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Journal of Invertebrate Pathology 95 (2007) 125–139

INVERTEBRATE

PATHOLOGY

Distribution of the entomopathogenic nematodesfrom La Rioja (Northern Spain)

Raquel Campos-Herrera a, Miguel Escuer a, Sonia Labrador a, Lee Robertson a,Laura Barrios b, Carmen Gutierrez a,*

a Departamento de Agroecologıa, Centro de Ciencias Medioambientales (CCMA,CSIC), c/ Serrano 115 bis, 28006 Madrid, Spainb Departamento de Estadıstica, Centro Tecnico de Informatica (CTI,CSIC), c/ Pinar 19, 28006 Madrid, Spain

Received 22 December 2006; accepted 8 February 2007Available online 22 February 2007

Abstract

Entomopathogenic nematodes (EPNs) distribution in natural areas and crop field edges in La Rioja (Northern Spain) has been stud-ied taking into account environmental and physical–chemical soil factors. Five hundred soil samples from 100 sites of the most repre-sentative habitats were assayed for the presence of EPNs. The occurrence of EPNs statistically fitted to a negative binomial distribution,which pointed out that the natural distribution of these nematodes in La Rioja was in aggregates. There were no statistical differences(p 6 0.05) in the abundance of EPNs to environmental and physical–chemical variables, although, there were statistical differences in thealtitude, annual mean air temperature and rainfall, potential vegetation series and moisture percentage recovery frequency. Twenty-sevensamples from 14 sites were positive for EPNs. From these samples, twenty isolates were identified to a species level and fifteen strainswere selected: 11 Steinernema feltiae, two S. carpocapsae and two S. kraussei strains. S. kraussei was isolated from humid soils of cooland high altitude habitats and S. carpocapsae was found to occur in heavy soils of dry and temperate habitats. S. feltiae was the mostcommon species with a wide range of altitude, temperature, rainfall, pH and soil moisture, although this species preferred sandy soils.The virulence of nematode strains were assessed using G. mellonella as insect host, recording the larval mortality percentage and the timeto insect die, as well as the number of infective juveniles produced to evaluate the reproductive potential and the time tooks to leave theinsect cadaver to determinate the infection cycle length. The ecological trends and biological results are discussed in relationship withtheir future use as biological control.� 2007 Elsevier Inc. All rights reserved.

Keywords: Steinernema carpocapsae; Steinernema feltiae; Steinernema kraussei; Biology; Distribution; Ecology; Entomopathogenic nematodes; Spain;Steinernematidae

1. Introduction

Entomopathogenic nematodes (EPNs) from Hetero-rhabtidae and Steinernematidae families have a symbioticassociation with enteric bacteria, forming the complexes:Xenorhabdus-Steinernematidae and Photorhabdus-Hetero-rhabditidae. These symbiotic associations result highly vir-ulent to insects, killing them rapidly (Boemare, 2002;Boemare et al., 1997). Entomopathogenic nematodes are

0022-2011/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jip.2007.02.003

* Corresponding author. Fax: +34 915640800.E-mail address: carmen.g@ccma.csic.es (C. Gutierrez).

widely distributed in soils throughout the world (Adamset al., 2006; Hominick, 2002; Hominick et al., 1996), andare considered one of the best non-chemical alternativesto insect pest control due to their ability to actively locateinsect-hosts as well as their high reproductive potential,capacity for mass production and the fact that they areharmless to vertebrates and plants (Burnell and Stock,2000; Gaugler, 2002; Gaugler and Kaya, 1990; Kaya andGaugler, 1993).

The application of non-native EPNs as biocontrolagents is used worldwide. Since environmental condi-tions influence survival, virulence and reproductive

126 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

potential of the EPNs strains, the efficacy of biologicalcontrol programs could be decreased. Many countriesand researchers are concerned about release of non-native nematodes, because they may have negativeeffects on non-target organisms and may partially orcompletely displace endemic EPNs (Bathon, 1996; Ehlersand Hokkanen, 1996; Lynch and Thomas, 2000; Millarand Barbercheck, 2001). In order to increase the efficacyof biological control programs and hence reducing theenvironmental risks, several countries are developingsurveys to isolate EPNs strains adapted to local ecolog-ical conditions, thereby restricting non-native nematodeimportations.

Recently, many surveys have been conducted in tem-perate areas of Europe: Austria (Hozzank et al., 2003)Belgium (Midituri et al., 1997), Bulgaria (Shishiniovaet al., 1998, 2000), Czechoslovakia (Mracek et al.,1999a, 2005), Denmark (Nielsen and Philipsen, 2003),Germany (Sturhan and Ruess, 1999), Poland (Bednarek,1998), Russia (Ivanova et al., 2000), Slovakia (Sturhanand Liskova, 1999), Switzerland (Steiner, 1996) and Uni-ted Kingdon (Gwynn and Richardson, 1996). Howeverthere is little information available about on EPNs fromMediterranean countries: Egypt (Shamseldean and Abd-Elgawad, 1994) Greece (Menti et al., 1997), Italy (Tarascoand Triggiani, 1997; Triggiani and Tarasco, 2000), Pales-tinian Territories (Iraki et al., 2000), Spain (De Doucetand Gabarra, 1994; Garcıa del Pino, 2005; Garcıa delPino and Palomo, 1996a,1997), and Turkey (Haziret al., 2003; Kepenekci, 2002; Susurluk et al., 2001,2003). Furthermore, studies of new strains about theirbiology, virulence and habitat preference can improvethe efficacy of field applications, thus it can be selectedvirulent strains with environmental and physical–chemicalsoil requirements compatible with the site for the EPNapplication. In order to contribute to the knowledgeabout these organisms for regional biological control pro-grams, the aim of this survey is to study the distribution,ecological requirements and virulence of EPNs from LaRioja (Northern Spain).

