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.

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.

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.

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.

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.

[Patients' understanding of the dietetic and pharmacological treatment of hypercholesterolaemia and its relationship with the achievement of objectives in patients receiving statins: OPINA study]. / Conocimiento sobre el tratamiento dietético y farmacológico de la hipercolesterolemia y su relación con el control de objetivos en pacientes que reciben estatinas: estudio OPINA.

OBJECTIVE: To evaluate the understanding of patients receiving statins about the dietary and therapeutic measures taken to control their cholesterol and to relate this to the achievement of therapy objectives. DESIGN: Transversal, multi-centre study, conducted between March and November, 2004. SETTING: Three-hundred seventy six primary care and specialist doctors from all over the country. PARTICIPANTS: A total of 2347 patients, with an average age of 59 (12) and 48% women, being treated with statins. METHOD: Survey to evaluate understanding of lipid-lowering diet and pharmacological treatment. The percentage of patients achieving therapy objectives in line with ATP III guidelines was determined. RESULTS: Understanding of diet was adequate only for foods with negative effects on the concentration of cholesterol. Overall, understanding was better in the young, those with a higher level of education, those with hyperlipaemia originating in the family, those under treatment for longer, and in those attended in primary care or internal medicine clinics; it was worse in diabetic patients and in those with cardiovascular disease. Patients with more understanding of their treatment reached their therapy objectives to a greater extent. CONCLUSION: Patients receiving statins treatment have an adequate understanding of diet, but little information on pharmacological treatment. This understanding is associated with the achievement of therapy objectives.