Trabajo presentado en el III Encuentro Phytoma La Vid y el Vino, celebrado en Logroño (España), los días 6 y 7 de noviembre de 2024, JDC 2022-048978-I(RBP), FPI-UR-2020(IVD),
PRE2023_ICVV_117(EMC), PID2022-1364870B
I00(RCH).

Entomopathogenic nematodes (EPNs) have emerged as a promising tool for controlling soil-dwelling crop pests. However, their efficacy varies according to EPN populations and targeted hosts. Wireworms are polyphagous insects causing significant crop losses, especially since the ban on pesticides previously used for their control. They are highly resistant to EPN populations and require high number of infective juveniles (IJs) to achieve optimal mortality rates. In this research, we collected and compared the virulence of 16 EPN populations, of foreign origin, purchased or collected from wireworms infested fields. Then, we have tested two hypotheses: (i) smaller nematodes induce heightened mortality rates against wireworms; (ii) virulence levels can be linked to nematodes-bacteria complex. Mortality rates scaled from three to 43 % after 56 days of continuous exposure across the 16 tested EPN populations (Heterorhabditis spp. and Steinernema spp.). Morphometric analysis of IJs revealed both intra- and interspecific variations in length and diameter among populations. Interestingly, while EPN length influence mortality at three days post-inoculation. We found leaner IJs (< 25 µm) to induce higher mortality rates at 56 days post-inoculation. To better determine the structure and dimensions of the primary entry routes utilized by EPNs, we provide optical microscope micrographs of wireworm Agriotes spp. spiracle, anal sclerotized coating anus and anal muscles. Symbiotic bacteria of each EPN population were identified, and a biochemical characterization was performed using Analytical Profile Index tests. The symbiotic bacteria belong to the species Photorhabdus antumapuensis, P. laumondii subsp. laumondii, P. thracensis, Xenorhabdus bovienii and X. nematophila. Bacteria biochemical profiles did not reflect the differences in virulence of nematodes-bacteria complex against wireworms. These findings highlight the importance of considering EPN morphometry and intraspecific variability in designing applications to control wireworms., This work was supported by Erasmus mundus grants: Brice Checconi (IN221021) and Salimata Drabo (FAMIENS01). Andrea Chacon Hurtado was awarded an FRIA grant (Fund for research training in industry and agriculture, FRIA/FC-2576 FC46977) to achieve a PhD thesis. Fanny Ruhland is supported by Attrack-and-kill Wallonia project (D65-1401/S1, Belgium). Raquel Campos-Herrera collaborated under the frame of the Grant PID2022-136487OB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and, by “ERDF A way of making Europe”., Peer reviewed

[Description of methods used for collection/generation of data] Experiment performed in the laboratory and semifield conditions, following details described in the publication., [Methods for processing the data] R software version 4.3.2 (R Core Team, 2023). All graphs were built using ggplot2 package., Viticulture is a global activity that annually yields over eighty million tonnes of grapes, 50 % used in winemaking. Traditional viticultural practices still rely on hazardous pesticides, posing environmental and health risks. Recently, Xenorhabdus and Photorhabdus, symbiotic bacteria of entomopathogenic nematodes, have arisen as eco-friendly solutions for pest and disease management in vineyards. However, whether their application can impact the quality attributes of grapes and their microbiota remains unexplored. To unravel the possible impact, we investigated (i) the impact of direct application on grapes' volatile composition and (ii) the combination with must and Saccharomyces cerevisiae fermentation. First, we applied unfiltered ferments (UFs) of Xenorhabdus nematophila and Photorhabdus laumondii to grapes of Vitis vinifera var. "Tempranillo". The volatile composition of the grapes was evaluated using solid-phase and gas chromatography-mass spectrometry (SPE-GC/MS). Second, microfermentations using reconstituted must media were supplemented (1:10) with the treatments (i) cell-free supernatants (CFS), (ii) UFs, and (iii) free-bacterial from X. nematophila and P. laumondii. No significant alterations in the total free volatile fraction were observed, although specific free volatiles (phenylethyl alcohol, hexanal and E-2-hexenal), exhibited significant variation under specific treatments. In the glycosidically bound fraction, phenylacetic acid concentrations increased markedly with X. nematophila UFs. In the fermentations, the treatments had no significant impact on CO2 production kinetics, only slightly affecting some fermentation parameters, underscoring their compatibility with yeast-driven fermentation processes. These findings suggest that Xenorhabdus and Photorhabdus can be safely integrated into vineyard pest management strategies without adversely affecting essential winemaking parameters or grape aroma quality., Ministry of Science and Innovation, grants PID2019-104112RB-I00 and PID2022-136487OB-I00 (MCIN/AEI/10.13039/50110001103)., Support for researchers: The predoctoral contract FPI-UR 2021 (University of La Rioja) support IVD., Peer reviewed