2. Material and methods

2.1. Samples collection and soil analysis

The survey was carried out from March to April of2003. The most representative areas were selected takinginto account environmental and physical–chemical soilfactors. Bioclimatic region in La Rioja include 10 potentialvegetation series (Fig. 1a): Oromediterranean region: 1,Vaccinio myrtilli-Junipereto nanae S.; Supramediterraneanregion: 2, Ilici-Fageto S.; 3, Festuco heterophyllae-

Querceto pyrenaicae S.; 4, Luzulo forsteri-Querceto pyrenai-

cae S.; 5, Junipero oxycedri-Querceto rotundifoliae S.; 6,Cephalanthero longifoliae-Querceto faginiae S.; 7, Juniperothuriferae-Querceto rotundifoliae S.; 8, Spiraeo obovaeta-

Quercetum rotundifoliae S.; and Mesomediterranean

region: 9, Asparago acutifolii-Quercetum rotundifoliae S.;10, Rhamno lycioidis-Quercetum cocciferae S. (Rivas-Martı-nez, 1987; Rivas-Martınez et al., 2001,2002). Soil type wasevaluated following the studies of Guerra and Monturiol(1970) and the Soil Survey Staff (1994) (Fig. 1b) obtaining11 soil types: 1, Haplic Calcisol; 2, Calcaric Cambisol; 3,Dystric Cambisol; 4, Calcaric Fluvisol; 5, Lithic Leptosol;6, Rendzic Leptosol; 7, Calcic Chromic, Luvisol; 8, Calca-ric Phaeozem; 9, Calcaric Regosol; 10, Dystric Regosol and11, Humic Cambisol. Data of annual average air tempera-ture and rainfall were recorded from the maps of Govern-ment of La Rioja (2001a,b) (Fig. 1c and d) and the altitudewas assessed in situ using the GPS system GARMIN�. Atotal of 500 soil samples were colleted from 100 sites partic-ularly those with a high incidence of insect populations(Hominick, 2002), such as natural areas (no. sites = 43),annual crop field edges of cereal and horticultural crops(no. = 38) and perennial crop field edges of fruit orchardand vineyards (no. = 19). Sampling habitats were classifiedas: natural grassland (NG), natural woodland (NW), natu-ral woodland scrub (NWS), natural scrub grassland(NSG), natural scrub (NS), annual crop edge-scrub (AS),perennial crop edge-scrub (PS), annual crop edge-scrub-grassland (ASG), annual crop edge-woodland-scrub(AWS), perennial crop edge-scrub-grassland (PSG), annualcrop edge-grassland (AG), and perennial crop edge-grass-land (PG).

Five soil samples were collected from each site, at leasttwo meters apart in transect sampling formation into an18–20 m2 plot. Each soil sample (approximately 1 kg) wastaken at a depth of 2–20 cm (Campbell et al., 1998; Yos-hida et al., 1998), placed in polyethylene bags to preventwater loss, transported to the laboratory under refrigeratedconditions, and stored at 12–15 �C until EPN evaluation.Each sampling site was characterised by bioclimatic regionswith the potential natural vegetation and soil types, habi-tat, altitude, annual average temperature and rainfall.For each soil sample, a 200 g portion was analysed for dif-ferent edaphological variables. Soil moisture was calcu-lated as: %H = (fresh weigh � dry weigh/dry weigh) ·100, where dry weigh was obtained after over dry soil at70 �C for 5 days. The pH was measured from a 1:2.5soil/mQ-water suspension. The sand, silt and clay contentswere evaluated by Bouyoucos method (MAPA, 1975). Inorder to compare the soil moisture trough different soil tex-ture types, the field capacity (FC) at pF 2.7 and wiltingpoint (WP) at pF 4.2 were determined by Richards’ method(Duchaufour, 1975) to calculate the available water in soil(FC-WP) in those positive samples for EPN presence. Allanalyses were performed by the Analytical Service of theEnvironmental Sciences Centre.

2.2. Isolation of entomopathogenic nematodes

Soil samples were roughly mixed and stored at roomtemperature for 24 h previous to the test for EPN occur-rence, using the insect baiting technique (Bedding and

Fig. 1. Environmental and edaphoecological characterization from La Rioja. (a) Potential vegetation series: 1, Vaccinio myrtilli-Junipereto nanae S.; 2,Ilici-Fageto S.; 3, Festuco heterophyllae-Querceto pyrenaicae S.; 4, Luzulo forsteri-Querceto pyrenaicae S.; 5, Junipero oxycedri-Querceto rotundifoliae S.; 6,Cephalanthero longifoliae-Querceto faginiae S.; 7, Junipero thuriferae-Querceto rotundifoliae S.; 8, Spiraeo obovaeta-Quercetum rotundifoliae S.; 9, Asparago

acutifolii-Quercetum rotundifoliae S.; 10, Rhamno lycioidis-Quercetum cocciferae S. (b) Soil type: 1, Haplic Calcisol; 2, Calcaric Cambisol; 3, DystricCambisol; 4, Calcaric Fluvisol; 5, Lithic Leptosol; 6, Rendzic Leptosol; 7, Calcic Chromic Luvisol; 8, Calcaric Phaeozem; 9, Calcaric Regosol; 10, DystricRegosol and 11, Humic Cambisol. (c) Annual average temperature. (d) Annual average rainfall.

R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139 127

Akhurst, 1975). Three soil subsamples from each samplewere placed in 90 mm · 15 cm diameter Petri dishes (Stuartand Gaugler, 1994), and the soil moisture was adjusted, ifrequired, by adding mQ-water (Milli-Q Water System, Mil-lipore S.A., Molsheim, France). Ten last instars larvae ofGalleria mellonella L. (Lepidoptera: Pyralidae) were placedon the soil surface of each subsamples. Petri dishes wereinverted and incubated in darkness at 24 ± 1 �C with55% relative humidity (R.H.). Insects were recovered after6 days and the dead larvae were washed and individuallyplaced on White traps to allow the emergence of infectivejuveniles stage (White, 1927). In the case of negativeresults, the isolation was repeated two-fold to confirmresults of the first experiment. Emerging infective juvenile(IJs) from White traps was used to infect fresh G. mellonella

to produce nematodes for establishment of cultures andproduce individuals for identification purposes.

2.3. Identification of entomopathogenic nematodes

Morphological and biometric studies were carried outwith 25 juveniles and 25 first generation males from eachisolate (Hominick et al., 1997; Stock et al., 2000). Nema-todes were fixed and processed to glycerine following themethod described by Steinhorst (1966). Semipermanentslides were prepared following the procedures describedby Gomez et al. (2004). The characters for the identifica-

tion were: total length, greatest width, distance from ante-rior end to excretory pore, distance from anterior end tonerve ring, distance from anterior end to base oesophagus,tail length, length of the spicules, length of the gubernacu-lums, and values of ratio a, b, c, d, e, GS and SW (Adamsand Nguyen, 2002; Hominick et al., 1997). As recom-mended by Stock and Reid (2004), the species identificationbased on morpho-biometric studies was confirmed bymolecular analysis, using the PCR amplification conditionsdescribed by Hominick et al. (1997) and the primers 18Sand 26S described by Vrain et al. (1992). The method forPCR-RFLP analysis of the ITS region was carried out asdescribed by Campos-Herrera et al. (2006). The restrictionenzymes used were AluI, HinfI, RsaI and HhaI, due to theirutility for these studies (Reid and Hominick, 1998), EcoRIand HaeIII, frequently used by others authors (Cutler andStock, 2003; Hominick et al., 1997; Mracek et al., 2003;Reid et al., 1997; Stock and Koppenhofer, 2003; Yoshida,2004) and DraI, HincII y MnlI used by Nguyen (2003) andYoshida (2003).

