ORIGINAL ARTICLE Studies on the interaction between tropical nematophagous fungi and entomopathogenic nematodes (Rhabditida) under laboratory conditions Anália Arêdesa, Liliana Parente Ribeiroa, Vicente Mussi Diasa and Claudia Dolinskia*

a Nematology Research Group, Laboratório de Entomologia e Fitopatologia (LEF), Centro de Ciências e Tecnologias Agropecuárias (CCTA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Campos dos Goytacazes (RJ) Brazil *[email protected]

HIGHLIGHTS • Arthrobotrys musiformis AM4 and Arthrobotrys sp. CO7 were the most aggressive tropical fungi tested against Heterorhabditis indica LPP30. • Water agar medium provided the best conditions to test nematode-fungus interaction with lowest environmental impact among the six media tested. • H. indica LPP30 was more apprehended than S. carpocapsae All by both tropical fungi tested. • In a soil environment, there was a repellent action from Arthrobotrys sp. CO7 against S. carpocapsae All.

ABSTRACT: This study settles a step toward to understanding the interaction between nematophagous fungi (NFs) generally found in the soil and entomopathogenic nematodes (EPNs) used in the control of agricultural pests. There are very few studies linking EPNs and their natural enemies around the world and none in Brazil. Therefore, we decided to test differences regarding aggressiveness and susceptibility among the fungi and EPNs found and used in this country. The aim of this work was to study the interactions between the tropical native NFs, Arthrobotrys oligospora AM2, Arthrobotrys musiformis AM4, Arthrobotrys sp. CO7, Arthrobotrys sp. CC7 and the EPNs, Heterorhabditis indica LPP30 (Heterorhabditidae), a native nematode, and Steinernema carpocapsae All (Steinernematidae), a exotic nematode, under laboratory conditions. The interaction was studied in four aspects: 1) NF aggressiveness against EPNs; 2) effect of culture medium on NF aggressiveness to infective juveniles (IJs); 3) EPN susceptibility to NFs; and 4) dispersion of IJs in two types of soil in the presence of NFs. The fungi A. musiformis AM4 and Arthrobotrys sp. CO7 were considered the most aggressive among the tested strains, since they trapped more IJs. Water agar medium provided the best conditions to test nematode-fungus interaction among the six media tested, since the fungi tested produced more trapping structures to apprehend IJs on it. Both fungi were considered very aggressive to EPNs due to their apprehension capacity. Regarding susceptibility, H. indica LPP30 IJs were more susceptible than IJs of S. carpocapsae ALL to both A. musiformis AM4 and Arthrobotrys sp. CO7 in the first 24 h of contact. However, after 48 h, the means of IJs captured by the fungi became statistically equal, except for S. carpocapsae All in Assay 2, which presented the highest mean capture rate. The fungus Arthrobotrys sp. CO7 repelled S. carpocapsae All IJs in sand at 15 cm. The more we learn about biotic factors that interfere in biocontrol, the more effective we can make it. Future studies will focus on the characterization of these interactions in the field under tropical conditions. Keywords: Arthrobotrys musiformis, Arthrobotrys oligospora, Steinernema carpocapsae All, Heterorhabditis indica LPP30, aggressiveness, repulsion, culture media. Cite as Arêdes A, Ribeiro LP, Dias VM, Dolinski C. Studies on the interaction between tropical nematophagous fungi and entomopathogenic nematodes (Rhabditida) under laboratory conditions. Nematoda. 2017;4:e112016. http://dx.doi.org/10.4322/ nematoda.01116.

Received: Oct. 17, 2016 Accepted: Feb. 04, 2017

Nematoda. ISSN 2358-436X. This work is licensed under a Creative Commons Attribution International License (https://creativecommons.org/ licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. http://dx.doi.org/10.4322/nematoda.01116 1 Nematoda, 2017;4:e112016 Arêdes et al. Interactions between tropical nematophagous fungi and entomopathogenic nematodes

