919) Chitwood, 1949 in potato plants provides P. chlamydosporia isolates with nutrients (different from those obtained from the host plant alone) that may enhance its reproduction and colonization of soil and rhizosphere.

Materials and methods

Fungal isolates from nematodes Pochonia chlamydosporia var. chlamydosporia isolate Pc280 (PCN biotype) and P. chlamydosporia var. catenulata isolate Pc392 (RKN biotype) were obtained from the Rothamsted culture collection. The original host for isolate Pc280 (Jersey, UK) is a Globodera sp. and for the Cuban isolate Pc392, a Meloidogyne sp. Paecilomyces lilacinus (labelled as isolate PL LINK) was used as a spore formulated wettable powder and prepared according to the manufacturers instructions (Biological Control Products SA, South Africa). The product, as supplied by the manufacturer, had a concentration of 4 109 spore g1 . M. cucumerina isolate CABI 380408 was obtained from CABI, UK. The original host for this isolate was PCN from Jersey, UK (Atkins et al., 2003). Production of inoculum (conidia and chlamydospores) The different fungi and isolates were grown in selective agar cultures as follows. Paecilomyces lilacinus A measure of 39 g of PDA (Oxoid, Basingstoke, UK), 10 g of sodium chloride and 28 mg pentachlornitrobendazole 99% (PCNB, Sigma-Aldrich, Milwaukee, MI, USA) were added to 800 mL of distilled water and
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autoclaved. Antibiotics included 50 mg of chlortetracycline hydrochloride (Sigma-Aldrich), 100 mg of streptomycin sulphate (Sigma-Aldrich) that were dissolved in 200 mL of tepid sterile distilled water (sdw) and 1 mL of tergitol type NP-10 (Sigma-Aldrich) prior to being added to the 800 mL of autoclaved agar. Monographella cucumerina A measure of 39 g of PDA (Oxoid), 10 g of sodium chloride and 37.5 mg PCNB were added to 800 mL of distilled water and autoclaved. Fifty microgram of chlortetracycline hydrochloride (Sigma-Aldrich), 100 mg of streptomycin sulphate (Sigma-Aldrich), 37.5 mg thiabendazole (2-[4-thiazolyl]benzimidazole) (Sigma-Aldrich) and 37.5 mg carbendazim 97% (Sigma-Aldrich) were dissolved in 200 mL of tepid sdw and 1 mL of tergitol type NP-10 before adding to 800 mL of autoclaved agar. Pochonia chlamydosporia selective agar, potato dextrose agar (PDA) and corn meal agar (CMA) were prepared according to Kerry & Bourne (2002). PDA medium was prepared for conidia production and CMA for chlamydospores. Mass production of chlamydospores was made using a rice culture. Rice culture Pochonia chlamydosporia isolates Pc280 and Pc392 were cultured and incubated at 25 C on rice substrate to produce chlamydospores (Kerry & Bourne, 2002; HidalgoD az, 2003). Twenty-ve days after inoculation, the rice containing the chlamydospores was tipped from the ask onto a sieve (250 m mesh pore) and rinsed with a jet of water to collect the substrate and chlamydospores onto a second sieve (10 m mesh pore). The sieve was blotted underneath with a sponge and chlamydospores were collected from the top surface of the mesh with a spatula. Chlamydospores were then mixed with ne sand (low iron; Fisher Scientic, Loughborough, UK) in a 10:1 w:w ratio (sand:chlamydospores). One gram of inoculum was added to 9 mL of water agar (0.05%) and thoroughly mixed before chlamydospores were counted using a haemocytometer (Marienfeld, Germany) and dilutions were made to produce a nal concentration of 5 103 chlamydospores mL1 . Chlamydospore viability and germination percentage were evaluated on sorbose agar with antibiotics (Esteves, 2007). Meloidogyne incognita culture Egg masses used in the experiments were taken from tomato plants infested with Me. incognita. Nematode cultures were started from a single egg mass and had been kept in the glasshouse of Rothamsted Research for at least 5 years.
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Inoculum preparation A 20-m pore sieve was rinsed with 70% ethanol and UV irradiated in a ow cabinet for 20 min. Conidia from M. cucumerina and Pa. lilacinus were harvested separately from selective agar cultures grown in Petri dishes. Each Petri dish was ooded with 5 mL of sdw and the mycelium was gently scraped using a sterile L-shaped glass rod. The conidia suspension was poured onto the sieve mesh and 10 mL of sdw were added into the sieve; the conidia suspension was then collected from a Petri dish placed underneath. Conidia were counted under the microscope using a haemocytometer and adjusted to a nal spore concentration of 5 103 mL1 . Experiment 1: fungal abundance (CFU) in potato rhizosphere and acid washed sand Quantication of P. chlamydosporia is difcult because of the fact that the different life stages are neither composed of approximately the same size units nor have the same genetic contents (Mauchline et al., 2002). Although quantitative PCR methods are increasingly used to quantify the fungus in soil (Mauchline et al., 2002; Atkins & Clark, 2004) and roots (Macia-Vicente et al., 2009), correlation, for example, between colony-forming unit (CFU) counts expressed as grams per dry weight to their equivalent DNA quantities is still difcult because of various factors including variable yields of DNA from samples and amplication of DNA from fungal moribund material that can give misleading results (Mauchline et al., 2002; Manzanilla-Lopez et al., 2009). The growth stage of the fungus (e.g. DNA replication, hyphal growth, sporulation) and root galling can also affect the number of gene copies detected by PCR (Mauchline et al., 2002). Considering that both methods can work up well to their theoretical limits in a sterile system (Mauchline et al., 2002), we measured fungal abundance using the classic approach of plate counting (CFU) that measures the abundance of viable propagules of the fungus. The abundance of two P. chlamydosporia isolates: Pc280 (P. chlamydosporia var. chlamydosporia), Pc 392 (P. chlamydosporia var. catenulata), and single isolates of Pa. lilacinus and M. cucumerina was compared when the carbon and nitrogen source for fungal growth was only provided through the root system of potato plants. To eliminate macro and micronutrients, 20 kg of acid washed coarse sand was saturated overnight with 1 M hydrochloric acid in a plastic container. Acid was washed away and the coarse sand was thoroughly rinsed with distilled water until pH 6 was reached. To prepare each experimental unit, 170 g of acid washed coarse sand was weighed and placed in a reclosable polythene plastic bag (180 200 mm) to which 5 mL of sdw was added
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to humidify the coarse sand before adding the chlamydospores (5 103 g1 coarse sand) of P. chlamydosporia. Coarse sand and chlamydospores were thoroughly mixed in the bag and then transferred into each experimental unit (20-cm long 4-cm diameter boiling tube). A similar procedure was followed for Pa. lilacinus and M. cucumerina except that inoculum for each fungus was added as 5 mL of spore suspension (5 103 conidia g1 coarse sand). Potato chits (cv. Cara) were planted in boiling tubes containing the inoculated sand, wrapped in aluminium foil and kept in a growth chamber for 4 weeks at 20 C and 16 h of light (300 mol m2 s1 ) per 24 h. Controls consisted of boiling tubes only containing fungus-inoculated coarse sand that were sealed with Paralm and watered regularly to keep them moist (Fig. 1). Boiling tubes containing the plants were watered daily. As soon as potato shoots had emerged, they were manually sprayed twice a day with foliar fertilizer (Phostrogen, pbi Home & Garden Limited, Hertfordshire, UK) prepared according to the manufacturers instructions, care being taken to avoid leakage to the coarse sand. Four weeks later, the fresh shoots and root system of each plant were measured and weighed. Fungal populations from sand and roots (i.e. rhizosphere) were isolated and 102 and 103 dilutions were prepared and plated in selective agar to count CFU (Kerry & Bourne, 2002) in triplicate from each boiling tube. The CFU g1 coarse sand values were corrected to the dry soil weight (Kerry & Bourne, 2002) according to weight differences between dry and wet sand obtained from 1 g of coarse sand taken per each experimental unit (i.e. boiling tubes). The experiment was laid out as a randomised block design with four blocks. There was a total of 10 treatments comprising a ve by two factorial set: four fungi and fallow (coarse sand) by two situations (plant or no plant). There were four replicates per treatment (a total of 40 experimental units). The analysis of CFU g1 coarse sand and g1 root was made using ANOVA with a square root transformation (to account for heterogeneity of variance across the treatments) using GenStat Release 8.2 (VSN international Ltd, Hemel Hempstead, UK). A stronger, natural log, transformation was used for the CFU g1 roots. Following ANOVA, biologically relevant comparisons of means were made using the least signicant difference (LSD) at the P = 0.05 level of signicance. Root and shoot variables were analysed similarly, but did not require transformation. Experiment 2: the ability of break crops to support selected fungal isolates On the basis of results obtained from Experiment 1, P. chlamydosporia isolates Pc392 and Pc280 as well as Pa. lilacinus were selected to be used in the second
Ann Appl Biol 159 (2011) 118129 2011 Rothamsted Research Ltd Annals of Applied Biology 2011 Association of Applied Biologists

