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Article

Steinernema australe Enhanced Its Efficacy against Aegorhinus nodipennis (Coleoptera: Curculionidae) Larvae in Berry Orchards after an Artificial Selection Process

by
Patricia D. Navarro
1,*,†,
Rubén Palma-Millanao
1,*,†,
Ricardo Ceballos
2 and
Almendra J. Monje
1
1
Laboratory of Insects Science, Instituto de Investigaciones Agropecuarias (INIA), Estación Experimental Carillanca, Km 10, Camino Cajón-Vilcún, Temuco 4800000, Región de La Araucanía, Chile
2
Laboratory of Insects Chemical Ecology, Instituto de Investigaciones Agropecuarias (INIA), Estación Experimental Quilamapu, Av. Vicente Méndez 515, Chillán 3800062, Región del Bío Bio, Chile
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agronomy 2022, 12(5), 1128; https://doi.org/10.3390/agronomy12051128
Submission received: 9 April 2022 / Revised: 27 April 2022 / Accepted: 29 April 2022 / Published: 7 May 2022
(This article belongs to the Special Issue Nematodes: Drivers of Agricultural Ecosystem Performance)
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Abstract

:
The entomopathogenic nematode (EPN) Steinernema australe was isolated from Isla Santa Magdalena in Chile and identified as a good alternative for controlling Aegorhinus nodipennis (Coleoptera: Curculionidae) larvae. This weevil is native to the south of Chile and some regions in Argentina, causing the decline and ultimate death of plants in berry orchards. The major problem brought about by the weevil is caused by the larvae, which spend between nine and eleven months below ground, feeding inside the roots of the plants. This study seeks to increase S. australe’s efficacy through an artificial selection process using an odor stimulus. We selected infective juveniles (IJs) that followed the stimulus in order to reach larvae at a depth of 30 cm to achieve this objective. Larvae infected with selected IJs and IJs from the original stock were compared under laboratory, greenhouse, and field conditions. The results showed a 20% increase in the efficacy of selected IJs compared with IJs from the original stock. We observed a higher proportion of selected IJs that reached the larvae faster during the first four days post-application. Moreover, larvae treated with selected IJs were depleted, with a mix of nematode stages emerging from the cadaver. Finally, a potential trade-off with regard to the recycling of nematodes into the soil is proposed.

