Entomopathogenic Nematode Compatibility with Vineyard Fungicides.

Vineyards, covering over seven million hectares worldwide, hold significant socio-cultural importance. Traditionally reliant on conventional practices and agrochemicals, this agroecosystem faces environmental challenges, including soil and water pollution. Sustainable viticulture, driven by eco-friendly practices and cost reduction, has gained prominence, underlining the importance of biological control agents such as entomopathogenic nematodes (EPNs). EPNs naturally occurr in vineyard soils and play a crucial role in controlling pest damage. Ensuring compatibility between EPNs and the commonly used vineyard fungicides is critical, as these applications constitute the predominant pest-management practice during the productive grapevine cycle. This study assessed the impact of authorized grapevine fungicides on EPNs, focusing on the survival of populations and sublethal effects on their virulence. We investigated the compatibility of two EPN populations (Steinernema feltiae 107 and S. carpocapsae 'All') with three organic production-approved products (Bacillus pumilus, sulfur, and copper oxychloride) and two synthetic chemicals (Trifloxystrobin and Mancozeb). Our findings revealed that the viability of S. feltiae 107 was reduced when exposed to sulfur and copper oxychloride, and its virulence was affected by copper oxychloride and Mancozeb, although only two days after exposure and with no significant differences for larval mortality at five days. In contrast, S. carpocapsae 'All' exhibited full compatibility with all five fungicides, with no impact on its viability or virulence. Consequently, our results suggested that the evaluated fungicides could be co-applied on both EPN populations if they were employed on the same day. However, further research on multi-target interactions is needed to ensure the successful implementation of this kind of co-application.

The deterrent ability of Xenorhabdus nematophila and Photorhabdus laumondii compounds as a potential novel tool for Lobesia botrana (Lepidoptera: Tortricidae) management.

The grapevine moth, Lobesia botrana (Lepidoptera: Tortricidae), is a critical pest for vineyards and causes significant economic losses in wine-growing areas worldwide. Identifying and developing novel semiochemical cues (e.g. volatile bacterial compounds) which modify the ovipositional and trophic behaviour of L. botrana in vineyard fields could be a novel control alternative in viticulture. Xenorhabdus spp. and Photorhabdus spp. are becoming one of the best-studied bacterial species due to their potential interest in producing toxins and deterrent factors. In this study, we investigated the effect of the deterrent compounds produced by Xenorhabdus nematophila and Photorhabdus laumondii on the ovipositional moth behaviour and the larval feeding preference of L. botrana. Along with the in-vitro bioassays performed, we screened the potential use of 3 d cell-free bacterial supernatants and 3 and 5 d unfiltered bacterial ferments. In addition, we tested two application systems: (i) contact application of the bacterial compounds and (ii) volatile bacterial compounds application. Our findings indicate that the deterrent effectiveness varied with bacterial species, the use of bacterial cell-free supernatants or unfiltered fermentation product, and the culture times. Grapes soaked in the 3 d X. nematophila and P. laumondii ferments had âˆ¼ 55% and âˆ¼ 95% fewer eggs laid than the control, respectively. Likewise, the volatile compounds emitted by the 5 d P. laumondii fermentations resulted in âˆ¼ 100% avoidance of L. botrana ovipositional activity for three days. Furthermore, both bacterial fermentation products have larval feeding deterrent effects (∼65% of the larva chose the control grapes), and they significantly reduced the severity of damage caused by third instar larva in treated grapes. This study provides insightful information about a novel bacteria-based tool which can be used as an eco-friendly and economical alternative in both organic and integrated control of L. botrana in vineyard.

When Competitors Join Forces: Consortia of Entomopathogenic Microorganisms Increase Killing Speed and Mortality in Leaf- and Root-Feeding Insect Hosts.