Figure 1 Boiling tubes lled with inoculated sand-grit, 4-week-old potato plants (left) and 4-week-old wheat plants (right).

experiment. Methods were similar to those described in Experiment 1. Modications included the use of acid washed sand-grit mix (1:1 w/w) instead of coarse sand. After the acid washed sand-grit had been inoculated with each fungus, 1 g of sand-grit was taken at random from 20 experimental units to assess initial CFU counts (T1). Along with potato (Solanum tuberosum cv. Maris Piper), for Experiment 2, spring cultivars of oilseed rape (Brassica napus cv. Heros), sugarbeet (Beta vulgaris cv. Dominica) and wheat (Triticum aestivum cv. Paragon) were included as break crops. Seeds were surface sterilised with commercial bleach (0.5%) and germinated at 25 C in Petri dishes containing nutritive agar [10 g L1 glucose
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(Sigma-Aldrich), 0.1 g L1 yeast extract (Merck, Darmstadt, Germany), 0.1 g L1 peptone (Sigma-Aldrich) and 12 g L1 technical agar (Oxoid)] to ensure that they were free from pathogens (Kerry et al., 1984). To synchronise plant development, seeds of the different species were germinated at 25 C for different lengths of time to provide seedlings with similar root length (3 cm long). Seedlings were taken from Petri dishes and planted into boiling tubes lled with sand-grit (170 g) previously inoculated with each fungus, and kept in a growth chamber as described for Experiment 1. Four weeks later, plants were removed from the boiling tubes and sand-grit was carefully removed from the roots. Shoots and root systems were measured and weighed and CFU were counted as in Experiment 1. The experiment was laid out as a randomised block design with three blocks. Treatments comprised the ve crops (including fallow) with each of the three fungi and control. There were three replicates per treatment, giving a total of 60 experimental units (i.e. boiling tubes). Data for CFU g1 sand-grit (i.e. soil) and CFU g1 root were recorded at the end of the experiment (T2) and analysed, along with plant variables, as described in Experiment 1. Root staining At the end of Experiment 2, sand-grit was removed from roots and rinsed in sdw. Roots were cut into 1-cm-long segments. Roots per sample were wrapped in an 11 11 cm piece of nylon voile, secured with a wire and plunged into a beaker containing a boiling solution of lactophenolethanol (1:2 v/v; Fisons and Fisher Scientic, Loughborough, UK) for 1015 min and left overnight in a hooded cabinet at room temperature. Afterwards, roots were transferred into another beaker containing Trypan blue (BDH Stain, Poole, UK) lactophenol (0.05%), stained for 45 min at 60 C and left for 24 h in a hooded cabinet (Menendez et al., 1997). Samples were then rinsed in sdw and left in water-glycerin (BDH, AnalaR; 1:1 v/v) within the hooded cabinet for 1 week to allow phenol evaporation. The nylon voile was then removed and the stained roots were rinsed in sdw, mounted in water-glycerin onto glass slides, covered with a cover glass (22 50 mm) and sealed with nail polish. Slides were examined for root endophytes under the microscope (Zeiss Axiophot, Carl Zeiss, Welwyn Garden City, UK) at 20, 40 and 63 magnication. Experiment 3: effect of nematode parasitism On the basis of CFU counts from Experiments 1 and 2, for isolates Pc280 and Pc392, a third experiment was carried out to assess the effect of nematode parasitism on fungal
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abundance. Potato chits (cv. Maris Piper) were planted in acid washed sand-grit (pH 6) contained in Sterilin (Sterilin Ltd, Aberbargoed, UK) skirted centrifuge tubes (50 mL vol., blue lid) and placed in a growth chamber for 1 week to allow roots to develop. Second-stage juveniles (J2) of Me. incognita were surface disinfected in 0.1% Malachite green (Sigma-Aldrich) and 0.1% streptomycin sulphate (Hooper, 1986). Each potato plant was inoculated with 1000 J2 and returned to the growth chamber. Ten days after J2 inoculation, plants were removed from the Sterilin tubes and roots were rinsed carefully to wash out those J2 that had not penetrated the roots. Plants were transplanted into sand-grit inoculated with chlamydospores as described before (Experiment 2) and returned to the growth chamber for another 6 weeks. One gram of sand was taken at random from 18 experimental units to assess initial CFU counts (T1). The experimental design was a randomised block with ve blocks. There were eight treatments comprising a three by two factorial set, being the two fungi and a control (no fungus) each with or without nematodes in the presence of potato, plus two further control treatments for the fungi in the absence of nematodes and potato. There were ve replicates of each treatment. Root and plant shoot lengths were recorded as well as CFU g1 sand-grit and CFU g1 root. Number of root galls and egg masses were also recorded. Egg masses were hand-picked with ne forceps under a stereo microscope and gently macerated in 2 mL of sdw contained in a sterile glass homogeniser (Fisher Scientic). Eggs were then plated in Petri dishes containing 0.08% water agar with antibiotics (Atkins et al., 2003; Esteves, 2007). Plates were incubated for 3 days at 25 C and the percentage of infected eggs was assessed. The percentage of eggs parasitised (P%) by the fungus was logit transformed, including an adjustment to account for zero recordings [log10 ((P% + 1)/(101 P%)], for ANOVA. Other variables recorded were analysed as for the previous experiments. Pearson correlations were calculated between the different variables.