1. Introduction

Aegorhinus nodipennis (Hope, 1834) (Coleoptera: Curculionidae), commonly known as the plum weevil, is a pest that is widely distributed in the south of Chile and some regions in the Andes mountains in Argentina [1,2,3,4]. This species affects a number of crops, including raspberry [5,6], blueberry [7,8,9], redberry (sarsaparilla) [5,8,10], cranberry [5,8], strawberry, gooseberry [5,8], and recently, hazelnut [11]. The larva mainly causes damage by feeding inside the root- and crown-making galleries [1]. Thus, within three years, highly infested orchards may die, while just one larva per plant may cause partial decline [12]. In blueberry orchards, losses of USD 5000/ha have been reported [13]. Although A. nodipennis is considered an economically important pest in the south of Chile [14,15,16], information about its biology and interactions with other players in the trophic chain is scarce. Thus far, the management of A. nodipennis has focused on adults by using insecticides; however, these products cannot reach the larva deep in the root, making it necessary to use alternative control methods.
The use of entomopathogenic nematodes (hereafter referred to as EPNs), an obligate lethal pathogen of insects, has been a promising tool for controlling soil-dwelling pests worldwide [17]. These EPNs have been shown to exert control over a wide range of insect pests in natural and agricultural systems [18,19] and are considered to be an eco-friendly tool for insect pest control [20], safe for vertebrates and plants [21], and compatible with many chemical insecticides [22]. The EPN third-stage infective juvenile (hereafter referred to as IJ) is the only soil-inhabiting stage of the pest, and—in the search of a suitable host—it may spend more or less time there, according to the (1) host’s habitat, (2) seeking ability of the EPN, (3) EPN–host recognition, and (4) EPN–host acceptance [23], dedicating their life as IJs to find and infect a new insect host. EPNs belonging to the Steinernematidae and Heterorhabditidae families have evolved a mutualistic relationship with bacteria from the Xenorhabdus and Photorhabdus genera, respectively. Both bacteria play a role in killing the insect host by septicemia between 24 and 48 h [24].
Steinernema australe (Edgington, 2009) (Panagrolaimomorpha: Steinernematidae) was evaluated against the raspberry weevil larvae, showing good results [25]. This nematode belongs to the glaseri group and was first isolated from a soil sample taken close to the beach on Isla Magdalena in the Austral region of southern Chile in 2006, with an in-situ soil temperature of 13 °C and a pH of 5.3 [26]. Later, the same species was reported by Lephoto and Gray [27,28] in South Africa. In both cases, S. australe was isolated from a loamy–sand soil. Its infective juvenile (IJ) has a long body (1162–1484 µm), which, according to Koppenhofer et al. [29], may be an adaptation to humid conditions. This EPN species survives and consistently infects in a temperature range between 7 and 23 °C and may complete its life cycle in 15 days [26,30].
Enhancing EPN traits through genetic selection has been studied over the last few decades to improve their biological control performance. More than 30 years ago, studies on different species of Steinernema demonstrated that their host-finding ability might be increased after several rounds of selection [31,32]. Later, new species were evaluated by Boff et al. and Santhi et al. [33,34]), new traits, such as cold tolerance, by Grewal et al. and Nimkingrat et al. [35,36], desiccation by Nimkingrat et al. and Salame et al. [37,38], heat tolerance by Mukuta et al. [39], and dispersal speed by Bal et al. [40]; all of these found similar results. Moreover, the ability of EPNs to respond to foraging cues emitted by damaged roots was also studied [41,42,43]. In this regard, a number of works have reported on the capacity of EPNs to perceive herbivore-induced root volatiles (HIPVs), defining HIPVs as volatiles emitted by plants when insects are feeding on them [44]. These HIPVs may recruit foraging EPNs [45] to infect an insect host, increasing their chances of finding, infecting, and successfully killing a suitable target [46].
France [25] reported that when S. australe was evaluated against Aegorhinus superciliosus (Guérin-Méneville) (Coleoptera: Curculionidae), its larvae showed 100% mortality in the laboratory and 72% in raspberry orchards. These findings reveal S. australe as a promising tool for the control of Aegorhinus larvae and prompt the question of whether this nematode’s efficacy may be increased. To address this question, we evaluated the hypothesis that S. australe may increase its efficacy against A. nodipennis in the field through artificial selection using a previously identified chemotactic attractant as a selective cue.

2. Materials and Methods

2.1. Nematodes

Infective juveniles of S. australe were obtained from a stock curated by the Microbiological Resources Bank at the Instituto de Investigaciones Agropecuarias (INIA), Chillán, Chile. These nematodes were propagated in vivo in the fifth instar Galleria mellonella (Linnaeus, 1756) (Lepidoptera: Pyralidae) following procedures described by Stock and Goodrich-Blair [47]. For all assays, IJs less than seven days old (storage time before initial emergence of IJs) were kept at 4 °C in 40 mL culture flasks (Thermo Scientific Nunc EasyFlask, Massachusetts, USA) and acclimated to room temperature before use.

2.2. Insects

Larvae of A. nodipennis were collected from the field and maintained in 20 × 10 cm plastic boxes containing autoclaved peat at 10 °C in the Insect Science Laboratory at INIA, Vilcún, La Araucanía Region, Chile. These larvae were fed on carrot chunks refreshed every five days until use. Larvae of A. nodipennis were used in all assays. A colony of Galleria Mellonella was reared in the same laboratory following the methodology described by Singh and Moore [48]. Larvae of G. mellonella larva were used for EPN rearing only.

2.3. Laboratory, Greenhouse, and Field Trials

Artificial selection assays and a greenhouse trial were conducted at the Insect Science Laboratory, INIA-Carillanca, Vilcún, Chile. Field assays were conducted in a blueberry orchard (var. Duke) in Negrete (37°35′09.3” S, 72°29′33.1” W, Bío Bio Region) in November 2021 and a sarsaparilla orchard (var Rovada) in Purranque (40°56′27.7” S, 73°06′39.6” W, Los Lagos Region) in January 2022. Both varieties are highly susceptible to A. nodipennis attack.

