Journal of Helminthology Natural occurrence of entomopathogenic www.cambridge.org/jhl nematodes (Steinernema and Heterorhabditis) and Pristionchus nematodes in black truffle soils from Spain Research Paper Cite this article: Julià I, Morton A and Garcia- Ivan Julià, Ana Morton and Fernando Garcia-del-Pino del-Pino F (2023). Natural occurrence of entomopathogenic nematodes (Steinernema Departament de Biologia Animal, Biologia Vegetal i Ecologia, Facultat de Biociències, Universitat Autònoma de Barcelona, and Heterorhabditis) and Pristionchus Bellaterra, Barcelona, Spain nematodes in black truffle soils from Spain. Journal of Helminthology, 97, e76, 1–10 Abstract https://doi.org/10.1017/S0022149X23000615 The European truffle beetle Leiodes cinnamomeus is the most important pest in black truffle Received: 04 July 2023 (Tuber melanosporum) plantations. Current control methods against it are inefficient, so ento- Revised: 05 September 2023 mopathogenic nematodes (EPNs) could play an important role in their population regulation due Accepted: 25 September 2023 to their efficacy against many soil-dwelling insect pests. A survey of EPNs and Pristionchus Keywords: nematodes was conducted in truffle soils of Spain, considering environmental and physical– Biodiversity; distribution; Steinernema feltiae; chemical soil factors. A total of 164 soil samples were collected from forests, productive Heterorhabditis bacteriophora; Pristionchus; plantations and null-low productive plantations, representing three distinct black truffle-growing necromenic; phoretic; Tuber melanosporum habitat types. EPNs were isolated from seven soil samples (4.3%); four nematodes were identified Corresponding author: as Steinernema feltiae and three as Heterorhabditis bacteriophora. Both species were sampled in Fernando Garcia-del-Pino; three types of soil texture (loam, sandy loam or sandy clay loam), characterized by alkaline pH Email: Fernando.Garcia@uab.cat (7.5 to 8.5) and high organic matter (2.1–11.04%). The presence of these EPNs was influenced by habitat type and organic matter content. Pristionchus nematodes were isolated from truffle soil, around truffle fruit bodies and under the elytra of L. cinnamomeus, with Pristionchus maupasi being the most commonly identified species. No significant associations were found between environmental and soil factors and the occurrence of Pristionchus nematodes. These nematodes were found in alkaline soils (pH 7.75 to 8.7), across all seven sampled soil textures, with variable organic matter content (0.73%–5.92%). The ecological trends and the presence of Pristionchus may affect the occurrence of EPNs and their prospective use as biological control agents against L. cinnamomeus in black truffle plantations. Introduction The black truffle, Tuber melanosporum Vittad. (Pezizales: Tuberaceae), is a hypogeal fungus that establishes a mycorrhiza relationship mainly with the Quercus genus (Bonito et al. 2010) that has been traditionally collected from wild forests and in the last decades cultivated due to its high gastronomic value and economic interest (Oliach et al. 2020). T. melanosporum usually starts to fructify when the host tree is about 6–7 years old and continues until it is 20–25 years old, when the production starts to drop (Martín-Santafé, 2020). During this period, the mycorrhiza develops a burnt area around the host tree where the herbaceous cover is scarce (Splivallo et al. 2011) due to the production of mainly two truffle volatiles (ethylene and indole-3-acetic acid) that act as potent herbicides at high concentrations (Hansen and Grossmann 2000; Grossmann 2003). Moreover, alkaline soils are required for its development, with pH that ranges from 7.0 to 8.9, with a median of 7.9 (Jaillard et al. 2016). This fungus is also benefited by high organic matter content and balanced textures, generating loamy soils that are well-structured, porous, aerated and without excess of water (Jaillard et al. 2016). The economic importance of this fungus has led to a monoculture situation that along with certain agricultural practices like irrigation have favoured the presence of some insect species that then become pests (Martín-Santafé 2020). The European truffle beetle, Leiodes cinnamomeus © The Author(s), 2023. Published by Cambridge (Panzer) (Coleoptera: Leiodidae), is one of the most serious pests in black truffle plantations University Press. This is an Open Access article, (Arzone 1971; Martín-Santafé et al. 2014; Navarro-Llopis et al. 2021). Adults and larvae feed on distributed under the terms of the Creative T. melanosporum fruiting bodies, causing galleries which reduce quality and can generate up to Commons Attribution licence (http:// 70% of economic losses in plantations (Barriuso et al. 2012). Cultural practices, such as frequent creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and collections of truffles and the use of traps for mass capture of adults, are recommended (Martín- reproduction, provided the original article is Santafé et al. 2014; Navarro-Llopis et al. 2021). However, these practices are not enough to reduce properly cited. the population of L. cinnamomeus to acceptable levels. Thus, alternative biological control methods are needed. Entomopathogenic nematodes (EPNs) are a group of species that have been studied and used as biological control agents for decades (Lacey and Georgis 2012; Shapiro-Illan et al. 2017). These https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press 2 Ivan Julià et al. nematodes, species of the genus Steinernema and Heterorhabditis, study their ecological requirements in truffle soils from the regions which are the most common, are widely distributed throughout the of Teruel and Catalonia (Spain); (2) assess the presence of species of world and have been reported from a wide variety of soils (Hominick Pristionchus nematodes that could interfere with the presence 2002). The presence and survival of EPNs are influenced by multiple of EPNs. factors, including geographical location, climatic conditions, habitat type and soil properties, such as pH, organic matter content and texture (Stuart et al. 2015). EPNs are believed to be adapted to the Material and methods soil-specific conditions where they were isolated (Kung et al. 1991). Field sampling and soil characterization Julià et al. (2023) have already observed the susceptibility of L. cinnamomeus adults and larvae to EPNs under laboratory condi- A total of 164 soil samples in 112 and 52 locations of Teruel and tions, suggesting that the presence of these nematodes in truffle Catalonia (Spain), respectively, were collected from different black plantations could naturally regulate the population of this beetle. truffle-growing areas (Figure 1) from autumn (October 2020) to Free-living bacterivorous nematodes (FLBNs) species can inter- spring (March 2021). Each soil sample weighed about 1 kg, resulted fere and compete with EPNs for host resources (Duncan et al. 2003; from the mixture of four subsamples of about 200 cm3 dug from 0 to Campos-Herrera et al. 2012). Some species of the genus Pris- 20 cm deep in soil around the host tree (Campbell et al. 1998). tionchus exhibit facultative insect parasitic, necromenic and nema- In 21 productive plantations in Teruel, both nonburnt areas tophagous behaviour (Félix et al. 2018). For example, Pristionchus (without the presence of T. melanosporum) and burnt areas were pacificus Sommer, Carta, Kim and Sternberg (Rhabditida: Diplo- sampled to assess whether the volatiles of T. melanosporum, which gastridae) can display dimorphic mouth structures, differing in the act as herbicides, influenced the occurrence of nematodes. The number and shape of teeth and in the complexity of other mouth sampling methodology employed was the same as explained above. armature. This dimorphism enables it to adopt a predator behav- The locations and altitudes of the sampled soils were recorded iour towards other nematodes when bacterial food is scarce (Meyer using global positioning system equipment. Data of annual average et al. 2017). Moreover, previous studies have also observed that air temperature and rainfall were recorded from maps of the species such as P. pacificus and Pristionchus maupasi (Potts) Government of Aragon and Government of Catalonia. The study (Rhabditida: Diplogastridae) form necromenic or phoretic associ- area in Teruel lies between 888 and 1760 m above sea level, with ations with various species of beetles (Herrmann et al. 2006, Hong mean annual temperatures of 9–13 ºC and mean annual rainfall of et al. 2008; Félix et al. 2018). 