INTRODUCTION

Entomopathogenic nematodes (EPNs) in the genera Heterorhabditis and Steinernema are useful biological control agents against soil-dwelling pests in different crops[1]. EPNs do not induce genetic resistance in insect pests, and do not contaminate water or soil, unlike chemical residues from pesticides. Moreover, they are easy to handle and do not require specialized application equipment or safety equipment, and they can be applied to the soil as an aqueous solution or using insect cadavers[2]. These nematodes are obligate insect pathogens, but individuals of the third juvenile stage (J3), also called infective juveniles (IJs), are free living, and they search the soil for susceptible hosts. As they find them, they enter through natural openings (mouth, anus and spiracles) or by puncturing the cuticle. IJs carry in their anterior intestine (Heterorhabditis sp.) or in an intestinal vesicle (Steinernema sp.) symbiotic bacteria that are released in the host’s hemolymph. Individuals in the genus Heterorhabditis carry bacteria from the genus Photorhabdus while Steinernema spp. carry Xenorhabdus. In general, the bacteria kill the host by septicemia within 24-48 h. The developing nematodes feed on the proliferating bacteria and digested host tissues, and reproduce[3]. Two to three generations occur in the insect cadaver, and when food is scarce, IJs leave the cadaver in order to reinitiate the infection cycle[4]. Their foraging strategies can be predicted from the behaviors they exhibit during host search. Infective juveniles either sit-and-wait (ambushers), actively search (cruisers), or exhibit a combination of these two strategies to locate hosts (intermediate)[5]. Infective juveniles that ambush potential hosts forage near the soil surface and are generally best adapted to attacking mobile insects. Ambusher IJs raise most of their bodies off the substrate, moving their bodies, and jump towards the host-associated volatile cues as a host passes nearby[6, 7]. Cruisers, when compared to ambushers, are typically found deeper in the soil profile, and are more effective at attacking sedentary hosts, because they move towards host-associated cues[8, 9]. Although there are clear differences in behavior between EPN species traditionally classified as ambushers and those classified as cruisers, it is becoming increasingly clear that S. carpocapsae, a so-called ambusher, can find and infect reasonably sedentary hosts at considerable distances from the application point and its behavior could be habitat-dependent, creating some doubt about the established classification[10]. Although excellent insect-pest controllers, IJs’ persistence and survival in the soil can be affected by abiotic (i. e. humidity, temperature, soil texture and pH) and biotic (i. e. pathogenic bacteria and nematophagous fungi) factors[2]. Nematophagous fungi (NFs) are an important group of natural enemies of EPNs[11, 12, 13]. The NFs are common in a wide range of ecologically diverse soil habitats that, regarding their different mechanisms of action, can be divided into two groups: endoparasitic and trapping fungi[14]. In the environment, nematophagous trapping fungi can coexist in two forms: as parasites, feeding on a variety of nematodes or as saprophytes, living from organic matter[15, 16]. While trapping fungi and those that are parasites of eggs can survive as saprophytes, endoparasites are mostly dependent on nutrients from nematodes, being obligate parasites[17]. Even saprophyte fungi, they may affect EPNs. The non-pathogenic soil-borne fungus, Fusarium oxysporum Schlecht (Ascomycota: Hypocreales), has been proved to affect Heterorhabditis sonorensis Stock, Rivera-Orduño & Flores-Lara in searching for a host, and regarding their virulence and reproductive fitness[18]. There are other studies on the interactions between EPNs and natural enemies[19, 20, 21]. According to El-Borai et al.[22], IJs can be attracted, repelled or neutral to fungi. So, they suggest the existence of different responses relating EPNs and NFs. We hypothesize that there are differences regarding aggressiveness and susceptibility among the fungi and EPNs found here, and that native strains are better adapted to local conditions; consequently, native EPNs could be more susceptible to native fungi. Our hypothesis is that differences in the habitat or climate might drive differences in both isolates and interactions, which is critical to understand and allow developing rational and ecologically sound biological control strategies in the country. Therefore, we tested the tropical native NFs Arthrobotrys oligospora Fresen AM2, A. musiformis Drechsler AM4, Arthrobotrys sp. CO7 and Arthrobotrys sp. CC7, against two EPN species, one native to Brazil, Heterorhabditis indica Poinar, Karunakar & David LPP30 (Heterorhaditidae) and the other exotic, Steinernema carpocapsae (Weiser) All (Steinernematidae). Specifically we tested: 1) native NF aggressiveness against exotic and native EPNs; 2) effect of different culture media on NF aggressiveness to infective juveniles (IJs); 3) exotic and native EPN susceptibility to native NFs; and 4) dispersion of IJs in two types of soil in the presence of NFs.