2.4. Entomopathogenic nematodes distribution in relation to

environmental and soil characteristics of La Rioja

To explore the ecology of native EPN isolates, the occur-rence were assessed as recovery frequency (no. positivesamples/no. total samples) and abundance (no. positive

128 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

sites/no. total sites) (Liu and Berry, 1995), expressed as per-centage. The distribution of the positive samples in each sitewas adjusted to a negative binomial distribution (Ludwigand Reynolds, 1988) using the XLSTAT 2006 program.The occurrence of EPN was related to the environmentalvariables, vegetation (potential vegetation series and habi-tat) and soil characteristics. To study the influence of thosevariables, Crostabs and Pearson’s v2 tests at p 6 0.05 levelwere carried out to asses significant differences in EPNoccurrence. Two hierarchical cluster analysis betweenEPN strains and environmental variables (altitude, averageof annual temperature and rainfall, potential vegetation ser-ies, habitat and soil type) and physical–chemical soil vari-ables (available water, pH, and percentage of sand, siltand clay), were carried out to explore the associationsamong different EPN strains, using squared Euclidean dis-tance and the values were standardized with Z transforma-tion. Statistics analysis was performed using SPSS 13.0software for Windows XP.

2.5. Virulence, infection cycle length and infective juveniles

production of entomopathogenic nematode native strains

The bioassays were carried out using strains that couldbe maintained and reproduced in laboratory conditions.In this survey, when two or more isolated from the samesite were identified as species level and all were corre-sponded to the same species, only was taken into accountone strain in this site, because it was considered that in thissite the natural occurrence of this EPN species had a widedistribution in the transect than other which only one iso-lated identified to species level per site. The bioassay wasdesigned to evaluate the virulence, infection cycle lengthand IJs production of EPN isolates. A mQ-water suspen-sion of 4900 IJs/mL of each native EPN strain was pre-pared, and 300 lL were applied in two filter paper diskscovering button and lid of 5 cm diameter Petri dishes,obtaining 75 IJs/cm2. Five Petri dishes were used in eachassay, and filter paper disks of control treatment weremoistened with 300 lL of mQ-water. Five G. mellonella lar-vae of 200 ± 13.5 mg fresh weigh were placed in each Petridish and incubated in the dark at 22 ± 1 �C and 55% R. H.The bioassay was repeated 3 times. Insect mortality wasdaily recorded, and dead larvae were washed, individuallyplaced in White traps and incubated at the same conditionsas above. Cadavers were daily observed recording the IJsemergence. Infective juveniles were counted after 10 daysto obtain the total IJs production (Wang and Bedding,1996), and reproductive potential was expressed as no.IJs/mg larva. The days to dead larvae and percent of larvalmortality were recorded to estimate the native EPN viru-lence. The infection cycle length was assessed countingnumber of days from start infection until the first IJs emer-gence. Insect mortality was corrected by Abbott’s formula(Abbott, 1925), and mortality percentages were arc sinetransformed, previous statistical analysis of biological databy SPSS 13.0 software for Windows XP. ANOVA analysis

and Tukey’s test at p 6 0.05 was carried out to assess differ-ences among EPN strains. One hieratical cluster analysisbetween EPN strains and variables: mortality percentage,days to dead larvae, days to IJs emergence and reproduc-tive potential was carried out to establish associationgroups among EPN strains, using squared Euclidean dis-tance and the values were standardized with Ztransformation.

3. Results

3.1. Entomopathogenic nematode distribution in relation to

environmental variables and soil physical–chemical

characteristic from La Rioja

Entomopathogenic nematodes were recovered from 27of 500 soil samples (5.4%) of 14 of 100 sites (14%) spreadwithin La Rioja (Fig. 2), although only 20 isolates couldbe identified at species level. The study of positive samplesfrequency showed that most the sites were negative forEPNs occurrence, some of them had only 1 positive sam-ple, and very few sites had 2 or 5 positive samples. Thusthe positive samples frequency adjusted to a negative bino-mial distribution, showing a patchiness distribution ofEPN.

Table 1 shows the abundance, recovery frequency andnumber of identified samples to species of EPN to eachenvironmental variable. Although EPN abundance didnot show significant differences among altitude ranges,the recovery frequency was significantly different(v2 = 28.410, df = 5, p = 0.001), recording the highestEPN presence at 501–800 m. Below 500 m of altitude therewere not recovered EPNs. Entomopathogenic nematodeabundance was not significantly different in annual averagetemperature or rainfall, although their recovery frequencywas significantly affected by temperature (v2 = 40.513,df = 7, p = 0.001) and rainfall (v2 = 25.038, df = 7,p = 0.003), recovering more nematodes from sites with arange of 9–12 �C, and rainfall ranging 400–700 mm. Thesoil type did not affect nematode occurrence, although sig-nificant differences were observed in the potential vegeta-tion series recovery frequency (v2 = 26.251, df = 9,p = 0.005), and EPN was not recovered from Spiraeo

obovaeta-Quercetum rotundifoliae S. and Rhamno lycioi-

dis-Quercetum cocciferae S. series. Nematode occurrencedoes not significantly affected by habitat type, but nema-todes were not recovered from scrub vegetation fromannual and perennial crop edges. Table 2 shows the abun-dance, recovery frequency and number of identified sam-ples to species of EPN to each physical–chemicalvariables. Soil texture and pH did not significantly affectEPNs occurrence, although they are easier isolated fromsoils with loam, sandy loam, silt, clay loam or loam/sandyloam textures and pH range 5.1–8.0. However the %humidity significantly affected nematode recovery fre-quency (v2 = 9.687, df = 3, p = 0.021), recovering few iso-lates from soil samples 610% humidity.