Results
Experiment 1 In this experiment, the proliferation of the different fungi in the presence or absence of a potato plant is considered. CFU data obtained from coarse sand and rhizosphere in order to assess the effect of the crop plant on the abundance of the fungal species revealed signicant (P < 0.001, F -test) main effects and interaction between absence/presence of a plant and the fungal species. The fungi reacted differently to the addition of a potato plant, M. cucumerina and P. chlamydosporia var. catenulata (Pc392)
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Table 1 Experiment 1: means of CFU g1 acid washed sand and CFU g1 roots from potato rhizosphere for Monographella cucumerina, Paecilomyces lilacinus, Pochonia chlamydosporia var. chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392)a Fungi Treatment No. potato (sand only) Potato and sand Potato rhizosphere
a Mean

Fallow (control) 0.0 0.0 0.0

M. cucumerina 0.0 15.0 3.2

Pa. lilacinus 193.4 185.2 13.5

P. chlamydosporia var. chlamydosporia (Pc280) 67.5 67.9 14.5

P. chlamydosporia var. catenulata (Pc392) 103.2 202.8 14.4

values are square root of CFU g1 of acid washed sand LSD (P = 0.05) = 35.53, SED = 17.32, df = 27, n = 4 and CFU log (CFU roots +1) g1 roots from potato rhizosphere LSD (P = 0.05) = 5.17, SED = 2.28, df = 9, n = 12.

being most different (Table 1). With a potato plant, Pa. lilacinus, Pc280 and Pc392 CFU coarse sand counts were higher and signicantly different (P < 0.05, LSD) from M. cucumerina but Pa. lilacinus and Pc392 were not signicantly (P > 0.05, LSD) different amongst themselves (Table 1). There was a signicant difference (P < 0.05, LSD) between treatments (potato versus no potato) only for isolate Pc392. Although CFU counts increased for Pc392 in coarse sand in the presence of a potato plant, CFU counts remained at similar levels for Pc280 regardless of presence/absence (Table 1). For the natural log of CFU counts for roots there was a signicant difference (P = 0.003, F -test) between isolates: Pa. lilacinus, Pc280 and Pc392 were signicantly different (P < 0.05, LSD) from M. cucumerina with higher number of CFU counts but were not signicantly different (P > 0.05, LSD) amongst themselves. There were no statistical differences (P > 0.05, F -test) between treatments for plants shoot and root variables (data not shown). Experiment 2 In this experiment, the effect of different break crops on the proliferation of the fungi is considered. First, in order to resolve if Pc280 levels remained much the same

because of survival of chlamydospores rather than minor increments of the isolate, CFU counts for both isolates were assessed at planting (T1) and at the end (T2) of the second experiment. At planting (T1) there was a signicant difference (P < 0.001, F -test) in square root CFU g1 of acid washed sand-grit between isolates, Pc280 being signicantly different from the other fungi (P < 0.05, LSD) but there was no signicant difference between Pa. lilacinus and Pc392 (P > 0.05, LSD). Signicant differences occurred between fungi (P < 0.001, F -test) for nal square root CFU g1 acid washed sand-grit at T2, but there was no signicant difference due to crops (P = 0.988, F -test) or due to an interaction between crops and fungus (P = 0.180, F -test). For the square root of CFU counts for roots there was a signicant interaction between crops and fungi (P = 0.003, F -test) with a strong main effect of fungus (P < 0.001, F -test) but not of crops (P = 0.460, F -test). Investigating this interaction, there was a strong effect of Pa. lilacinus for oilseed rape and sugarbeet and this fungus gave the highest CFU value in the potato rhizosphere. The isolate Pc392 was not assessed on sugarbeet because plants died before the experiment was completed, but this isolate produced the largest CFU counts in the wheat rhizosphere (Table 2).

