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Development and application of a PCR diagnostic assayfor the accurate and rapid identification of thenematophagous fungus Duddingtonia flagrans

Paula KELLYa,b, Barbara GOODa,*, Richard FITZPATRICKa, J. P. HANRAHANa,Theo D. T. DE WAALb

aTeagasc, Animal Production Research Centre, Mellows Campus, Athenry, Co. Galway, IrelandbUniversity College Dublin, School of Agriculture Food Science and Veterinary Medicine, Belfield, Dublin 4, Ireland

a r t i c l e i n f o

Article history:

Received 29 June 2007

Received in revised form

13 December 2007

Accepted 14 February 2008

Corresponding Editor: Judith K. Pell

Keywords:

Biocontrol

Nematodes

Nematogenous fungi

Soil fungi

Species-specific primers

* Corresponding author. Tel.: þ353 91 845 8E-mail address: barbara.good@teagasc.ie

0953-7562/$ – see front matter ª 2008 The Bdoi:10.1016/j.mycres.2008.02.005

a b s t r a c t

The nematophagous fungus, Duddingtonia flagrans is a potential biocontrol agent against

nematode parasites of ruminants. Improved methods for the rapid and accurate detection

of D. flagrans would aid the evaluation of this fungus as a biocontrol agent and its suitability

for environmental release. To date, detection and identification of D. flagrans is reliant on

morphological methods, which can be laborious, time-consuming, and error prone. In this

study, a PCR assay using species-specific primers located in the ITS regions was developed

for the rapid and accurate identification of D. flagrans. The PCR assay was specific to five

different isolates of D. flagrans and was capable of detecting a minimum concentration

of 100 chlamydospores per gram of soil. In contrast to cultured-based detection and iden-

tification methods, this assay is amenable to high throughput screening of environmental

samples. The assay detected D. flagrans in faecal, leaf litter, and soil samples collected from

80 % of the Irish farms tested indicating that the fungus is abundant in Ireland.

ª 2008 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Introduction (Chandrawathani et al. 2004; Fontenot et al. 2003; Larsen et al.

Parasitic gastrointestinal nematodes impose a significant pro-

duction penalty in pasture-based livestock systems world-

wide. Since the 1960s, parasite control has been enhanced

with the availability of broad-spectrum anthelmintics. How-

ever, anthelmintic resistance has now emerged as a worldwide

phenomenon, and therefore, there is a need to develop alter-

native strategies to control parasitic nematodes. The use of

nematophagous fungi is now emerging as an alternative nem-

atode control method. In particular, Duddingtonia flagrans

(Ascomycota: Ascomycetes: Orbiliaceae) has shown promising

results in controlling the main ruminant endoparasites

48; fax: þ353 91 845 847.

ritish Mycological Society

1998; Terrill et al. 2004; Waller et al. 2004). D. flagrans traps

and digests the nematode larvae using three-dimensional ad-

hesive nets. Several studies have shown the beneficial effects

of feeding chlamydospores of D. flagrans to animals on the

subsequent level of nematode challenge on pasture and ulti-

mately on host worm burden (Faedo et al. 1998; Larsen 1999;

Mendoza de Gives et al. 1998; Waller et al. 1994). It was first de-

scribed by Duddington (1950) as Trichothecium flagrans and was

later renamed as D. flagrans (Cooke 1969).

If D. flagrans is to be used as a biocontrol agent in a country,

it is important to establish that it is indigenous to that country

before being released into the environment. D. flagrans has

. Published by Elsevier Ltd. All rights reserved.

Table 1 – Fungal species, source material, country oforigin and Centraalbureau voor Schimmelcultures (CBS)accession number

Species Source material Countryof origin

CBSref. no.

Arthrobotrys oligospora – USA 111.37

Drechmeria coniospora Nematodes Sweden 615.82

Duddingtonia flagrans Solidago canadensis,

dead stem

USA 343.94

D. flagrans Leaf litter UK 143.83

D. flagrans Solidago canadensis,

dead stem

Germany 342.94

D. flagrans – – 561.92

D. flagransa Greenhouse soil,

Camellia house

Germany 583.91

Fusarium coeruleum Nematode egg Germany 618.76

F. incarnatum Medicago sativa, seed Denmark 621.87

F. oxysporum Cucumis melo

(Cucurbitaceae)