The extensive use of fungicides raises significant environmental and health concerns, including biodiversity loss and risks to agricultural workers, while the emergence of new fungal diseases exacerbates reliance on these chemical agents. Recent research highlights the potential of Xenorhabdus and Photorhabdus, symbiotic bacteria of entomopathogenic nematodes (EPN), as alternative biocontrol agents against soilborne and aerial fungal pathogens. This study aimed to evaluate the efficacy of EPN-symbiotic bacteria and their by-products against selected fungi and their potential non-target effects on biocontrol agents used in vineyards as a model agroecosystem. Specifically, this study investigated (i) the antifungal effect of Xenorhabdus nematophila cultured in three different nutrient media (Tryptone Soya Broth- TSB-, Nutrient Broth –NB, and Luria-Bertani –LB-) and under varying fermentation durations (3 and 10 days) against Botrytis cinerea; (ii) the antifungal activity of X. nematophila and Photorhabdus laumondii subsp. laumondii against the soilborne pathogen Armillaria mellea; and (iii) the antibacterial activity of X. nematophila, X. bovienii, P. laumondii subsp. laumondii and Photorhabdus luminescens subsp. kayaii by-products against Bacillus spp., including strains used as commercial biocontrol products. Our results demonstrated that X. nematophila cultured in TSB and NB produced the highest inhibition of B. cinerea, with inhibitory effects ranging from 63.5 % to 74.3 %, depending significantly on the fermentation duration. For A. mellea, both cell-free supernatants (CFS) and unfiltered ferments (UFs) from X. nematophila and P. laumondii subsp. laumondii significantly reduced colony numbers and colonized areas, with UFs exhibiting superior efficacy. Non-target effect assays revealed selective antibacterial activity, with significant inhibition observed only against Bacillus thuringiensis var. kurstaki ABTS-351, while no effect was detected against B. thuringiensis PB-54 or B. amyloliquefaciens QST-713. These findings highlight the dual potential of Xenorhabdus and Photorhabdus bacteria as biocontrol agents for fungal pathogens and for promoting selective microbial interactions. However, further studies are needed to optimize their efficacy and assess potential non-target effects in integrated pest management in vineyards and other agroecosystems., IVD is supported by FPI-UR (2021) fellowship from the University of La Rioja (Spain). JDH is supported by the Programa Investigo from the Government of La Rioja and the “European Union NextGenerationEU/PRTR” and MCIN/AEI/10.13039/501100011033. The results are part of the grant PID2022-136487OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by “ERDF A way of making Europe” and by the Instituto de Estudios Riojanos (IER), Government of La Rioja, Spain, ref. 11/2023., Peer reviewed