2.4. Artificial Selection of IJs Using an Odor Stimulus

The T-shaped modified olfactometer: We designed a PVC pipe (25 mm OD) T-shape modified olfactometer (Figure 1A). The pieces, assembly, and functioning are described as follows: the central section consisted of one piece of 40 mm PVC (Figure 1A) and two pieces of 160 mm length PVC (Figure 1B,C); both pieces were assembled using Parafilm. The sections were connected to a PVC Tee pipe (Figure 1D), totaling 360 mm in length. One 40 mm length of PVC piece was connected to each side of the arm (Figure 1E,F) and taped with Parafilm and masking tape to the other side. All pieces mentioned before were necessary to set up the T-shaped modified olfactometer.
The entire device was filled with moisturized sand using sterilized water (10% w/w). The sand was previously sieved, washed with tap and distilled water, and finally oven-dried at 250 °C for 150 min. The cleaned sand was stored in plastic recipients until use. In order to build a uniform environment, PVC pieces were filled little by little with 5 g of humid sand per 10 mm of pipe length. Finally, the Tee was filled with 12 g of humid sand, approximately. To charge the stimulus, 100 µL of 2-Carene solution (1000 µg mL−1, double-distilled hexane used as a solvent) was added to five grams of sand inside one of the arms. In order to allow for solvent evaporation, the pipe was air-ventilated for 15 min before adding the subsequent 15 g of sand. Then, the arm containing the stimulus was connected to the Tee (Figure 2A), and the whole system remained upright, attached to a universal support (Figure 2B). The opposite arm received solvent.
The selection process: For the EPN selection process, we adapted procedures previously reported by Boff et al. [33] and Santhi et al. [34], with some modifications. The T-shaped modified olfactometer, previously described, was designed with the objective that the IJs would move down it in order to reach an odor stimulus (thus named the stimulus), allocated perpendicularly with respect to the IJs’ trajectory. The stimulus corresponded to 2-Carene, a compound previously identified from blueberry roots that has been found to elicit the positive chemotaxis of Steinernema australe (non-published results). The stimulus, allocated at the end of one arm of the T-shaped device, had the role of attracting IJs to be collected later. The IJ collection process was performed at intervals of times starting at 24 h post-inoculation of IJs, and up to 5% of the initially inoculated IJs reached the stimulus [49]. As the new generations (G1 to G6) performed in the experiment, the time intervals were reduced to 16, 8, and 4 h, because 5% of the IJs reached the stimulus faster. The stimulus was replaced every time the IJs were collected from the modified olfactometer. In the first generation (G1), five percent of IJs that first reached the stimulus were collected and used to produce the next generation of nematodes (G2) through a new infection using G. mellonella. This process was repeated over time up to the sixth generation (G6), according to the results reported by Hiltpold et al. [49] for Heterorhabtis bacteriophora (Poinar, 1976).
Close to 7500 IJs were inoculated into the modified olfactometer in three groups of approximately 2500 IJs. Nematodes were counted the day before the olfactometer was set up and remained at 4 °C in culture flasks until use. Three modified olfactometers were used to run the assay, and each group of IJs was inoculated into the top of the pipe. Nematodes were suspended in 1000 µL of sterilized water. Once the EPNs were inoculated, this section was covered with a piece of Parafilm in order to reduce water evaporation. The three modified olfactometers were covered with black fabric to avoid light leaks. In a climate chamber, these olfactometers were kept at 25 °C and 65% humidity.
Nematodes were recovered from the arms of the T-shaped modified olfactometers at the intervals described above and replaced with fresh ones using a new load of attractant. Subsequently, sand from each section containing IJs was individually recovered using 50 mL of tap water and kept in 500 mL plastic cups until use. These cups were gently hand-shaken for 10 s and then remained for 45 min at a 30° inclination for IJ decantation. Finally, water containing the IJs was carefully recovered using a 1 mL plastic dropper. A second wash of the sand was performed using 30 mL of tap water, repeating the procedure described before. Collected IJs were counted under a scope (Nexius Zoom Euromex, Arnhem, The Netherlands) and gently transferred to a culture flask filled with sterilized water and stored at 4 °C. These nematodes were used to infect G. mellonella larvae and obtain each new generation of selected nematodes.