Israel 420.90

Haptocillium balanoides – – 250.82

H. sphaerosporum Soil in potato field Canada 589.92

Harposporium helicoides Nematodes Canada 944.70

H. lilliputanum Nematode in soil Japan 111353

Paecilomyces lilacinus Meloidogyne, egg

mass

Philippines 431.87

Pochonia chlamydosporia

var. cantenulata

Heterodera

avenae, egg

Sweden 250.83A

Pyricularia higginsii Cyperus rotundus Israel 665.79

Pochonia chlamydosporia

(syn. Verticillium

chlamydosporium)

Egg of slug (Mollusca) Brazil 101244

V. coccospora Lymantria dispar, egg USA 691.86

V. sphaerosporum Ditylenchus triformis

on compost

Germany 522.80

– No information available.

a Isolate used in testing the sensitivity of the PCR assay.

Development and application of a PCR diagnostic assay 1027

been detected in Denmark (Larsen et al. 1992), France (Chartier

& Pors 2003), Germany (CBS 342.94, isolated by A. Rubner), UK

(CBS 143.83, isolated by R. C. Cooke), South Africa (Durand et al.

2005), Australia (Larsen et al. 1994), USA (CBS 343.94, isolated

by A. Rubner), Mexico (Mendoza de Gives et al. 1998), Malaysia

(Chandrawathani et al. 2002), New Zealand (Skipp et al. 2002),

and Iran (Ghahfarokhi et al. 2004). However, it has not been

detected in Ireland (Gray 1983). In addition, for regulatory pur-

poses, it is important to have methods that permit the reliable

and sensitive monitoring of biocontrol agents after release

into the environment (Avis et al. 2001).

Routine detection and identification of nematophagous

fungi typically involves nematode baiting, repeated incuba-

tions, and re-isolations in order to obtain a pure culture, fol-

lowed by microscopic examination of hyphae and spores.

Discrimination between closely related species requires addi-

tional detailed microscopic examination of particular phases

of the life cycle. Morphological identification is, therefore, la-

borious, can take several weeks, requires specialised training

in mycology, and is prone to contamination and error, which

delays definitive identification. Therefore, a simple and reli-

able method for the detection and identification of D. flagrans

would be highly desirable in the evaluation of this fungus as

a potential biocontrol agent.

Molecular approaches are increasingly being used to de-

velop PCR-based species-specific diagnostic assays for a broad

range of organisms (Atkins & Clark 2004). Molecular tools have

been employed to identify and monitor a number of fungi,

namely, Pochonia chlamydosporia var. catenulata (Atkins et al.

2003a), Plectophaerella cucumerina (Atkins et al. 2003b), Paecilo-

myces lilacinus (Atkins et al. 2005), Pochonia chlamydosporia

(syn. Verticillium chlamydosporium) (Hirsch et al. 2000), and Fusa-

rium sp. (Abd-Elsalam et al. 2003). These molecular approaches

are rapid, sensitive, easy to interpret, and provide a useful

alternative to culture-based detection and identification

methods. The ITS regions are commonly used as target re-

gions to design species-specific primers for PCR amplification.

The ITS regions, located between the 18S and 28S ribosomal

sequences, are hypervariable DNA regions, which make

them ideal target sequences for the generation of species-

specific primers. There are also multiple copies of the ITS re-

gion per genome, which increases the template number for

the PCR assay, thereby improving assay sensitivity of the diag-

nostic assay.

This study describes the development and application of

a PCR diagnostic assay for the rapid detection and identifica-

tion of D. flagrans in the environment.

Materials and methods

Fungal cultures

The 15 fungal species and five Duddingtonia flagrans isolates

used in these study were obtained from the Centraalbureau

voor Schimmelcultures (CBS), Utrecht, The Netherlands and

are listed in Table 1. All fungi were grown on oatmeal agar

with the exception of Cantenaria anguillulae and Hirsutella rhois-

siliensis, which were grown on archimycetes and malt extract

agar, respectively (CBS 2007: www.cbs.knaw.nl).

Field samples

To determine whether Duddingtonia flagrans was present in

Ireland, faecal (fresh) (n¼ 5), leaf litter (n¼ 2), and soil

(n¼ 5) samples were collected at random from permanent

sheep pastures on each of ten Irish farms, between 5–6

September, 2006. The criterion for inclusion of the farm in

the survey was that it was a well-established sheep enter-

prise. The three habitats were chosen based on previous ev-

idence, which reported the detection of D. flagrans in these

habitats, with the greatest occurrence in fresh faecal sam-

ples (Chandrawathani et al. 2002; Larsen et al. 1994; Skipp

et al. 2002) followed by decaying vegetation or leaf-litter

(Durand et al. 2005; Skipp et al. 2002). Samples were kept

in air-tight plastic bags at 4 �C before processing. Samples

collected from each farm were pooled and mixed by type

prior to processing.

