Can Photoselective Nets' Influence Pollen Traits? A Case Study in 'Matua' and 'Tomuri' Kiwifruit Cultivars.

The increasing use of photoselective nets (PNs) raises the question of their influence on pollen traits. We aimed to evaluate the effect of PNs (yellow, pearl, and grey) on the pollen of 'Matua' and 'Tomuri' Actinidia deliciosa cultivars. The pollen size and the exine were studied with a light microscopy and a scanning electron microscopy, and the fertility was analysed by a viability assay and in vitro germination. The total soluble proteins (TSPs) and sugars (TSSs) were quantified by colorimetric assays. The molecular structure of the pollen grain's wall was analysed by a Raman spectroscopy. The pollen from the plants under the PNs had a larger width and area and a lower germination rate. No significant changes were observed in the exine's microperforations. The TSP and TSS contents were influenced by the cultivar and PNs (particularly the pearl PN). The Raman spectra of the pollen from the plants grown under the nets presented some bands that significantly shifted from their original position, indicating differences in the vibration modes of the molecules, but no overall changes at their structural or organisation level were found. Our study showed that the PNs could influence several pollen traits, with the pearl PN inducing greater modifications. Our results also support the idea that cultivars affect the outcome of some characteristics.

Satureja montana and Mentha pulegium essential oils' antimicrobial properties against Pseudomonas syringae pv. actinidiae and elicitor potential through the modulation of kiwifruit hormonal defenses.

Pseudomonas syringae pv. actinidiae (Psa) is responsible for the kiwifruit bacterial canker, the most severe disease of Actinidia spp. The use in agriculture of antibiotics and cooper-based compounds is increasingly being restricted, demanding for new sustainable alternatives to current agrochemicals. We aimed to characterize the anti-Psa potential of essential oils (EOs) of Mentha pulegium and Satureja montana and investigate if they elicit the plant-host hormonal defenses. The EOs were characterized through gas-chromatography with flame ionization detector (GC-FID) and mass spectrometry (MS). Pulegone (78.6%) and carvacrol (43.5%) were the major constituents of M. pulegium and S. montana EO, respectively. Only S. montana EO showed relevant anti-Psa activity in vitro. To evaluate if the EOs also elicited host defenses, in vitro shoots were treated with 2 mg shoot-1 of EO-solution and subsequently inoculated with Psa three days later. Shoots were analyzed 10 min, three days (and 10 min after Psa-inoculation), four and ten days after EO application. The up/down regulation of RNA-transcripts for hormone biosynthesis, Psa biofilm production and virulence genes were quantified by real-time quantitative PCR (RT-qPCR). Phytohormones were quantified by High-Performance Liquid Chromatography (HPLC). S. montana EO showed the most promising results as a defense elicitor, increasing 6-benzylaminopurine (BAP) by 131.07% and reducing indole-3-acetic acid (IAA) levels by 49.19%. Decreases of salicylic acid (SA), and gibberellic acid 3 (GA3) levels by 32.55% and 33.09% respectively and an increase of abscisic acid (ABA) by 85.03%, in M. pulegium EO-treated shoots, revealed some protective post-infection effect. This is the most comprehensive research on the Psa's impact on phytohormones. It also unveils the protective influence of prior EO exposure, clarifying the plant hormonal response to subsequent infections. The results reinforce the hypothesis that carvacrol-rich S. montana EO can be a suitable disease control agent against Psa infection. Its dual action against pathogens and elicitation of host plant defenses make it a promising candidate for incorporation into environmentally friendly disease management approaches. Nonetheless, to fully leverage these promising results, further research is imperative to elucidate the EO mode of action and evaluate the long-term efficacy of this approach.

Peptaibiotics: Harnessing the potential of microbial secondary metabolites for mitigation of plant pathogens.

Agricultural systems are in need of low-cost, safe antibiotics to protect crops from pests and diseases. Peptaibiotics, a family of linear, membrane-active, amphipathic polypeptides, have been shown to exhibit antibacterial, antifungal, and antiviral activity, and to be inducers of plant resistance against a wide range of phytopathogens. Peptaibiotics belong to the new generation of alternatives to agrochemicals, aligned with the United Nations Sustainable Development Goals and the One Health approach toward ensuring global food security and safety. Despite that, these fungi-derived, non-ribosomal peptides remain surprisingly understudied, especially in agriculture, where only a small number has been tested against a reduced number of phytopathogens. This lack of adoption stems from peptaibiotics' poor water solubility and the difficulty to synthesize and purify them in vitro, which compromises their delivery and inclusion in formulations. In this review, we offer a comprehensive analysis of peptaibiotics' classification, biosynthesis, relevance to plant protection, and mode of action against phytopathogens, along with the techniques enabling researchers to extract, purify, and elucidate their structure, and the databases holding such valuable data. It is also discussed how chemical synthesis and ionic liquids could increase their solubility, how genetic engineering and epigenetics could boost in vitro production, and how omics can reduce screenings' workload through in silico selection of the best candidates. These strategies could turn peptaibiotics into effective, ultra-specific, biodegradable tools for phytopathogen control.