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MATERIAL AND METHODS Maintenance of entomopathogenic nematodes and nematophagous trapping fungi S. carpocapsae All was isolated from North Carolina, U.S.A. and H. indica LPP30 from a guava orchard indigenous to Brazil[23]. The two EPN species were multiplied in last-instar Galleria mellonella L. (Lepidoptera: Pyralidae) larvae. G. mellonella larvae were reared in the laboratory as described by Dolinski et al.[24]. These larvae were placed in 9-cm Petri dishes lined with two sheets of filter paper moistened with 2 mL of a nematode suspension containing 200 IJs. The dishes were sealed with Parafilm M, identified and placed in a climate-controlled chamber at 25 ± 1 °C and 80 ± 10% relative humidity (RH) for 48 h. The dead larvae were transferred to modified White traps[25]. These traps were made of Petri dishes (9 cm in diameter) with a PVC ring (3 cm diameter × 1 cm height), with a piece of filter paper placed on the top of the ring (2 cm x 4 cm). The dead G. mellonella larvae were placed on the top of the filter paper, and distilled water was added to the base of the dish until reaching half way up the PVC ring. The traps were then placed in the climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH until IJ emergence. IJs were collected with Pasteur pipettes every other day for 12 days, placed in cell culture bottles (40 mL), identified regarding collection date and stored in a climate‑controlled chamber at 16 ± 1 °C and 80 ± 10% RH for no more than one week before the experiments. In the experiments we used four nematophagous trapping fungal isolates: Arthrobotrys oligospora AM2, A. musiformis AM4, Arthrobotrys sp. CO7 and Arthrobotrys sp. CC7. The first two were isolated from an Atlantic Forest fragment in São Francisco do Itabapoana, RJ, Brazil, and the other two from a guava orchard in Campos dos Goytacazes, RJ, Brazil. They were identified based on morphometrics by Dr. Clóvis de Paula Santos, UENF. They were kept on 1.7% cornmeal agar, in Petri dishes (9 cm diameter) sealed with Parafilm M, and placed in a climate-controlled chamber at 25 ± 1° C and 80 ± 10% RH in the dark for one week at the most.

Aggressiveness of nematophagous fungi to Heterorhabditis indica LPP30 To test the fungi’s aggressiveness, the experiment was conducted under laboratory conditions, comparing four fungal isolates, A. oligospora AM2, A. musiformis AM4, Arthrobotrys sp. CO7 and Arthrobotrys sp. CC7 against H. indica LPP30. The treatments were: LPP30/AM2, LPP30/AM4, LPP30/CO7, and LPP30/CC7. One disk of 1.7% cornmeal agar containing each fungi (1 cm diameter) was transferred to the center of 2% water agar Petri dishes (9 cm diameter). The dishes were sealed with Parafilm M and kept in a climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH in the dark for four days. After that, 0.5 mL of a suspension containing 50 IJs of H. indica LPP30 was added to each dish. The dishes were sealed again and placed in the climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH in the dark. There were five replicates for each treatment, and the experimental unit was a Petri dish. The aggressiveness was evaluated after 48 h from the addition of IJs, observing each dish under a stereoscope microscope and counting the number of IJs caught in the traps. The entire experiment was repeated after the first assay, with new nematode batches and fungal colonies, thus being called Assays 1 and Assay 2.

Aggressiveness of nematophagous fungi to entomopathogenic nematodes in different nutrient media To check whether the nutrients in the media would affect the aggressiveness of Arthrobotrys sp. AM4 and Arthrobotrys sp. CO7 against H. indica LPP30 and S. carpocapsae All, the fungi were maintained in pure culture in 1.7% cornmeal agar, as mentioned above, and a disk of 1.7% cornmeal agar with the fungi (1 cm diameter) was transferred to the center of the Petri dishes (9 cm diameter) containing the following media: i) 2% water agar (WA); ii) 1.7% cornmeal agar (CA); iii) 3.9% potato-dextrose  agar (PDA); iv) V8 juice agar (V8) made of 200 mL of V8 juice plus 3 g of CaCO3, 20 g of agar and 1 L of distilled water; v) soil extract agar (SE) made of 1 kg of clay-silty soil (46.7% clay, 7.7% sand, 45.6% silt, pH 5.8) autoclaved with 1 L of water, filtered in filter paper; then 100 mL of the filtered soil solution

was added to 900 mL of distilled water, 1 g of dextrose, 0.5 g of K2 HPO4 and 20 g of agar; vi) nematode medium (NM) made of 20 g of agar plus 1 L of distilled water and 250 mL of concentrated solution containing approximately live 1.5 × 105 IJs of H. indica LPP30. Petri dishes were kept in a climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH in the dark for one week. After this growing period, we added to each dish 0.1 mL of suspension containing 1,000 IJs