Combining different biocontrol agents (BCA) is an approach to increase efficacy and reliability of biological control. If several BCA are applied together, they have to be compatible and ideally work together. We studied the interaction of a previously selected BCA consortium of entomopathogenic pseudomonads (Pseudomonas chlororaphis), nematodes (Steinernema feltiae associated with Xenorhabdus bovienii), and fungi (Metarhizium brunneum). We monitored the infection course in a leaf- (Pieris brassicae) and a root-feeding (Diabrotica balteata) pest insect after simultaneous application of the three BCA as well as their interactions inside the larvae in a laboratory setting. The triple combination caused the highest mortality and increased killing speed compared to single applications against both pests. Improved efficacy against P. brassicae was mainly caused by the pseudomonad-nematode combination, whereas the nematode-fungus combination accelerated killing of D. balteata. Co-monitoring of the three BCA and the nematode-associated Xenorhabdus symbionts revealed that the four organisms are able to co-infect the same larva. However, with advancing decay of the cadaver there is increasing competition and cadaver colonization is clearly dominated by the pseudomonads, which are known for their high competitivity in the plant rhizosphere. Altogether, the combination of the three BCA increased killing efficacy against a Coleopteran and a Lepidopteran pest which indicates that this consortium could be applied successfully against a variety of insect pests.

Organic mulching modulated native populations of entomopathogenic nematode in vineyard soils differently depending on its potential to control outgrowth of their natural enemies.

The entomopathogenic nematodes (EPNs) are biological control agents that are widespread in crop soils. However, traditional agricultural management practices such as cultivation and agrochemical usage can alter the soil balance that enables their occurrence and activity. Alternative strategies like mulching are commonly employed to prevent weed growth, enhance below-ground biodiversity by improving soil, organic matter content, fertility, and moisture. We hypothesized that organic mulches would favor biotic conditions for nematofauna development in crop soil, including EPNs, compared to herbicide application or tillage. Traditional (insect baits) and molecular (qPCR analysis) tools were used in this study to assess the abundance and activity of native EPNs, and the abundance of potential natural enemies, such as free-living nematode (FLN) competitors, nematophagous fungi (NF), and ectoparasitic bacteria, in soils managed with different organic mulches or traditional practices. As a model agroecosystem, we selected the vineyard, one of the most intensively managed crop systems. We compared mulches of grape pruning debris (GPD-M), straw (Str-M), and spent mushroom compost (SMC-M) in two commercial vineyards, which employed either integrated or organic pest and disease management. Following a completely randomized design, we retrieved two composite samples per plot (n = 3 per treatment in each vineyard) in April, June, and October 2020. Numbers of EPNs and selected members of their soil food web were higher in the organic than the integrated managed vineyard. Supporting our hypothesis, organic mulching overall favored nematode occurrence in both vineyards. We found higher NF abundance for Str-M, and GPD-M in the organic vineyard, which plausibly explained the lower EPN activity and occurrence compared to SMC-M in both vineyards. We conclude that the organic mulches can provide appropriate conditions for increasing nematofauna numbers but, depending on the mulch type, may also adversely affect EPNs by increasing their natural enemies. Our findings highlight the need to explore alternative farming practices to unravel complex biotic interactions that affect beneficial soil organisms in agroecosystems.

Potent Ant Deterrents Emitted from Nematode-Infected Insect Cadavers.

Most known species of entomopathogenic nematodes (EPNs) are generalist obligate parasites of insects. They kill their hosts within days after infection and mortality is mainly caused by toxins produced by bacteria that co-infect the hosts and serve as food for the nematodes. EPNs can infect a very broad spectrum of insects and these insects can therefore be expected to have evolved strategies to avoid infection. Indeed, ants are known to avoid feeding on EPN-infected insect cadavers, most likely because they are repelled by semiochemicals that emanate from the cadavers. The source and nature of these repellents are so far unknown. In a series of behavioral and chemical analytical experiments we identified hexadecanal and 2-heptadecanone as two compounds that are emitted by insect larva that are infected by the EPN Steinernema feltiae, but not by uninfected larvae. When spiking honey water with the two semiochemicals, they were confirmed to be highly deterrent to the ant Lasius niger. The environmentally benign hexadecanal and 2-heptadecanone could be employed to ward off ants and possibly other pests. Additional experiments are needed to fully determine their application potential.