Fig. 2. EPNs sampling sites map in La Rioja (Northern Spain). d, sites with nematodes. , sites without nematodes. The numbered sites indicate thelocation of sites with nematodes.

R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139 129

3.2. Entomopathogenic nematodes species and their

ecological trends

Twenty-seven soil samples were positive for Steinerne-matids populations of EPNs in this survey, although 7 iso-lates could not be reproduced to be identified at the specieslevel. Twenty isolated were identified as: 11 Steinernema

feltiae (Filipjev), 7 S. carpocapsae (Weiser), 2 S. krausei

(Steiner). Morphological and biometrical characteristicsof isolates are in agreement with information given byAdams and Nguyen (2002). A PCR-RFLP analysis wascarried out to confirm the species identification (Fig. 3).All the S. feltiae isolates belonged to RFLP type A2 asshowed the profile obtained from ITS digestion with HinfIand RsaI enzymes (Hominick et al., 1997; Reid et al.,1997). The S. carpocapsae RFLP profile was the sameobtained by several authors (Cutler and Stock, 2003; Haziret al., 2003; Reid and Hominick, 1998; Reid et al., 1997;Stock et al., 1998) after the digestion with the AluI, EcoRI,HaeIII, HhaI, HinfI and RsaI enzymes. The S. kraussei

RFLP profile obtained from ITS digestion with AluI, HinfIy HhaI was the same recorded by Reid and Hominick(1998) and Reid et al. (1997), and EcoRI produced twobands as in Hominick et al. (1997).

Environmental and soil characteristics of positive sitesand samples for EPN species isolated in La Rioja areshowed in the Table 3. Steinernema feltiae was the mostabundant species representing 55% of positive samples

identified. It was highly distributed in sites ranging 639–1754 m altitude, 5–12 �C and 400–1100 mm annual averagerainfall, with soil types: Calcaric, Dystric and HumicCambisol, Calcaric and Dystric Regosol, Calcaric Pha-eozem, and Haplic Calcisol. The nematode was present inthe Oromediterranean region with Vaccinio myrtilli-

Junipereto nanae S. as potential vegetation series, in theSupramediterranean region with Ilici-Fageto S., Luzulo

forsteri-Querceto pyrenaicae S., Junipero oxycedri-Querceto

rotundifoliae S., Junipero thuriferae-Querceto rotundifoliaeS., Cephalanthero longifoliae-Querceto faginiae S. andAsparago acutifolii-Quercetum rotundifoliae S. series fromthe Mesomediterranean region. It was isolated from natu-ral areas and annual and perennial crop field edges withgrassland, scrub or woodland vegetation. The availablewater in soil samples ranged as 5.88–16.58, the pH as4.9–8.3, and the soil texture was sandy loam, loam orloam/sandy loam. Thus, S. feltiae can be considered as ahighly adaptable species.

Steinernema carpocapsae was finding in 35% of positivesamples associated to crop valley areas. It was recovered ingrassland habitats of annual crop edges 677–602 m alti-tude, 11 �C annual average temperature, and 500–400 mmannual average rainfall. The potential vegetation was Cep-

halanthero longifoliae-Querceto faginiae S. associated toSupramediterranean region, with soil types: CalcaricCambisol and Calcaric Phaeozem. Soil samples showedranges from pH 7.6 to 7.9 and available water from 11.01

Table 2Distribution of EPNs in La Rioja at different physical–chemical variables

Categories(total sites)

Abundance(%)

Recoveryfrequency (%)

No. of identifiedsamples to species

Texture soil type

Loam (48) 8.3 3.3 5a

Sandy Loam(22)

27.3 9.1 7a

Silt (4) 25.0 5.0 0Clay Loam

(12)16.7 11.7 7

Loamy Sand(2)

0 0 0

Silty ClayLoam (1)

0 0 0

Clay (3) 0 0 0Silt Loam (2) 0 0 0Loam/Slit

Loam (2)0 0 0

Loam/ClayLoam (2)

0 0 0

Loam/SandyLoam (2)

50.0 10.0 1

pH

65.0 (7) 14.3 8.6 2a

5.1–6.0 (13) 23.1 6.2 26.1–7.0 (7) 28.6 11.4 27.1–8.0 (50) 14.0 6.0 13a

P8.1 (23) 4.3 0.9 1

Moisture %

610% (16) 18.8 3.8 311–20% (54) 7.4 3.7 921–30% (20) 20.0 7.0 5a

P31% (10) 30.0 14.0 3a

a Presence of two EPN species in the same site, in different samples.

Table 1Distribution of EPNs in La Rioja at different environmental variables

Categories(total sites)

Abundance(%)

Recoveryfrequency (%)

No. of identifiedsamples to species

Altitude (masl)

6500 (15) 0 0 —501–800 (42) 21.4 7.1 14801–1100 (33) 6.1 1.8 11101–1400 (4) 25.0 15.0 2a

1401–1700 (3) 33.3 26.7 2a

P1701 (3) 33.3 13.3 1

Annual averages temperature (�C)

66 (4) 50.0 30.0 3a

7 (7) 0 0 —8 (4) 0 0 —9 (9) 22.2 8.9 2a

10 (25) 12.0 2.4 311 (20) 25.0 11.0 1012 (20) 10.0 3.0 213 (11) 0 0 —

Annual averages rainfall (mm)

300 (9) 0 0 —400 (26) 7.7 4.6 6500 (28) 25.0 7.2 8600 (16) 6.3 1.3 1700 (6) 33.3 13.3 2a

800 (1) 0 0 —900 (8) 0 0 —P1000 (6) 33.3 20.0 3a

Soil typeb

1 (13) 23.1 4.6 32 (24) 8.3 5.0 4a

3 (15) 13.3 5.3 2a

4 (3) 0 0 —5 (3) 0 0 —6 (1) 0 0 —7 (3) 0 0 —8 (9) 22.2 13.3 69 (13) 7.7 1.5 110 (11) 27.3 10.9 311 (5) 20.0 4.0 1

Vegetation Serie typec

1 (2) 50.0 20.0 12 (7) 14.3 11.4 2a

3 (10) 10.0 2.0 04 (14) 21.4 8.6 4a

5 (10) 10.0 2.0 16 (15) 26.7 13.3 97 (9) 11.1 2.2 18 (2) 0 0 —9 (25) 08.0 1.6 210 (6) 0 0 —

Habitat typed

NG (5) 20.0 20.0 1NW (8) 12.5 10.0 2a

NWS (19) 10.0 4.0 1NSG (13) 7.7 1.5 0NS (7) 14.3 8.6 2a

AS (5) 0 0 —PS (5) 0 0 —ASG (12) 8.3 3.3 1AWS (5) 20.0 4.0 1PSG (6) 16.7 3.3 1

Table 1 (continued)

Categories(total sites)

Abundance(%)

Recoveryfrequency (%)

No. of identifiedsamples to species

AG (16) 25.0 11.3 9PG (8) 25.0 5.0 2

a Presence of two EPN species in the same site, in different samples.b Soil type code correspond with Fig. 1b.c Potential vegetation code correspond with Fig. 1a.d Abbreviations are corresponded with Natural (N), Annual crop edge

(A), Perennial crop edge (P), Grassland (G), Woodland (W), Scrub (S).

