Disclosing the native blueberry rhizosphere community in Portugal-an integrated metagenomic and isolation approach.

Backgorund: The production of red fruits, such as blueberry, has been threatened by several stressors from severe periods of drought, nutrient scarcity, phytopathogens, and costs with fertilization programs with adverse consequences. Thus, there is an urgent need to increase this crop's resilience whilst promoting sustainable agriculture. Plant growth-promoting microorganisms (PGPMs) constitute not only a solution to tackle water and nutrient deficits in soils, but also as a control against phytopathogens and as green compounds for agricultural practices. Methods: In this study, a metagenomic approach of the local fungal and bacterial community of the rhizosphere of Vaccinium corymbosum plants was performed. At the same time, both epiphytic and endophytic microorganisms were isolated in order to disclose putative beneficial native organisms. Results: Results showed a high relative abundance of Archaeorhizomyces and Serendipita genera in the ITS sequencing, and Bradyrhizobium genus in the 16S sequencing. Diversity analysis disclosed that the fungal community presented a higher inter-sample variability than the bacterial community, and beta-diversity analysis further corroborated this result. Trichoderma spp., Bacillus spp., and Mucor moelleri were isolated from the V. corymbosum plants. Discussion: This work revealed a native microbial community capable of establishing mycorrhizal relationships, and with beneficial physiological traits for blueberry production. It was also possible to isolate several naturally-occurring microorganisms that are known to have plant growth-promoting activity and confer tolerance to hydric stress, a serious climate change threat. Future studies should be performed with these isolates to disclose their efficiency in conferring the needed resilience for this and several crops.

Winter Is (Not) Coming: Is Climate Change Helping Drosophila suzukii Overwintering?

Anthropogenic challenges, particularly climate change-associated factors, are strongly impacting the behavior, distribution, and survival of insects. Yet how these changes affect pests such as Drosophila suzukii, a cosmopolitan pest of soft-skinned small fruits, remains poorly understood. This polyphagous pest is chill-susceptible, with cold temperatures causing multiple stresses, including desiccation and starvation, also challenging the immune system. Since the invasion of Europe and the United States of America in 2009, it has been rapidly spreading to several European and American countries (both North and South American) and North African and Asian countries. However, globalization and global warming are allowing an altitudinal and latitudinal expansion of the species, and thus the colonization of colder regions. This review explores how D. suzukii adapts to survive during cold seasons. We focus on overwintering strategies of behavioral adaptations such as migration or sheltering, seasonal polyphenism, reproductive adaptations, as well as metabolic and transcriptomic changes in response to cold. Finally, we discuss how the continuation of climate change may promote the ability of this species to survive and spread, and what mitigation measures could be employed to overcome cold-adapted D. suzukii.

Towards a Sustainable Management of the Spotted-Wing Drosophila: Disclosing the Effects of Two Spider Venom Peptides on Drosophila suzukii.

The spotted-wing drosophila (Drosophila suzukii) is a polyphagous pest that causes severe damage and economic losses to soft-skinned fruit production. Current control methods are dominated by inefficient cultural practices and broad-spectrum insecticides that, in addition to having toxic effects on non-target organisms, are becoming less effective due to acquired resistance. The increasing awareness of the real impact of insecticides on health and the environment has promoted the exploration of new insecticidal compounds, addressing novel molecular targets. This study explores the efficacy of two orally delivered spider venom peptides (SVPs), J-atracotoxin-Hv1c (Hv1c) and µ-theraphotoxin-Hhn2b (TRTX), to manage D. suzukii, through survival assays and the evaluation of gene expression associated with detoxification pathways. Treatment with TRTX at 111.5 µM for 48 h enhanced fly longevity compared with the control group. Gene expression analysis suggests that detoxification and stress-related mechanisms, such as expression of P450 proteins and apoptotic stimuli signaling, are triggered in D. suzukii flies in response to these treatments. Our results highlight the potential interest of SVPs to control this pest, shedding light on how to ultimately develop improved target-specific formulations.

Drosophila suzukii energetic pathways are differently modulated by nutritional geometry in males and females.