400–600 mm. In the case of Catalonia, the study area lies between The discovery of new strains and species of EPNs has been 514 and 1484 m above sea level, with mean annual temperatures of important in their commercial success as biocontrol agents against 8–12 ºC and mean annual rainfall of 600–800 mm. The samples pests (Shapiro-Ilan et al. 2002; Lacey and Georgis 2012) due to the were categorized based on habitat type, with 20.1% obtained from importance of being adapted to the environmental conditions of the forests known to naturally produce T. melanosporum, 67.1% from site of application (Bedding 1990). There is currently a lack of productive truffle plantations and 12.8% from low-productive or studies examining the presence of EPNs in truffle soils. Therefore, nonproductive plantations. This last category includes plantations the main objectives of this research were: (1) to isolate EPNs and less than 6 years old or more than 25 years old. Figure 1. Geographical distribution of EPN sampling locations in the regions of A. Teruel and B. Catalonia (Spain). Green triangles: sites with S. feltiae. Red triangles: sites with H. bacteriophora. White circles: sites without nematodes. https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press Journal of Helminthology 3 For each collected sample, pH, soil organic content and soil Identification of nematodes particle size were measured. The soil pH was measured from a 1:2.5 EPNs and Pristionchus nematodes isolated were identified using soil/mQ-water suspension and the total organic matter was deter- molecular techniques. For each sample, a PCR reaction was per- mined by wet oxidation (MAPA 1975). To determine the soil formed to amplify an internal transcribed spacer (ITS) region from texture, particle size analysis was performed to calculate the per- genomic DNA extracted from a single female. PCR amplification centage of silt, sand and clay using the Bouyoucos method (MAPA conditions followed procedures described by Hominick et al. (1997) 1975). using the primers 18s (5-AAAGATTAAGCCATGCATG-3) and 26s (5-CATTCTTGGCAAATGCTTTCG-3) (Vrain et al. 1992). Isolation of nematodes Amplified samples were purified and sequenced before being com- pared with GenBank database sequences of Steinernema, Hetero- Isolation of EPNs rhabditis and Pristionchus using Blastn (NCBI; http:// The insect bait method (Galleria trap) described by Bedding and www.ncbi.nlm.nih.gov), searching for sequence similarity matches Akhurst (1975) was used to isolate EPNs. For each soil sample, five at ≥ 98%. Petri dishes containing five last-stage larvae of Galleria mellonella An alignment of the ITS rDNA sequences was generated (Linnaeus) (Lepidoptera: Pyralidae) were covered with soil. After using Clustal W (Thompson et al. 1997) for Steinernema and incubating for one week at 24 ºC, dead insects that showed symp- Heterorhabditis species separately. Phylogenetic analyses were toms of infection were rinsed in sterile Ringer solution and placed performed using the maximum parsimony (MP) method with individually into modified White traps (White 1927) to collect the MEGA X software (Kumar et al. 2018). The calculated phylo- emerged infective juveniles (IJs). The assay was conducted twice. To genetic trees were evaluated by bootstrap analysis based on 1000 confirm their pathogenicity, harvested nematode strains were replicates. Caenorhabditis elegans (Maupas) (X03680) was used reared at 24 °C in last instar larvae of G. mellonella, according to as outgroup during calculation of the trees based on ITS the method of Woodring and Kaya (1988). They were maintained sequences. at 9 °C until their molecular identification. Isolation of Pristionchus nematodes Data analysis The presence of Pristionchus and other FLBNs was determined using a similar methodology. For each soil sample, one Petri dish The data for EPNs and Pristionchus were assessed in relation to containing three frozen dead last-stage larvae of G. mellonella was environmental and soil factors, using the recovery frequency (num- covered with soil. After incubating for one week at 24 ºC, nema- ber positive samples/number total samples), and the number and todes detected around the G. mellonella cadavers were collected in percentage of positive samples per variable category. The data Eppendorf tubes with ethanol 70%. The assay was conducted