Table 2 Experiment 2: means of CFU g1 acid washed sand-grit and g1 root of Pochonia chlamydosporia var. chlamydosporia (Pc280), P. chlamydosporia var. catenulata (Pc392) and Paecilomyces lilacinus from break crops at initial assessment (planting, T1) and at nal assessment (4 weeks after planting, T2)a Mean for isolates (T2) Root 0.0 957 292 1488 Sand-grit 1.2 407.5 163.1 432.5

T1 (n) Fungus Control Pa. lilacinus Pc280 Pc392

a Means

Fallow (T2) Sand-grit 0.0 382.7 152.7 488.7 Root 0.0 0.0 0.0 0.0

Oilseed rape (T2) Sand-grit 0.0 410.9 179.5 435.1 Root 0.0 2152 372 574

Potato (T2) Sand-grit 6.1b 441.2 155.7 420.2 Root 0.0 669.0 570.0 573.0

Sugarbeet (T2) Sand-grit 0.0 386.4 162.5 438.4 Root 0 1916 261 ND

Wheat (T2) Sand-grit 0.0 416.5 165.3 380.2

Sand-grit 0.0 (18) 30.2 (15) 84.2 (15) 39.5 (9)

are square root values of CFU g1 acid washed sand-grit and g1 root. T1: CFU sand-grit: LSD (Pa. lilacinus versus Pc280, P = 0.05) = 16.74, SED = 7.84, df = 15; LSD (Pa. lilacinus versus Pc392 or Pc280 versus Pc392, P = 0.05) = 19.33, df = 15. T2: CFU sand-grit LSD (only for comparison of means for isolates, in bold, P = 0.05) = 32.30, SED = 15.95, df = 38, n = 45; CFU roots (for all comparisons) LSD (P = 0.05) = 897.70, df = 38, n = 9; ND = not determined because of plants having died. b Possible contamination.

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Table 3 Experiment 2: means of root length (cm) for combinations of isolates of Pochonia chlamydosporia var. chlamydosporia (Pc280), P. chlamydosporia var. catenulata (Pc392) and Paecilomyces lilacinus with crops Oilseed rape Root length Control (no fungus) Pa. lilacinus Pc280 Pc392 Means for crops
a Shoot b

Potato Shoot length 4.2a 4.6 8.3 4.6 5.4 Root length 30.5 26.8 26.3 28.2 27.8 Shoot length 7.7 7.9 6.9 7.2 7.4

Sugarbeet Root length 4.8 15.8 21.2 17.4 14.8 Shoot length 2.1 3.2 3.7 2.5 2.9

Wheat Root length 43.2 31.2 56.2 40.0 42.6 Shoot length 14.7 17.2 13.7 14.7 15.1

Means for isolates Root length 25.2b 23.5 32.4 26.8 Shoot length 7.2 8.2 8.2 7.2

22.5 20.7 25.8 21.6 22.6b

length: LSD (comparisons between two-way table of means, P = 0.05) = 2.98; SED = 1.45, df = 25, n = 3. Root length: LSD (comparison of means for crops or for isolates only, in bold, P = 0.05) = 6.53; SED = 3.17, df = 26, n = 12.

There was a main effect of crop (P < 0.001, F -test) and fungi (P = 0.030, F -test) on root length (Table 3). Isolates Pc280 and Pc392 were associated with the longest roots, but root length for Pc280 and Pa. lilacinus was not significantly different from the control (P > 0.05, LSD). Only root length for isolate Pc280 was signicantly different from the control (P < 0.05, LSD). Overall, there was little real effect on root length due to the presence of fungus. Following a marginally signicant main effect of fungus (P = 0.029, F -test) for root weight, there were, however, no signicant differences (P > 0.05, LSD) in root weight (data not shown) when comparing fungi to control. Finally, for shoot length, a marginally signicant interaction was found to occur between fungal isolates and crops (P = 0.036, F -test). In particular, isolate Pc280 was associated with greater shoot length for oilseed rape, and Pa. lilacinus was associated with greater shoot length for wheat, than the other two fungi and the control (Table 3). Fungus endophytic behaviour Chlamydospores were observed on the surface of the roots of all crops. However, microscopical observations of the endophytic root behaviour of the two isolates of Pochonia were made only for potato and wheat (a total of 12 root samples plus controls because of the poor growth of plants from the other crops). Hyphae of the fungus were found on the rhizoplane of both crops, often associated with chlamydospores as well as intercellular hyphae (Fig. 2), and forming steps along cell walls, as reported for barley (Hordeum vulgare) by different authors (Bordallo et al., 2002; Lopez-Llorca et al., 2002; Montfort et al., 2005). Conidia and conidiophores were also observed in epidermal cells. Experiment 3 Here the effect of crop nematode parasitism on fungal abundance is investigated. The ANOVA of the square
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root of sand-grit CFU at T1, showed a statistically marginal effect of fungus (P = 0.056, F -test), with a higher inoculum level for isolate Pc280 (82.5) on the inoculated sand-grit in comparison with isolate Pc392 (36.1) and control treatments (0.0) [LSD (P = 0.05) = 62.94, SED = 19.78, df = 3, n = 6]. Final sand-grit CFU counts (T2) showed that, despite the difference in CFU at T1, isolate Pc392 reached greater CFU numbers than isolate Pc280 (Table 4). ANOVA of the square root of sand-grit CFU at T2, partitioning the various sources of variation, showed a signicant difference between the two isolates in the absence of potato (P < 0.001, F -test), a signicant effect of fungus overall (P < 0.001, F -test), a weak effect of the presence of nematodes (P = 0.089, F -test), and a weak difference between the two fungi (P = 0.073, F -test). Most importantly, there was a signicant interaction between fungus and presence of Me. incognita (P = 0.005, F -test) having accounted for the control treatments without potato, so the presence of the nematode affected the two isolates in different ways. For Pc280 no difference was found in presence/absence of nematodes (132.6 vs 144.5). For Pc392 a difference was found (198.6 vs 128.7; Table 4). On roots, the presence of the nematode was associated with lower square root CFU g1 for both isolates, with mean values of 406 for Pc392 and 212.5 for Pc280, in comparison with 467.6 (Pc392) and 272.9 (Pc280) in the absence of the nematode [LSD (P = 0.05) = 75.52, SED = 36.20, df = 20]. A similar result was obtained for Pc280 CFU in sand-grit, but a contrary result was obtained for Pc392, which had higher CFU in sand-grit in the presence than in the absence of the nematode (Table 4). Longer roots and shoots were produced by potato plants in the presence of the nematode (Table 5). On average, the highest number of galls (64.2 17.87, n = 5) and egg masses (29 10.23, n = 5) per root occurred in control plants (i.e. without the fungus) followed by Pc392 (60 16.11 galls and 28.6 7.25 egg masses, n = 5) and Pc280 (57.4 23.45 galls and 28.4 12.97 egg masses,
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Figure 2 Micrographs of Pochonia chlamydosporia on wheat roots. (A) control without fungus, (B) Pc392 mycelium inside root cells, (C) Pc280 chlamydospores and (D) Pc392 hyphae.