DNA extraction from fungi

Genomic DNA was isolated from pure cultures using mechan-

ical disruption and enzymatic digestion. Briefly, approximately

500 mg frozen (liquid nitrogen) fungal mycelium was

1028 P. Kelly et al.

homogenized (Heidolph Diax 900, Sigma, UK) for 30 s in 3 ml

phosphate buffer saline (PBS, Sigma, UK, pH 7.0) containing

3 mg of lysing enzyme from Trichoderma harzianum (Sigma,

UK) and incubated at 37 �C for 3 h. This was followed by 1 h in-

cubation at 65 �C in 3 ml extraction buffer [50 mM Tris–HCl (pH

8), 100 mM EDTA, 100 mM NaCl2, 1 % sodium dodecyl sulphate

(SDS)] containing 100 mg proteinase K (Sigma, UK). RNA was

digested by the addition of 100 mg RNase (QIAGEN, UK) and

the sample incubated for a further 30 min at 37 �C. Genomic

DNA was then extracted with an equal volume of phenol and

chloroform and precipitated using 0.6 volumes of isopropanol.

The precipitated DNA was washed with 70 % ethanol, resus-

pended in 50 ml of nuclease-free water and stored at �20 �C.

The quantity and quality of the isolated DNA was evaluated

by absorbance at 260 and 280 nm.

DNA extraction from field samples

Total DNA was extracted from 5 g samples using the Ultra-

Clean� Mega Soil Kit (Mo Bio Laboratories, CA , USA) according

to manufacturer’s instructions. In order to remove humic acid

and other PCR inhibitors, the recovered DNA was then cleaned

using the PowerClean� DNA Clean-up Kit (Mo Bio Laborato-

ries, CA, USA) and subjected to PCR analysis as described

below. The identity of the PCR product was confirmed by

sequencing (Macrogen) Universal fungal primers, as described

by Turenne et al. 1999, were used as a positive control.

Design of selective primers and optimizationof PCR conditions

The previously published Duddingtonia flagrans ITS1–5.8S–ITS2

DNA sequence (GenBank accession no. U51961) was aligned

using ClustalW (EMBL-EBI) with homologous DNA sequences

from five closely related species, namely, Arthrobotrys

oligospora (accession no. AY773462), Pyricularia higginsii

(accession no. AB274438), Fusarium incarnatum (accession no.

EF158029), Pochonia chlamydosporia (accession no. AM412780),

and Paecilomyces lilacinus (accession no. AY213668). From this

alignment, D. flagrans species specific primers were designed;

forward primer: DFITS1F (50-CTT CGT CCG GTC TTG ACC G-30)

and reverse primer: DFITS2R (50-TTC GGG TTC AAA ACC AGC

GTT-30). The optimum PCR reaction consisted of 100 ng geno-

mic DNA, 1� PCR buffer (Promega, WI), 0.2 mM dNTPs, 4 mM

MgCl2, 100 ng of each primer and 2.5 U of Taq DNA polymerase

(Promega, WI) in a final reaction volume of 50 ml. Optimised

PCR amplification conditions consisted of 34 cycles of dena-

turation at 95 �C for 30 s, annealing at 66.6 �C for 30 s, and ex-

tension at 72 �C for 30 s. PCR products were separated on 1.5 %

ethidium bromide-stained agarose gels and visualised using

a Molecular Imager FX (Bio Rad Laboratories, CA, USA). The

identity of the PCR product was confirmed by sequencing

(Macrogen). The primers and PCR reaction conditions used

for universal fungal amplification were as previously de-

scribed (Turenne et al. 1999).

PCR assay specificity

The specificity of the Duddingtonia flagrans PCR assay using

primers DFITS1 and DFITS2 was tested on 15 different fungal

species and five D. flagrans isolates with different geographical

origins (Table 1).

PCR assay sensitivity

The sensitivity of the PCR assay was determined in two ways.