Intraspecific virulence of entomopathogenic nematodes against the pests Frankliniella occidentalis (Thysanoptera: Thripidae) and Tuta absoluta (Lepidoptera: Gelechiidae).

Entomopathogenic nematodes (EPN) are excellent biocontrol agents against various insect pests. Novel biotechnological approaches can enhance their utility against insects above-ground, opening a new venue for selecting superior EPN against certain insects. We hypothesize that different populations of the same species but from different origins (habitat, ecoregion) will differ in their virulence. This study aimed to evaluate the virulence of various EPN populations against two pests of worldwide incidence and damage to high value crops: Frankliniella occidentalis (Thysanoptera: Thripidae) and Tuta absoluta (Lepidoptera: Gelechiidae). We tested 10 EPN populations belonging to three EPN species: Heterorhabditis bacteriophora (Koppert, MG-618b, AM-203, RM-102), Steinernema feltiae (Koppert, RS-5, AM-25, RM-107), and Steinernema carpocapsae (Koppert, MG-596a). Each EPN population was tested at two concentrations. Frankliniella occidentalis was tested at 160 and 80 IJs/cm2 and T. absoluta at 21 and 4 IJs/cm2. Control treatments followed the same experimental procedure but only adding distilled water. Overall, whenever different, higher IJs concentration resulted in lower adult emergence, higher larval mortality, and shorter time to kill the insects. Considering the low concentration, S. feltiae provided the best results for both insects and instars investigated, while H. bacteriophora and S. carpocapsae required a high concentration to reach similar or slightly better results. Differences among populations of each of the species were detected, but only the native populations of H. bacteriophora populations showed consistently higher control values against both insects/instar compared with the commercial one. Differences among S. feltiae and S. carpocapsae populations depended on the IJs concentration, insect, and instar. We consider S. feltiae a very promising species for their application against F. occidentalis and T. absoluta, with the Koppert population as the most consistent among the populations tested. Specific EPN-populations of S. carpocapsae and H. bacteriophora were good candidates against certain instar/insects at high concentrations. This study emphasized the importance of intraspecific variability for EPN virulence.

Exploring the Use of Entomopathogenic Nematodes and the Natural Products Derived from Their Symbiotic Bacteria to Control the Grapevine Moth, Lobesia botrana (Lepidoptera: Tortricidae).

The European grapevine moth (EGVM) Lobesia botrana (Lepidoptera: Tortricidae) is a relevant pest in the Palearctic region vineyards and is present in the Americas. Their management using biological control agents and environmentally friendly biotechnical tools would reduce intensive pesticide use. The entomopathogenic nematodes (EPNs) in the families Steinernematidae and Heterorhabditidae are well-known virulent agents against arthropod pests thanks to symbiotic bacteria in the genera Xenorhabdus and Photorhabdus (respectively) that produce natural products with insecticidal potential. Novel technological advances allow field applications of EPNs and those bioactive compounds as powerful bio-tools against aerial insect pests. This study aimed to determine the viability of four EPN species (Steinernema feltiae, S. carpocapsae, S. riojaense, and Heterorhabditis bacteriophora) as biological control agents against EGVM larval instars (L1, L3, and L5) and pupae. Additionally, the bioactive compounds from their four symbiotic bacteria (Xenorhabdus bovienii, X. nematophila, X. kozodoii, and Photorhabdus laumondii subsp. laumondii, respectively) were tested as unfiltered ferment (UF) and cell-free supernatant (CFS) against the EGVM larval instars L1 and L3. All of the EPN species showed the capability of killing EGVM during the larval and pupal stages, particularly S. carpocapsae (mortalities of ~50% for L1 and >75% for L3 and L5 in only two days), followed by efficacy by S. feltiae. Similarly, the bacterial bioactive compounds produced higher larval mortality at three days against L1 (>90%) than L3 (~50%), making the application of UF more virulent than the application of CFS. Our findings indicate that both steinernematid species and their symbiotic bacterial bioactive compounds could be considered for a novel agro-technological approach to control L. botrana in vineyards. Further research into co-formulation with adjuvants is required to expand their viability when implemented for aboveground grapevine application.

