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.

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.

Variability within a clonal population of Erwinia amylovora disclosed by phenotypic analysis.

Background: Fire blight is a destructive disease of pome trees, caused by Erwinia amylovora, leading to high losses of chain-of-values fruits. Major outbreaks were registered between 2010 and 2017 in Portugal, and the first molecular epidemiological characterization of those isolates disclosed a clonal population with different levels of virulence and susceptibility to antimicrobial peptides. Methods: This work aimed to further disclose the genetic characterization and unveil the phenotypic diversity of this E. amylovora population, resorting to MLSA, growth kinetics, biochemical characterization, and antibiotic susceptibility. Results: While MLSA further confirmed the genetic clonality of those isolates, several phenotypic differences were recorded regarding their growth, carbon sources preferences, and chemical susceptibility to several antibiotics, disclosing a heterogeneous population. Principal component analysis regarding the phenotypic traits allows to separate the strains Ea 630 and Ea 680 from the remaining. Discussion: Regardless the genetic clonality of these E. amylovora strains isolated from fire blight outbreaks, the phenotypic characterization evidenced a population diversity beyond the genotype clonality inferred by MLSA and CRISPR, suggesting that distinct sources or environmental adaptations of this pathogen may have occurred. Conclusion: Attending the characteristic clonality of E. amylovora species, the data gathered here emphasizes the importance of phenotypic assessment of E. amylovora isolates to better understand their epidemiological behavior, namely by improving source tracking, make risk assessment analysis, and determine strain-specific environmental adaptations, that might ultimately lead to prevent new outbreaks.

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.

CRISPR genotyping as complementary tool for epidemiological surveillance of Erwinia amylovora outbreaks.

Fire blight is a destructive plant disease caused by Erwinia amylovora affecting pome fruit trees, and responsible for large yield declines, long phytosanitary confinements, and high economic losses. In Portugal, the first major fire blight outbreaks occurred in 2010 and 2011, and although later considered eradicated, the emergence of other outbreaks in recent years stressed the need to characterize the E. amylovora populations associated with these outbreaks. In this regard, CRISPR genotyping, assessment of three virulence markers, and semi-quantitative virulence bioassays, were carried out to determine the genotype, and assess the virulence of thirty-six E. amylovora isolates associated with outbreaks occurring between 2010 and 2017 and affecting apple and pear orchards located in the country central-west, known as the main producing region of pome fruits in Portugal. The data gathered reveal that 35 E. amylovora isolates belong to one of the widely-distributed CRISPR genotypes (5-24-38 / D-a-α) regardless the host species, year and region. Ea 680 was the single isolate revealing a new CRISPR genotype due to a novel CR2 spacer located closer to the leader sequence and therefore thought to be recently acquired. Regarding pathogenicity, although dot-blot hybridization assays showed the presence of key virulence factors, namely hrpL (T3SS), hrpN (T3E) and amsG from the amylovoran biosynthesis operon in all E. amylovora isolates studied, pathogenicity bioassays on immature pear slices allowed to distinguish four virulence levels, with most of the isolates revealing an intermediate to severe virulence phenotype. Regardless the clonal population structure of the E. amylovora associated to the outbreaks occurring in Portugal between 2010 and 2017, the different virulence phenotypes, suggests that E. amylovora may have been introduced at different instances into the country. This is the first study regarding E. amylovora in Portugal, and it discloses a novel CRISPR genotype for this bacterium.

In Vitro Evaluation of Five Antimicrobial Peptides against the Plant Pathogen Erwinia amylovora.

Fire blight is a major pome fruit trees disease that is caused by the quarantine phytopathogenic Erwinia amylovora, leading to major losses, namely, in pear and apple productions. Nevertheless, no effective sustainable control treatments and measures have yet been disclosed. In that regard, antimicrobial peptides (AMPs) have been proposed as an alternative biomolecule against pathogens but some of those AMPs have yet to be tested against E. amylovora. In this study, the potential of five AMPs (RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) together with BP100, were assessed to control E. amylovora. Antibiograms, minimal inhibitory, and bactericidal concentrations (minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC), growth and IC50 were determined and membrane permeabilization capacity was evaluated by flow cytometry analysis and colony-forming units (CFUs) plate counting. For the tested AMPs, the higher inhibitory and bactericidal capacity was observed for RW-BP100 and CA-M (5 and 5-8 µM, respectively for both MIC and MBC), whilst for IC50 RW-BP100 presented higher efficiency (2.8 to 3.5 µM). Growth curves for the first concentrations bellow MIC showed that these AMPs delayed E. amylovora growth. Flow cytometry disclosed faster membrane permeabilization for CA-M. These results highlight the potential of RW-BP100 and CA-M AMPs as sustainable control measures against E. amylovora.