Table 4 Experiment 3: means of square root CFU (sand-grit) for combinations of Pochonia chlamydosporia var. chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392) and presence (+) and absence () of Meloidogyne incognita Isolate Pc280 Pc280 Pc280 Pc392 Pc392 Pc392 Control (no fungus)
a For

Table 5 Experiment 3: means of root and shoot length (cm) in presence of Meloidogyne incognita and Pochonia chlamydosporia var. chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392) combinations Me. incognita Me. incognita Me. incognita Me. incognita (+) root () root (+) shoot () shoot Pc280 Pc392 Control (no fungus) Means
a Root

Plant + + + + +

Nematode + +

CFU 166.2a 144.5 132.6 398.8 128.7 198.6 0.0

29.5 26.0 31.2 28.9a

20.9 25.0 26.1 24.0

8.7 8.1 7.9 8.2b

7.4 7.6 6.0 7.0

comparison of means: LSD (P = 0.05) = 38.99, SED = 19.04, df = 28, n = 15.

n = 5). The number of eggs produced in each egg mass ranged between 0 and 60 (data not shown). Using only data from plants in the presence of the nematode for the two fungal isolates, there were significant (P < 0.05, F -test) negative correlations (r, Pearson,
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length comparing Me. incognita (+ versus ) means in bold over fungi (P = 0.019, F-test, LSD (P = 0.05) = 3.96, SED = 1.89, df = 9, n = 15). b Shoot length comparing Me. incognita (+ versus ) means in bold over fungi (P = 0.063, F-test, LSD (P = 0.05) = 1.3, SED = 0.62, df = 19, n = 15).

n = 15 pairs) of the sand-grit CFU with root weight, root length and shoot weight. Therefore, when the nematode was present, as CFU values in the sand-grit went up, the
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plant measures went down. Egg masses were only significantly correlated (r = 0.524, P = 0.045, n = 15, F -test) with root length. In the absence of nematodes the correlations of CFU with plant measures were not signicant (P > 0.05, F -tests). For data where egg masses and fungal infection were present, a positive correlation (r = 0.791, P = 0.020, n = 8, F -test) was found between percentage of egg infection and numbers of CFU in the root. The correlation of egg infection with CFU in the sand-grit was not signicant for the two isolates (r = 0.582, P = 0.130, n = 8, F -test) but also indicative of a positive relationship between fungus and nematode. However, care should be taken as these results are only based on eight pairs of values. CFUs incremented over time (T1 to T2) of the experiment (6 weeks) but there was no signicant effect of the presence of the nematode on the rate of increase of Pochonia (P = 0.558, F -test). However, across both Pochonia isolates, the presence of the nematode gave a higher rate of CFU acquisition (52 vs 31 square root CFU week1 , s = 39.62, df = 4), and isolate Pc392 had a higher, although not signicantly different (P = 0.209, F -test) rate of increase in CFU over time in comparison with Pc280 (76.3 vs 47.6 square root CFU week1 , s = 18.0, df = 3).