130 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

to 13.48. This species is remarkable for being isolated inagricultural clay loam soils with 30–32% clay content.

Steinernema kraussei was isolated from 2 soil samples,representing 10% of positive samples. It was found associ-ated to natural woodland and scrub habitat areas at 1115–1596 m altitude, 6–9 �C annual average temperature and700–1000 mm annual average rainfall. It was associatedto potential vegetation Ilici-Fageto S. and Luzulo forsteri-Querceto pyrenaicae S. from Supramediterranean region.The soil types were Calcaric Cambisol and Dystric Camb-isol respectively. The pH was higher, ranging from slightlybasic (7.8) to acid (4.9), 13.09 and 11.07 of available water,and soil texture were loam and sandy loam, respectively.

Fig. 3. RFLP-PCR analysis of Steinernematid species from La Rioja, digested with nine restriction enzymes in a 2% (w/v) TBE buffered agarose gel. M1marker 1 kb ladder (band sizes 10,000–500 base pairs) and M2 marker 100 bp ladder (band sizes 1000–80 base pairs). (a) RFLP-PCR S. feltiae type. (b)RFLP-PCR S. carpocapsae type. (c) RFLP-PCR S. kraussei type.

R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139 131

Four association groups were established between EPNstrains occurrence and altitude, annual average tempera-ture and rainfall, potential vegetation series, habitat andsoil type (Fig. 4a). Strains S. feltiae 88A and S. kraussei

88C strains were included in group 1, isolated from a nat-ural woodland habitats at 1115 m altitude, 9 �C annualaverage temperature, 700 mm annual average rainfall, Dys-tric Cambisol soil type and Luzulo forsteri-Querceto pyre-

naicae S. as potential vegetation type. Group 2 includedby 58C of S. kraussei, and 66 and 58A of S. feltiae strains,which were recovered at high altitude (1569–1754 m) innatural grassland and woodland habitats, 5–6 �C annualaverage temperature and 1000–1100 mm annual average

rainfall. The potential vegetation was representative ofOromediterranean (Vaccinio myrtilli-Junipereto nanae S.)and Supramediterranean (Ilici-Fageto S.) regions and soiltypes were Dystric Regosol and Calcaric Cambisol. Group3 is included by S. carpocapsae 98 strain and S. feltiae 23,38 and 75 strains. The strains were isolated at 677–699 maltitude 10–12 �C annual average temperature and 400–500 annual average rainfall from annual and perennial cropedges with grassland and woodland-scrub habitats. Theassociated potential vegetation types were Cephalanthero

longifoliae-Querceto faginiae S., Junipero thuriferae-Quer-ceto rotundifoliae S., Asparago acutifolii-Quercetum rotun-

difoliae S, and soil types were Haplic Calcisol and

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Fig. 4. Hierarchical cluster analysis between EPN species occurrence andenvironmental, and soil type variables. (a) Environmental variables(altitude, annual mean temperature and rainfall, potential vegetationseries, habitat and soil type). (b) Physical–chemical soil variables(available water, pH, and percentage of sand, silt and clay. Strain codenumber in blood, corresponded with S. kraussei strain, underlined, with S.

carpocapsae and normal, with S. feltiae.

132 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

Calcaric Cambisol. The group 4 include 6 strains: S. carpo-

capsae 96 strain and S. feltiae 17, 37, 63, 91 and 100 strains,which were recovered in natural woodland-scrub habitatand in annual and perennial crop edges of grassland, wood-land, scrub-grassland and woodland-scrub habitats, rang-ing their altitude, annual average temperature andrainfall as: 602–812 m, 10–12 �C and 400–600 mm,respectively. The potential vegetation types were Luzuloforsteri-Querceto pyrenaicae S., Junipero oxycedri-Querceto

rotundifoliae S. and Cephalanthero longifoliae-Querceto

faginiae S. and soil types Calcaric Phaeozem, CalcaricRegosol, Dystric Regosol and Humic Cambisol.

Fig. 4b shows the dendrogram which presents theassociation between EPN strain occurrence and the physi-cal–chemical soil variables (available water, pH, and per-centage of sand, silt and clay). Steinernema feltiae 66 and23 strains are regarded as groups A and B out of range,both groups were finding in sandy loam soil texture, pH

Fig. 5. Hierarchical cluster analysis between EPN species and biologicaldata of virulence (days to dead larvae and percentage of larval mortality),days to JIs emergence and reproductive potential. Strain code number inblood, corresponded with S. kraussei strain, in underlined, with S.

carpocapsae and normal, with S. feltiae.

R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139 133

6.6 and 7.9, but with very different available water content:16.58 and 5.88. The S. kraussei 58C, S. carpocapsae 96 and98, and S. feltiae 58A, 63 and 100 strains were joining ingroup 1. The pH appeared ranging 6.5–8.3 and availablewater from 10.53 to 13.90. Steinernema feltiae and S. kraus-

sei strains were isolated from loamy soils with 15–18% claycontent; however, S. carpocapsae was isolated from clayloam soils with high clay content (30–32%). The group 2include S. kraussei 88C and S. feltiae 17, 23, 37, 38, 66,75, 88A and 91 strains. They were isolated from soils withpH range 4.9–7.8 and 7.58–11.77 of available water con-tent, being the soil texture loam, sandy loam, and loam/sandy loam with a 5–13% clay content.