Insecticidal Effect of Entomopathogenic Nematodes and the Cell-Free Supernatant from Their Symbiotic Bacteria against Philaenus spumarius (Hemiptera: Aphrophoridae) Nymphs.

The meadow spittlebug Philaenus spumarius (Hemiptera: Aphrophoridae) is the primary vector of Xylella fastidiosa (Proteobacteria: Xanthomonadaceae) in Europe, a pest-disease complex of economically relevant crops such as olives, almonds, and grapevine, managed mainly through the use of broad-spectrum pesticides. Providing environmentally sound alternatives to reduce the reliance on chemical control is a primary challenge in the control of P. spumarius and, hence, in the protection of crops against the expansion of its associated bacterial pathogen. Entomopathogenic nematodes (EPNs) are well-known biocontrol agents of soil-dwelling arthropods. Recent technological advances in field applications, including improvements in obtaining cell-free supernatant from their symbiotic bacteria, allow their successful implementation against aerial pests. Thus, this study aimed to evaluate, for the first time, the efficacy of EPN applications against nymphal instars of P. spumarius. We tested four EPN species and the cell-free supernatant of their corresponding symbiotic bacteria: Steinernema feltiae-Xenorhabdus bovienii, S. carpocapsae-X. nematophila, S. riojaense-X. kozodoii, and Heterorhabditis bacteriophora-Photorhabdus laumondii subsp. laumondii. First, we showed that 24 and 72 h exposure to the foam produced by P. spumarius nymphs did not affect S. feltiae virulence. The direct application of steinernematid EPNs provided promising results, reaching 90, 78, and 53% nymphal mortality rates after five days of exposure for S. carpocapsae, S. feltiae, and S. riojaense, respectively. Conversely, the application of the cell-free supernatant from P. laumondii resulted in nymphal mortalities of 64%, significantly higher than observed for Xenorhabdus species after five days of exposure. Overall, we demonstrated the great potential of the application of specific EPNs and cell-free supernatant of their symbiont bacteria against P. spumarius nymphs, introducing new opportunities to develop them as biopesticides for integrated management practices or organic vineyard production.

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi.

Earthworms are ecological engineers that can contribute to the displacement of biological control agents such as the entomopathogenic nematodes (EPNs) and fungi (EPF). However, a previous study showed that the presence of cutaneous excreta (CEx) and feeding behavior of the earthworm species Eisenia fetida (Haplotaxida: Lumbricidae) compromise the biocontrol efficacy of certain EPN species by reducing, for example, their reproductive capability. Whether this phenomenon is a general pattern for the interaction of earthworms-entomopathogens is still unknown. We hypothesized that diverse earthworm species might differentially affect EPN and EPF infectivity and reproductive capability. Here we investigated the interaction of different earthworm species (Eisenia fetida, Lumbricus terrestris, and Perionyx excavatus) (Haplotaxida) and EPN species (Steinernema feltiae, S. riojaense, and Heterorhabditis bacteriophora) (Rhabditida) or EPF species (Beauveria bassiana and Metarhizium anisopliae) (Hypocreales), in two independent experiments. First, we evaluated the application of each entomopathogen combined with earthworms or their CEx in autoclaved soil. Hereafter, we studied the impact of the earthworms' CEx on entomopathogens applied at two different concentrations in autoclaved sand. Overall, we found that the effect of earthworms on entomopathogens was species-specific. For example, E. fetida reduced the virulence of S. feltiae, resulted in neutral effects for S. riojaense, and increased H. bacteriophora virulence. However, the earthworm P. excavates increased the virulence of S. feltiae, reduced the activity of H. bacteriophora, at least at specific timings, while S. riojaense remained unaffected. Finally, none of the EPN species were affected by the presence of L. terrestris. Also, the exposure to earthworm CEx resulted in a positive, negative or neutral effect on the virulence and reproduction capability depending on the earthworm-EPN species interaction. Concerning EPF, the impact of earthworms was also differential among species. Thus, E. fetida was detrimental to M. anisopliae and B. bassiana after eight days post-exposure, whereas Lumbricus terrestris resulted only detrimental to B. bassiana. In addition, most of the CEx treatments of both earthworm species decreased B. bassiana virulence and growth. However, the EPF M. anisopliae was unaffected when exposed to L. terrestris CEx, while the exposure to E. fetida CEx produced contrasting results. We conclude that earthworms and their CEx can have positive, deleterious, or neutral impacts on entomopathogens that often coinhabit soils, and that we must consider the species specificity of these interactions for mutual uses in biological control programs. Additional studies are needed to verify these interactions under natural conditions.