Discussion
Plants and their rhizodeposits sensu lato are an important source of C and N for soil microbiota to maintain some entomopathogenic and nematophagous fungi, in a saprophytic stage in soil (Bruck, 2010). In the present study, we have used a simple and economical approach to assess, in the absence of other source of N, C and other nutrients, except for the plant (and, later on, the nematode), the effect of different host plants on the abundance of three different species of nematophagous fungi in the sand and rhizosphere. The approach developed worked better for potato and wheat plants, which produced larger foliage surfaces earlier in their development and throughout the duration of the experiments, than sugarbeet or oilseed rape. Foliar feeding alone was not enough to sustain further development of the young plants of oilseed rape and sugarbeet. CFU data from Experiment 1 showed that the plant had an important effect in increasing abundance of the different fungal species and isolates in coarse sand and roots in comparison with fallow (i.e. no plant). Of the three fungal species tested, Pa. lilacinus was the most abundant and M. cucumerina was the least abundant in coarse sand. This result supports a previous report for the latter species as a poor competitor in an assay to control PCN that included Pa. lilacinus and P. chlamydosporia (Jacobs et al., 2003). Comparison of isolates of
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the two varieties of P. chlamydosporia, revealed that isolate Pc280 (P. chlamydosporia var. chlamydosporia) was less abundant than isolate Pc392 (P. chlamydosporia var. catenulata) despite evidence of higher Pc280 CFU initial counts related to chlamydospore germination (data not shown). CFU counts of Pc280 at the beginning and the end of the experiment remained almost at the same level (or had a negligible increment). Hence it was not affected by presence of the plant, and remained viable in the absence of plants, as has been also reported by Mauchline et al. (2002). The poor saprophytic behaviour shown by Pc280 in comparison with Pc392, agrees with Mauchline et al. (2004) who also found that Pc280 was present in similar numbers at the start and end of experiments, when applied to healthy and PCN-infested tomato plants. However, differences between colonization of soil, rhizosphere and eggs parasitism can vary between Pochonia isolates. According to Siddiqui et al. (2009), although Pc280 was a less effective soil and rhizosphere colonizer, it was the most virulent isolate on RKN and PCN eggs. There was no signicant difference in sand-grit CFU counts for Pa. lilacinus and Pc392 due to break crop species as shown by Experiment 2 but an interaction was found to occur between fungal isolates and crops. Of the crops tested, potato (Solanaceae) has been reported as a good host for the fungus, with up to 14 125 CFU g1 soil on sandy loam with potato (Bourne et al., 2004), whereas wheat (Gramineae) has been reported as a poor host (Kerry, 2000). However, the status of wheat as a poor host of Pochonia will need to be revised in view of the high CFU counts obtained on roots for Pc392 as they were more abundant in the rhizosphere of wheat, rather than that of potato. Cereals such as wheat release C and N in good quantities in their rhizodeposits and these may range between 4.3% and 56% of total plant N (Wichern et al., 2008). Such crops are more likely than other to support the fungus in higher CFU numbers in soil and rhizosphere but wheat and its residues can also inuence weeds, pests, diseases and other soil microbes because of the allelochemical compounds produced (Bertin et al., 2003; Bais et al., 2006). Of the four break crops tested, there is scant information available on the effect of sugarbeet and oilseed rape on P. chlamydosporia isolates and abundance of other nematophagous fungi. However, one study, on the inuence of green manuring on egg pathogens of Heterodera schachtii with three intercrops in crop rotation with sugarbeet, showed that the antagonistic potential of the egg pathogenic fungi was much greater in a rotation (sugarbeetwheat) than in a sugarbeet monoculture (Pyrowolakis et al., 1999). Our data showed that there was a strong effect of oilseed rape (Brassicaceae) and sugarbeet (Chenopodiaceae) on Pa. lilacinus abundance. Reports on
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the effect of brassicas on the fungus have shown both positive and negative growth results on P. chlamydosporia isolates, situations that ultimately affect both the nematode and fungus (Bourne et al., 1996, 2004). Biotypes of P. chlamydosporia from cyst- and rootknot nematodes can have important differences in their biology, host preference (at the plant and nematode level), physiology and ecology requirements (Mauchline et al., 2004; Siddiqui et al., 2009). Some isolates of P. chlamydosporia grow rapidly while others grow poorly in different soils following application (Kerry et al., 1993; Siddiqui et al., 2009). Our results showed that RKN biotype Pc392 had a higher growth rate than PCN biotype Pc280 and that this, under the experimental conditions used, may be linked to host preference as Meloidogyne spp. is the preferred host for RKN biotype Pc392 and PC280 is a poor parasite of RKN (Mauchline et al., 2002). According to Siddiqui et al. (2009) marked biotype differences in abundance in soil and CFU numbers can be generally greater in nematode-infested soils than in non-infested soils. Our results showed the opposite effect with lower CFU counts in soil in the presence of the nematode, thus giving support to the hypothesis that low levels of nematode eggs will maintain the fungi in the parasitic, rather than the saprophytic, phase. There was little correlation between CFU and the potato plant measurement data, but our results also showed that when the fungus and nematodes occurred together there was a negative effect on plant growth variables (i.e. root and shoot length), in contrast to results obtained from Experiment 2 where plants had longer roots and shoots in the presence of the fungi but without nematodes. The negative correlation of the sand-grit CFU counts of the fungus and the plant growth found in the presence of the nematode could be explained by the fungus proliferating more in the roots than in the sand-grit when there were nematode eggs in the vicinity of the rhizosphere for it to infect; however, it may also be related to the tness cost of saprophytic versus parasitic growth and virulence (Siddiqui et al., 2009). Different isolates from biotypes of RKN and PCN (such as Pc280) of P. chlamydosporia var. chlamydosporia can increase the fresh weights of the shoots and roots of potato plants to differing degrees (Siddiqui et al., 2009), a phenomenon also observed in Experiment 2. However, nematode infestation, although perhaps not affecting shoot weight, may reduce mean root biomass (Siddiqui et al., 2009). Colonisation of the root surface by the fungus is closely linked to egg mass production and is thought to be related to changes in root exudation and systemic effects on the plant because of nematode infection of the root system (Bourne & Kerry, 1999; Yeates, 1999). Mauchline et al. (2004) pointed out that differential growth of Pochonia
Ann Appl Biol 159 (2011) 118129 2011 Rothamsted Research Ltd Annals of Applied Biology 2011 Association of Applied Biologists