3.3. Virulence, infection cycle length and infective juveniles

production of entomopathogenic nematode native strains

The bioassay results carried out with native strains areshown in Table 4. The virulence was assessed as percentageof larval mortality and time to die larvae from nematodeinfection. The virulence was very similar and significantdifferences were not observed among strains, mortality per-centage (86.25–97.62%) and days to dead larvae (1.94–2.00days). Significant differences were observed in the infectioncycle length, that was measured from larval exposure to JIsemergence (F = 19.635, gl = 12, p = 0.000). The S. feltiae

cycle ranged from 8.44 to 9.84 days, 10.23–10.36 days forS. carpocapsae and 12.83 days for the S. kraussei 88C strainwhich is the largest infection cycle. Significant differenceswere also observed in the reproductive potential(F = 33.050, gl = 12, p = 0.000). The S. carpocapsae 96and 98 strains showed the highest values with 626.29 and831.72 IJs/mg larvae, respectively, in S. feltiae these valueranged from 124.94 to 444.23 IJs/mg larvae, showingS. kraussei 88C strain the lowest reproductive potential84.07 IJs/mg larvae. Fig. 5 shows the dendogram of theassociation groups hierarchical cluster analysis among

Table 4Biological characteristics1,2 of native entomoathogenic nematode isolated based

Species Straincode

Days to deadlarvae

% Larvmortali

S. feltiae 17 1.94 ± 0.035 90.90 ±23 2.00 ± 0.000 88.83 ±37 2.00 ± 0.206 90.67 ±38 2.00 ± 0.000 95.22 ±58A 2.00 ± 0.000 90.91 ±63 2.00 ± 0.000 97.62 ±66 2.00 ± 0.000 86.25 ±75 2.00 ± 0.000 90.73 ±91 2.00 ± 0.000 91.65 ±100 2.00 ± 0.022 95.29 ±

S. kraussei 88C 2.00 ± 0.000 94.20 ±

S. carpocapsae 96 2.00 ± 0.000 95.73 ±98 2.00 ± 0.000 90.91 ±

1 Values are means ± SE.2 Means in a column followed by the same letter are not significantly differe

EPN strains and the biological variables. The group 1include by S. feltiae 23, 37, 58A, 66, 75, 91 and 100 strainswith the following ranging values: 1.98–2.00 days to deadlarvae, 86.25–95.29% larval mortality, 8.28–9.84 days fromthe infection cycle, and 124.94–362.20 IJs/mg larvae for thereproductive potential. The group 2 includes S. feltiae 38strain (with 2.00 days to dead larvae, 95.22% larval mortal-ity, 9.91 days from the infection cycle and 444.23 IJs/mglarvae) and S. carpocapsae 96 and 98 strains (with 2.00days to dead larvae, 95.73–90.91% larval mortality,10.36–10.23 days from the infection cycle and 626.24–831 IJs/mg larvae). Three strains were out of range: Aand C were S. feltiae 63 and 17 strains, respectively, andvalues of 2.00–1.94 days to dead larvae, 97.62–90.90% lar-val mortality, 8.77–9.57 days from the infection cycle, and

on data recorded from Galleria mellonella larvae infected with 75 JIs/cm2

alty

Days from infectionto JIs emergence

Reproductive potentialno. JIs/mg larvae

0.236 9.57 ± 0.211 bcd 196.73 ± 24.286 bcd1.426 9.84 ± 0.284 cd 225.39 ± 24.286 abcd4.864 9.33 ± 0.185 abcd 261.77 ± 20.413 bcd0.248 9.91 ± 0.232 d 444.23 ± 21.748 e5.248 9.76 ± 0.285 cd 124.94 ± 21.015 ab1.374 8.77 ± 0.164 abc 301.56 ± 44.752 cde4.087 8.44 ± 0.284 ab 362.20 ± 40.389 de2.973 8.28 ± 0.141 a 350.97 ± 26.142 de0.202 9.57 ± 0.285 cd 268.20 ± 34.509 bcd0.710 9.62 ± 0.349 cd 166.01 ± 27.316 abc

3.833 12.83 ± 0.356 e 84.07 ± 9.243 a

0.159 10.36 ± 0.181 d 626.24 ± 61.180 f5.248 10.23 ± 0.159 d 831.72 ± 63.003 g

nt, Tukey test at p 6 0.05.

134 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

301.56–196.78 IJs/mg larvae; and B with S. kraussei 88Cstrain with values of 2.00 days to dead larvae, 94.20% lar-val mortality, 12.83 days from the infection cycle, and84.07 IJs/mg larvae.

4. Discussion

The EPNs abundance, distribution and habitat prefer-ence are related to host-parasite relationships, environmen-tal conditions and soil characteristics (Barbercheck, 1992;Mracek et al., 2005; Nielsen and Philipsen, 2003; Peters,1996; Puza and Mracek, 2005). The study of EPN occur-rence, distribution and biology taking into account theenvironmental and soil characteristics in a geographicalarea provide a base knowledge to increase biological con-trol program efficacy in those region. Few studies had beencarried out on this topic through the world, although Gar-cıa del Pino and Palomo (1996a) began these kind of stud-ies in our country in Catalonia, it is the first time that EPNare bio-ecological studied in La Rioja, an important agri-cultural region in Spain with an increasing organic produc-tion (MAPA, 2005).

Over one month (March–April), 500 soil samples werecollected in order to avoid seasonal fluctuations in climaticconditions of the Mediterranean region, which affect EPNspopulations (Garcıa del Pino and Palomo, 1997) andensure a high presence of EPNs (Mracek, 1980; Puza andMracek, 2005; Rio and Cameron, 2000). The positive sam-ples distribution can be adjusted to a negative binomial dis-tribution. This natural patchiness distribution has beenrecorded by other authors (Boag et al., 1992; Bohan,2000; Cabanillas and Raulston, 1994; Campbell et al.,1998; Stuart and Gaugler, 1994). This large variabilityEPN occurrence can be also increased due to insect hostaggregations (Mracek and Becvar, 2000). Consequently,the EPNs occurrence in soil samples can be variable in dif-ferent surveys, ranging the recovery frequency from 0.7 to70.1% (Bruck, 2004; Mracek and Becvar, 2000). PreviousEPN surveys carried out in Spain showed a variable recov-ery frequency, in Catalonia 3.3% (De Doucet and Gabarra,1994) and 11.9% (Garcıa del Pino, 1994), in Galicia 26%, inMallorca Island 6.5%, and in Canary Islands 0.66–11%(Garcıa del Pino, 2005). The recovery frequency valuerecorded in La Rioja (5.4%) was also similar to some valuesobtained in other Mediterranean countries: Greece: 5%(Menti et al., 1997), Egypt: 10% (Shamseldean and Abd-Elgawad, 1994), Mediterranean Turkey region: 5,8% (Kep-enekci, 2002), Italy: 5% (Ehlers et al., 1991), 14% (Tarascoand Triggiani, 1997), and 6% or 15.5% from pinewood orholm-oak wood habitat, respectively (Triggiani and Taras-co, 2000). In La Rioja was recorded a 14% of EPNs abun-dance being this value similar to 12.2% obtained in theUnited Kingdon by Gwynn and Richardson (1996),although in most surveys are recorded higher abundancevalues: 23.0% in Catalonia (Spain) (Garcıa del Pino,1994; Garcıa del Pino and Palomo, 1996a), 23.7% in Ore-gon (USA) (Liu and Berry, 1995), 28.5% in Southeastern