This article is part of a special issue entitled: ‘EPN 100 years’ published in Journal of Invertebrate Pathology, The field of entomopathogenic nematology has grown exponentially since the discovery of the first species, Steinernema kraussei (=Aplecatna kraussei), in 1923. Initially, entomopathogenic nematodes (EPN) were solely viewed as a curiosity. The discovery of the nematode-bacteria association in 1965 and the incipient research for mass production motivated their recognition as biological control agents for agricultural pests. Subsequent studies were focused on the discovery of new species and/or populations, the early studies to understand the biotic and abiotic factors that contribute to their performance in the field and success in insect pest management. However, as we entered the 21st century, and with the advent of molecular biology, research on these organisms took a fascinating turn, unraveling a deeper understanding of the complex symbiotic relationship EPN has with their bacterial symbionts and the insect host. Furthermore, because of their experimental tractability, EPNs have proven to be model organisms that are used among various biological sciences to gain further insights into host-symbiont, host-pathogen interactions, population dynamics, and as resources for pharmaceutical bioprospecting. This special issue commemorates the first 100 years of research in entomopathogenic nematology and summarizes the contributions of ten symposia and presentations at the 100th Anniversary of the First EPN Discovery Congress in Logroño, Spain (https://www.icvv.es/english/epn). This specific article focuses on the historical review of EPN, their bacterial partners and the numerous and diverse applications in disciplines in basic such as phylogeny, biogeography, symbiosis, and soil biology and ecology, or more applied venues such as formulation and mass production, application technology, commercialization and regulation, from 1923 to the present time., SPS acknowledges the Agricultural Research Institute (ARI) from California State University for providing partial support to attend the EPN 100 Anniversary Conference in Logroño Spain. RCH was also supported by the frame of the Grant PID2022-136487OB-I00 funded by MCIN/AEI/ 10.13039/501100011033 and, by “ERDF A way of making Europe”., Peer reviewed

Soil organisms are key to plant growth and ecosystem functions. Earthworms (EWs) enhance soil and indirectly affect plant growth, while their cutaneous excreta (CEx) contain bioactive compounds capable of eliciting plant responses. However, their role in plant immunity is still not well understood. We hypothesized that EWs and their CEx enhance plant defense against foliar pathogens by activating induced resistance. To test this, we evaluated the effect of Eisenia fetida and their CEx on Solanum lycopersicum (tomato), focusing on growth, physiology, and defense response against Botrytis cinerea. Plants were exposed to EWs, CEx, or water (control), followed by B. cinerea infection after two weeks. Gene expression of defense markers was assessed at 24 and 48 h post-inoculation (hpi), while physiological parameters and disease severity were evaluated at 72 hpi. EWs increased shoot biomass compared to CEx, while both treatments reduced root dry weight, suggesting a possible shift in resource allocation. CEx significantly reduced B. cinerea-induced leaf damage and showed a trend for flavonoid accumulation, a known marker of induced resistance. Both treatments, EWs and CEx, activated the jasmonic acid (JA) signaling pathway, with CEx specifically upregulating genes involved in fungal pathogen defense, sustaining their expression over time. The present study offers, for the first time, clear evidence that EW-derived CEx can induce resistance by stimulating plant defense responses. Further biochemical, transcriptomic, and metabolomic analyses are needed to confirm indirect results, along with field validation. Nonetheless, the findings underscore the crucial role of soil biodiversity in enhancing crop resilience., MC was supported by the mobility program established by the CSIC i-coop + 2018 grant (COOPA20231). IVD is supported by FPI-UR (2021) fellowship from the University of La Rioja (Spain). RBP is supported by the contract Juan de la Cierva (JDC2022-048978-I) from the “European Union NextGenerationEU/PRTR” and MCIN/AEI/10.13039/501100011033. JDH is supported by the Programa Investigo from the Government of La Rioja and the “European Union NextGenerationEU/PRTR. The results were funded by CSIC i-coop + 2018 grant (COOPA20231), by the Instituto de Estudios Riojanos (IER), Government of La Rioja, Spain, ref. 8/2020, December 29th 2020, and by the grant PID2022-136487OB-I00 funded by MCIN/ AEI/10.13039/501100011033 and by “ERDF A way of making Europe”., Peer reviewed