Distinct phenotypic behaviours within a clonal population of Pseudomonas syringae pv. actinidiae.

Bacterial canker of the kiwifruit caused by the etiological agent Pseudomonas syringae pv. actinidiae is the most severe disease in kiwifruit production. Since 2008 a hypervirulent Psa biovar 3 has spread rapidly worldwide. Different genomic and phenotypic approaches have been used to understand the origin of the dissemination and geographical evolution of populations associated with this pandemic. This study aimed to characterize the genetic and phenotypic diversity of 22 Psa isolates collected in different regions of Portugal between 2013 and 2017. Genotypic and phenotypic characterization was based on Multi-Locus Sequence Analysis (MLSA), motility, IAA production, Biolog GEN III, and copper sensitivity. No polymorphisms were detected for the concatenated sequence (1950 bp) of the housekeeping genes gltA, gapA, gyrB, and rpoD. Results support the analysed Portuguese Psa isolates (2013-2017) belonging to Psa3, and MLSA indicates high genetic clonality and stability of these populations. The phenotypic analysis through Biolog revealed a heterogeneous pattern in the Psa collection and its position in the Pseudomonas complex. This heterogeneity reflects a genomic diversity that may reflect distinct adaptive trends associated with the environmental conditions and widespread. The Portuguese Psa collection showed no resistance to copper. This information is relevant to kiwi producers that predominantly use Cu-treatments to control kiwifruit bacterial canker.

In vitro modulation of gilthead seabream (Sparus aurata L.) leukocytes by Bacillus spp. extracellular molecules upon bacterial challenge.

Stimulation of the fish immune system using immunostimulants is an environmentally friendly strategy to minimize bacterial outbreaks in aquaculture. Different biological and synthetic immunostimulants can enhance non-specific innate immune responses by directly activating immune cells. An example are Bacillus spp., known for their immunostimulatory effects, although the exact mechanisms by which Bacillus spp. offer protection against diseases remains to be elucidated. Furthermore, most studies have focused on Bacillus spp. cells, while the immunostimulant effect of their extracellular metabolome, known to harbour biologically important metabolites, including antimicrobial molecules, has been scarcely evaluated. Here, we evaluated the in vitro immune-modulatory properties of extracellular extracts of three Bacillus spp. strains (B. subtilis FI314, B. vezelensis FI436 and B. pumilus FI464), previously isolated from fish-guts and characterized for their in vitro and in vivo antimicrobial activity against a wide range of fish pathogens. Bacillus spp. extracellular extracts did not affect immune cells viability, but remarkably increased pathogens' phagocytosis when seabream head-kidney leukocytes were challenged with Vibrio anguillarum and Edwardsiella tarda. All extracts significantly increased the engulfment of bacterial pathogens 1 h post-infection. Cells stimulated with the extracellular extracts showed an up-regulation of the expression of immune-relevant genes associated with inflammation, including IL-1ß, IL-6, and COX-2. In cells challenged with E. tarda, FI314 extracellular extract significantly increased the expression of IL-1ß, IL-6, and COX-2, while FI436 and FI464 significantly increased IL-6 expression. The results of this study revealed that the extracellular molecules from Bacillus spp. fish isolates improved the in vitro response of gilthead seabream immune cells and are thus promising candidates to act as immunostimulants, helping fish fight diseases.

Low levels of TiO2-nanoparticles interact antagonistically with Al and Pb alleviating their toxicity.

The contamination and bioavailability of deleterious metals in arable soils significantly limits crop development and yield. Aiming at mitigating Pb- and Al-induced phytotoxicity, this work explores the use of P25 titanium dioxide nanoparticles (nTiO2) in soil amendments. For that, Lactuca sativa L. plants were germinated and grown in the presence of 10 ppm Pb or 50 ppm Al, combined or not with 5 ppm nTiO2. Growth parameters, as well as endpoints of the redox state [cell relative membrane permeability (RMP), thiobarbituric acid reactive substances content, total phenolic content and photosynthesis (sugars and pigments levels, chlorophyll a fluorescence and gas exchange), were evaluated. Concerning Al, nTiO2 treatment alleviated the impairments induced in germination rate, seedling length, water content, RMP, stomatal conductance (gs), intercellular CO2 (Ci), and net CO2 assimilation rate (PN). It increased anthocyanins contents and effective efficiency of photosystem II (ΦPSII). In Pb-exposed plants, nTiO2 amendment mitigated the effects in RMP, PN, gs, and Ci. It also increased the pigment contents and the transpiration rate (E) comparatively to the control without nTiO2. These results clearly highlight the high potential of low doses of nTiO2 in alleviating metal phytotoxicity, particularly the one of Pb. Additionally, further research should explore the use of these nanoparticles in agricultural soil amendments.