twice. corresponding with EPNs and Pristionchus were analysed separ- The presence of Pristionchus nematodes was also determined ately. Generalized linear model (GLM), with negative binomial directly from the surface of black truffle fruit bodies to confirm their distribution and a log link function, was used to examine the presence on this fungus. Eight fruit bodies of T. melanosporum were relationships between the nematodes and the environmental/soil sampled in Teruel. The nematodes around them were rinsed and variables. The variables were categorized into groups, as shown in collected in Eppendorfs with ethanol 100%. Their presence was also Tables 1 and 2. The recovery frequency of nematodes obtained in determined under the elytra of L. cinnamomeus to confirm the the regions of Catalonia and Teruel and in the burnt and nonburnt phoretic relationship between nematode and insect. Twenty Petri areas were also compared using GLM with negative binomial dishes with nutrient agar were used, with two elytra per dish. After distribution. All data were analysed with the R software (version 7–10 days of incubation, the percentage of Petri dishes with Pris- 4.2.2) (R Core Team 2022). Any comparison was considered sig- tionchus nematodes was calculated. nificant if the p-value was less than 0.05. Table 1. Distribution of EPNs at different environmental variables Positive samples Categories (total samples) Recovery frequency (%) S. feltiae No (%) H. bacteriophora No (%) Habitat type Productive plantation (110) 0 0 0 Null-low productive plantation (21) 14.3 2 (50%) 1 (33.3%) Truffle forest (33) 12.1 2 (50%) 2 (66.6%) Altitude (masl) 500–800 (22) 9.1 0 2 (66.6%) 800–1100 (90) 3.3 2 (50%) 1 (33.3%) 1100–1400 (41) 2.5 1 (25%) 0 >1400 (5) 20 1 (25%) 0 https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press 4 Ivan Julià et al. Table 2. Distribution of EPNs at different physical–chemical variables Positive samples Categories (total samples) Recovery frequency (%) S. feltiae No (%) H. bacteriophora No (%) Soil pH 7–7.5 (1) 0 0 0 7.5–8 (11) 18.2 2 (50%) 0 8–8.5 (129) 3.9 2 (50%) 3 (100%) ≥8.5 (23) 0 0 0 Organic matter ≤1 (7) 0 0 0 1–2 (48) 0 0 0 2–3 (31) 9.7 2 (50%) 1 (33.3%) 3–4 (38) 0 0 0 4–5 (21) 4.8 1 (25%) 0 ≥5 (19) 15.8 1 (25%) 2 (66.6%) Texture Sandy loam (24) 16.7 3 (75%) 1 (33.3%) Loam (59) 3.4 0 2 (66.6%) Sandy clay loam (37) 2.7 1 (25%) 0 Silt loam (4) 0 0 0 Clay loam (36) 0 0 0 Sandy clay (1) 0 0 0 Clay (3) 0 0 0 Results and 804 masl) than S. feltiae (899, 942, 1336 and 1417 masl) (χ2 = 6.41, df = 1, p < 0.05). Isolation of entomopathogenic nematode species EPN distribution was also examined according to soil pH, organic Entomopathogenic nematodes were isolated from 7 of 164 soil sam- matter and texture. Both EPN species isolated were found in alkaline ples (4.3%) distributed across plantations and wild truffle-producing soil samples with pH ranging from 7.58 to 8.47 (Table 2). However, forests. EPNs were recovered from 3 of 112 samples from Teruel there was no significant effect of soil pH to EPNs occurrence (χ2 = (2.6%) and 4 of 52 samples from Catalonia (7.7%), with no significant 4.83, df = 3, p = 0.18). Instead, the recovery frequency of S. feltiae and differences between both regions (χ2 = 1.92, df = 1, p = 0.17). H. bacteriophora was significantly influenced by the content of Morphologic and molecular examinations revealed the presence organic matter (χ2 = 12.85, df = 5, p < 0.05), as these nematodes were of four isolates of Steinernema and three isolates of Heterorhabditis. predominantly isolated from soils with higher organic matter content BLASTn analysis of the ITS region showed that the three steiner- (2.1–11.04%). The occurrence of EPNs was not significantly influ- nematid recovered from Teruel and one from Catalonia shared enced by soil texture (χ2 = 9.06, df = 6, p = 0.17), although they were sequence similarity of >99% with Steinernema feltiae (Filipev) detected in sandy loam (16.7%), loam (3.4%) and sandy clay loam (Panagrolaimida: Steinernematidea) (Figure 2). The second species (2.7%) soils but not in silt loam, clay loam, sandy clay and clay soils isolated from three samples in Catalonia shared sequence similarity (Table 2). of >99% with Heterorhabditis bacteriophora (Poinar) (Rhabditida: Heterorhabditidae) (Figure 3). Presence of Pristionchus