In the first approach, a 5 g soil sample taken from a recrea-

tional site (lawn) was spiked with 106 Duddingtonia flagrans

chlamydospores and subjected to DNA extraction as described

above (Table 1). The extracted DNA was diluted to provide the

equivalent of 120, 50, ten, five, and one chlamydospores and

then subjected to PCR analysis. In the second approach, DNA

was extracted from 5 g soil samples that had been spiked

with either 5� 105, 5� 104, 5� 103, 5� 102, 50 or five chla-

mydospores. Control samples consisted of soil from the

same source with no chlamydospores added. The extracted

DNA was subjected to PCR and the presence or absence of

the D. flagrans specific 299 bp PCR product determined by aga-

rose gel electrophoresis.

Statistical analysis

Fisher’s exact test (SAS, 2000) was used to compare the fre-

quency of Duddingtonia flagrans between the three habitats

examined.

Results

PCR assay specificity

The primers, DFITS1 and DFITS2, were highly specific when

tested against 15 different fungal species (Fig 1A). A single

PCR product of the expected size (299 bp) was exclusively am-

plified from Duddingtonia flagrans demonstrating the specific-

ity of the PCR assay. The control PCR reaction using

universal fungal primers was positive for all fungal isolates

(Fig 1B). The PCR assay was positive when tested on five differ-

ent D. flagrans isolates (data not shown).

PCR assay sensitivity

The first approach which examined a serial dilution of DNA

extracted from a specific number of chlamydospores demon-

strated that Duddingtonia flagrans could be detected in a soil

sample containing ten chlamydospores (data not shown).

The second approach using DNA from a range of spiked

samples demonstrated that a minimum concentration of 100

chlamydospores per gram of soil was necessary for the reli-

able amplification and detection of D. flagrans (Fig 2). No PCR

product was detected in the negative control (no chlamydo-

spores added).

PCR detection in environmental samples

The PCR assay detected Duddingtonia flagrans in material from

eight out of the ten Irish farms surveyed. The fungus was

found in faecal, leaf litter, and soil samples, from seven, five,

and three farms respectively (Table 2). There was no

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17A

B

299 bp

320 bp

Fig 1 – Specificity of the DFITS1F and DFITS2R primers using

genomic DNA isolated from 16 different fungal species.

(A) PCR assay using Duddingtonia flagrans species-specific

primers DFITS1F and DFITS2R. The 299 bp PCR product was

specific to D. flagrans. (B) Control PCR assay using fungal

universal primers. The 300 bp universal PCR product was

amplified in all isolates. Lane 1, 100 bp marker; 2, Dudding-

tonia flagrans; 3, Arthrobotrys oligospora; 4, Harposporium

lilliputanum; 5, H. helicoides; 6, Haptocillium sphaerosporum; 7,

Paecilomyces lilacinus; 8, H. balanoides; 9, Verticillium sphaer-

osporum; 10, Drechmeria coniospora; 11, Pochonia chlamydo-

sporia var. cantenulata; 12, Fusarium coeruleum; 13,

Verticillium coccospora; 14, F. incarnatum; 15, P. chlamydo-

sporia (syn. Vertcillium chlamydosporium); 16, Pyricularia

higginsii; 17, F. oxysporum.

Table 2 – Information on permanent sheep pasturessampled: farm reference number, location and presenceor absence of Duddingtonia flagrans in the three habitats

Farmreference no.

Location(country)

Habitat

Fresh faeces Leaf litter Soil

1 Wicklow þ þ2 Wicklow þ þ3 Wicklow

4 Wicklow þ5 Wicklow þ þ6 Wicklow þ þ7 Kilkenny þ þ þ8 Kilkenny þ9 Kilkenny þ þ10 Kilkenny

þ, Indicates Duddingtonia flagrans was detected, blank indicates D.

flagrans was not detected.

Development and application of a PCR diagnostic assay 1029

significant difference between in the frequency of D. flagrans

between habitats (P¼ 0.26). The control PCR reaction, using

universal fungal primers, was positive for all soil samples

tested (data not shown).

1 2 3 4 5 6 7 8

299 bp

Fig 2 – Sensitivity assessment of the PCR assay using DNA

isolated from soil samples containing absolute quantities of

Duddingtonia flagrans chlamydospores (per gram). Lane 1.

100 bp marker; 2, control (0 chlamydospores) 3, 1 3 105

chlamydospores; 4, 1 3 104 chlamydospores; 5, 1 3 103

chlamydospores; 6, 1 3 102 chlamydospores; 7, 1 3 10 chla-

mydospores; 8, one chlamydospore.