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of H. indica LPP30 or S. carpocapsae All, arranged in 24 treatments: six different media, two fungi and two entomopathogenic nematodes. There were five replicates for each treatment and the experimental unit was a Petri dish. The fungi aggressiveness measured by trapped nematodes was evaluated after 72 h from the addition of IJs, washing each dish with 10 mL of distilled water and collecting the IJs in tubes. Live IJs were counted using a Peters chamber. The entire experiment was repeated after the first assay, with new nematode batches and fungal colonies, so these were called Assay 1 and 2 and the analysis was kept separated for better visualization.

Entomopathogenic nematode susceptibility to nematophagous fungi in vitro In this experiment we used the most aggressive fungi from the previous test, which were A. musiformis AM4 and Arthrobotrys sp. CO7, against H. indica LPP30 and S. carpocapsae All aiming to test their susceptibility. The treatments were arranged as: LPP30/AM4, LPP30/CO7, All/AM4, All/CO7. Fungi grown in 1.7% cornmeal agar, as first described, were transferred as 0.5 cm-disks to 2% water agar 5-cm-Petri dishes. Each dish received one disk with fungi, was sealed with Parafilm M and kept in a climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH in the dark for 48 h. After that, when the fungi had covered the entire dish, we added 0.1 mL suspension with 500 IJs of H. indica LPP30 or 500 IJs of S. carpocapsae All, uniformly throughout the dish and around the disk. The dishes were sealed again and taken to the same climate-controlled chamber. There were 10 replicates for each treatment, and the evaluation was performed by observing and counting the number of IJs apprehended in the traps after 24 and 48 h, after adding the nematodes. The entire experiment was repeated after the first assay, with new nematode batches and fungal colonies, so we called them Assay 1 and Assay 2.

Infective juvenile dispersion in different soil types in the presence of a nematophagous fungus This experiment was conducted using the strain Arthrobotrys sp. CO7, because it was isolated from the same guava orchard as H. indica LPP30, and also because it had shown aggressive behavior towards different EPNs in previous studies. Therefore, the nematodes used were H. indica LPP30 and S. carpocapse All arranged in the treatments: CO7/LPP30, LPP30 (Control 1), CO7/All, All (Control 2), in which the controls had no fungi added, only nematodes. To evaluate the dispensability of the nematodes in different types of soils in the presence of the fungus, an apparatus was set up consisting of two pieces of PVC tube (15 cm long × 6 cm in diameter) joined by masking tape and connected to another identical set by a 8-cm-long pipe connector of the same diameter. At each end, a 9-cm-Petri dish containing the fungus in water agar 2% media was placed (Figure 1). In one set of PVC tubes about 1200 g of sandy soil (0.7% clay, 99.3% sand, pH 6.1 and 1.9 g/dm3 OM) was added, and in the other set 1200 g of clay-silty soil (46.7% clay, 7.7% sand, 45.6% silt, pH 5.8 and 12.6 g/dm3 OM). Previously, the soils had been solarized for three days, in which the temperature reached 70 °C and RH adjusted for 10% (w/v). Forty-eight h after adding the fungus to the end of the apparatus,

Figure 1. Apparatus developed for dispensability evaluation of the nematodes in sand and clay soil. The tubes were made of PVC with Petri dishes containing Arthrobotrys sp. CO7 colonies in agar 2% medium at each end.