species Species of genus Pristionchus were detected in 46 of 164 soil samples EPN distribution in relation to environmental and soil (28%) distributed across plantations and truffle forests surveyed. characteristics Individuals were recovered from 35 of 112 samples in Teruel The habitat type significantly affected the recovery frequency of (31.3%) and 11 of 52 samples in Catalonia (21.2%), with no signifi- EPNs (χ2 = 16.25, df = 2, p < 0.05). Both species were isolated in cant differences between both regions (χ2 = 1.85, df = 1, p = 0.17). truffle forest (12.1% of this habitat) and null-low productive plant- BLASTn analysis of the ITS region showed that most of these ations (14.3% of this habitat), but no EPNs were detected in isolates (39 of 46) shared sequence similarity of >98% with productive plantations (Table 1). Altitude did not affect the occur- P. maupasi. The other isolates (7 of 46) were not similar to pub- rence of EPNs (χ2 = 2.99, df = 3, p = 0.39). However, lished ITS sequences of any specific species of this genus. Hence, H. bacteriophora strains were isolated at lower altitudes (694, 767 they were considered as Pristionchus sp. Other free-living https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press Journal of Helminthology 5 Figure 2. Phylogenetic relationship of isolated S. feltiae strains from Teruel (TE15, TE104 and TE112) and Catalonia (CT3) and published ITS sequences of Steinernema species using maximum parsimony tree. C. elegans was used as the outgroup. Numbers before species names correspond to GenBank accession numbers. bacteriophage nematode species were also recovered and identified Pristionchus distribution was also examined according to soil from nine samples in Teruel (8%) and six samples in Catalonia pH, organic matter and texture. None of these variables signifi- (11.5%) (Table 3). cantly influenced the presence of Pristionchus (χ2 = 3.55, df = 3, p = BLASTn analysis also identified P. maupasi species around the 0.31; χ2 = 6.47, df = 5, p = 0.26 and χ2 = 4.1, df = 6, p = 0.66, eight fruit bodies of T. melanosporum sampled, sharing sequence respectively). All nematodes were found in alkaline soil samples similarities of >99% with this species. Moreover, Pristionchus nema- (7.75 to 8.7 pH), in all seven types of texture sampled with variable todes were identified under the elytra of 45% of L. cinnamomeus organic matter content (0.73–5.92%) (Table 5). adults. Some other nonidentified nematodes were also detected. Pristionchus distribution in relation to environmental and soil Presence of EPNs and Pristionchus in burnt and nonburnt areas characteristics The number of isolated EPNs was not statistically analysed because no Pristionchus nematodes were isolated in all three sampled habitat nematodes were recovered from the 42 samples (21 from burnt areas types (χ2 = 1.15, df = 2, p = 0.56), including productive plantations and 21 from nonburnt areas). In the case of Pristionchus, its recovery (28.2%), null-low productive plantations (33.3%) and truffle forests frequency was found to be significantly higher in burnt areas (38.1%) (21.2%) (Table 4). Furthermore, these nematodes were found compared to nonburnt areas (4.8%) (χ2 = 7.69, df = 1, p < 0.05) across all altitudes sampled (χ2 = 1.77, df = 3, p = 0.62). (Table 6). https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press 6 Ivan Julià et al. Figure 3. Phylogenetic relationship of isolated H. bacteriophora strains from Catalonia (CT2, CT47 and CT52) and published ITS-sequences of Heterorhabditis species using maximum parsimony tree. C. elegans was used as the outgroup. Numbers before species names correspond to GenBank accession numbers. Discussion Table 3. Number of soil samples with other identified species of free-living bacteriophage nematodes This study represents the first survey aimed to isolate native EPN species and necromenic Pristionchus nematodes in truffle soils. Species Teruel Catalonia Global Despite a relatively low prevalence of EPNs (7 of 164 samples; Rhabditis terricola 3 1 4 4.3%), our findings are consistent with previous studies. Globally, EPN prevalence in soil samples has been reported to range from Oscheius sp. 3 0 3 0.7% to 50% (Hominick 2002). In Spain, Morton and Garcia del Panagrolaimus sp. 2 1 3 Pino (2009) observed an incidence of 5.2% in stone