The role of chitinase from Lecanicillium antillanum B-3 in parasitism to root-knot nematode Meloidogyne incognita eggs

B-3 fungal isolate was isolated from soil samples of Gwangju in Korea. Based on morphological and phylogenetic analysis, it was designated as Lecanicillium antillanum B-3 (syn. Verticillium antillanum B-3). The fungus was a chitinolytic-nematophagous microorganism. B-3 chitinase activity from 0.5% swollen chitin broth medium reached the highest level on the sixth day and then plateaued until 12 days. B-3 isolate showed the high rate of parasitism on Meloidogyne incognita eggs with more than 90% infection rate on the third day after treatment. B-3 crude chitinase damaged the eggshell structures more than 78% based on lactoglycerol staining data at a final protein concentration of 14.6 µg mL^?1 on the fourth day following treatment. Partially purified chitinase with molecular 37 kDa from DEAE-Sephadex chromatography also showed damaging effect on the eggs. These results suggested that chitinase from B-3 isolate was responsible for degradation of M. incognita eggshell structures.     

Alginate films for delivery of root-knot nematode inoculum and evaluation of microbial interactions

A method was developed for utilizing alginate films to deliver inoculum into soil and evaluate microbial antagonistic activity against nematode eggs. Eggs of Meloidogyne incognita were harvested from galled tomato roots (Lycopersicon esculentum), surface disinfested, suspended in 2% (w/v) aqueous sodium alginate, and applied to 2.5 × 5.0 cm polyvinyl chloride coated fiberglass screens (1.5 mm² mesh size) at a uniform thickness of 0.5 mm. The alginate solution was gelled by dipping in 0.25 M CaCl₂. Films containing eggs were observed in vitro and egg development was evaluated. The number of immature eggs and eggs with first stage juveniles declined linearly over time while the number of empty eggs shells, and hatched juveniles increased over time, indicating that the alginate gel did not inhibit development and motility of M. incognita juveniles. In a greenhouse experiment using cucumber (Cucumis sativus) the number of galls g^-1 root was correlated with the number of eggs in alginate films placed in each pot at planting. Films containing M. incognita eggs were buried in field soil containing organic amendments, incubated, removed from soil, rinsed with water, and observed. The number of immature eggs in grids from soil amended with chitin or flax seed meal were lower than in untreated soil; percent parasitized eggs was also greater in films from amended soil than from untreated soil.     

Nematicidal effect of an Amaranthus hypochondriacus L. cystatin (AhCPI) on the root-knot nematode Meloidogyne incognita (Kofoid and White) Chitwood

The root-knot nematode Meloidogyne incognita is one of the most damaging plant parasitic nematodes in the world. In this study, the effect of cystatin from Amaranthus hypochondriacus (AhCPI) as a potential control agent for M. incognita was explored. In vitro bioassays demonstrated that AhCPI affects the growth and development of eggs and the infectivity of juveniles (J2) of M. incognita, such as mortality and slower development, showing characteristic tissue damage. Mortality levels were quantified by Probit analysis, estimating LC₅₀s of 1.4 mg/mL for eggs and 0.028 mg/mL for J2. In planta bioassays showed that infected tomato seedlings treated with 0.056 mg/mL of AhCPI showed a 60% reduction in the number of galls, as compared with untreated J2-inoculated seedlings. Under greenhouse conditions, three applications of 10 mL of AhCPI (1.4 mg/mL) in the soil around the stem of M. incognita-infected tomato plants, reduced the number of galls by 93 ± 8%, as compared to the control M. incognita-infected plants. The application of AhCPI to the infected plants increased the yield (10.7%) of harvested tomato fruits, as compared to infected plants. These results show the potential of AhCPI for the control of M. incognita in tomato plants.     