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a hole was made in the center of the pipe connector and an aqueous solution containing 10,000 IJs/mL of H. indica LPP30 or S. carpocapsae All was added. The pieces of PVC joined by masking tape were used to evaluate the IJ dispersion at 15 and 30 cm. The apparatuses were covered by Parafilm M to avoid desiccation and kept in a horizontal position in the climate-controlled chamber at 25 ± 1 °C and 80 ± 10% RH in the dark. For each treatment there were 10 replicates or apparatuses. The number of IJs were counted in each piece of tube 10 days after nematode addition, emptying the tube and processing the soil by Jenkin’s method[26]. The entire experiment was repeated after the first with new nematode batches and fungal colonies, so we called them Assay 1 and Assay 2.

Statistical analysis The data were submitted to Lilliefors test of normality and Cochran & Bartlett’s homoscedasticity test at 5% probability using SigmaPlot[27]. Non transformed data were submitted to analysis of variance (P≤ 0.05) and the means compared by Tukey’s test (P< 0.05)[28]. When the data were not normally distributed, the parameters were compared by the nonparametric Kruskal-Wallis (P< 0.05).

RESULTS AND DISCUSSION Aggressiveness of nematophagous fungi to Heterorhabditis indica LPP30

All four NFs tested were able to apprehend H. indica LPP30 IJs in both assays (H(0.05, 3) = 10.25;

P= 0.017 and F(0.05, 3) = 4.67; P= 0.016; Assays 1 and 2, respectively). The number of IJs apprehended by A. musiformis AM4 was significantly higher for the strains A. oligospora AM2, in both assays, and significantly higher for strain Arthrobotrys sp. CC7 in Assay 2 (Figure 2). Although this assay was performed in water agar and not in the soil, we may infer that our data can be corroborated by El-Borai et al.[13], who observed a reduction in the number of H. indica IJs by A. musiformis, while A. oligospora had no effect on this EPN. Actually, they observed that neither S. diaprepsi Nguyen & Duncan nor S. glaseri Steiner were affected by any species of Arthrobotrys, whereas A. musiformis reduced recovery of all other EPNs tested, and A. oligospora reduced numbers of all other species except H. indica, which reinforces the hypothesis of specificity between fungal isolates and EPNs. Therefore, due to their high virulence against this EPN, we opted to continue the experimental studies with A. musiformis AM4 and Arthrobotrys sp. CO7.

Figure 2. Mean percentage of Heterorhabditis indica LPP30 infective juveniles apprehended by Arthrobotrys oligospora AM2 (AM2), Arthrobotrys musiformis AM4 (AM4), Arthrobotrys sp. CO7 (CO7), Arthrobotrys sp. CC7 (CC7). Means followed by the same lower-case letter are not significantly different (P< 0.05) according to the Kruskal-Wallis test for Assay 1 and the Tukey test for Assay 2.

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Aggressiveness of nematophagous fungi to entomopathogenic nematodes in different nutrient media Infective juveniles of both EPN species, H. indica LPP30 and S. carpocapsae All, were more apprehended by fungi Arthrobotrys sp CO7 and A. musiformes AM4 in water agar medium (WA), herein considered the control treatment (Figure 3, Table 1). The WA medium was used as control because

Figure 3. Mean number of Heterorhabditis indica LPP30 and Steinernema carpocapsae All infective juveniles recovered (non-apprehended) from different culture media colonized by Arthrobotrys sp. CO7 and Arthrobotrys musiformes AM4. The experiment was performed twice (Assays 1 and 2). Water agar (WA), Cornmeal agar (CA), Potato-dextrose agar (PDA), V8 juice agar (V8), Soil extract agar (SE), nematode media agar (NM). Means followed by the same lower-case letter are not significantly different according to the Tukey test (P≤ 0.05). ns: not significative.

Table 1. ANOVA analysis followed by Tukey’s test for the aggressiveness of nematophagous fungi, Arthrobotrys sp. CO7 and A. musiformes AM4 to entomopathogenic nematodes Heterorhabditis indica LPP30 and Steinernema carpocapsae All (P= 0.05 gl= 5). H. indica LPP30 S. carpocapsae ALL Nematodes Assays Assays 1 2 1 2 Fungi Arthrobotrys sp. CO7 F= 3.93, P=0.014 F= 8.84, P<0.001 F= 4.10, P=0.012 F= 1.98, P=0.130 A. musiformes AM4 F= 4.15, P=0.011 F= 9.63, P<0.001 F= 5.89, P=0.002 F= 5.90, P=0.002