fruit orchards in Cruznema tripartitum 1 2 3 Catalonia, which aligns with the results of our study. Similar results were obtained by Campos-Herrera et al. (2007) in soils of La Rioja Pratylenchus goodeyi 0 2 2 (5.4%). In contrast, Garcia del Pino and Palomo (1996) recovered Total 9 6 15 EPNs in 28.2% of cultivated soils and in 16.3% of woodlands in Catalonia. https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press Journal of Helminthology 7 Table 4. Distribution of Pristionchus at different environmental variables Positive samples Categories (total samples) Recovery frequency (%) P. maupasi Pristionchus sp. Habitat type Productive plantation (110) 29.1 28 (71.8%) 4 (57.1%) Null-low productive plantation (21) 33.3 6 (15.4%) 1 (14.3%) Truffle forest (33) 21.2 5 (12.8%) 2 (28.6%) Altitude (masl) 500–800 (22) 18.2 2 (5.1%) 2 (28.6%) 800–1100 (90) 31.1 26 (66.7%) 2 (28.6%) 1100–1400 (41) 29.3 10 (25.6%) 2 (28.6%) >1400 (5) 40 1 (2.6%) 1 (14.3%) Table 5. Distribution of Pristionchus at different physical–chemical variables Positive samples Categories (total samples) Recovery frequency (%) P. maupasi Pristionchus sp. Soil pH 7–7.5 (1) 0 0 0 7.5–8 (11) 27.3 2 (5.1%) 1 (14.3%) 8–8.5 (129) 21.7 24 (61.5%) 4 (57.1%) ≥8.5 (23) 65.2 13 (33.3%) 2 (28.6%) Organic matter ≤1 (7) 42.9 3 (7.7%) 0 1–2 (48) 22.9 10 (25.6%) 1 (14.3%) 2–3 (31) 38.7 11 (28.2%) 1 (14.3%) 3–4 (38) 28.9 8 (20.5%) 3 (42.9%) 4–5 (21) 9.5 2 (5.1%) 0 ≥5 (19) 31.6 4 (10.3%) 2 (28.6%) Texture Sandy loam (24) 29.2 7 (17.9%) 0 Loam (59) 20.3 12 (30.8%) 3 (50%) Sandy clay loam (37) 29.7 11 (28.2%) 1 (16.7%) Silt loam (4) 25 1 (2.6%) 0 Clay loam (36) 16.7 6 (15.4%) 2 (33.3%) Sandy clay (1) 100 1 (2.6%) 0 Clay (3) 33.3 1 (2.6%) 0 Table 6. Distribution of Pristionchus at burnt and nonburnt areas Positive samples Categories (total samples) Recovery frequency (%) P. maupasi Pristionchus sp. Burnt area (21) 38.1 6 (85.7%) 2 (100%) Nonburnt area (21) 4.8 1 (14.3%) 0 (0%) https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press 8 Ivan Julià et al. In our study, the diversity of EPNs was found to be low, with EPN species were significantly more prevalent in soils with higher only two species isolated: S. feltiae and H. bacteriophora. These organic matter. Additionally, all strains of S. feltiae and findings are consistent with previous studies conducted in Medi- H. bacteriphora were isolated from alkaline soils because terranean countries, which have also reported low diversity of EPNs T. melanosporum requires soils with high pH levels, ranging from (Garcia del Pino and Palomo 1996; Campos-Herrera et al. 2007; 7.0 to 8.9 (Jaillard et al. 2016). Our results agree with Campos- Morton and Garcia del Pino 2009; Noujeim et al. 2011; Valadas Herrera et al. (2007), who also isolated EPNs from alkaline soils, et al. 2013; Tarasco et al. 2009, 2015; Benseddik et al. 2020). Four while Khatri-Chhetri et al. (2010) detected most of EPNs in acidic new strains of S. feltiae were isolated, being the most common soils, particularly steinernermatids. Kung et al. (1990) reported that species detected globally (57%) and in the region of Teruel (100%). pH values within the range of 4–8 did not significantly affect the In fact, S. feltiae is frequently reported as the dominant EPN species survival and pathogenicity of EPNs, suggesting that the pH toler- in Mediterranean countries, accounting for 38% in Italy (Tarasco ance of indigenous nematodes may vary depending on the region of et al. 2015), 55% (Campos-Herrera et al. 2007) and 70% in Spain isolation (Khathwayo et al. 2021). In fact, EPNs are believed to be (Garcia del Pino and Palomo, 1996), 75% in Portugal (Valadas et al. adapted to the specific soil conditions of the region they were 2013), 71–85% in Turkey (Hazir et al. 2003; Yuksel and Canhilal isolated (Kung et al. 1991). 