Isolation of nematophagous fungi from eggs and females of Meloidogyne spp. and evaluation of their biological control potential

Fungi were isolated from Meloidogyne spp. eggs and females on 102 field-collected root samples in China. Of the 235 fungi isolated (representing 18 genera and 26 species), the predominant fungi were Fusarium spp. (42.1% of the isolates collected), Fusarium oxysporum (13.2%), Paecilomyces lilacinus (12.8%), and Pochonia chlamydosporia (8.5%). The isolates were screened for their ability to parasitise Meloidogyne incognita eggs in 24-well tissue culture plates in two different tests. The percentage of eggs parasitised by the fungi, the numbers of unhatched eggs and alive and dead juveniles were counted at 4 and 7 days after inoculation. The most promising fungi included five Paecilomyces isolates, 10 Fusarium isolates, 10 Pochonia isolates and one Acremonium isolate in test 1 or test 2. Paecilomyces lilacinus YES-2 and P. chlamydosporia HDZ-9 selected from the in vitro tests were formulated in alginate pellets and evaluated for M. incognita control on tomato in a greenhouse by adding them into a soil with sand mixture at rates of 0.2, 0.4, 0.8 and 1.6% (w/w). P. lilacinus pellets at the highest rate (1.6%) reduced root galling by 66.7%. P. chlamydosporia pellets at the highest rate reduced the final nematode density by 90%. The results indicate that P. lilacinus and P. chlamydosporia as pellet formulation can effectively control root-knot nematodes.     

Evaluation of Pochonia chlamydosporia var. chlamydosporia as a control agent of Meloidogyne javanica on pistachio

The potential of isolates of Pochonia chlamydosporia var. chlamydosporia as biocontrol agents for root-knot nematodes was investigated in vitro and on pistachio plants. On potato dextrose agar, growth of all isolates started at temperatures above 10°C, reached maximum between 25 and 28°C and slowed down at 33°C. On water agar, all isolates parasitized more than 85% of the eggs of Meloidogyne javanica at 18°C after 3 weeks. Filtrates of isolates grown on malt extract broth did not cause more than 5% mortality on second-stage juveniles of M. javanica after 48 h of incubation. A single application of 10×10³ chlamydospores (produced on sand–barley medium) g^–1 soil, was applied to unsterilised soil planted with pistachio cv. Kalehghochi, and plants were inoculated with 3000 nematode eggs. After 120 days in the glasshouse, nematode multiplication and damage were measured. Ability of fungus isolates to survive in the soil and to grow on roots were estimated by counting colony forming units (cfu) on semi-selective medium. Fungal abundance in soil increased nearly 3-fold and 10×10³ and 20×10³ cfu g^–1 root of pistachio were estimated in pots treated with isolates 40 and 50, respectively. Strain 50 was more abundant in soil and on the roots, infected more eggs (40%) on the roots and controlled 56% of total population of M. javanica on pistachio roots, whereas isolate 40 parasitized 15% of the eggs on the roots and controlled ca. 36% of the final nematode population.     

Nematicidal screening of essential oils and potent toxicity of Dysphania ambrosioides essential oil against Meloidogyne incognita in vitro and in vivo

It is known that some plant essential oils have pesticide activities. Among the 29 oils evaluated in this study, 14 showed nematicidal activities of 8 to 100% at the concentration of 1,000 μg/ml, compared with a control of 0.01 g/ml Tween 80^®. At a lower concentration of 500 μg/ml, only Dysphania ambrosioides oil caused >90% mortality of second‐stage juveniles (J2) of Meloidogyne incognita. The LC₅₀ and LC₉₅ values for D. ambrosioides oil were 307 μg/ml and 580 μg/ml, respectively. M. incognita eggs placed in D. ambrosioides oil solutions had a significant reduction in J2 hatching compared with controls. Therefore, the oil had a toxic effect on both eggs and J2 of M. incognita. This was in contrast to nematicides on the market that act efficiently only on J2. When J2 were placed in D. ambrosioides oil at its LC₅₀ concentration and inoculated onto tomato plants, the reduction in numbers of galls and eggs was 99.5% and 100%, respectively. Dysphania ambrosioides oil applied to the potting substrate of plants at a concentration of 1,100 μg/ml significantly reduced the number of galls and eggs of M. incognita, whereas a concentration of 800 μg/ml only reduced the number of eggs compared with the controls (Tween 80^® and water). The main components of the D. ambrosioides oil detected by gas chromatography–mass spectrometry were (Z)‐ascaridole (87.28%), E‐ascaridole (8.45%) and p‐cymene (3.35%), representing 99.08% of the total oil composition. Given its nematicidal activity, D. ambrosioides oil represents an exciting raw material in the search for new bioactive molecules for the pesticide industry.     