Discussion

This study describes a rapid PCR assay for the detection of

Duddingtonia flagrans using species-specific primers designed

from the highly variable ITS1 and ITS2 rDNA regions. The mo-

lecular assay developed, specifically detects D. flagrans and

showed no cross-reaction with its closest related species

Arthrobotrys oligospora and Pyricularia higginsii. In addition to

being highly specific, the PCR assay is rapid (<8 h), easy to in-

terpret, and amenable to high throughput screening analysis.

The assay was capable of detecting as little as 100 chlamydo-

spores using DNA directly extracted from 1 g soil avoiding the

need for fungal culture.

Serial dilution of DNA is the most common method used to

determine the sensitivity of a PCR assay (Abd-Elsalam et al.

2003). In this study sensitivity was examined in two ways, first

using serial dilutions of DNA and second using DNA extracted

from soil samples containing known quantities of D. flagrans

chlamydospores. A ten-fold difference in sensitivity between

the two methods was observed. In accounting for the lower

quantity of DNA recovered from samples taken from the envi-

ronment, it is suggested that the second method represents

a more appropriate measure of assay sensitivity for field sam-

ples than a method based on serial dilution of DNA, which

overestimates sensitivity. The overall usefulness of the PCR

assay was also confirmed by its ability to detect five D. flagrans

isolates obtained from different geographical locations.

Previous studies using conventional methods for detecting

D. flagrans have shown that D. flagrans was more frequently

found in fresh faecal samples (Chandrawathani et al. 2002;

Larsen et al. 1994; Skipp et al. 2002) followed by decaying veg-

etation or leaf litter (Durand et al. 2005; Skipp et al. 2002). Using

the PCR diagnostic assay developed in this study, there was no

evidence to support any difference between habitats. D. fla-

grans was confirmed as abundant in Ireland in contrast to an

extensive survey carried out by Gray (1983), that used conven-

tional culturing and microscopic detection methods on a large

number of samples (n¼ 161). In this study 30 samples were ex-

amined for the presence of D. flagrans and 50 % were found to

be positive.

1030 P. Kelly et al.

r e f e r e n c e s

Abd-Elsalam KA, Aly IN, Abdel-Satar MA, Khalil MS, Verreet JA, 2003.PCR identificationofFusariumgenus based onnuclearribosomal-DNA sequence data. African Journal of Biotechnology 2: 82–85.

Atkins S, Clark IM, 2004. Fungal molecular diagnostics: a minireview. Journal of Applied Genetics 45: 3–15.

Atkins S, Hidalgo-Diaz L, Clark IM, Norton CO, Montes de Oca N,Gray PA, Kerry B, 2003a. Approaches for monitoring the releaseof Pochonia chlamydosporia var. catenulata, a biocontrol agent ofroot-knot nematodes. Mycological Research 107: 206–212.

Atkins SD, Clark IM, Pande S, Hirsch PR, Kerry BR, 2005. The use ofreal-time PCR and species-specific primers for the identifica-tion and monitoring of Paecilomyces lilacinus. FEMS MicrobiologyEcology 51: 257–264.

Atkins SD, Clark IM, Sosnowska D, Hirsch PR, Kerry BR, 2003b.Detection and quantification of Plectophaerella cucumerina,a potential biological control agent of potato cyst nematodes,by using conventional PCR, real-time PCR, selective media andbaiting. Applied and Environmental Microbiology 69: 4788–4793.

Avis TJ, Hamelin RC, Belamger RR, 2001. Approaches to molecularcharacterization of fungal biocontrol agents: some casestudies. Canadian Journal of Plant Pathology 23: 8–12.

Chandrawathani P, Jamnah O, Adran M, Waller PJ, Larsen M,Gillespie AT, 2004. Field study on the biological control ofnematode parasites of sheep in the tropics, using microfungusDuddingtonia flagrans. Veterinary Parasitology 120: 177–187.

Chandrawathani P, Jamnah O, Waller PJ, Hoglund J, Larsen M,Zahari WM, 2002. Nematophagous fungi as a biological controlagent for nematode parasites of small ruminants in Malaysia:a special emphasis on Duddingtonia flagrans. VeterinaryParasitology 33: 685–696.

Chartier C, Pors I, 2003. Effects of nematophagous fungus,Duddingtonia flagrans, on larval development of goat parasiticnematodes: plot study. Veterinary Research 34: 221–230.

Cooke RC, 1969. Two nematode-trapping hyphomycetes,Duddingtonia flagrans gen. et comb. nov. and Monacrosporiummutabilis sp. nov. Transactions of the British Mycological Society53: 315–318.