isolates indicated both the great variation in the ability of P. chlamydosporia isolates to use root exudates saprophytically, and the qualitative and/or quantitative difference in nutrients available in the rhizosphere of plants. Abundance may be related to C and N provided alone by the plant in rhizodeposits that are used by the fungus to support its saprophytic behaviour. In the present study, fungi were more abundant in the plant rhizosphere than in soil and, under our experimental conditions, another source of nutrients (macro/micronutrients) could have been provided through root leakage/exudates induced by the nematode whose feeding sites (i.e. giant cells) act as a metabolic sink for nutrients withdrawn from the plant (Bais et al., 2006). Attraction by the fungus to a richer source of energy (e.g. carbohydrates) such as plant rhizodeposits, nematode gelatinous matrix (i.e. glycoproteins) and nitrogen from eggs may support the hypothesis that nutrition (use of C and N) is one of the factors involved in switching from saprophytic to parasitic behaviour. The presence of Me. incognita was associated with higher CFU mean values for isolate Pc392 in the soil in comparison with isolate Pc280, but CFU differences may also be due to differential saprophytic and parasitic abilities of the two isolates of P. chlamydosporia varieties. Two of the fungal species included in our study have been reported as endophytes. Pochonia is a facultative parasite that can also behave as an endophyte with species such as P. rubescens increasing root length of barley (H . vulgare) seedlings and reducing Gaeumannomyces graminis var. tritici root colonization (Montfort et al., 2005; LopezLlorca et al., 2008). Pa. lilacinus endophytic behaviour has been reported elsewhere (Rumbos & Kiewnik, 2006) but there is no information available in the literature regarding the endophytic potential of P. chlamydosporia isolates for break crops such as oilseed rape, potato, sugarbeet and wheat. In the present study, preliminary results using light microscopy showed P. chlamydosporia var. chlamydosporia (Pc280) and P. chlamydosporia var. catenulata (Pc392) to be a root endophyte in the wheat and potato roots. This observation deserves further investigation using different approaches, including molecular (Schulz & Boyle, 2006). Potential endophytic root colonization by egg-parasitic fungi such as Pochonia may open an opportunity to infect eggs of plant endoparasitic nematodes inside the roots and also to explore new application methods of the fungus to the plant (i.e. seed) and to the soil (Lopez-Llorca et al., 2008). Bio-management strategies for control of nematodes that incorporate the use of P. chlamydosporia in combination with selected cultivars of host plants that are less susceptible or resistant to the nematode and support extensive growth of the fungus in their rhizosphere, can be improved by taking into consideration not only the
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inclusion of a poor host for the nematode, but also a host that releases rhizodeposits that could support fungal growth in the rhizosphere and an endophytic behaviour. This bio-management strategy can be combined with other IPM practices and biological control agents incorporated as part of an IPM for PCN. According to Jacobs et al. (2003), some potato growers already apply two control measures for PCN, a fumigant in the autumn followed by a granular nematicide in the spring (at a cost of approximately 900 ha1 ) and so separate applications of two biological control agents, such as Pa. lilacinus and Pochonia spp., may be feasible (Jacobs et al., 2003). Tobin et al. (2006) have also shown the potential of using P. chlamydosporia to control PCN in potato crops grown under commercial eld conditions.

Acknowledgements
Rothamsted Research is an institute of the Biotechnology and Biological Science Research Council of the UK. This project was funded by DEFRA Link Project LK0966. The authors thank Dr Penny R. Hirsch and Mr Ian Clark for the technical advice and the Bioimaging and Visual Communications Unit (Rothamsted Research) for preparing the gures.

References
Araujo J.M., Araujo J.V., Braga F.R., Carvalho R.O., Ferreira S.R. (2009a) Activity of the nematophagous fungi Pochonia chlamydosporia, Duddingtonia agrans and Monacrosporium thaumasium on egg capsules of Dipylidium caninum. Veterinary Parasitology, 166, 8689. Araujo J.M., Araujo J.V., Braga F.R., Carvalho R.O., Silva A.R., Campos A.K. (2009b) Interaction and ovicidal activity of nematophagous fungus Pochonia chlamydosporia on Taenia saginata eggs. Experimental Parasitology, 121, 338341. Atkins S.D., Clark I.M., Sosnowska D., Hirsch P.R., Kerry B.R. (2003) Detection of Plectosphaerella cucumerina, a potential biological control agent of potato cyst nematodes, by using conventional PCR, real-time PCR, selective media, and baiting. Applied and Environmental Microbiology, 69, 47884973. Atkins S.D., Clark I.M. (2004) Fungal molecular diagnostics: a mini review. Journal of Applied Genetics, 45, 315. Atkins S.D., Clark I.M., Hirsch P.R., Kerry B.R. (2005) The use of real-time and species specic primers for the identication and monitoring of Paecilomyces lilacinus. FEMS Microbiology Ecology, 51, 257264. Bais H.P., Weir T.L., Perry L.G., Gilroy S., Vivanco J.M. (2006) The role of root exudates in the rhizosphere interactions with plant and other organisms. Annual Review of Plant Biology, 57, 233266.

Bertin C., Yang X., Weston L.A. (2003) The role of root exudates and allelochemicals in the rhizosphere. Plant and Soil, 256, 6783. Bordallo J.J., Lopez-Llorca L.V., Hansson H.B., Salinas J., Persmark L., Asensio L. (2002) Colonization of plant roots by egg-parasitic and nematode trapping-fungi. New Phytologist, 154, 491499. Bourne J.M., Kerry B.R., de Leij F.A.A.M. (1996) The importance of the host plant on the interaction between root-knot nematodes (Meloidogyne spp.) and the nematophagous fungus, Verticillium chlamydosporium Goddard. Biological Control Science and Technology, 6, 539548. Bourne J.M., Kerry B.R. (1999) Effect of the host plant on the efcacy of Verticillium chlamydosporium as a biological control agent of root-knot nematodes at different nematode densities and fungal application rates. Soil Biology and Biochemistry, 31, 7584. Bourne J.M., Karanja P.K., Kalisz H., Karanja D.K., Mauchline T.H., Kerry B.R. (2004) Incidence and severity of damage caused by Meloidogyne spp. and isolation and screening of the nematophagous fungus Pochonia chlamydosporia from some of the main vegetable growing areas in Kenya. International Journal of Nematology, 14, 111122. Braga F.R., Araujo J.V., Carvalho R.O., Silva A.R., Araujo J.M., Soares F.E.F., Andre G.L.H., Ferreira S.R., Queiroz J.H. (2010) Ovicidal action of a crude enzymatic extract of the fungus Pochonia chlamydosporia against cyathostomin eggs. Veterinary Parasitology, 72, 264268. Bruck D.J. (2010) Fungal entomopathogens in the rhizosphere. Biocontrol, 55, 103112. Esteves I. (2007) Factor affecting the performance of Pochonia chlamydosporia as a biological control agent of nematodes. PhD Thesis. Craneld University, UK, pp. 190191. Frassy L.N., Braga F.R., Silva A.R., Araujo A.R., Ferreira S.R., de Freitas L.G. (2010) Destruic ao de ovos de Toxocara canis pelo fungo nematofago Pochonia chlamydosporia. Revista da Sociedade Brasileira de Medicina Tropical, 43, 102104. Hidalgo-D az L. (2003) Standard Operating Procedure for Mass Production Pochonia chlamydosporia. Havana, Cuba: OCIC CENSA. Hooper D.J. (1986) Extraction of nematodes from plant material. In Laboratory Methods for Work with Plant and Soil Nematodes, pp. 5158. Ed J.F. Southey. London, UK: MAFF/ADAS [Reference Book No. 402]. Jacobs H., Gray S.N., Crump D.H. (2003) Interactions between nematophagous fungi and consequences for their potential as biological agents for the control of potato cyst nematodes. Mycological Research, 107, 4756. Kerry B.R. (2000) Rhizosphere interactions and the exploitation of microbial agents for the biological control

128

Ann Appl Biol 159 (2011) 118129 2011 Rothamsted Research Ltd Annals of Applied Biology 2011 Association of Applied Biologists

 R.H. Manzanilla-Lopez et al.