2.5. Comparison of Selected IJs vs. the Original Stock

A laboratory assay was carried out using the same devices and procedure used for selection to compare the efficacy of the selected nematodes with respect to those from the original stock (unselected) following the procedure described above. In this case, three olfactometers were loaded with the G6 of selected IJs, and the other three were loaded with IJs from the original stock. The nematodes arriving at the arm with the stimulus were recovered every 24 h for seven days. IJs were counted and stored at 4 °C in culture flasks.

2.6. Efficacy of the Selected IJs in the Greenhouse

In this study, we sought to determine the effect of the selection process using VOCs on the efficacy of S. australe against A. superciliosus larvae. For this purpose, we compared the larval mortality obtained by the selected IJs, IJs from the original stock, and the control without EPNs on 3 years of blueberry plants var. Duke. These plants were contained in 35 L pots (40 cm diameter and 36 cm deep). Each pot containing one plant was suitably watered every two days and maintained at 15 °C previous to the assay. The soil contained in the pots was autoclaved to avoid the previous presence of EPNs. Ten A. nodipennis 5th instar (40 mg) larvae were introduced per plant, separated into two groups of three larvae and one of four larvae. Each group of larvae was introduced into a metallic jar with holes (Figure 3A,B), each containing autoclaved soil and a chunk of carrot for food (Figure 3C). The use of these metallic jars was necessary to avoid the escape of the larvae (observation made during previous assays) and to keep the larvae at different depths between 0 and 30 cm. The dose of nematodes applied was 290.000 IJs/pot diluted in 1 L of tap water. The IJ solution was gently poured into the pot with a watering can. All pots were maintained at 75% RH, and 14:10 L:D. Larval mortality was observed 72 h after inoculation of IJs, and nematode virulence (efficacy) was measured as the percentage of dead larvae per treatment. Cadavers showing typical EPN infection symptoms (i.e., brownish color and floppy texture) were separated and thoroughly rinsed in distilled water to remove adhering IJs. Each cadaver was individually placed into a 5 cm Petri dish with 9 mL of pepsin and opened with a scalpel to determine the number of IJs that penetrated the larva 72 h after IJ application [50,51]. Larvae with IJs inside were considered successfully infected by S. australe. The number of IJs that penetrated each larva was determined. The greenhouse trial was arranged in a completely randomized design with eighteen replicates per treatment. Independent trials were conducted at 16 and 25 °C.

2.7. Efficacy of the Selected IJs in the Field

Field trials were conducted independently in November (blueberry orchard) and December (sarsaparilla orchard) of 2021. The objective of these trials was to compare the efficacy of the selected IJs with those from the original stock. Each orchard consisted of seven-year-old blueberry plants and five-year-old sarsaparilla plants, with a spacing of 3 × 1 m and 3 × 1.5 m, respectively. The soil in the blueberry orchard was a silty loam with 20 °C and 5.8 pH, and the sarsaparilla orchard was a Trumao soil (derived from volcanic ashes) with 14 °C and 5.5 pH. In both trials, plants were artificially infested with A. nodipennis larvae as previously described. Soil temperature was considered the main factor in selecting the location of the orchards (see Section 2.3). Thus, the blueberry trial was located 370 km north of the sarsaparilla trial, with a difference in soil temperature of 6 °C.
For both trials, plants receiving the treatments were randomly selected and separated by 10 m on each side to avoid a mixture of treatments (treatment IJs). Before trials, soil samples were taken from both orchards to discount the previous presence of EPNs. EPNs were applied once per orchard in a dose of 290.000 IJs/plant diluted in 2 L of tap water. The IJ solution was gently poured on the plant with a watering can. As previously described in the greenhouse trial, ten larvae were artificially inoculated per plant and distributed into three metallic jars. Metallic jars were randomly buried into the soil within the first 40 cm of depth. Larval mortality was observed 96 h after the application of IJs. Nematode virulence (efficacy) was measured as the percentage of dead larvae per treatment. Cadavers showing the typical EPN infection symptoms (i.e., brownish color and floppy texture) were separated and thoroughly rinsed in distilled water to remove adhering IJs. S. australe-infected larvae were determined as described before. The number of IJs that penetrated each larva was determined. Field trials were arranged in a completely randomized design, with ten replicates (plants) per treatment.