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.

A Synergic Potential of Antimicrobial Peptides against Pseudomonas syringae pv. actinidiae.

Pseudomonas syringae pv. actinidiae (Psa) is the pathogenic agent responsible for the bacterial canker of kiwifruit (BCK) leading to major losses in kiwifruit productions. No effective treatments and measures have yet been found to control this disease. Despite antimicrobial peptides (AMPs) having been successfully used for the control of several pathogenic bacteria, few studies have focused on the use of AMPs against Psa. In this study, the potential of six AMPs (BP100, RW-BP100, CA-M, 3.1, D4E1, and Dhvar-5) to control Psa was investigated. The minimal inhibitory and bactericidal concentrations (MIC and MBC) were determined and membrane damaging capacity was evaluated by flow cytometry analysis. Among the tested AMPs, the higher inhibitory and bactericidal capacity was observed for BP100 and CA-M with MIC of 3.4 and 3.4-6.2 µM, respectively and MBC 3.4-10 µM for both. Flow cytometry assays suggested a faster membrane permeation for peptide 3.1, in comparison with the other AMPs studied. Peptide mixtures were also tested, disclosing the high efficiency of BP100:3.1 at low concentration to reduce Psa viability. These results highlight the potential interest of AMP mixtures against Psa, and 3.1 as an antimicrobial molecule that can improve other treatments in synergic action.

Antioxidant Adjustments of Olive Trees (Olea Europaea) under Field Stress Conditions.

Extreme climate events are increasingly frequent, and the 2017 summer was particularly critical in the Mediterranean region. Olive is one of the most important species of this region, and these climatic events represent a threat to this culture. However, it remains unclear how olive trees adjust the antioxidant enzymatic system and modulate the metabolite profile under field stress conditions. Leaves from two distinct adjacent areas of an olive orchard, one dry and the other hydrated, were harvested. Tree water status, oxidative stress, antioxidant enzymes, and phenolic and lipophilic metabolite profiles were analyzed. The environmental conditions of the 2017 summer caused a water deficit in olive trees of the dry area, and this low leaf water availability was correlated with the reduction of long-chain alkanes and fatty acids. Hydrogen peroxide (H2O2) and superoxide radical (O2•-) levels increased in the trees collected from the dry area, but lipid peroxidation did not augment. The antioxidant response was predominantly marked by guaiacol peroxidase (GPOX) activity that regulates the H2O2 harmful effect and by the action of flavonoids (luteolin-7-O-glucuronide) that may act as reactive oxygen species scavengers. Secoiridoids adjustments may also contribute to stress regulation. This work highlights for the first time the protective role of some metabolite in olive trees under field drought conditions.

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.

Lead induces oxidative stress in Pisum sativum plants and changes the levels of phytohormones with antioxidant role.

The interaction of lead (Pb) with plant hormonal balance and oxidative stress remains under discussion. To evaluate how Pb induces oxidative stress, and modulates the antioxidant enzymes and the phytohormones pool, four-week old Pisum sativum plants were exposed during 28 days to 10, 100 and 500 mg kg-1 Pb in soil. In comparison to leaves, roots showed higher Pb accumulation, oxidative damages and changes in phytohormone pools. Contrarily to leaves, where glutathione reductase (GR) and ascorbate peroxidase (APX) activities were more stimulated than catalase (CAT) and superoxide dismutase (SOD), roots showed a stimulation of SOD, GR and APX in all doses, and of CAT in the highest dose. While protein oxidation occurred in roots even at lower Pb-doses, lipid peroxidation and membrane permeability also occurred but at 500 mg kg-1 and in both organs, accompanied by increases of H2O2. Jasmonic acid (JA) responded in both organs even at lowest Pb-doses, while salicylic acid (SA) and abscisic acid (ABA, only in leaves), increased particularly at the concentration of 500 mg Pb kg-1. In conclusion, and compared with leaves, roots showed oxidative damage even at 10 mg Pb Kg-1, being proteins a first oxidative-target, although there is a stimulation of the antioxidant enzymes. Also, JA is mobilized prior to oxidative stress changes are detected, and may play a protective role (activating antioxidant enzymes), while the mobilization of SA is particularly relevant in cells expressing oxidative damage. Other hormones, like indolacetic acid and ABA may have a low protective role against Pb toxicity.

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.