Activity of Steinernema colombiense in plant-based oils.

Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.Entomopathogenic nematodes (EPNs) are excellent biological control agents. Although traditionally EPN application targeted belowground insects, their aboveground use can be supported if combined with adjuvants. We hypothesized that EPN infective juveniles (IJs) could be combined with plant-based oils as adjuvants, without decreasing their efficacy against insect larvae under various scenarios. Specifically, our objectives were to evaluate the activity of Steinernema colombiense (Nematoda: Steinernematidae) when mixed with two plant-based oils (coconut and olive oils) and maintained at different temperatures and times, or combined with entomopathogenic fungi. First, we evaluated how these oils affected IJ survival and virulence against last instar Galleria mellonella (Lepidoptera: Pyralidae) larvae when maintained at five different temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days), using water as control treatment. Second, we evaluated virulence when combined with these two oils as well as with water (control) and combined with the entomopathogenic fungi (EPF), Beauveria bassiana (Hypocreales: Clavicipitaceae). Infective juvenile survival was higher in coconut than olive oil and water mixtures up to 7 days at 4°C. Conversely, olive oil supported higher larval mortality than coconut oil at 4 to 20°C and 14 days. Similarly, the number of days needed to kill insect larvae increased at extreme temperatures (4 and 24°C) after 14 days. Finally, the EPN + EPF combination showed an additive effect compared to EPN and EPF single treatments. Our findings indicate that our plant-based oil mixtures maintain viable IJs at moderate temperatures and up to 7 to 14 days, and can be used in single EPN mixtures or combined with EPF.

Patterns of Occurrence and Activity of Entomopathogenic Fungi in the Algarve (Portugal) Using Different Isolation Methods.

Entomopathogenic fungi (EPF) are distributed in natural and agricultural soils worldwide. To investigate EPF occurrence in different botanical habitats and soil-ecoregions, we surveyed 50 georeferenced localities in the spring of 2016 across the Algarve region (South Portugal). Additionally, we compared three EPF isolation methods: insect baiting in untreated or pre-dried-soil and soil dilution plating on a selective medium. We hypothesized that forest habitats (oak and pine semi-natural areas) and the acidic soil ecoregion may favor EPF occurrence. Overall, EPF species were present in 68% of sites, widely distributed throughout the Algarve. The use of selective media resulted in higher recovery of EPF than did either soil-baiting method. Contrary to our hypothesis, neither vegetation type nor ecoregion appeared to influence EPF occurrence. Traditional and molecular methods confirmed the presence of five EPF species. Beauveria bassiana (34% of sites), was the most frequently detected EPF, using pre-dried soil baiting and soil dilution methods. However, baiting untreated soil recovered Fusarium solani more frequently (26% of sites), demonstrating the utility of using multiple isolation methods. We also found Fusarium oxysporum, Purpureocillium lilacinum and Metarhizium anisopliae in 14%, 8% and 2% of the sites, respectively. Three abiotic variables (pH, soil organic matter and Mg) explained 96% of the variability of the entomopathogen community (EPF and entomopathogenic nematodes) in a canonical correspondence analysis, confirming the congruence of the soil properties that drive the assemblage of both entomopathogens. This study expands the knowledge of EPF distribution in natural and cultivated Mediterranean habitats.