2.8. Data Analysis

To estimate the effect of the artificial selection process in the laboratory, a comparison between treatments was made using the percentage (%) of nematodes collected in each sampling calculated for both groups: (1) selected IJs and (2) original stock. The values obtained were compared using the Student’s t-test for independent samples. Larval mortality was considered a response variable in both the greenhouse and field trials. Treatments and soil temperature were evaluated as explanatory variables in both cases (i.e., greenhouse and field). Treatment effects were analyzed using one-way ANOVA in the greenhouse and field trials. Differences among averages were determined using Tukey’s range test (p = 0.05). Data from the greenhouse and field trials were analyzed using JMP®, Version 16.0.0. (SAS Institute Inc., Cary, NC, USA, 1989–2021) and plotted using GraphPad Prism version 9.0.0 (GraphPad Software, San Diego, CA, USA).

3. Results

3.1. Comparison of Selected IJs vs. the Original Stock in the Laboratory

At 96 h post-treatment, significant differences were observed in the proportion of S. australe IJs that quickly responded to the 2-Carene odor stimuli when the selected IJs (8.65% ± 2.65%) and IJs from the original stock (3.23% ± 1.29%) were compared. Selected IJs significantly reduced the time required to search for and reach the larva (t = 3.5; p = 0.0248). Infective juveniles collected from G6 were considered as the selected IJs for further assays. As each generation occurred (G1 up to G6), it took less time for IJs to travel from the starting point in the T-shaped modified olfactometer to the stimulus area. Initially, the time for G1 was 144 h, while for the successive generations, it was 120, 72, 54, and finally, 48 h in G6 (Figure 4), reducing the foraging time to reach the stimulus by three times.
For selected IJs, a larger proportion (number of IJs collected/number of IJs inoculated) of recovered IJs was observed during the first four days of collection from the T-shaped modified olfactometers (Figure 3A) when compared with the remaining time. The advantage taken by the selected IJs during the first four days post-application was unattainable for the IJs from the original stock. Even when the difference narrowed as the days went by, it was confirmed that selected IJs were faster to reach the stimulus than IJs from the original stock (Figure 3B).

3.2. Efficacy of the Selected IJs in the Greenhouse

The results from the greenhouse trial revealed that the efficacy of S. australe selected IJs against A. nodipennis was significantly higher than that for IJs from the original stock at 16 °C (one-way ANOVA; F2,51 = 14.6; p ≤ 0.0001) and 25 °C (one-way ANOVA; F2,51 = 81.6; p ≤ 0.0001). Selected IJs reached 100% mortality at 25 °C and 79% at 16 °C, compared to the original stock (Figure 5), where IJs showed 70% of larval mortality at 25 °C and 49% at 16 °C. For both temperatures, control mortality did not exceed 15%.
Larvae treated with the selected IJs resulted in flat cadavers with a group of mixed stages (J2, females and males) that emerged from the cadaver. The latter was observed at both temperatures in all replicates.
Flat cadavers with a mix of nematode stages were not observed in larvae treated with IJs from the original stock. At 16 and 25 °C, larvae treated with IJs from the original stock showed 88% dead larvae at 16 °C and 98% at 25 °C, with at least two or three IJs alive inside. Selected IJs showed a faster life cycle in A. nodipennis larvae than in IJs from the original stock.

