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

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.Os fungos entomopatogénicos (EPF) são organismos benéficos e bem conhecidos de ocorrência natural no solo. Estudar a sua distribuição natural em habitats representativos e as suas interações com outros organismos do solo pode estabelecer um melhor cenário ecológico para a atividade como agentes de biocontrolo. Além disso, a co-formulação de dois fungos entomopatogénicos com produtos à base de plantas pode ser uma abordagem acessível para os agricultores. Tendo a região do Algarve (Portugal) como quadro de investigação, os objetivos desta tese de doutoramento foram: (1) investigar a distribuição natural dos EPF em solos do Algarve em quatro habitats (carvalho, pinheiro, palmeira anã e citrinos) e duas ecorregiões de solo (calcário e não calcário), (2) avaliar se a presença de nemátodes entomopatogénicos (EPN) ou/e fungos nematófagos (NF) pode afetar o potencial biocontrolo dos EPF, e (3) investigar o impacto na atividade de biocontrole dos EPF de adjuvantes de óleo à base de plantas, EPF e EPN individual ou em combinação, e o impacto do tempo e da temperatura na sobrevivência e atividade da EPN. Em primeiro lugar, combinámos três métodos de isolamento (solo fresco, solo seco e meios selectivos) para investigar a distribuição natural dos EPF no Algarve, utilizando ferramentas tradicionais e moleculares para a identificação. Estabelecemos a relação dos EPF com as propriedades do solo e dos EPN por meio de análise multivariada. Em segundo lugar, investigamos a interações multitróficas em combinações simples (EPF, EPN, NF), duplas (EPN + EPF, EPF + NF, EPN + NF) e triplas (EPN + EPF + NF) de um EPF (Beauveria bassiana), um EPN (Steinernema feltiae) e dois NF (Arthrobotrys musiformis e Purpureocillium lilacinum). Três aplicações diferentes de fungos (contacto com micélios, imersão e injeção) simularam tempos diferentes de chegada do fungo. Além disso, avaliamos a interação no crescimento de micélios entre espécies de EPF e NF. Testamos o impacto de dois óleos vegetais (óleo de coco e azeite) no biocontrolo, quando combinados o EPF B. bassiana e o EPN Steinernema colombiense. Além disso, foi também avaliada a viabilidade e virulência do EPN incubado a cinco temperaturas (4, 8, 14, 20 e 24 ° C) e cinco tempos de incubação (1, 3, 7, 14 e 21 dias) com ambos os adjuvantes à base de plantas. A distribuição dos EPF na região do Algarve não foi determinada por grupo botânico ou ecorregião (calcário versus não calcário). Foram identificadas cinco espécies de EPF, sendo B. bassiana o EPF dominante (34% das localidades), seguida de Fusarium solani (26%) e F. oxysporum (14%), ambas reportadas como EPF pela primeira vez em Portugal. As espécies P. lilacinus (8%) e Metarhizium anisopliae (2%) foram as menos representadas. O pH do solo, a matéria orgânica e o conteúdo de Mg explicaram a maior parte da variabilidade da comunidade dos EPF e da distribuição específica das espécies dos EPN. Em segundo lugar, observámos que o EPF B. bassiana limitou o crescimento das espécies de NF e viceversa. O EPF B. bassiana dominou a interação tripla aquando da exposição dos micélios. O EPN S. feltiae dominou a interação tripla na exposição por imersão. O NF A. musiformis causou mortalidade larval quando introduzido dentro do hospedeiro. Finalmente, os óleos vegetais testados foram compatíveis com os EPF e EPN. A combinação de EPN + EPF produziu um efeito aditivo. A sobrevivência de EPN foi maior em misturas de coco do que de azeite e água por até 7 dias a 4ºC. Além disso, o azeite suportou maior mortalidade larval causada por EPN do que o óleo de coco a 4-20ºC e 14 dias. No geral, esta tese fornece novas perspetivas sobre as interações que ocorrem no solo, as quais podem modular a atividade dos EPF como agentes de controlo biológico

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

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.Research Subject Categories::FORESTRY, AGRICULTURAL SCIENCES and LANDSCAPE PLANNING::Soil science::Soil biology; Research Subject Categories::NATURAL SCIENCES::Biology::Terrestrial, freshwater and marine ecology::Ethology and behavioural ecology; Research Subject Categories::NATURAL SCIENCES::Biology::Terrestrial, freshwater and marine ecology::Terrestrial ecologyinfo:eu-repo/semantics/publishedVersio

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.The Government of Portugal, thanks to the ‘Starting Grant’ associate funds (grant number IF/00552/2014/CP1234/CT0007), supported this work. RCH was awarded an Investigator Program contract (grant number IF/00552/2014) and currently is supported by the Ramón y Cajal Program (Spanish Government, RYC-2016-19939). GCE was supported by a Master Fellowship from the government of Mexico (CONACYT CVU 717361). RBP and FBP were financed by the scientific assistantship fellowships associated with the grant IF/00552/2014/CP1234/CT0007 (BI calls UAlg-2016/004 and UAlg-2016/003, respectively). RBP is currently supported by the Department of Economic Development and Innovation of the Government of La Rioja (Spain) with a pre-doctoral contract (CAR-2018).Peer reviewe