The collaboration among academia, industry, and government is crucial for scientific progress and innovation. Academia generates fundamental knowledge, which industry translates into sound applications, considering government policies. This partnership is vital to feed progress and constant development and address global challenges like climate change and food security. Sustainable crop protection is a topical theme, with efforts to reduce pesticide reliance and promote alternatives to chemical pest management, and it continues to grow and be accepted worldwide. In this respect, biopesticides such as entomopathogenic nematodes (EPNs) offer a promising solution for pest and disease management as an eco-friendly alternative. However, EPN continue to face adoption barriers due to regulatory, commercialization and basic and applied knowledge gaps. Thus, stronger collaborations are needed to unlock their full potential, as highlighted in the 2024 congress organized in La Rioja (Spain) to commemorate the 100 years since the discovery of the first EPN. This review examines the gap between academia and industry, suggesting strategies to bridge it, thereby promoting the advancement of EPN in 21st-century agriculture. Despite decades of research demonstrating their efficacy, EPN commercialization remains limited by production, formulation, and application challenges. Universities and government research agencies have driven fundamental innovation through the discovery of new EPN-bacteria partnerships in new regions of the world, which are helping us understand their distribution and habitant adaptations which are required for their registration and in establishing global regulations. Research conducted both in academia and the private sector (both big and small start-up companies) has and continues to play a key role in the characterization of EPN and in assessing their performance for their subsequent formulation, product optimization, and commercialization. These are fundamental steps to reach the ultimate goal, which is to provide growers with reliable products that are cost-effective and sustainable. In this review, we summarize key findings that have led to the commercialization and application of EPN, spanning from the characterization of EPN and their symbiotic bacteria to production, formulation, and the requirements for their registration. We also highlight critical knowledge gaps and opportunities for collaboration between academia, government agencies, and industry. Strengthening these partnerships will drive EPN adoption in agriculture, establishing them as a desirable biocontrol solution., RCH acknowledges grant PID2022-136487OB-I00 funded by MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe” associated with the ideas presented in this manuscript., Peer reviewed

Viticulture is a global activity producing over eighty million tonnes of grapes annually, 50 % used in winemaking. Traditional viticultural practices still rely on hazardous pesticides, posing environmental and health risks. Recently, Xenorhabdus and Photorhabdus, symbiotic bacteria of entomopathogenic nematodes, have arisen as eco-friendly solutions for pest and disease management in vineyards. However, whether their application can impact the quality of grapes and their microbiota remains unexplored. To unravel the possible impact, we investigated the effect of (i) their direct application on grapes’ volatile composition and (ii) their combination with Saccharomyces cerevisiae in must fermentation. First, we applied unfiltered ferments (UFs) of Xenorhabdus nematophila and Photorhabdus laumondii subsp. laumondii to grapes of Vitis vinifera var. “Tempranillo”. The volatile composition of the grapes was evaluated using solid-phase and gas chromatography-mass spectrometry (SPE-GC/MS). Second, microfermentations using two S. cerevisiae in reconstituted must media were supplemented (1:10) with the treatments (i) cell-free supernatants (CFS), (ii) UFs, and (iii) free-bacterial from X. nematophila and P. laumondii. No significant alterations in the total free volatile fraction were observed, although specific free volatiles (phenyl ethyl alcohol, hexanal, and (E)-2-hexenal) exhibited significant variation under specific treatments. In the glycosidically bound fraction, phenylacetic acid concentrations increased markedly with X. nematophila UFs. In the fermentations, the treatments had no significant impact on CO<inf>2</inf> production kinetics and only slightly affected some fermentation parameters, underscoring their compatibility with yeast-driven fermentation processes. These findings suggest that Xenorhabdus and Photorhabdus can be integrated into vineyard pest management strategies without adversely affecting winemaking parameters or grape aroma quality., The authors thank M. Cristina Juez Ojeda and Bianca Souza da Costa for their invaluable assistance in the laboratory. We also thank the ICVV analytical service for GC-MS analysis. The pre-doctoral contract FPI-UR 2021 (University of La Rioja) supported IVD. This study was also funded by the Ministry of Science and Innovation, grants PID2019-104112RB-I00 (MCIN/AEI/10.13039/50110001103) and PID2022-136487OB-I00 (MCIN/AEI/10.13039/50110001103 and European Union NextGenerationEU/PRTR)., Peer reviewed