Entomopathogenic nematodes from Mexico that can overcome the resistance mechanisms of the western corn rootworm.

Natural enemies of herbivores are expected to adapt to the defence strategies of their preys or hosts. Such adaptations may also include their capacity to cope with plant metabolites that herbivores sequester as a defence. In this study, we evaluated the ability of Mexican entomopathogenic nematodes (EPN) to resist benzoxazinoids that are sequestered from maize roots by the western corn rootworm (WCR, Diabrotica virgifera virgifera; Coleoptera: Chrysomelidae), an important maize pest in America and Europe. From maize fields throughout Mexico, we retrieved 40 EPN isolates belonging to five different species, with a majority identified as Heterorhabditis bacteriophora. In the laboratory, all nematodes readily infected non-sequestering larvae of the banded cucumber beetle (D. balteata), while infectivity varied strongly for WCR larvae. While some H. bacteriophora isolates seemed negatively affected by benzoxazinoids, most showed to be resistant. Thus, EPN from Mexican maize fields can cope with these plant defence metabolites, but the results also indicate that WCR larvae possess other mechanisms that help to resist EPN. This work contributes to a better understanding of the capacity of herbivore natural enemies to resist plant defence metabolites. Furthermore, it identifies several benzoxazinoid-resistant EPN isolates that may be used to control this important maize pest.

A global database of soil nematode abundance and functional group composition.

As the most abundant animals on earth, nematodes are a dominant component of the soil community. They play critical roles in regulating biogeochemical cycles and vegetation dynamics within and across landscapes and are an indicator of soil biological activity. Here, we present a comprehensive global dataset of soil nematode abundance and functional group composition. This dataset includes 6,825 georeferenced soil samples from all continents and biomes. For geospatial mapping purposes these samples are aggregated into 1,933 unique 1-km pixels, each of which is linked to 73 global environmental covariate data layers. Altogether, this dataset can help to gain insight into the spatial distribution patterns of soil nematode abundance and community composition, and the environmental drivers shaping these patterns.

Effects of Ground Cover Management on Insect Predators and Pests in a Mediterranean Vineyard.

Conservative techniques, such as ground cover management, could help promote viticulture sustainability, which is a goal of conservation biological control, by providing shelter and food sources for predatory insects. A field experiment was conducted in a Mediterranean vineyard to evaluate ground cover management impacts on predatory insect and potential grapevine pest abundance and diversity, both on the ground and in the grapevine canopy. Three different ground cover management techniques (tillage, spontaneous cover and flower-driven cover) were tested for two years (2016 and 2017). Overall, the ground cover management significantly affected the abundance of important epigeal predators, of which carabids, forficulids and staphylinids were the most captured. The carabid abundances under both the cover crop treatments were found to be approximately three times higher compared with that under the tillage treatment. In contrast, the canopy insect abundance in the vineyard was similar among the treatments for both the predators and the potential grapevine pest species. These results indicate that cover crop vegetation can be used in vineyards to enhance predatory insect abundance and may improve agroecosystem resilience.

Soil nematode abundance and functional group composition at a global scale.

Soil organisms are a crucial part of the terrestrial biosphere. Despite their importance for ecosystem functioning, few quantitative, spatially explicit models of the active belowground community currently exist. In particular, nematodes are the most abundant animals on Earth, filling all trophic levels in the soil food web. Here we use 6,759 georeferenced samples to generate a mechanistic understanding of the patterns of the global abundance of nematodes in the soil and the composition of their functional groups. The resulting maps show that 4.4 ± 0.64 × 1020 nematodes (with a total biomass of approximately 0.3 gigatonnes) inhabit surface soils across the world, with higher abundances in sub-Arctic regions (38% of total) than in temperate (24%) or tropical (21%) regions. Regional variations in these global trends also provide insights into local patterns of soil fertility and functioning. These high-resolution models provide the first steps towards representing soil ecological processes in global biogeochemical models and will enable the prediction of elemental cycling under current and future climate scenarios.