3.3. Efficacy of the Selected IJs in the Field

In the field, selected IJs showed higher efficacy than IJs from the original stock in both blueberry (one-way ANOVA; F2,8 = 3.9; p≤0.0001; Figure 6A) and sarsaparilla (one-way ANOVA; F2,87 = 166.6; p ≤ 0.0001; Figure 6B) orchards. In both localities, selected IJs reached approximately 90% of S. australe larvae mortality, showing almost 20% more efficacy than treatments evaluated with IJs from the original stock (70% approximately). In both localities, larvae from the control resulted in less than 9% mortality.
With respect to the number of IJs that penetrated the larva, we found that 85% and 93% of the tested larvae were infected by at least one female and one male in treatments with the selected IJs in blueberry and sarsaparilla, respectively. An average of 64% of larvae were infected with IJs from the original stock in both localities. No nematodes were found in larvae from the control.
Additionally, we observed a higher number of nematodes inside the cadavers treated with selected IJs (a mix of stages impossible to count) than in cadavers treated with IJs from the original stock. It was evidenced that selected IJs reached the host larva before IJs from the original stock, resulting in a life cycle that started and finished before the original stock IJs arrived. No nematodes were found in larvae from the control.
For all assays and field trials conducted in our study, where selected IJs were applied, we found that cadavers were flat and depleted of food 72 h post-infection (Figure 7A). In fact, we only recovered the cephalic capsule (Figure 7B) and some portions of the skin. Evidence of a mix of nematode stages was found on the cadaver skin and in the surrounding soil.

4. Discussion

During EPN foraging, the decision of whether to infect one host or another is informed by olfactory and mechanosensory signals [52]. Taking advantage of these cues, we enhanced the foraging of S. australe through artificial selection using an odor stimulus. Our results showed that the selection process increased the efficacy of S. australe selected IJs against A. nodipennis larvae in blueberry and sarsaparilla orchards by 20%. Similar studies have been reported by Gaugler et al. [31]; Hiltpold et al. [49,53], and Santhi et al. [34]. As far as we know, this is the first report focusing on the effect of artificial selection to increase the efficacy of S. australe in the field.
In this study, we found that the most outstanding effect of the selection process on S. australe was the increase in the proportion of IJs that reached the larva faster at the location of at least 30 cm deep in the soil and vertically downwards, similar to results found by Hiltpold et al. [49]. However, based on these results only, we cannot yet confirm that it was 2-Carene that triggered the IJ trait enhancement. We speculate that the selection process could increase the proportion of the so-called sprinter nematodes corresponding to IJs that go to find the far away host [54].
Our results suggest that S. australe has some foraging characteristics and olfaction capacities that helped improve their ability to find the host [55,56]. Both traits were addressed by Baiocchi et al. [52] in a study of EPNs’ host-seeking abilities using specific odors to assess host resources. These authors evaluated the response of Steinernema carpocapsae (Weiser, 1955), Steinernema feltiae (Filipjev, 1934), Steinernema glaseri (Steiner, 1929), and Steinernema riobrave (Cabanillas, Poinar, and Raulston, 1994) for behavioral decisions regarding attraction to or avoidance of previously infected hosts. Although the objectives were different from our study, their results support the idea that S. glaseri, a cruiser-type nematode characterized by Glaser et al. [57], is a good candidate for chemotaxis assays, while not observing the same results in the other EPN species. We found similar results for S. australe, an EPN that belongs to the glaseri group, sharing similar characteristics with S. glaseri, such as IJs’ size and cruiser searching behavior [26,30].
The higher efficacy obtained after six rounds of artificial selection on S. australe apparently did not result in detrimental effects on nematode fitness (i.e., penetration and virulence). Similar results were observed by Hiltpold et al. [49], where the EPN Heterorhabditis bacteriophora was selected for higher responsiveness towards (E)-β-caryophyllene, enhancing EPN efficacy against Diabrotica virgifera LeConte (Coleoptera: Chrysomelidae). These authors did not find a reduction in the establishment or persistence of the nematodes after field application. However, in other studies reported by Gaugler et al. and Stuart et al. [58,59], trade-offs to the EPN have been evaluated, for example, between efficacy and storage stability, and between efficacy and emergence from the host cadaver, or between enhanced dispersal and reduction in reproduction capacity [54]. Further studies are recommended to determine the potential trade-off between efficacy and nematode reproduction in S. australe [60,61].
Initially, we planned to carry-out a field trial on blueberry orchards only. Later, and based on our interesting efficacy results in the greenhouse and the field, the consistent behavior of selected IJs, and the non-negative effects on nematode fitness, we decided to run a second field trial in a different berry species to expand our observations. Although sarsaparilla is not as important as the blueberry in southern Chile, this crop is known to be even more attacked by Aegorhinus larvae than are blueberry plants [8,10,62]. For sarsaparilla, we found similar results (>20% efficacy using selected IJs than IJs from the stock) in the field at 16 °C soil temperature, confirming the positive effect of the selection process on the efficacy of this biocontrol agent.
Species belonging to the glaseri group are recognized by having IJs with large body sizes and a preference for humid–sandy soils, and S. australe is no exception [26,27]. This EPN species showed high specificity for Aegorhinus larvae and other curculionids, whose efficacy was briefly described by France [25] and Luppichini et al. [63]. Edgington et al. [26,30] also mentioned that in S. australe, the emergence of J2 and a mix of other stages might occur seven days after infection in small insect hosts. Larvae of A. nodipennis are relatively large, ranging from 1.8 to 2 cm in length when fully grown [64], and weighing 40 mg on average (non-published data).
We believe that depletion did not occur due to the host size [56] and poor food availability, but rather due to the more significant proportion of selected IJs that reached the larva faster, competing for resources, and increasing in number and size, which drastically reduced food availability [65,66]. We also speculate that these conditions triggered the switch to endotokia matricida, a phenomenon observed on S. australe in previous studies in our lab (non-published results), where females retain eggs that hatch in the uterus, and these eggs consume the mother [67].
We did not observe cadaver depletion or a mixture of nematode stages in any trial using IJs from the original stock. Observations about cadaver depletion caused by selected IJs of S. australe were even more dramatic in the greenhouse trial at 25 °C soil temperature and field trial at 20 °C, where limited and ephemeral cadavers were observed [56]. Finally, we did not observe a trade-off in S. australe fitness; however, a potential cost of recycling nematodes in the soil may occur, requiring further studies to confirm.