As a polyphagous pest, Drosophila suzukii has a variety of host fruits available for feeding and oviposition, but how the nutritional geometry of different hosts influences its metabolism is still poorly understood. This work aimed to evaluate how D. suzukii metabolic and transcriptional pathways are influenced by feeding on different host fruits, and how sex influences these responses. Adult flies were allowed to feed on five different fruit-based media. Lipids, glucose, glycogen, and energy pathways-associated gene expression, were quantified. Females showed an energetic metabolism easily adaptable to the food's nutritional characteristics; in contrast, males' energetic metabolism was particularly influenced by food, predominantly those fed on raspberry media who showed changes in glucose levels and in the expression of genes associated with metabolic pathways, suggesting activation of gluconeogenesis and trehaloneogenesis as a result of nutritional deficiency. Here we present novel insight into how D. suzukii's energetic pathways are modulated depending on fruits' nutritional geometry and sex. While the females showed high adaptability in their energetic metabolism to the diet, males were more feeding-sensitive. These findings might be used not only to control this pest population but to better advise producers to invest in less suitable fruits based on the hosts' nutritional geometry.

Evaluation of Three Antimicrobial Peptides Mixtures to Control the Phytopathogen Responsible for Fire Blight Disease.

Fire blight is a severe bacterial plant disease that affects important chain-of-value fruit trees such as pear and apple trees. This disease is caused by Erwinia amylovora, a quarantine phytopathogenic bacterium, which, although highly distributed worldwide, still lacks efficient control measures. The green revolution paradigm demands sustainable agriculture practices, for which antimicrobial peptides (AMPs) have recently caught much attention. The goal of this work was to disclose the bioactivity of three peptides mixtures (BP100:RW-BP100, BP100:CA-M, and RW-BP100:CA-M), against three strains of E. amylovora representing distinct genotypes and virulence (LMG 2024, Ea 630 and Ea 680). The three AMPs' mixtures were assayed at eight different equimolar concentrations ranging from 0.25 to 6 µM (1:1). Results showed MIC and MBC values between 2.5 and 4 µM for every AMP mixture and strain. Regarding cell viability, flow cytometry and alamarBlue reduction, showed high reduction (>25%) of viable cells after 30 min of AMP exposure, depending on the peptide mixture and strain assayed. Hypersensitive response in tobacco plants showed that the most efficient AMPs mixtures and concentrations caused low to no reaction of the plant. Altogether, the AMPs mixtures studied are better treatment solutions to control fire blight disease than the same AMPs applied individually.

Physiological, Biochemical and Molecular Assessment of UV-A and UV-B Supplementation in Solanum lycopersicum.

Daily UV-supplementation during the plant fruiting stage of tomato (Solanum lycopersicum L.) growing indoors may produce fruits with higher nutraceutical value and better acceptance by consumers. However, it is important to ensure that the plant's performance during this stage is not compromised by the UV supplement. We studied the impact of UV-A (1 and 4 h) and UV-B (2 and 5 min) on the photosynthesis of greenhouse-grown tomato plants during the fruiting/ripening stage. After 30 d of daily irradiation, UV-B and UV-A differently interfered with the photosynthesis. UV-B induced few leaf-necrotic spots, and effects are more evidenced in the stimulation of photosynthetic/protective pigments, meaning a structural effect at the Light-Harvesting Complex. UV-A stimulated flowering/fruiting, paralleled with no visible leaf damages, and the impact on photosynthesis was mostly related to functional changes, in a dose-dependent manner. Both UV-A doses decreased the maximum quantum efficiency of photosystem II (Fv/Fm), the effective efficiency of photosystem II (ΦPSII), and gas exchange processes, including net carbon assimilation (PN). Transcripts related to Photosystem II (PSII) and RuBisCO were highly stimulated by UV supplementation (mostly UV-A), but the maintenance of the RuBisCO protein levels indicates that some protein is also degraded. Our data suggest that plants supplemented with UV-A activate adaptative mechanisms (including increased transcription of PSII peptides and RuBisCO), and any negative impacts on photosynthesis do not compromise the final carbohydrate balances and plant yield, thus becoming a profitable tool to improve precision agriculture.

DNA screening of Drosophila suzukii predators in berry field orchards shows new predatory taxonomical groups.

Drosophila suzukii (spotted wing drosophila, SWD) is a pandemic quarantine pest that attacks mostly red fruits. The high number of life cycles per year, its ability to rapidly invade and spread across new habitats, and highly polyphagous nature, makes this a particularly aggressive invasive species, for which efficient control methods are currently lacking. The use of native natural predators is particularly promising to anchor sustainable and efficient measures to control SWD. While several field studies have suggested the presence of potential predatory species in infested orchards, only a few confirmed the presence of SWD DNA in predators' gut content. Here, we use a DNA-based approach to identify SWD predators among the arthropod diversity in South Europe, by examining the gut content of potential predator specimens collected in SWD-infested berry fields in North Portugal. These specimens were morphologically identified to the family/order, and their gut content was screened for the presence of SWD DNA using PCR. New SWD predatory taxonomical groups were identified, as Opiliones and Hemerobiidae, in addition to known SWD predators, such as Hemerobiidae, Chrysopidae, Miridae, Carabidae, Formicidae and Araneae. Additionally, the presence of a spider family, Uloboridae, in the orchards was recorded for the first time, posing this family as another SWD-candidate predator. This study sets important bases to further investigate the potential large-scale use of some of these confirmed predator taxa for SWD control in South Europe.