Scavenging behavior and interspecific competition decrease offspring fitness of the entomopathogenic nematode Steinernema feltiae.

Entomopathogenic nematodes (EPNs) are well-studied biocontrol agents of soil-dwelling arthropod pests. The insecticidal efficiency of EPNs is modulated by food web dynamics. EPNs can reproduce in freeze-killed insect larvae, even in competition with free-living bacterivorous nematodes (FLBNs) in the genus Oscheius. The objective of this study was to assess the efficiency of EPNs as scavengers when competing with free-living saprophagous nematodes and fungi, and to determine the possible impact on subsequent EPN offspring fitness. Live and freeze-killed larvae of Galleria mellonella were used to evaluate the reproduction rate and progeny fitness of two EPN species, Heterorhabditis bacteriophora and Steinernema feltiae, applied individually or combined with the FLBN species Oscheius onirici or Pristionchus maupasi, or Aspergillus flavus, an opportunistic saprophytic fungus. We hypothesized that (1) EPN scavenging behaviors previously observed (for H. megidis and S. kraussei) apply to other EPN species, (2) infective juveniles (IJs) emerging from freeze-killed larvae will display reduced pathogenicity and reproduction, and (3) fitness reduction will be amplified by exposure to other organisms competing for the resources. The reproduction rate of S. feltiae was lower in freeze-killed larvae than in larvae infected and killed by the nematode, whereas H. bacteriophora failed to reproduce as a scavenger. The S. feltiae F1 IJs that emerged from freeze-killed larvae exhibited lower pathogenicity rates than IJs resulting from entomopathogenic activity, and also lower reproductive rates if they experienced high FLBN competitive pressure during development. This study illustrates that scavenging is a suboptimal alternative pathway for EPNs, especially in the face of scavenger competition, even though it provides a means for some EPN species to complete their life-cycle.

Entomopathogenic nematology in Latin America: A brief history, current research and future prospects.

Since the 1980s, research into entomopathogenic nematodes (EPNs) in Latin America has produced many remarkable discoveries. In fact, 16 out of the 117 recognized species of EPNs have been recovered and described in the subcontinent, with many more endemic species and/or strains remaining to be discovered and identified. In addition, from an applied perspective, numerous technological innovations have been accomplished in relation to their implementation in biocontrol. EPNs have been evaluated against over 170 species of agricultural and urban insects, mites, and plant-parasitic nematodes under laboratory and field conditions. While much success has been recorded, many accomplishments remain obscure, due to their publication in non-English journals, thesis dissertations, conference proceedings, and other non-readily available sources. The present review provides a brief history of EPNs in Latin America, including current findings and future perspectives.

Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes.

Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR), is the most destructive pest of maize in North America, and has recently spread across central Europe. Its subterranean larval stages are hard to reach with pesticides and it has evolved resistance to conventional management practices. The application of beneficial soil organisms is being considered as a sustainable and environmental friendly alternative. In a previous study, the combined application in wheat fields of arbuscular mycorrhizal fungi, entomopathogenic Pseudomonas bacteria, and entomopathogenic nematodes was found to promote growth and protection against a natural pest infestation, without negative cross effects. Because of the insect-killing capacity of the bacteria and nematodes, we hypothesized that the application of these organisms would have similar or even greater beneficial effects in WCR-infested maize fields. During three consecutive years (2015-2017), we conducted trials in Missouri (USA) in which we applied the three organisms, alone or in combinations, in plots that were artificially infested with WCR and in non-infested control plots. For two of the three trials, we found that in plots treated with entomopathogenic nematodes and/or entomopathogenic Pseudomonas bacteria, roots were less damaged than the roots of plants in control plots. During one year, WCR survival was significantly lower in plots treated with Pseudomonas than in control plots, and the surviving larvae that were recovered from these plots were lighter. The bacterial and nematodes treatments also enhanced yield, assessed as total grain weight, in one of the trials. The effects of the treatments varied considerable among the three years, but they were always positive for the plants.