USA (Shapiro-Ilan et al., 2003), 20.3% in Indonesia (Grif-fin et al., 2000) and 33% in Hungria, Estonia and Denmark(Griffin et al., 1999) and sometimes the abundance canraise to 92.3% as in New Jersey (USA) (Stuart and Gau-gler, 1994).

Environmental and soil characteristics had not signifi-cant affect on EPNs abundance; however, altitude, temper-ature, rainfall, potential vegetation series and soil humidityshowed statistical differences in their recovery frequency.Similar effect was recorded for altitude, temperature andrainfall (Constant et al., 1998; Garcıa del Pino and Palomo1996a; Mracek et al., 2005). The soil characteristics alsoinfluence the EPN occurrence being important the moisture(Garcıa del Pino and Palomo, 1996a) and texture (Black-shaw, 1988), producing more EPN isolations from sandysoils (Hazir et al., 2003; Liu and Berry, 1995; Ruedaet al., 1993). Mracek et al. (2005) suggested that clay con-tent of heavy soils could difficult the EPNs movement,affecting their recovery from soil samples. The pH alsohas importance in the EPNs occurrence as reported byConstant et al. (1998) and Stock et al. (1999). The potentialvegetation series has a significant effect nematode distribu-tion and none EPN was recovered from series 8, Spiraeo

obovaeta-Quercetum rotundifolia S., and 10, Rhamno lycioi-

dis-Quercetum cocciferae. Although the habitat did notaffect significantly the nematode occurrence, none EPNwas recovered from soils with scrub vegetation fromannual and perennial crop edges. Moreover, there weremore positive samples in human manipulated habitats thanin natural ones. This trend could be statistically improvedincreasing the sampling effort in the studied sites, betterthan increase the number soil samples per site (Stocket al., 1999). Garcıa del Pino and Palomo (1996a) and Mra-cek and Webster (1993) also observed similar trend, sug-gesting that habitat preference is related to insect hostdistribution. Natural areas have large insect diversity thanagricultural areas, although their population levels arehardly controlled by natural enemies. In highly intensivemonoculture areas can produce outbreaks of insect pestspecies susceptible to EPNs attack, which could leave thecrop field looking for shelter in the crop edge. Thus inedges the EPN population can increase more easily enhanc-ing their recovery frequency from soil samples, so that thecrop field edges could be a good natural stock for EPNspopulations.

In this survey, only the genus Steinernenma (Filipjev)was recovered. It could be probably due to the low preva-lence of Heterohabditis genus as compared with Stein-

ernenma genus, which is adapted to many environments(Hominick, 2002). This fact can be observed through Med-iterranean area too: in Catalonia (Northeast Spain) Garciadel Pino and Palomo (1996a) recorded 1.3% positive sitesfor Heterohabditis vs 22% for Steinernema, in Italy (Taras-co and Triggiani, 1997) 5.3% Heterohabditis vs 8.5% forSteinernema, in Turkey (Hazir et al., 2003) 0.7% Hetero-

habditis vs 1.4% for Steinernema. However, in some cases,when many sampling sites are adjacent to the sea, Hetero-

R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139 135

habditis genus has higher prevalence than Steinernenma

genus: 2.5% Heterohabditis vs 1.4% for Steinernema in theAzores islands (Rosa et al., 2000), 17% Heterohabditis vs

9% for Steinernema in Oregon (USA) (Liu and Berry,1995).

In the distribution surveys sometimes several isolates arenot identified to species level due to they cannot be repro-duced successfully in the laboratory (Blackshaw, 1988; Sali-nas, 2002). In this study were identified 15 strains of 3 speciesfrom 20 Steinernematids isolates, although another 7 isolatescould not be identified to species level. Our survey recovered3 steinernematids species from 500 soil samples of 100 sam-pling sites, being this recovery value similar to another sur-veys (Hominick, 2002). In Spain, Garcıa del Pino andPalomo (1996a) also isolated 3 steinernematids species from750 soil samples of 150 catalonian sites, although De Doucetand Gabarra (1994) isolated 2 Steinernematids species from60 soil samples of 1 site from these region. Similar valueswere recorded from other Mediterranean regions. Triggianiand Tarasco (2000) identified 4 EPN species from 203 Italiansites, in Turkey Kepenekci (2002) identified 2 species from 52samples of 15 sites but Hazir et al. (2003) only identified 3species from 1080 samples of 6 regions. The number of iden-tified species were very variable in others surveys, with arange to one to 13 species (Boag et al., 1992; Mracek et al.,2005; Spiridonov et al., 2004; Stock, 1995; Sturhan, 1999;Sturhan and Liskova, 1999).

In agreement with previous results obtained by Adamsand Nguyen (2002), Hominick et al. (1997), Reid et al.(1997), and Reid and Hominick (1998), Riojan steinerne-matids strains were identified as: 11 populatios of S. feltiae,2 of S. carpocapsae and 2 of S. kraussei. These species hadbeen previously isolated in our country, although also havebeen isolated S. affine, S. intermedium, S. arenarium, andsome isolates unidentified to species level of S. glasseri, S.

feltiae and S. bicornotum groups (Garcıa del Pino, 2005).S. feltiae was the most widely distributed species in La Riojaand this result is agreed with Garcıa del Pino (2005) obser-vations in other Spanish regions. Steinernema feltiae and S.