5. Conclusions

Artificial selection using an odor stimulus was shown to be an effective method to increase S. australe efficacy against A. nodipennis larvae in blueberry and sarsaparilla orchards in the south of Chile. The use of 2-Carene allowed for an increase in the proportion of IJs that reached the larva faster at a location of at least 30 cm deep in the soil, vertically downwards, resulting in a 20% increase in larval mortality in the field. The higher efficacy obtained after six rounds of artificial selection on S. australe did not result in detrimental effects on the penetration and virulence of IJs; however, a potential cost of recycling nematodes in the soil may occur, requiring further studies to confirm.

Author Contributions

Conceptualization, P.D.N. and R.P.-M.; data curation, P.D.N., R.P.-M. and A.J.M.; funding acquisition, P.D.N. and R.P.-M.; investigation, P.D.N., R.P.-M., R.C. and A.J.M.; methodology, P.D.N., R.P.-M. and A.J.M.; project administration, P.D.N.; supervision, P.D.N.; writing—original draft, P.D.N. and R.P.-M.; writing—review and editing, R.C. and A.J.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by ANID and Fondef/Concurso IDeA, I+D 18I10005. We thanks to Agrícola Giddings and Biofuturo for their interest in this project, collaboration and financial support.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Schematic representation of a modified T-shaped olfactometer. (A) 40 mm PVC piece, (B) and (C) 160 mm length pieces, (D) PVC Tee pipe, and (E,F) left and right arms (40 mm length PVC).
Figure 1. Schematic representation of a modified T-shaped olfactometer. (A) 40 mm PVC piece, (B) and (C) 160 mm length pieces, (D) PVC Tee pipe, and (E,F) left and right arms (40 mm length PVC).
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Figure 2. Modified T-shaped olfactometer. (A) Arm containing the odor stimulus and (B) universal support where the olfactometer was attached during the assays.
Figure 2. Modified T-shaped olfactometer. (A) Arm containing the odor stimulus and (B) universal support where the olfactometer was attached during the assays.
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Figure 3. Metallic jar with holes for assays with entomopathogenic nematodes in the greenhouse and the field. (A) Schematic sections, (B) empty metallic jar sealed at both ends, (C) filled with autoclaved soil, host larvae, and a chunk of carrot as a food source.
Figure 3. Metallic jar with holes for assays with entomopathogenic nematodes in the greenhouse and the field. (A) Schematic sections, (B) empty metallic jar sealed at both ends, (C) filled with autoclaved soil, host larvae, and a chunk of carrot as a food source.
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Figure 4. Percentage of S. australe IJs recovered over the time (hours) from a T-shaped modified olfactometer with an odor stimulus. Olfactometers were loaded with the treatments: (1) selected IJs (G6) (blue bars) and (2) IJs from the original stock (orange bars) and kept for 24 h. The Figure shows (A) the percentage of daily IJ recovery and (B) accumulated IJ recovery for both treatments. The asterisk shows significant differences in the percentage of IJs recovered between both treatments, according to the Student’s t-test (p ≤ 0.05). * Bars with asterisk show statistically significant differences.