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it is currently known as a poor media, with low C and N sources, that stimulates sporulation and suppresses vegetative growth[29]. In predation tests, WA is used so trapping rings are highly stimulated when nematodes are added to the medium[30,31]. Maciel et al.[32] studied the conidial production in Arthrobotrys robusta Dudd. I31, Duddingtonia flagrans (now Arthrobotrys flagrans (Dudd.) Sidorova, Gorlenko & Nalepina) CG768 and Monacrosporium thaumasium (now Arthrobotrys thaumasium (Drechsler) Schenck, Kendr. & Pramer) NF34A in four different culture media. They found that sporulation varied among different tested media and fungi, and in WA the lowest number of conidia were produced, and it was in this culture media that more Ancylostoma spp. larvae were predated. Therefore, WA, the poorest medium, was the one producing the most traps. Regarding the low nutrient fact, we observed the same in this study. The data herein presented suggests that in poorer nutrient media these fungi tend to be more aggressive, relying on the nematodes as a source of nutrients, forming more trapping rings. Although these fungi can live as saprophytes, they have the ability to change their morphology and increase their parasitic ability according to their physiological necessities[17].

Susceptibility of entomopathogenic nematodes to nematophagous fungi H. indica LPP30 IJs were more susceptible than IJs of S. carpocapsae All to both A. musiformis AM4 and Arthrobotrys sp. CO7 in the first 24 h of contact. However, after 48 h, the means of IJs captured by the fungi became statistically equal, except for S. carpocapsae All in Assay 2, which presented the highest mean capture rate (214.7 IJs) carried out by Arthrobotrys sp. CO7, which was therefore

considered more susceptible than H. indica LPP30 (Figure 4) (F(0.05, 3)= 4.52, P= 0.009 at 24 h; F(0.05, 3)= 1.73,

P= 0.178 at 48 h in Assay 1; and F(0.05, 3)= 4.28, P= 0.011 at 24 h F(0.05, 3)= 5.86, P= 0.002 at 48 h in Assay 2).

Figure 4. Heterorhabditis indica LPP30 and Steinernema carpocapse All infective juveniles apprehended by Arthrobotrys musiformes AM4 and Arthrobotrys sp. CO7, 24 and 48 h after inoculation of juveniles. The experiment was performed twice (Assays 1 and 2). Means followed by the same lower-case letter are not significantly different according to the Tukey test (P≤ 0.05).

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Timper & Kaya[33] demonstrated the protective role of the second-stage cuticle, which is retained as a sheath by IJs of heterorhabditids. We may infer that this extra cuticle may play a role in avoiding fungus recognition and therefore trapping formation. The exsheathment of S. carpocapsae All IJs occurred later than in H. indica LPP30 (data not shown). At 24 h few traps were formed and as time passed, the number of exsheathed IJs grew, as did the number of traps (Figure 5). On the other hand, El-Borai et al.[22]. tested two species and observed differential susceptibility between them, opening up the discussion about the presence of a nematode-fungus chemical interaction. El Borai et al.[13] suggested that the larger IJs, such as S. diaprepesi and S. glaseri, possess greater strength, thus being able to escape from the capture organs of predatory fungi. Since H. indica LPP30 and S. carpocapsae are about the same size (528 µm (479-573) vs. 558 µm (438-650), respectively), we believe that after exsheathment, or at 48 h, H. indica LPP30 IJs were better able to escape from the traps due to their smaller circumference, when compared to S. carpocapsae All IJs. S. carpocapsae All IJs are 25 µm (20-30) in diameter, slightly larger than H. indica LPP30 IJs with 20 µm (19-22) in diameter. Another point to be raised regarding the evaluation at 48 h is the foraging behavior. H. indica LPP30 is considered a cruiser forager, and these individuals could have moved around the plate escaping from the traps, while S. carpocapsae All, with ambusher behavior and low mobility, could have stayed static in the dish, becoming an easier prey. During the whole experiment, intense movement could be seen among IJs of H. indica LPP30, all over the Petri dish, including at the edges and on the lids. Furthermore, the assay evaluation time was short (up to 48 h), due to the difficulty in directly observing EPNs captured on the Petri dish, since they decompose rapidly after being captured, and because the fungal mycelia grows quickly all over the dish, making observation difficult. Therefore, the Baermann funnel technique could have been better used to recover IJs, as was done by El Borai et al.[13], rather than using direct observation of EPNs on the Petri dish by means of a stereoscopic microscope, as was done in the present study (Figure 5). Few studies involving the susceptibility of EPNs to nematophagous fungi are available, but the data from the current study are consistent with the hypothesis that these fungi can negatively affect the efficiency of EPNs as agents in the biological control of insect pests. On the other hand, the hypothesis that native fungi would be more aggressive to native EPNs was not confirmed. We shall test more exotic EPNs to confirm that hypothesis.