Bio-control activity of Purpureocillium lilacinum strains in managing root-knot disease of tomato caused by Meloidogyne incognita

The potential of 24 indigenous isolates of Purpureocillium lilacinum (Paecilomyces lilacinus) (Thom) Samson collected from different agro-climatic zones of India was investigated against the root-knot nematode, Meloidogyne incognita. The studies were conducted in vitro (larvicidal, ovicidal and egg-parasitising capacity) and under naturally infested field conditions with selected strains. Repeated field trials were conducted with talc-based preparations of fungal strains at 10 kg ha^?1, which were applied mixed in farm yard manure (FYM) at 1.5 t ha^?1. Results (in vitro) showed that all tested isolates were capable to parasitise eggs, inhibit egg hatching and cause juvenile mortality of M. incognita at various levels. Based on the performance under in vitro studies, eight isolates (NDPL-01, ANDPL-02, SHGPL-03, HYBPL-04, AHDPL-05, PTNPL-06, SNGPL-07 and VNSPL-08) were re-tested to confirm the results. HYBDPL-04 was found causing highest mortality (80%), inhibition of egg hatching (90%) as well as parasitisation of M. incognita eggs (75%). Under field trials also, the best protection of root-knot disease of tomato (Lycopersicon esculentum L.), in terms of reduction of galls (61%) and reproductive factor (P_f/P_i (RF) = 0.2) was achieved through application of HYBDPL-04 + FYM compared to control and other tested isolates. It also enhanced marketable yield of tomato up to 43%. It is concluded that the HYBDPL-04 strain of P. lilacinum is highly effective for management of root-knot disease of tomato under naturally infested field conditions. It is the isolate which produced the maximum number of metabolites which were extracted through high pressure liquid chromatography.     

Nematicidal activity of fervenulin isolated from a nematicidal actinomycete, <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CMU-MH021, on <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

An isolate of the actinomycete, Streptomyces sp. CMU-MH021 produced secondary metabolites that inhibited egg hatch and increased juvenile mortality of the root-knot nematode Meloidogyne incognita in vitro. 16S rDNA gene sequencing showed that the isolate sequence was 99% identical to Streptomyces roseoverticillatus. The culture filtrates form different culture media were tested for nematocidal activity. The maximal activity against M. incognita was obtained by using modified basal (MB) medium. The nematicidal assay-directed fractionation of the culture broth delivered fervenulin (1) and isocoumarin (2). Fervenulin, a low molecular weight compound, shows a broad range of biological activities. However, nematicidal activity of fervenulin was not previously reported. The nematicidal activity of fervenulin (1) was assessed using the broth microdilution technique. The lowest minimum inhibitory concentrations (MICs) of the compound against egg hatch of M. incognita was 30 μg/ml and juvenile mortality of M. incognita increasing was observed at 120 μg/ml. Moreover, at the concentration of 250 μg/ml fervenulin (1) showed killing effect on second-stage nematode juveniles of M. incognita up to 100% after incubation for 96 h. Isocoumarin (2), another bioactive compound produced by Streptomyces sp. CMU-MH021, showed weak nematicidal activity with M. incognita.     

Phythonematoxic properties and nematicidal potential of Tithonia diversifolia extract and residue on Meloidogyne incognita infecting yam (Discoria rotundata)

A laboratory study and a screen house experiment were conducted to determine the phythonematoxic properties and nematicidal potential of extract and residue of Tithonia diversifolia (Hemsl.) A. Gray on Meloidogyne incognita, (Kofoid and White) Chitwood infecting yam. Alkaloids and saponins were found to be present in the constituents of Tithonia ethanol extract. Also, Tithonia water extract significantly (P ≤ 0.05) inhibits M. incognita egg hatch by 98% from 2 days after incubation (DAI) and was more evidenced with 100% inhibition at 9 DAI in the in vitro studies. M. incognita (5000 eggs/plant) reproduction, number of eggs and juveniles and galling were significantly (P ≤ 0.05) suppressed by Tithonia residue treatment at a rate of 30 tons/ha on yam (Discoria rotundata) in the screen house. The extract and residue of T. diversifolia have potential for the control of root-knot nematodes.     

