Background and aims As part of a research consortium that explores ways to improve soil health, we study how entomopathogenic nematodes (EPNs) can be better exploited for the biological control of soil-dwelling insect pests in annual crops. Methods We evaluated how tillage might affect belowground interactions in two 30-year running Swiss field trials by combining traditional (insect bait) and molecular (novel real-time qPCR protocols) methods. Soil samples (April and October 2013) were evaluated for the presence and activity of EPN soil food web assemblage comprising 13 EPN species, six nematophagous fungi, one ectoparasitic bacterium, and the free-living nematodes (FLN) of the Acrobeloides group. Results Mortality of sentinel larvae, as well as qPCR analyses (for which we provide seven new primers/ probes sets) found only trace levels of six EPN species, dominated by heterorhabditids species. Analysis of nematode progeny revealed that EPN compete intensely with FLN for insect cadavers. Overall, it appears that temperate annual cropping systems provide poor environments for EPN and that tillage does not negatively affect the natural occurrence of EPN. Conclusions Natural occurrence of EPN in Swiss tillage soils was very low, and augmentation may be a promising strategy to improve the control of root pests of annual crops.

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In agricultural ecosystems, pest insects, pathogens, and reduced soil fertility pose major challenges to crop productivity and are responsible for significant yield losses worldwide. Management of belowground pests and diseases remains particularly challenging due to the complex nature of the soil and the limited reach of conventional agrochemicals. Boosting the presence of beneficial rhizosphere organisms is a potentially sustainable alternative and may help to optimize crop health and productivity. Field application of single beneficial soil organisms has shown satisfactory results under optimal conditions. This might be further enhanced by combining multiple beneficial soil organisms, but this remains poorly investigated. Here, we inoculated wheat plots with combinations of three beneficial soil organisms that have different rhizosphere functions and studied their effects on crop performance. Plant beneficial Pseudomonas bacteria, arbuscular mycorrhizal fungi (AMF), and entomopathogenic nematodes (EPN), were inoculated individually or in combinations at seeding, and their effects on plant performance were evaluated throughout the season. We used traditional and molecular identification tools to monitor their persistence over the cropping season in augmented and control treatments, and to estimate the possible displacement of native populations. In three separate trials, beneficial soil organisms were successfully introduced into the native populations and readily survived the field conditions. Various Pseudomonas, mycorrhiza, and nematode treatments improved plant health and productivity, while their combinations provided no significant additive or synergistic benefits compared to when applied alone. EPN application temporarily displaced some of the native EPN, but had no significant long-term effect on the associated food web. The strongest positive effect on wheat survival was observed for Pseudomonas and AMF during a season with heavy natural infestation by the frit fly, Oscinella frit, a major pest of cereals. Hence, beneficial impacts differed between the beneficial soil organisms and were most evident for plants under biotic stress. Overall, our findings indicate that in wheat production under the test conditions the three beneficial soil organisms can establish nicely and are compatible, but their combined application provides no additional benefits. Further studies are required, also in other cropping systems, to fine-tune the functional interactions among beneficial soil organisms, crops, and the environment.

Agricultural management practices can modify soil properties in ways that may disrupt the abundance and activity of beneficial organisms in the soil. We assessed the impact of different soil management practices on entomopathogenic nematodes (EPN), which have great potential as biological control agents against root-feeding insects. Soils were sampled during spring and autumn 2013 in all 96 plots of a long-term Swiss field trial (DOK experiment) in a seven-year grass-clover ley rotation. By combining a traditional insect-baiting technique and real-time qPCR analyses, we identified and quantified over 20 soil-dwelling species (or genera). This allowed us to investigate how communities of natural EPN populations and their associated natural enemies and competitors are affected by (i) three crop types (wheat, maize and grass-clover ley) and (ii) farming systems, i.e. conventional, organic and biodynamic, which differed in fertilization, and pesticide use. We also determined the effects on soils' microbial biomass in terms of carbon (C mic) and nitrogen (N mic) and applied spatial distribution analysis (SADIE) to uncover patterns of aggregations and associations of the study organisms. Although manure based farming systems increased microbial biomass, the systems did not influence the presence of EPN or their antagonists. EPN were more abundant in winter-wheat plots after potatoes than in maize and grass-clover ley plots. Overall, very low numbers of EPN were recorded, implying that their natural presence would not be sufficient to have a satisfactory suppressive effect on root-feeding pests and the application of EPN would therefore be an appropriate measure to protect yields in case of root pest outbreaks.

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13 14Entomopathogenic nematodes (EPNs) and their bacterial partners are well-studied insect 15 pathogens, and their persistence in soils is one of the key parameters for successful use as 16 biological control agents in agroecosystems. Free-living bacteriophagous nematodes (FLBNs) in 17 the genus Oscheius, often found in soils, can interfere in EPN reproduction when exposed to live 18 insect larvae. Both groups of nematodes can act as facultative scavengers as a survival strategy. 19Our hypothesis was that EPNs will reproduce in insect cadavers under FLBN presence, but their 20 reproductive capacity will be severely limited when competing with other scavengers for the same 21 niche. We explored the outcome of EPN -Oscheius interaction by using freeze-killed larvae of 22Galleria mellonella. The differential reproduction ability of two EPN species (Steinernema 23 kraussei and Heterorhabditis megidis), single applied or combined with two FLBNs (Oscheius 24 onirici or Oscheius tipulae), was evaluated under two different infective juvenile (IJ) pressure: 25 low (3 IJs/host) and high (20 IJs/host). EPNs were able to reproduce in insect cadavers even in 26 the presence of potential scavenger competitors, although EPN progeny was lower than that 27 recorded in live larvae. Hence, when a highly susceptible host is available, exploiting cadavers 28 by EPN might limit the adaptive advantage conferred by the bacteria partner, and might result in 29 an important trade-off on long-term persistence. Contrary to our hypothesis, for most of the 30 combinations, there were not evidences of competitive relationship between both groups of 31 nematodes in freeze-killed larvae, probably because their interactions are subject to interference 32 by the microbial growth inside the dead host. Indeed, evidences of possible beneficial effect of 33 FLBN presence were observed in certain EPN-FLBN treatments compared with single EPN 34 exposure, highlighting the species-specific and context dependency of these multitrophic 35 interactions occurring in the soil. 36 37

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.