2019; Gümüş Askar et al. 2022) and 87% in Algeria (Tarasco et al. Biotic factors, such as FLBNs species, may also affect the pres- 2009). In the region of Catalonia, three strains of H. bacteriophora ence of EPNs (Stuart et al. 2015). Previous studies observed that were identified and were the most common species detected (75%). these nematodes are able to interfere and compete with EPNs This species was isolated in inland areas at lower altitudes (694–807 species for host resources. Laboratory experiments observed that masl) compared to S. feltiae (899–1417 masl). Most studies have Pellioditis sp. can compete with and even displace EPNs species that reported that H. bacteriophora is commonly found in maritime had previously killed the insect host (Duncan et al. 2003). Another environments, where it tends to be more prevalent than steinerne- study demonstrated the ability of Acrobeloides maximum and matids (Rosa et al. 2000; Emelianoff et al. 2008). However, our Rhabditis rainaispecies to interfere with the development of various results align with other reports, suggesting that H. bacteriophora species of EPNs inside weevil Diaprepes abbreviatus hosts, reducing can also be the dominant species in inland areas (Benseddik et al. the production of new progeny (Campos-Herrera et al. 2012). The 2020). genus Pristionchus sp. is known to be associated with different The measured prevalence of EPN is influenced by various factors, substrates, such as soil, humus, compost, moss, around roots of including sampling method, insect bait and environmental and soil several species, rotten wood, stems and fruits, and decomposing characteristics such as habitat type, altitude, soil texture, moisture fungi (Sudhaus and Fürst von Lieven 2003; Félix et al. 2018). In our level, organic matter, pH and biotic factors (Garcia del Pino and study, Pristionchus nematodes, particularly P. maupasi, were fre- Palomo 1996; Benseddik et al. 2020). In our study, we found that quently found in truffle soils (28% of all samples) and on the fruit habitat type significantly influenced the recovery frequency of EPNs. bodies of these truffles (100%). These results agree with Kilian et al. Although productive plantations are subjected to more frequent (2022), who also observed the presence of unidentified nematodes irrigation compared to null-low productive plantations and truffle around the fruit bodies of T. melanosporum. Additionally, we forests, EPNs were only isolated from the latter two habitats, and detected the presence of this nematode under the elytra of none were isolated from productive plantations. T. melanosporum is L. cinnamomeus (45%), which is the first report that has confirmed associated with the development of burnt area around the host tree the necromenic/phoretic relationship between P. maupasi and where the herbaceous cover is scarce (Splivallo et al. 2011). This L. cinnamomeus. Previous studies reported that some Pristionchus characteristic is more prominent in productive plantations, poten- species can prey on other nematodes due to the mouth dimorphism tially leading to a loss of diversity among potential insect hosts developed by these species (Serobyan et al. 2014; Wilecki et al. (Campos-Herrera et al. 2007). Although highly intensive monocul- 2015). Moreover, we also observed predatory behaviour in ture areas can produce outbreaks of insect pest species susceptible to P. maupasi towards EPNs under laboratory conditions (unpub- EPNs infection (Campos-Herrera et al. 2007), the beetle lished data). The co-occurrence of several other FLBN species with L. cinnamomeus develops during the coldest period of the year when P. maupasi in truffle soil could indicate that these nematodes may low temperatures are suboptimal for EPN infection (Julià et al. serve as a food source in addition to bacteria. Therefore, the 2023). Moreover, the high recovery rates of the genus Pristionchus presence of P. maupasi may be a factor that has contributed to observed in burnt areas could indicate that the herbicides emitted by the low prevalence of EPNs in truffle soils. T. melanosporum may not have a nematicidal effect on nematodes, In our study, P. maupasi was not significantly affected by envir- including EPNs. Thus, the greater presence of vegetation in null-low onmental and soil variables. Félix et al. (2018) observed that both productive plantations and wild truffles could positively influence dauer and feeding stages of various Pristionchus species were fre- the presence of EPNs by providing increased diversity of potential quently and abundantly present on different rotting vegetal matter insect hosts during spring and summer. and decomposing fungi. They also highlight the need for more In our survey, EPNs were recovered from soils with moderate to detailed studies to confirm whether the