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.The Government of Portugal, thanks to the “Starting Grant” associate funds (Grant Number IF/00552/2014/CP1234/CT0007), supported this work. R.C.-H. was awarded an Investigator Program contract (Grant Number IF/00552/2014) and currently is supported by the Ramón y Cajal Program (Spanish Government, RYC-2016-19939). R.B.-P. and F.A.B.-P. were financed by the scientific assistantship fellowships associated with the grant IF/00552/2014/CP1234/CT0007 (BI calls UAlg-2016/004 and UAlg-2016/003, respectively). R.B.-P. is currently supported by the Department of Economic Development and Innovation of the Government of La Rioja (Spain) with a pre-doctoral contract (CAR-2018). I.V.-D. is currently supported by an ADER postgraduate grant (Government of La Rioja, Spain)Peer reviewe

The presence of earthworm mucus secretion could altered entomopathogenic nematodes activity as biological control agents

Trabajo presentado en el 33rd European Society of Nematologist meeting, celebrado en Ghent (Bélgica), del 9 al 13 de septiembre de 2018Peer reviewe

The earthworm mucus and their feeding activity can decrease the biological control action by entomopathogenic nematodes and entomopathogenic fungi.

Trabajo presentado en el 52nd Annual Meeting of the Society for Invertebrate Pathology y 17th Meeting of the IOBC‐WPRS Working Group “Microbial and Nematode Control of Invertebrate Pests”, celebrado en Valencia (España), del 28 de julio al 1 de agosto de 201

Steinernema feltiae scavenging behavior: offspring fitness is modulated by various cadaver scenarios

Trabajo presentado en el 52nd Annual Meeting of the Society for Invertebrate Pathology y 17th Meeting of the IOBC‐WPRS Working Group “Microbial and Nematode Control of Invertebrate Pests”, celebrado en Valencia (España), del 28 de julio al 1 de agosto de 201

Earthworms and their cutaneous excreta can modify the virulence and reproductive capability of entomopathogenic nematodes and fungi [Dataset]

Experiment performed in the lab, following details described in the publication: http://dx.doi.org/10.1016/j.jip.2021.107620 / http://hdl.handle.net/10261/261936Grants ICVV-CSIC: 1) CSIC I-COOP + 2018 grant (COOPA20231) 2) Associated funds of Ramon y Cajal contract award MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”: Grant RYC-2016-19939 from the Government of Spain Support for researchers: 1) MC was supported by the travel assistance is associated with the CSIC I-COOP+ 2018 grant (COOPA20231) and a Moroccan scholarship for the Ministry of National Education, Vocational Training, Higher Education, and Scientific Research 2) RBP was supported by the pre-doctoral contracts CAR-2018 (Department of Economic Development and Innovation of the Government of La Rioja). 3) IVD was funded by ADER I + D + i (2019) fellowship by the Rioja Agency of Economic Development (La Rioja, Spain) and by FPI-UR (2021) fellowship (Universidad de La Rioja, Spain). 4) RCH was awarded by Ramon y Cajal contract award MCIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”: Grant RYC-2016-19939 from the Government of SpainPeer reviewe

Vegetation drives assemblages of entomopathogenic nematodes and other soil organisms: evidence from the Algarve, Portugal

Entomopathogenic nematodes (EPNs) are widely distributed in soils throughout the world. Their activity as biological control agents is modulated by abiotic and biotic factors (e.g. soil type, climatic fluctuation and natural enemies). We sought to identify soil properties in a Mediterranean region, which might be managed to enhance biological control agents’ services provided by EPNs. We hypothesized that responses of EPN soil food web assemblages to abiotic factors in such a Mediterranean region would be consistent with previous observations in other biomes in subtropical and temperate regions, in which pH and variables related to water content were main drivers of such association. We also expected that EPN abundance and species composition would differ between stable botanical habitats (citrus groves, palmetto areas, oaks and pines), with EPNs and associated organisms favoured in cultivated sites (citrus). In spring 2016, 50 georeferenced localities, representing four botanical habitats and two soil-ecoregions (calcareous versus non-calcareous), were surveyed. Using published and de novo real time qPCR tools, we evaluated the frequency and abundance of 10 EPN species and 13 organisms associated with EPNs: 6 nematophagous fungi (NF), 5 free-living nematodes (FLN), and 2 ectoparasitic bacteria. EPN activity was also assessed by traditional insect-baiting, allowing the evaluation of FLN-EPN mixed progeny. EPNs were detected by qPCR in 50% of localities, and strongly correlated with EPN activity. Steinernema feltiae was the dominant EPN species measured by both techniques (qPCR and insect-bait), being widespread in all Algarve, while Heterorhabditis bacteriophora was detected mainly in citrus groves. The species S. arenarium and H. indica were detected by qPCR for the fi rst time in continental Portugal. The molecular analysis of insect cadaver progeny revealed novel FLN-EPN associations with Pristionchus maupasi and P. pacificus. EPN, FLN and NF abundance differed among botanical groups, with citrus groves supporting high numbers of all trophic guilds. Oaks also favoured EPNs. Similarly, calcareous soil-ecoregion supported higher NF, FLN and EPN abundance. Two abiotic variables (pH, and clay content) explained the community variation in multivariate analysis, consistent with key abiotic variables described for other subtropical and temperate regions. The results supported the hypothesis that cultivated perennial habitats favour EPNs and soil organisms that can limit EPN activity as biological control agents.info:eu-repo/semantics/publishedVersio