Reproduction and Development of Meloidogyne incognita and M. javanica on Guardian Peach Rootstock

Guardian peach rootstock was evaluated for susceptibility to Meloidogyne incognita race 3 (Georgia-peach isolate) and M. javanica in the greenhouse. Both commercial Guardian seed sources produced plants that were poor hosts of M. incognita and M. javanica. Reproduction as measured by number of egg masses and eggs per plant, eggs per egg mass, and eggs per gram of root were a better measure of host resistance than number of root galls per plant. Penetration, development, and reproduction of M. incognita in Guardian (resistant) and Lovell (susceptible) peach were also studied in the greenhouse. Differences in susceptibility were not attributed to differential penetration by the infectivestage juveniles (J2) or the number of root galls per plant. Results indicated that M. incognita J2 penetrated Guardian roots and formed galls, but that the majority of the nematodes failed to mature and reproduce.     

Relationships between Meloidogyne incognita resistance genes in Lycopersicon peruvianum differentiated by heat sensitivity and nematode virulence

Resistance to Meloidogyne incognita (Kofoid and White) Chitwood in clones of Lycopersicon peruvianum (L.) Mill. PI 126443-1MH, 270435-2R2 and 2704353MH, their F₁, a field-produced F₂, and their test-cross (TC₁) populations, was evaluated based on egg masses and eggs produced on root systems. Reactions to M. incognita isolates differing in virulence to gene Mi were determined at 25°C (Mi expressed) and 32°C (Mi not expressed). PI 126443-1MH, 270435-2R2, 270435-3MH, and their F₁ progenies were resistant to Mi-virulent and Mi-avirulent isolates. At 32°C with a Mi-avirulent isolate and at 25°C with a Mi-virulent isolate, four TC₁ generations segregated into resistant: susceptible (RS) ratios close to 31. These results indicated resistance to Mi-(a)virulent M. incognita isolates is conferred by different non-allelic dominant genes in PI 126443-1MH, 270435-2R2 and 270435-3MH. The F₂ progeny of PI 126443-1MH x EPP-1, challenged with Mi-avirulent M. incognita at 32°C and with Mi-virulent M. incognita at both 25°C and 32°C, segregated with a ratio of 31 (RS), indicating expression of a single dominant resistance gene in PI 126443-1MH in each case. In dual screenings on clones of the same individual plants from the TC₁ and F₂ segregating populations, some individual plants were susceptible at 32°C to a Mi-avirulent isolate but resistant to the Mi-virulent isolate, and vice versa, suggesting that different but linked genes confer heat-stable resistance to Mi-avirulent M. incognita and resistance to Mi-virulent M. incognita. We propose the symbol Mi-5 for the gene in PI 126443 clone 1MH and the symbol Mi-6 for the gene in PI 270435 clone 3MH which both confer resistance to Mi-avirulent M. incognita isolates at high temperature. We propose the symbol Mi-7 for the gene in PI 270435 clone 3MH and the symbol Mi-8 for the gene in PI 270435 clone 2R2 that both confer resistance to the Mi-virulent M. incognita isolate 557R at moderate (25°C) temperature. The novel resistance genes are linked and reside in a genomic region in each parental clone that is independent from the Mi locus.     

Plantago lanceolata and Plantago rugelii Extracts are Toxic to Meloidogyne incognita but not to Certain Microbes

Extracts from the plants Plantago lanceolata and P. rugelii were evaluated for toxicity to the root-knot nematode Meloidogyne incognita, the beneficial microbes Enterobacter cloacae, Pseudomonas fluorescens and Trichoderma virens, and the plant-pathogenic fungi Fusarium oxysporum f. sp. gladioli, Phytophthora capsici, Pythium ultimum, and Rhizoctonia solani. Wild plants were collected, roots were excised from shoots, and the plant parts were dried and ground to a powder. One set of extracts (10% w/v) was prepared in water and another in methanol. Treatments included extract concentrations of 25%, 50%, 75% and 100%, and water controls. Meloidogyne incognita egg hatch was recorded after 7-day exposure to the treatments, and second-stage juvenile (J2) activity after 48 hours. All extracts were toxic to eggs and J2, with P. lanceolata shoot extract tending to have the most activity against M. incognita. Numbers of active J2 remained the same or decreased in a 24-hour water rinse following the 48-hour extract treatment, indicating that the extracts were lethal. When data from water- and methanol-extracted roots and shoots of both plant species were combined for analysis, J2 tended to be more sensitive than eggs to the toxic compounds at lower concentrations, while the higher concentrations (75% and 100%) were equally toxic to both life stages. The effective concentrations causing 50% reduction (EC₅₀) in egg hatch and in J2 viability were 44.4% and 43.7%, respectively. No extract was toxic to any of the bacteria or fungi in our assays.     