Genotoxic endpoints in a Pb-accumulating pea cultivar: insights into Pb2+ contamination limits.

Lead (Pb) persists among the most hazardous contaminant metals. Pb-induced genotoxic effects remain a matter of debate as they are a major cause of plant growth impairment, but assessing Pb genotoxicity requires the selection of Pb-sensitive genotoxic biomarkers. Seedlings of the ecotoxicological model species Pisum sativum L. were exposed to Pb2+ (≤ 2000 mg L-1). Flow cytometry (FCM) revealed that 28 days after, Pb2+ arrested root cell cycle at G2 but no eu/aneuploidies were found. Comet assay and FCM-clastogenicity assays showed that Pb2+ increased DNA breaks in roots at concentrations as low as 20 mg L-1. Leaves showed no variation in DNA-ploidy or cell cycle progression but had increased DNA breaks at the highest Pb2+ dose. We conclude that both Comet assay and the full-peak coefficient of variation (FPCV) were the most relevant endpoints of Pb-phytogenotoxicity. Also, the Pb-induced DNA breaks may be related with the arrest at the G2-checkpoint. Data will be relevant to better define Pb2+ ecogenotoxicological effects and their measuring tools and may contribute to a regulatory debate of this pollutant limits.

Titanium dioxide nanoparticles: Toxicity and genotoxicity in Drosophila melanogaster (SMART eye-spot test and comet assay in neuroblasts).

Titanium dioxide nanoparticles (TiO2NP) are used in the food, drug, and cosmetics industries and evaluation of their human and environmental toxicity is required. We have tested the toxicity of TiO2NP (anatase) with respect to developmental effects and DNA damage in Drosophila melanogaster strain Ok, using the eye-spot Somatic Mutation and Recombination Test (SMART) and the comet assay (neuroblasts). For the survival assay, TiO2NP were supplied to adult flies for 72 h and no adverse effects were seen. TiO2NP were supplied chronically for the prolificacy, SMART, and comet assays. TiO2NP increased fly prolificacy. With regard to genotoxicity, an increase was observed in the eye-spot SMART assay at 8 µg/mL dose, but not in the neuroblast comet assay for DNA damage.

Inorganic Hg toxicity in plants: A comparison of different genotoxic parameters.

Inorganic Mercury (Hg) contamination persists an environmental problem, but its cyto- and genotoxicity in plants remains yet unquantified. To determine the extent of Hg-induced cyto- and genotoxicity, and assess most sensitive endpoints in plants, Pisum sativum L. seedlings were exposed for 14 days to different HgCl2 concentrations up to 100 µM. Shoots and roots from hydroponic exposure presented growth impairment and/or morphological disorders for doses >1 µM, being the roots more sensitive. Plant growth, ploidy changes, clastogenicity (HPCV), cell cycle dynamics (G1-S-G2), Comet-tail moment (TM), Comet-TD, Mitotic-index (MI) and cell proliferation index (CPI) were used to evaluate Hg-induced cyto/genotoxicity. Both leaf and root DNA-ploidy levels, assessed by flow cytometry (FCM), remained unaltered after exposure. Root cell cycle impairment occurred at lower doses (≥1 µM) than structural DNA damages (≥10 µM). Cytostatic effects depended on the Hg concentration, with delays during S-phase at lower doses, and arrests at G1 at higher ones. This arrest was paralleled with decreases of both mitotic index (MI) and cell proliferation index (CPI). DNA fragmentation, assessed by the Comet assay parameters of TD and TM, could be visualized for conditions ≥10 µM, while FCM-clastogenic parameter (FPCV) and micronuclei (MNC) were only altered in roots exposed to 100 µM. We demonstrate that inorganic-Hg induced cytostaticity is detectable even at 1 µM (a value found in contaminated sites), while structural DNA breaks/damage are only visualized in plants at concentrations ≥10 µM. We also demonstrate that among the different techniques tested for cyto- and genotoxicity, TD and TM Comet endpoints were more sensitive than FPCV or MNC. Regarding cytostatic effects, cell cycle analysis by FCM, including the difference in % cell cycle phases and CPI were more sensitive than MI or MNC frequency. Our data contribute to better understand Hg cyto- and genotoxicity in plants and to understand the information and sensitivity provided by each of the genotoxic techniques used.