Effects of crop plants on abundance of Pochonia chlamydosporia and other fungal parasites

of plant-parasitic nematodes. Annual Review of Phytopathology, 38, 423441. Kerry B.R., Simon A., Rovira A.D. (1984) Observations on the introduction of Verticillium chlamydosporium and other parasitic fungi into soil for control of the cereal cyst-nematode Heterodera avenae. Annals of Applied Biology, 105, 509516. Kerry B.R., Kirkwood I.A., de Leij F.A.A.M., Barba J., Leijdens M.B., Brookes P.C. (1993) Growth and survival of Verticillium chlamydosporium Goddard, a parasite of nematodes in soil. Biocontrol Science and Technology, 3, 355365. Kerry B.R., Bourne J.M. (2002) Analysis. In A Manual for Research on Verticillium chlamydosporium a Potential Biological Control Agent for Root-Knot Nematode, pp. 2829. Gent, Belgium: International Organization for Biological and Integrated Control of Noxious Animals and Plants, West Palaearctic Regional Section (IOBC/WPRS). Lopez-Llorca L.V., Bordallo J.J., Monfort E., Lopez-Serna M.L. (2002) Use of light and scanning electron microscopy to examine colonisation of barley rhizosphere by the nematophagous fungus Verticillium chlamydosporium. Micron, 33, 6167. Lopez-Llorca L.V., Macia-Vicente J.G., Jansson H.B. (2008) Mode of action and interactions of nematophagous fungi. In Integrated Management and Biocontrol of Vegetable and Grains Crops Nematodes, pp. 5176. Eds A. Ciancio and K.G. Mukerji. Dordrecht, The Netherlands: Springer. Macia-Vicente J.G., Jansson H.B., Talbot N.J., Lopez-Llorca L.V. (2009) Real-time PCR quantication and live-cell imaging of endophytic colonization of barley (Hordeum vulgare) roots by Fusarium equiseti and Pochonia chlamydosporia. New Phytologist, 182, 213228. 10.1111/j.1469-8137.2008.02743.x. Manzanilla-Lopez R.H., Atkins S.D., Clark I.M., Kerry B.R., Hirsch P.R. (2009) Measuring abundance, diversity and parasitic ability in the populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia. Biocontrol Science and Technology, 19, 391406. Mauchline T.H., Kerry B.R., Hirsch P.R. (2002) Quantication in soil and the rhizosphere of the nematophagous fungus Verticillium chlamydosporium by competitive PCR and comparison with selective plating. Applied and Environmental Microbiology, 68, 18461853. Mauchline T.H., Kerry B.R., Hirsch P. (2004) The biocontrol fungus Pochonia chlamydosporia shows nematode host preference at the infraspecic level. Mycological Research, 108, 106169. Menendez A., Bertoni M.D., Cabral D. (1997) Endotos en Juncus imbricatus var. chamissonis: fungicos

identicacion de los patrones de colonizacion. Revista Iberoameriacana de Micolog a, 14, 125128. Monfort E., Lopez-Llorca L.V., Jansson H.B., Salinas J., On Park J., Sivasithamparam K. (2005) Colonisation of seminal roots of wheat and barley by egg-parasitic nematophagous fungi and their effects on Gaeumannomyces graminis var. tritici and development of root-rot. Soil Biology and Biochemistry, 37, 12291235. Pyrowolakis A., Schuster R.P., Sikora R. (1999) Effect of cropping pattern and green manure on the antagonistic potential and the diversity of egg pathogenic fungi in elds with Heterodera schachtii infection. Nematology, 1, 165171. Rumbos C.I., Kiewnik S. (2006) Effect of plant species on persistence of Paecilomyces lilacinus strain 251 in soil and root colonization by the fungus. Plant and Soil, 283, 2531. Schulz B., Boyle C. (2006) The endophytic continuum. Mycological Research, 109, 661686. Siddiqui I.A., Atkins S.D., Kerry B.R. (2009) Relationship between saprotrophic growth in soil of different biotypes of Pochonia chlamydosporia and the infection of nematode eggs. Annals of Applied Biology, 155, 131141. Singh B.K., Millard P., Whiteley A.S., Murrell J.C. (2004) Unravelling rhizospheremicrobial interactions: opportunities and limitations. Trends in Microbiology, 12, 386393. Tobin J.D., Haydock P.P.J., Hare M.C., Woods S.R., Crump D.H. (2008) Effect of the fungus Pochonia chlamydosporia and fosthiazate on the multiplication rate of potato cyst nematodes (Globodera palllida and G. rostochiensis) in potato crops grown under UK eld conditions. Biological Control, 46, 194201. Whitehead A., Turner S.J. (1998) Management and regulatory control strategies for potato cyst nematodes (Globodera rostochiensis and Globodera pallida). In Potato Cyst Nematodes Biology, Distribution and Control. pp. 135152. Eds R.J. Marks and B.B. Brodie. Wallingford, UK: CAB International. Wichern F., Eberhardt E., Mayer J., Georg J.R., Muller T. (2008) Nitrogen rhizodeposition in agricultural crops: methods, estimates and future prospects. Soil Biology & Biochemistry, 40, 3048. Yeates G.W. (1999) Effects of plants on nematode community structure. Annual Review of Phytopathology, 37, 127149. Zare R., Gams W., Evans H.C. (2001) A revision of the Verticillium section Prostrata. V. The genus Pochonia, with notes on Rotiferophthora. Nova Hedwigia, 73, 5186.

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