Duddington CL, 1950. A new predacious species of Trichothecium.Transactions of the British Mycological Society 32: 284–287.

Durand DT, Boshoff HM, Micheal LM, Krecek RC, 2005. Survey ofnematophagous fungi in South Africa. Onderstepoort Journal ofVeterinary Research 72: 185–187.

Faedo M, Barnes EH, Dobson RJ, Waller PJ, 1998. The potential ofnematophagous to control the free-living stages of nematodeparasites of sheep: pasture plot study with Duddingtoniaflagrans. Veterinary Parasitology 76: 129–135.

Fontenot ME, Miller JE, Pena MT, Larsen M, Gillespie A, 2003.Efficiency of feeding Duddingtonia flagrans chlamydospores tograzing ewes on reducing availability of parasitic nematodeslarvae on pasture. Veterinary Parasitology 118: 203–213.

Ghahfarokhi SM, Abyaneh MR, Bahadori SR, Eslami A, Zare A,Ebrahimi M, 2004. Screening of soil and sheep samples forpredacious fungi: isolation and characterisation of the

nematode-trapping fungus Arthrobotrys oligospora. Iranian Bio-medical Journal 8: 135–142.

Gray NF, 1983. Ecology of nematophagous fungi: distribution andhabitat. Association of Applied Biologists 102: 501–509.

Hirsch PR, Mauchline TM, Mendum TA, Kerry B, 2000. Detectionof nematophagous fungus Verticillium chlamydosporium innematode-infested plant roots using PCR. Mycological Research104: 435–439.

Larsen M, 1999. Biological control of helminths. InternationalJournal of Parasitology 29: 139–146.

Larsen M, Faedo M, Waller PJ, 1994. The potential of nematoph-agous fungi to control the free-living stages of nematodeparasites of sheep: survey for the presence of fungi in freshfaeces of grazing livestock in Australia. Veterinary Parasitology53: 275–281.

Larsen M, Faedo M, Waller PJ, Hennessy DR, 1998. The potential ofnematophagous fungi to control the free-living stages ofnematode parasites of sheep: studies with Duddingtoniaflagrans. Veterinary Parasitology 76: 121–128.

Larsen M, Wolstrup J, Henriksen SA, Gronvold J, Nansen P, 1992. Invivo passage through calves of nematophagous fungi selectedfor biocontrol of parasitic nematodes. Journal of Helminthology66: 137–141.

Mendoza de Gives P, Crespo JF, Rodriguez DH, Prats VV,Hernandez EL, Fernandez GEO, 1998. Biological control ofHaemonchus contortus infective larvae in ovine faeces byadministering an oral suspension of Duddingtonia flagranschlamydospores to sheep. Journal of Helminthology 72:343–347.

SAS Institute, Inc., 2000. SAS/STAT User’s Guide, Version 8. SASInstitute, Inc., Cary, North Carolina, USA.

Skipp RA, Yeates GW, Chen LY, Glare TR, 2002. Occurrence,morphological characteristics and ribotyping of New Zealandisolates of Duddingtonia flagrans, a candidate for biocontrol ofanimal parasitic nematodes. New Zealand Journal of AgriculturalResearch 45: 187–196.

Terrill TH, Larsen M, Samples O, Husted S, Miller JE, Kaplan RM,Gelaye S, 2004. Capability of nematode-trapping fungus Dud-dingtonia flagrans to reduce infective larvae of gastrointestinalnematodes in goat faeces in the south-eastern United States:dose titration and dose time interval studies. VeterinaryParasitology 120: 285–296.

Turenne CY, Sanche SE, Hoban DJ, Karlowsky JA, Kabani AJ,1999. Rapid identification of fungi by using the ITS2genetic region and an automated fluorescent cappilaryelectrophoresis system. Journal of Clinical Microbiology 37:1846–1851.

Waller PJ, Larsen M, Faedo M, Hennessy DR, 1994. The potential ofnematopaghous fungi to control the free-living stages ofnemtode parasites of sheep: in vitro and in vivo studies.Veterinary Parasitology 51: 289–299.

Waller PJ, Schwan O, Ljungstorm B-L, Rydzik A, Yeates GW,2004. Evaluation of biological control of sheep parasitesusing Duddingtonia flagrans under commercial farmingconditions on the island of Gotland, Sweden. VeterinaryParasitology 126: 299–315.