beetles that have been high sand content. Previous studies observed that EPNs were associated with Pristionchus spp. visit places with rotting vegetal commonly found in soils with high sand content (Stock et al. matter, considering the seasonality of the beetle’s life cycle. In our 1999; Valadas et al. 2013; Tarasco et al. 2015), suggesting that light study, L. cinnamomeus is a beetle that develops during the coldest textured soils improve the mobility and survival of EPNs (Stock period of the year, as it is adapted to the life cycle of T. melanosporum et al. 1999) compared to heavy textured soils, which clay content (Martin-Santafé et al. 2014). Despite the low temperatures, could difficult the EPNs movement and affect their recovery P. maupasi nematodes were not only isolated from truffle soils but (Mráček et al. 2005). Furthermore, organic matter content has also also from T. melanosporum fruit bodies and the elytra of been observed to positively correlate with the occurrence of EPNs adult L. cinnamomeus beetles during this period. Adults of (Hominick and Briscoe 1990; Alumai et al. 2006; Canhilal and L. cinnamomeus frequently move between truffles to reproduce Carner 2006). Our results agree with these studies, in which both (Martin-Santafé 2020), visiting the fruit bodies where Pristionchus https://doi.org/10.1017/S0022149X23000615 Published online by Cambridge University Press Journal of Helminthology 9 populations are present. Therefore, our results confirm the phoretic/ and long-distance dispersal. Molecular Ecology 19(22), 4994–5008. https:// necromenic relationship between these organisms. This is supported doi.org/10.1111/j.1365-294X.2010.04855.x. by the higher presence of Pristionchus in burnt areas (38.1%) than in Cambefort Y (1991) From saprophagy to coprophagy. pp. 22–35. In Hanski I nonburnt areas (4.8%). These results are consistent with previous and Cambefort Y (Eds.), Dung Beetle Ecology. Princeton, Princeton Univer- sity Press. studies that have observed scarab beetle species associated with Campbell JF, Orza G, Yoder F, Lewis EE and Gaugler R (1998) Spatial and Pristionchus feeding as adults on mature and rotting vegetal matter, temporal distribution of endemic and released entomopathogenic nematode such as Geotrupes stercorosus and Exomala orientalis (Cambefort populations in turfgrass. Entomologia Experimentalis et Applicata 86(1), 1991; Choo et al. 2002; Herrmann et al. 2006). 1–11. https://doi.org/10.1046/j.1570-7458.1998.00260.x. To sum up, this study has revealed the natural occurrence of Campos-Herrera R, El-Borai FE and Duncan LW (2012) Wide interguild S. feltiae and H. bacteriophora in truffle soils but at relatively low relationships among entomopathogenic and free-living nematodes in soil frequency, suggesting these EPNs might have specific adaptations as measured by real time qPCR. Journal of Invertebrate Pathology 111(2), to local conditions, which make them potential candidates for the 126–135. https://doi.org/10.1016/j.jip.2012.07.006. development of novel biological pest control agents. However, their Campos-Herrera R, Escuer M, Labrador S, Robertson L, Barrios L and absence in productive plantations, where the herbaceous cover Gutiérrez C (2007) Distribution of the entomopathogenic nematodes from La Rioja (Northern Spain). Journal of Invertebrate Pathology 95(2), 125–139. around the host tree is reduced due to T. melanosporum, might https://doi.org/10.1016/j.jip.2007.02.003. impact EPNs by limiting the diversity of available insect hosts. Canhilal R and Carner GR (2006) Natural occurrence of entomopathogenic The high recovery frequency of P. maupasi indicates that this nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) in South nematode is closely associated with truffles, having a phoretic/ Carolina. Journal of Agricultural and Urban Entomology 23(3), 159–166. necromenic relationship with the beetle L. cinnamomeus. More- Choo HY, Lee DW, Park JW, Kaya HK, Smitley DR, Lee SM and Choo YM over, its presence may affect the occurrence of natural populations (2002) Life history and spatial distribution of oriental beetle (Coleoptera: of other nematodes, including EPNs, through competition and Scarabaeidae) in golf courses in Korea. Journal of Economic Entomology 95 predation. 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