Relationships of Initial Population Densities of Meloidogyne incognita and M. hapla to Yield of Tomato

Microplots 80 × 100 cm, infested with varying initial population densities (P_i) of Meloidogyne incognita or M. hapla, were planted to tomato at two locations. Experiments were conducted in a sandy loam soil at Fletcher, N. C. (mountains) where the mean temperature for May to September is ca 20.7 C, and in a loamy saml at Clayton, N. C. (coastal plain) where the mean temperature for May to Septemher is ca 24.8 C. In these experimentally infested plots, M. incognita and M. hapla caused maximunt yield losses of 20-30%, at lhe mountain site with Pi of 0-12,500 eggs and larvae/500 cm³ of soil. In the coaslal plain, M. incognita suppressed yields up to 85%, and M. hapla suppressed yields up to 50% in comparison with the noninfested control. A part of the high losses at this site apparently was due to M. incognita predisposing tomato to the early blight fungus. In a second experintent, in which a nematicide was used to obtain a range of P_is (with P_i as high as 25,000/50 cm³ of soil) at Fletcher, losses due to M. incognita were as great as 50%, but similar densities of M. hapla suppressed yields by only 10-25%. Approximate threshold densities for both species ranged from 500 to 1,000 larvae and eggs (higher for surviving larvae) for the mountain site, whereas nutnbers as low as 20 larvae/500 cm³ of soil of either species caused signiticant damage in the coastal plain. Chemical soil treatments proved useful in obtaining various initial population densities; however, problems were encountered in measuring effective inoculum after such treatments, especially in the heavier soil.     

Studies on the disease complex incidence by Meloidogyne incognita and Fusarium solani on Poi,Basella rubra

A survey of Poi crop in Ghaziabad (UP) exhibited a disease complex incidence by Meloidogyne incognita and Fusarium solani causing synergistic effect on the host. Paecilomyces lilacinus was found from the egg masses of M. incognita and Aspergillus niger and Aspergillus terreus from the rhizosphere of root-knot infected Poi crop. Paecilomyces lilacinus parasitised the eggs to a greater extent. The level of parasitism was highest (65%) by P. lilacinus while Aspergillus spp. did not colonise the eggs. Fusarium solani which in the present investigation has been established to be pathogenic to Poi plant.     

Broad spectrum action of phenazine against active and dormant structures of fungal pathogens and root knot nematode

Antifungal antibiotic from Pseudomonas chlororaphis isolate PA23 was identified as Phenazine using TLC and HPLC. Phenazine recorded the highest inhibition zone of 21?mm with 35.55% percent inhibition of mycelial growth of Pythium aphanidermatum over control. It had a significant effect on the hyphal morphology of P. aphanidermatum and on spore germination of Botryodiplodia theobromae and Alternaria solani. Disorganization of hyphal morphology of P. aphanidermatum includes vacuolization, cell content degeneration and hyphal lysis. Similarly interaction of phenazine with Rhizoctonia solani resulted in abnormal swelling of hyphal tips was noticed in the hyphal tips. Similarly the germination of sclerotia of Macrophomina phaseolina, R. solani and Sclerotium rolfsii were completely inhibited by phenazine at a concentration 50?μl. Incubation of the eggs of the root knot nematode Meloidogyne incognita in 30?μl concentration of phenazine, completely suppressed the hatching of juveniles.     

Effect of organic fertiliser and Chromolaena odorata residue on the pathogenicity of Meloidogyne incognita on maize

Pot experiments laid out in a complete randomised design were conducted in the screen house of the Department of Crop Protection, University of Agriculture, Abeokuta, Ogun State, Nigeria to determine the effects of organic fertiliser and Chromolaena odorata residue at 1% w/w on the pathogenicity of Meloidogyne incognita infecting maize. M. incognita significantly reduced the plant height, number of leaves per plant, leaf area, cob weight and grain yield of maize by 6.89, 15.18, 20, 63.92 and 56.16% respectively. C. odorata residue and organic fertiliser significantly suppressed M. incognita galling, inhibited the nematode fecundity and reduced the number of eggs and juveniles on maize. A remarkable increase in plant height, number of leaves per plant, leaf area, cob weight and grain yield were observed on maize plants treated with the mixture of C. odorata and organic fertiliser despite the nematode infection. The observation from this study suggests that C. odorata in combination with organic fertiliser is a viable option for the control of M. incognita on maize.     

