Mediterranean ecosystem as a model in the development of molecular and ecological approaches for the study of multitrophic interactions in the soil: ecosystem services and biocontrol enhancement

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Publications

Entomopathogenic fungi from Algarve region (Portugal): studies on their natural occurrence in the soil and multitrophic interactions that shape their biocontrol potential

Publication . Bueno Pallero, Francisco Ángel; Neto, Luís Mascarenhas; Dionísio, Lídia Pó Catalão; Herrera, Raquel Campos

The entomopathogenic fungi (EPF) are well-known beneficial soil organisms naturally occurring in the soil. Learning about their natural distribution in representative habitats and their interactions with other soil organisms can establish the best ecological scenario for the activity as biocontrol agents. Also, the co-formulation of two entomopathogens with plant-based products can be an affordable approach for farmers. With the Algarve region (Portugal) as a research frame, the objectives of this PhD thesis were: (1) to investigate the natural distribution of EPF in soils from the Algarve in four habitats (oak, pines, palmettoes, and citrus groves) and two soil eco-regions (calcareous and noncalcareous), (2) to evaluate whether the presence of entomopathogenic nematodes (EPN) or/and nematophagous fungi (NF) can affect the biocontrol potential of EPF, and (3) to investigate the impact of plant-based oil adjuvants on biocontrol activity of single or combined EPF and EPN, and the impact of time and temperature in the EPN survival and activity. Firstly, we combined three isolation methods (fresh soil, dry soil, and selective media) to investigate the natural EPF distribution in the Algarve, using traditional and molecular tools for the identification. We established the assemblage of the EPF with the soil properties and EPN by using multivariate analysis. Second, we investigated the multitrophic interactions in single (EPF, EPN, NF), dual (EPN+EPF, EPF+NF, EPN+NF) and triple (EPN+EPF+NF) combinations of one EPF (Beauveria bassiana), one EPN (Steinernema feltiae), and two NF (Arthrobotrys musiformis and Purpureocillium lilacinum). Three different fungal applications (contact with mycelia, immersion, and injection) simulated different timing of fungal arrival. Also, we evaluated the interaction in the mycelia growth between EPF and NF species. We tested the impact on biocontrol of two plant-based oils (coconut and olive oils) when combined the EPF B. bassiana and the EPN Steinernema colombiense. Also, the viability and virulence of EPN incubated at five temperatures (4, 8, 14, 20, and 24°C) and five incubation times (1, 3, 7, 14, and 21 days) with both plant-based adjuvants was also evaluated. The distribution of EPF in the Algarve region was not driven by botanical group or by ecoregion (calcareous versus non-calcareous). Five EPF species were identified, with B. bassiana as the dominant EPF (34% of the localities), followed by Fusarium solani (26%), and F. oxysporum (14%), both reported as EPF for the first time in Portugal. The species P. lilacinus (8%) and Metarhizium anisopliae (2%) were minority. Soil pH, organic matter, and Mg content explained most of the variability of the EPF community and EPN species-specific distribution. Second, we observed that EPF B. bassiana limited both NF species growth and viceversa. The EPF B. bassiana dominated triple-interaction when mycelia were exposed. The EPN S. feltiae dominated the triple-interaction in immersion exposure. The NF A. musiformis caused larval mortality if vectored inside the host. Finally, the plant-based oils tested wer compatible with the EPF and EPN. The combination of EPN+EPF produced an additive effect. EPN survival was higher in coconut than olive oil and water mixtures up to 7 days at 4ºC. Moreover, olive oil supported higher larval mortality caused by EPN than coconut oil at 4-20ºC and 14 days. Overall, this thesis provides new insights on the interactions occuring in the soil that can modulate their activity of EPF as biological control agents.

2023-03-09Doctoral thesis

Open Access

Reproductive efficiency of entomopathogenic nematodes as scavengers. Are they able to fight for insect’s cadavers?

Publication . Blanco-Pérez, Rubén; Bueno-Pallero, Francisco Ángel; Neto, Luis; Campos-Herrera, Raquel

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

2017Journal article

Open Access

Combined field inoculations of pseudomonas bacteria, arbuscular mycorrhizal fungi, and entomopathogenic nematodes and their effects on wheat performance

Publication . Imperiali, Nicola; Chiriboga, Xavier; Schlaeppi, Klaus; Fesselet, Marie; Villacres, Daniela; Jaffuel, Geoffrey; Bender, S. Franz; Dennert, Francesca; Blanco-Perez, Ruben; van der Heijden, Marcel G. A.; Maurhofer, Monika; Mascher, Fabio; Turlings, Ted C. J.; Keel, Christoph J.; Campos-Herrera, Raquel

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 Pseudornonas, 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.

2017-10Journal article

Open Access

Cutaneous excreta of the earthworm Eisenia fetida (Haplotaxida: Lumbricidae) might hinder the biological control performance of entomopathogenic nematodes

Publication . Chelkha, Maryam; Blanco-Perez, Ruben; Bueno-Pallero, Francisco Angel; Amghar, Souad; El Harti, Abdellatif; Campos-Herrera, Raquel

Earthworms enhance soil structure, the decomposition of organic matter and the dissemination of beneficial soil organisms such as the entomopathogenic nematodes (EPNs). Nevertheless, the effects of earthworm feeding behavior or cutaneous excreta (CEx) on the performance of EPNs as biological control agents is poorly understood. We hypothesized that the presence of earthworms or their excreta reduces EPN fitness, measured in terms of pathogenicity and reproductive success. In laboratory experiments we first evaluated the killing ability of EPNs against Galleria mellonella (Lepidoptera: Pyralidae) larvae when inoculated in autoclaved soil alone or in combination with the earthworm Eisenia fetida (Haplotaxida: Lumbricidae) or their excreta. We also evaluated EPN efficacy and reproduction when exposed to CEx derived from E. fetida at two nematode concentrations (1.5 and 10 IJs/cm(2)). For both experiments, we tested four steinernematids (Steinernema carpocapsae, S. feltiae, S. glaseri, and S. khuongi) and two heterorhabditids (Heterorhabditis bacteriophora and H. zealandica). The presence of earthworms or their excreta resulted in significant reduction of the larval mortality caused by some of the steinernematids at certain timings depending of the species (P < 0.015), while heterorhabditids were mainly not affected. Both S. feltiae and H. zealandica progeny production was significantly reduced (P < 0.01) when exposed to CEx. Hence, we showed that the presence of CEx might alter the biocontrol performance of certain EPN species, especially steinernematids bigger than 600 mu m in size (S. feltiae, S. glaseri, and S. khoungi), by affecting their pathogenicity and reproductive success.

2020-02Journal article

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