Molecular detection and quantification of slug parasitic nematodes from the soil and their hosts.

Terrestrial gastropod molluscs are widely distributed and are well known as pests of many types of plants that are notoriously difficult to control. Many species of nematodes are able to parasitize land snails and slugs, but few of them are lethal to their host. Species and/or populations of mollusc-parasitic nematodes (MPNs) that kill their hosts are promising for biological control purposes. The recent discovery of new nematode species of the genus Phasmarhabditis in Europe and the associations between Alloionema spp. and slugs are expanding the possibilities of using MPNs as control agents. However, very little is known about the distribution and ecology of these species. Using molecular techniques based on qPCR methods for quick identification and quantification of various species of MPN isolated directly from the soil or from infected hosts can assist in providing information on their presence and persistence, as well as the composition of natural assemblages. Here, we developed new primers and probes for five species of the genus Phasmarhabditis and one species of the genus Alloionema. We employed these novel molecular techniques and implemented a published molecular set to detect MPN presence in soil samples coming from natural and agricultural areas in Switzerland. We also developed a method that allows the detection and quantification of Phasmarhabditis hermaphrodita directly from the tissues of their slug host in a laboratory experiment. The new molecular approaches were optimized to a satisfactory limit of detection of the species, with only few cross-amplifications with closely related species in late cycles (>32). Using these tools, we detected MPNs in 7.5% of sampled sites, corresponding to forest areas (P. hermaphrodita and Alloionema appendiculatum) and wheat-oriented agricultural areas (Phasmarhabditis bohemica). Moreover, we confirmed that the method can be used to detect the presence of P. hermaphrodita inside slug hosts, with more detections in the susceptible slug Deroceras larvae compared to the resistant Arion vulgaris. These primers/probe sets provide a novel and quick tool to identify MPNs from soil samples and infected slugs without having to culture and retrieve all nematode life stages, as well as a new tool to unravel the ecology of nematode-slug complexes.

Simultaneous exposure of nematophagous fungi, entomopathogenic nematodes and entomopathogenic fungi can modulate belowground insect pest control.

Entomopathogenic nematodes (EPNs) and fungi (EPF) are well known biological control agents (BCAs) against insect pests. Similarly, the nematophagous fungi (NF) are considered good BCA candidates for controlling plant parasitic nematodes. Because NF can employ EPNs as food and interact with EPF, we speculate that the simultaneous application of EPNs and EPF might result in higher insect mortality, whereas the triple species combination with NF will reduce the EPN and EPF activity by predation or inhibition. Here we evaluated single, dual (EPN + EPF, EPF + NF, EPN + NF) and triple (EPN + EPF + NF) combinations of one EPN, Steinernema feltiae (Rhabditida: Steinernematidae), one EPF, Beauveria bassiana (Hypocreales: Clavicipitaceae), and two NF, Arthrobotrys musiformis (Orbiliales: Orbiliaceae) and Purpureocillium lilacinum (Hypocreales: Ophiocordycipitaceae) under laboratory conditions. First, we showed that EPF reduced the growth rate of NF and vice versa when combined in both rich and limiting media, suggesting a negative interaction when combining both fungi. Three different fungal applications (contact with mycelia-conidia, immersion in conidial suspension, and injection of conidial suspension) were tested in single, dual and triple species combinations, evaluating Galleria mellonella (Lepidoptera: Pyralidae) larval mortality and time to kill. When mycelia was presented, the EPF appeared to be the dominant in combined treatments, whereas in immersion exposure was the EPN. In both types of exposure, NF alone did not produce any effect on larvae. However, when A. musiformis was injected, it produced larval mortalities >70% in the same time span as EPN. Overall, additive effects dominated the dual and triple combinations, with the exception of injection method, where synergisms occurred for both NF species combined with EPN + EPF. This study illustrates how differences in species combination and timing of fungal arrival can modulate the action of BCAs when augmented in the soil. Further studies are required to fine-tune these multitrophic interactions to provide successful, sustainable and resilient pest management in agroecosystems.