El proyecto genera bases de datos (datasets) experimentales y observacionales sobre biocontrol de plagas y patógenos de la vid y su impacto en la vid, incluyendo bioensayos de control biológico, imágenes de daño, medidas de crecimiento (planta, hongo, bacteria, nematodo), resultados de qPCR de detección y cuantificación de varios agentes, datos de calidad de uva, perfiles químicos (UHPLC, GC‑MS), metabolómica (targeted/untargeted), nutrición vegetal y ensayos de interacción raíz‑microorganismo. Los datos de los bioensayos se registrarán conforme a una hoja de resumen de ensayos normalizada del grupo IN-Vid, en la que se incluyen de forma sistemática los siguientes datos: investigador responsable, título del experimento, hipótesis, número de repetición del experimento (trial), fecha de inicio y de fin del experimento, material empleado, metodología general, diseño experimental y observaciones. Todos los datos en formato papel se custodian en carpetas definidas por el laboratorio según la tipología de estudios, principalmente organizadas según los objetivos del proyecto. Estas carpetas están a disposición del grupo de investigación/trabajo en todo momento para garantizar la transparencia y la accesibilidad. Los datos se procesan principalmente en formato de hoja de cálculo (Excel) para su análisis en software estadístico (R environment y SPSS, principalmente)., Peer reviewed

Experiment performed in the laboratory, following details described in the publication, Ministry of Science and Innovation, grant PID2022-136487OB-I00 (MCIN/AEI/10.13039/50110001103 and and by “ERDF A way of making Europe").

Wallonia project (D65-1401/S1, Belgium)

Interreg (0100226 - Trans-Pest)

The predoctoral contract for Andrea Chacón-Hurtado FRIA/FC-2576 FC46977, Peer reviewed

Root-knot nematodes (RKNs) cause major yield losses in horticultural systems worldwide. With increasing restrictions on chemical nematicides, environmentally sustainable alternatives such as entomopathogenic nematodes (EPNs) and derivatives of their symbiotic bacteria are gaining attention. However, it remains unclear how EPNs and their bacterial products might influence RKN behavior. This study investigated the behavioral responses of the RKN Meloidogyne incognita to EPN-derived treatments, testing the hypothesis that these cues induce directional avoidance when RKNs are given a choice, and that such responses may be modulated by plant presence. Experiments were conducted using two-choice horizontal tube systems filled with moistened sand with and without tomato seedlings ( Solanum lycopersicum cv. Moneymaker). Three EPN Steinernema species were assessed: S. feltiae , S. carpocapsae , and S. riojaense . Treatments included (i) EPN infective juveniles (IJs), (ii) EPN-infected Galleria mellonella cadavers, and (iii) bacterial cell-free supernatant (CFS) derived from Xenorhabdus spp. After inoculation of 1500 M. incognita second-stage juveniles (J2s) into the central tube section, nematode movement was quantified after seven days. Although overall J2 mobility was generally low, with most individuals remaining near the inoculation point, clear patterns still emerged: J2 showed a significant avoidance of EPNs and their derivatives, with the strongest responses observed for cadaver and CFS treatments. Plant presence did not markedly change J2 behavioral patterns; repellency occurred regardless of plant cues, although the presence of plants slightly amplified the effect. These findings advance our understanding of how EPNs and by-products influence RKN behavior, revealing early behavioral barriers that may shape rhizosphere interactions., This work was supported by the Erasmus+ Traineeship Program for LB and is part of the grant PID2022-136487OB-I00 funded by MCIN/AEI/10.13039/501100011033 and by the European Regional Development Fund (ERDF) “A way of making Europe” granted to RCH. The use of the entomopathogenic nematodes described in this study was conducted under permit reference number ESNC 273, corresponding to the Internationally Recognized Certificate of Compliance (IRCC) with unique identifier ABSCH-IRCC-ES-281207-1. This certificate was issued in accordance with the Nagoya Protocol and is registered in the Access and Benefit-sharing Clearing-House. Also, RBP is funded by the Consejería de Innovación (Government of La Rioja, Spain), and MMGT was awarded a FPI-CAR-2022 fellowship (University of La Rioja, Spain)., Peer reviewed