Competition of Meloidogyne incognita and Rotylenchulus reniformis on Cotton Following Separate and Concomitant Inoculations

It has been hypothesized Rotylenchulus reniformis (Rr) has a competitive advantage over Meloidogyne incognita (Mi) in the southeastern cotton production region of the United States. This study examines the reproduction and development of Meloidogyne incognita (Mi) and Rotylenchulus reniformis (Rr) in separate and concomitant infections on cotton. Under greenhouse conditions, cotton seedlings were inoculated simultaneously with juveniles (J2) of M. incognita and vermiform adults of R. reniformis in the following ratios (Mi:Rr): 0:0, 100:0, 75:25, 50:50, 25:75, and 0:100. Soil populations of M. incognita and R. reniformis were recorded at 3, 6, 9, 14, 19, 25, 35, 45, and 60 days after inoculations. At each date, samples were taken to determine the life stage of development, number of egg masses, eggs per egg mass, galls, and giant cells or syncytia produced by the nematodes. Meloidogyne incognita and R. reniformis were capable of initially inhibiting each other when the inoculum ratio of one species was higher than the other. In concomitant infections, M. incognita was susceptible to the antagonistic effect of R. reniformis. Rotylenchulus reniformis affected hatching of M. incognita eggs, delayed secondary infection of M. incognita J2, reduced the number of egg masses produced by M. incognita, and reduced J2 of M. incognita 60 days after inoculations. In contrast, M. incognita reduced R. reniformis soil populations only when its proportion in the inoculum ratio was higher than that of R. reniformis. Meloidogyne incognita reduced egg masses produced by R. reniformis, but not production of eggs and secondary infection.     

Interaction between Pseudomonas fluorescens and Meloidogyne incognita on tomato plant as influenced by the different levels of phosphorus

A pot experiment was conducted on tomato (Solanum lycopersicum cv. Pusa Ruby) to assess the effect of different phosphorus (P) levels (0, 125, 250 and 500 mg/pot) and the plant growth promoting rhizobacterium, Pseudomonas fluorescens, on the growth of tomato and on the reproduction of Meloidogyne incognita. Maximum growth of tomato occurred at P rates of 125 mg/kg soil, irrespective of whether plants were uninoculated or inoculated with P. fluorescens or M. incognita or inoculated with both the agents. Nematodes per gram of roots, egg masses per root, eggs per egg mass and galls per root significantly increased by increasing levels of P. P. fluorescens performed better than other treatments and different P levels in improving tomato growth and reducing galling and multiplication of M. incognita.     

Measuring abundance,diversity and parasitic ability in two populations of the nematophagous fungus Pochonia chlamydosporia var. chlamydosporia

Abundance, genetic diversity and parasitic ability in the facultative nematode parasite Pochonia chlamydosporia var. chlamydosporia were compared in soils from two sites in Portugal under long-term tomato cultivation where root-knot nematodes (Meloidogyne sp.) were present. Fungal abundance assessed by selective agar or real-time quantitative PCR with specific primers was similar in both soils. PCR fingerprinting of isolates with ERIC primers indicated that the dominant P. c. var. chlamydosporia biotypes (profiles A and B) in both soils were very closely related, although a second biotype (profile C) was detected in one soil. When tomato plants infected with M. incognita were grown in the two soils, only profiles A and B were recovered from eggs. Primers based on polymorphisms in vcp1 demonstrated that isolates with profiles A and B were likely to prefer root-knot nematodes, whereas profile C preferred cyst nematodes. In the soil containing profiles A, B and C, egg parasitism by P. chlamydosporia was estimated at 1% using water agar plates with antibiotics but fewer than 0.2% of M. incognita eggs were shown to be infected with P. c. var. chlamydosporia when using species-specific β-tubulin-PCR primers. In contrast, the soil containing only profile B showed 22% egg parasitism on water agar plates and more than 2.5% of eggs were confirmed as P. c. var. chlamydosporia by species-specific β-tubulin-PCR primers. The results, which reveal limited diversity within the fungus at the two sites, are discussed in relation to biological control of plant-parasitic nematodes.