carpocapsae have a much extended distribution through theworld (Hominick, 2002). Peters (1996) suggested that thisfact could be due to these species are generalist and havea broad range of insect hosts. Studying the environmentaland soil characteristics related to distribution of these spe-cies in La Rioja, it was observed, in agreement with previ-ous results from Hominick’s revision (2002), that S. feltiaeshowed a broad distribution isolating it from fresh-humidhabitats to temperate-dry ones with grassland, scrub orwoodland vegetation from natural areas or crop field edges.The soil texture ranged from sandy to loam, a 5.88–16.58available water, and acid to basic pH. These data showthe high adaptability of this species, thus some of theirstrains, with a good virulence level against local insect pests,could be valuable to develop bioinsecticide products. Veryinteresting result were obtained with S. carpocapsae becauseboth strains were related to temperate crop field edges andheavy soils with a 30% of clay content. Mracek et al. (2005)

suggested that heavy soils decrease the nematode mobilityand thus it can affect their infectivity. Consequently, themost EPN strains used in biological control had been iso-lated from sandy soils (Griffin et al., 1991; Kung et al.,1990; Mracek et al., 2005), so the S. carpocapsae Riojanstrains could be of a great interest to produce commercialproducts to apply in heavy soils. Finally, in agreement withothers authors, S. kraussei showed a more restricted arearelated to Holartic distribution (Hominick, 2002; Mraceket al., 2005; Spiridonov et al., 2004; Stock et al., 2000)occurring in fresh-humid natural habitats with loam andsandy loam soil texture. In Spain, it was recovered fromCatalonian Pyrenees with similar habitat type (Garcıa delPino and Palomo, 1996b). In this survey, it was observedthat both S. kraussei and S. feltiae were isolated in the samesite, so it is recommended to use S. kraussei isolates for bio-insecticide development only if their virulence is higher thanS. feltiae isolates.

The bioassay carried out with Riojan strains showedthat they were highly virulent to G. mellonella larvae pro-ducing a 86.25–97.62% larval mortality in 1.94–2.00 daysafter 75 JIs/cm2 exposition. These results are in agreementwith several authors, although the dose used could changebetween assays. Caroli et al. (1996) observed a 100% of lar-val mortality in the S. feltiae UK strain using 168.4 IJs/cm2, reaching the same result after 12 h of 113 IJs/cm2

applications or 28.3 IJs/cm2 for 48 h (Ricci et al., 1996).Chen and Glazer (2005) observed that S. feltiae IS-6 strainfrom several storage mediums produced 100% larval mor-tality at 113 JIs/cm2. Our study did not showed statisticaldifferences for larval mortality percentage among Riojanstrains. However, Tarasco (1997) observed high intraspe-cific variability in the larval percentage mortality, rangingfrom 37.7 to 71.1%, after applying 3 IJs/ cm2 of ItalianS. feltiae strains. Simoes et al. (2000) observed that S.

carpocapsae Bretain strain was more virulent than AZ27strain, and Bretain strain has also a lethal time twentytimes lower than Az27. Mracek et al. (1999b), carryingout the bioassays at 10 �C, observed that an applicationof 2 JIs/cm2 S. kraussei Canadian strain produced a larvalmortality percentage as high as those produced by the Rio-jan S. kraussei 88C strain.

The days to complete the infection cycle were statisti-cally different among Riojan strains. The S. feltiae strainsranged from 8.44–9.84 days followed to S. carpocapsae

strains that ranged from 10.23 to 10.26 days, showing S.kraussei 88C strain the largest infection cycle with 12.83days. These results are in agreement with the usual timedescribed to the Steinernematid species (Garcıa del Pino,1994), although specific studies are not available yet. Thehigher reproductive potential was recorded by the S. carpo-

capsae 96 and 98 strains, which ranged from 626.29 to831.72 IJs/mg larvae, respectively. They were closed tothe values obtained by Dutky et al. (1964) and Wang andBedding (1996) with the S. carpocapsae DD-136 andLeningrado A24 strains, that produced approximately1000 IJs/mg larvae. However, Sandner and Stanuszek

136 R. Campos-Herrera et al. / Journal of Invertebrate Pathology 95 (2007) 125–139

(1971) observed a wide range among strains, producingsome of them 3091 IJs/mg larvae. The reproductivepotential of the S. feltiae Riojan strains ranged from124.74 to 444.23 IJs/mg larvae, which is into the valuesobserved to the S. feltiae UK strain (226.6–359.2 IJs/mglarvae) by Schirocki and Hague (1997). This is the first timethat the infection cycle length and reproductive potential ofS. kraussei is evaluated. The S. kraussei Riojan 88C strainshowed the largest infection cycle and the lowest reproduc-tive potential recorded in the present study. As the naturaldistribution of S. kraussei is Holartic (Stock et al., 2000),and their occurrence is related to cool-temperate conditions(Hominick, 2002; Spiridonov et al., 2004), thus it ispossible that the normal development of the S. kraussei88C strain could be slow down, due to the bioassay wascarried out at 22 �C. A study using lower temperaturewould provide more accurate information about the infec-tion cycle length and reproductive potential of this species.The best results obtained from this study were for the S.

feltiae strains, adapted to a broad range of environmentaland soil characteristics, proposing to 38 and 63 strainsfor bioinsecticide development. Steinernema carpocapsae96 and 98 strains were related to temperate conditionsand heavy soils, so it could be interesting to explore theirutility as biological control agents. More bioassays withS. kraussei 88C carried out with their optimal temperaturewill be developed to assess their utility as bioinsecticide.

The results of this survey extend the knowledge onEPNs in Spain and provided some ecological observationspotentially useful to develop new commercial strainsadapted to Mediterranean environment for biologicalcontrol of insects. Future studies on the virulence of indig-enous EPNs against local insect pests will be developed.

Acknowledgments

We thank to Antonio Bello and Avelino Garcıa-Alvarezfor their comments on the manuscript and Ana Piedra Bue-na for their English corrections; Milagros Herrera forassisting in the soil collection; Ruben Blanco-Perez forhis help is the EPNs biometric measurements; OctavioCedenilla and Margarita Perez-Penasco for the soil analy-sis and Manuel Fernandez for soil samples preparation.This research was supported by funds of: Spanish Scienceand Technology Ministry (Grants: REN2002-02550 andRTA3-004-C4-4), Riojan Sudies Institute, Consejerıa deEducacion, Cultura, Juventud y Deportes del Gobiernode La Rioja (Grant: 2002/3461) and Union de Agricultoresy Ganaderos de La Rioja-Coordinadora de Agricultores yGanaderos (UAGR-COAG) (Grant: 2003/672). We thankthe Ministerio de Educacion, Cultura y Deporte for theFPU fellowship.

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