Root-knot nematodes (RKNs) cause major yield losses in horticultural systems worldwide. With increasing restrictions on chemical nematicides, environmentally sustainable alternatives such as entomopathogenic nematodes (EPNs) and derivatives of their symbiotic bacteria are gaining attention. However, it remains unclear how EPNs and their bacterial products might influence RKN behavior. This study investigated the behavioral responses of the RKN Meloidogyne incognita to EPN-derived treatments, testing the hypothesis that these cues induce directional avoidance when RKNs are given a choice, and that such responses may be modulated by plant presence. Experiments were conducted using two-choice horizontal tube systems filled with moistened sand with and without tomato seedlings ( Solanum lycopersicum cv. Moneymaker). Three EPN Steinernema species were assessed: S. feltiae , S. carpocapsae , and S. riojaense . Treatments included (i) EPN infective juveniles (IJs), (ii) EPN-infected Galleria mellonella cadavers, and (iii) bacterial cell-free supernatant (CFS) derived from Xenorhabdus spp. After inoculation of 1500 M. incognita second-stage juveniles (J2s) into the central tube section, nematode movement was quantified after seven days. Although overall J2 mobility was generally low, with most individuals remaining near the inoculation point, clear patterns still emerged: J2 showed a significant avoidance of EPNs and their derivatives, with the strongest responses observed for cadaver and CFS treatments. Plant presence did not markedly change J2 behavioral patterns; repellency occurred regardless of plant cues, although the presence of plants slightly amplified the effect. These findings advance our understanding of how EPNs and by-products influence RKN behavior, revealing early behavioral barriers that may shape rhizosphere interactions., Ministry of Science and Innovation, grant PID2022-136487OB-I00 (MCIN/AEI/10.13039/50110001103 and and by “ERDF A way of making Europe").

The use of the entomopathogenic nematodes described in this study was conducted under permit reference number ESNC 273, corresponding to the Internationally Recognized Certificate of Compliance (IRCC) with unique identifier ABSCH-IRCC-ES-281207-1

RBP is funded by the Consejería de Innovación (Government of La Rioja, Spain, (Resolution 198/2025, call 2025–2028)
MMGT was awarded a FPI-CAR-2022 fellowship (University of La Rioja, Spain), Peer reviewed

Analysis from laboratory and semifield derived data described in the material and method section in the article, The grant PID2022-136487OBI00 funded by MCIN/AEI/10.13039/501100011033 and by the European Regional Development Fund (ERDF) “A way of making Europe”
The use of the entomopathogenic nematodes described in this study was conducted under permit reference number ESNC 273, corresponding to the Internationally Recognized Certificate of Compliance (IRCC) with unique identifier ABSCH-IRCC-ES-281207-1. This certificate was issued in accordance with the Nagoya Protocol and is registered in the Access and Benefit- haring Clearing-House., Peer reviewed

Analysis from laboratory and semifield derived data described in the material and method section in the article, The grant PID2019-104112RB-I00 (MCIN/AEI/10.13039/50110001103)
The grant PID2022-136487OBI00 (MCIN/AEI/10.13039/501100011033 and by the European Regional Development Fund (ERDF) “A way of making Europe”)
The use of the entomopathogenic nematodes described in this study was conducted under permit reference number ESNC 273, corresponding to the Internationally Recognized Certificate of Compliance (IRCC) with unique identifier ABSCH-IRCC-ES-281207-1. This certificate was issued in accordance with the Nagoya Protocol and is registered in the Access and Benefit- haring Clearing-House.

Support for researchers:

RBP is supported by the Consejería de Innovación (Government of La Rioja, Spain)
MP is funded by the FPI-UR (2022) from the University of La Rioja
JM is funded by PTA2023-022748-I (MCIN/AEI/10.13039/50110001103 and “European Union NextGenerationEU/PRTR"), Peer reviewed