Figure 5. Infective juveniles of Heterorhabditis indica LPP30 captured by the nematophagous fungus Arthrobotrys musiformis AM4 in a Petri dish after 48 h of inoculation.

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Entomopathogenic nematode dispersion in different soil types in the presence of nematophagous fungi Both in sandy soil and clay-silty soil, the fungus Arthrobotrys sp. CO7 did not show any effect (P= 0.05) on the spread of the IJs of H. indica LPP30. However, the same fungus showed an effect (P= 0.05) on the spread of S. carpocapsae All in sand at a distance of 15 cm. The means remained below the control treatment, indicating that there was a possible repellent action of the fungus for this species

of EPN (Figure 6) (sand 30 cm: H(0.05, 3)= 0.99, P= 0.801; sand 15 cm: H(0.05, 3)= 12.72, P= 0.005; soil 15 cm:

H(0.05, 3)= 25.45, P< 0.001; soil 30 cm: H(0.05, 3)= 8.93, P= 0.030 for Assay 1, and sand 30 cm: H(0.05, 3)= 2.93,

P= 0.403; sand 15 cm: H(0.05, 3)= 9.36, P= 0.025; soil 15 cm: H(0.05, 3)= 17.91, P< 0.001; soil 30 cm: H(0.05, 3)= 6.72, P= 0.081 for Assay 2). These results demonstrated that there is a specificity in the responses of EPNs to nematophagous fungi, as was observed by El-Borai et al.[22], according to whom all the evaluated EPNs (S. diaprepesi, S. glaseri, S. riobrave, H. zealandica and H. indica) were repelled by Arthrobotrys gephyropaga (Drechsler) Subram., but were attracted by the endoparasitic fungi Myzocytium sp. and Catenaria sp. The exceptions were H. indica, which remained neutral for Myzocytium sp., and S. glaseri, which

Figure 6. Dispersal of Heterorhabditis indica LPP30 and Steinernema carpocapsae All in sandy and clay-silty soil at 15 and 30 cm from the infective juveniles application site in the presence or absence of the fungus Arthrobotrys sp. CO7. Means followed by the same lower-case letter are not significantly different according to the Kruskal-Wallis test (P≤ 0.05). ns: not significant.

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remained neutral for Catenaria sp. in sand columns. However, in Petri dishes with water-agar medium, all the species of EPNs were attracted by the nematophagous fungi, except for S. riobrave. The results of this study also demonstrated that the IJs of EPNs can be repelled or showed neutral responses to Arthrobotrys sp. CO7 in tests involving the choice of different substrates and distances. As was observed by El Borai et al.[22], the results indicated that there was a specificity and a movement to aggregate the group in the responses of the EPNs to the fungus. It is thus of vital importance to use different substrates to evaluate properly the role of semiochemicals in the interactions between nematodes and fungi. Robinson & Jaffee[34] demonstrated that the phytoparasitic nematode Meloidogyne incognita was repelled by the fungus A. gephyropaga in assays with sand, while Jansson & Nordbring-Hertz[35] had previously reported that this fungus attracted M. incognita in Petri dishes with water agar medium. These results demonstrate that the substrate used may be an important factor, and it is fundamental to use substrates that are similar to those that exist in the natural environments of these organisms during experimental assays. The repellence of S. carpocapsae to the isolate Artrhobotrys sp. CO7 suggests that these EPNs present the ability to avoid this highly aggressive fungus, at least in some circumstances as in the soil. It is still not clear if the attraction responses of the EPNs to fungi have a direct relationship with their susceptibility, and it is therefore necessary to carry out more detailed studies on the chemistry between them.

ACKNOWLEDGEMENTS The authors are grateful to the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a scholarship given to the first author.

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