Figure 4. Percentage of S. australe IJs recovered over the time (hours) from a T-shaped modified olfactometer with an odor stimulus. Olfactometers were loaded with the treatments: (1) selected IJs (G6) (blue bars) and (2) IJs from the original stock (orange bars) and kept for 24 h. The Figure shows (A) the percentage of daily IJ recovery and (B) accumulated IJ recovery for both treatments. The asterisk shows significant differences in the percentage of IJs recovered between both treatments, according to the Student’s t-test (p ≤ 0.05). * Bars with asterisk show statistically significant differences.
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Figure 5. Efficacy (mean ± SEM) of selected S. australe against A. nodipennis larvae in 35 L pots under two different soil temperatures. Mortality was recorded 72 h after nematode application. Different letters represent differences according to the Tukey HSD test (p ≤ 0.05).
Figure 5. Efficacy (mean ± SEM) of selected S. australe against A. nodipennis larvae in 35 L pots under two different soil temperatures. Mortality was recorded 72 h after nematode application. Different letters represent differences according to the Tukey HSD test (p ≤ 0.05).
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Figure 6. Efficacy (mean ± SEM) of the selected S. australe IJs (blue), original stock S. australe IJ (red), and control without nematode (green) in blueberry and sarsaparilla orchards, measured as a percentage of larval mortality (A,B); the percentage of larvae with a confirmed infection by EPN (C,D). Different letters indicate significant differences according to the Tukey HSD test (p ≤ 0.05).
Figure 6. Efficacy (mean ± SEM) of the selected S. australe IJs (blue), original stock S. australe IJ (red), and control without nematode (green) in blueberry and sarsaparilla orchards, measured as a percentage of larval mortality (A,B); the percentage of larvae with a confirmed infection by EPN (C,D). Different letters indicate significant differences according to the Tukey HSD test (p ≤ 0.05).
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Figure 7. A. superciliosus larvae cadavers. (A) Larvae treated without EPN, with the selected IJs and IJs from the stock and (B) cephalic capsules obtained after 96 h of treatment of the larvae with the selected IJs.
Figure 7. A. superciliosus larvae cadavers. (A) Larvae treated without EPN, with the selected IJs and IJs from the stock and (B) cephalic capsules obtained after 96 h of treatment of the larvae with the selected IJs.
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Navarro, P.D.; Palma-Millanao, R.; Ceballos, R.; Monje, A.J. Steinernema australe Enhanced Its Efficacy against Aegorhinus nodipennis (Coleoptera: Curculionidae) Larvae in Berry Orchards after an Artificial Selection Process. Agronomy 2022, 12, 1128. https://doi.org/10.3390/agronomy12051128

AMA Style

Navarro PD, Palma-Millanao R, Ceballos R, Monje AJ. Steinernema australe Enhanced Its Efficacy against Aegorhinus nodipennis (Coleoptera: Curculionidae) Larvae in Berry Orchards after an Artificial Selection Process. Agronomy. 2022; 12(5):1128. https://doi.org/10.3390/agronomy12051128

Chicago/Turabian Style

Navarro, Patricia D., Rubén Palma-Millanao, Ricardo Ceballos, and Almendra J. Monje. 2022. "Steinernema australe Enhanced Its Efficacy against Aegorhinus nodipennis (Coleoptera: Curculionidae) Larvae in Berry Orchards after an Artificial Selection Process" Agronomy 12, no. 5: 1128. https://doi.org/10.3390/agronomy12051128

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