Wheat is an important cereal crop that often suffers from osmotic stress under various growing conditions. The objective of this study was to investigate the effects of PEG-induced osmotic stress at the phytomer level on root growth and root hair morphology of 22 hydroponically grown wheat varieties. Two treatments, 0% and 10% PEG, were imposed for 15 days duration at 20 days old wheat seedlings. PEG stress significantly reduced plant height, number of live leaves per tiller, chlorophyll content, shoot dry weights, number of root bearing phytomers and roots per tiller. By contrast, PEG stress significantly increased leaf injury scores, root dry weight, main axis length and diameter of developed roots, length and diameter and density of both first and second order lateral roots, density and length of root hairs. Principal component analysis was conducted taking three less-affected, high yielding varieties and three highly-affected, low yielding varieties under 10% PEG stress compared to control. PC1 separated PEG-treated wheat genotypes from control treated genotypes for high and positive co-efficient of density of lateral roots and root hairs, length and diameter of main axis and first order later roots and leaf injury scores indicating that these traits are associated with osmotic stress responsive root traits. PC2 separated high-yielding and tolerant wheat genotypes from low-yielding and susceptible genotypes for high co-efficient of root dry weight, density of root hairs and second order lateral roots, length of main axis and first order lateral roots indicating that these traits are associated with osmotic stress tolerance of high-yielding wheat genotypes. Evolved information of this research could be exploited for identifying osmotic stress tolerant QTL and in developing abiotic tolerant cultivars of wheat.

In plants, there are several thousands of different types of proteins. All and each have different functions and, therefore, must be correctly located to a specific subcellular compartment. This study focuses on plant vacuolar proteins and their sorting pathways to the vacuole. The conventional vacuolar sorting route is already well described and research teams are now more interested in understanding mechanisms behind how unconventional sorting routes work. Our laboratory has been working for a long time with two very similar aspartic proteinases from Cynara cardunculus: cardosin A and cardosin B. Recently, we found that a 100 amino acid domain inside cardosins’ sequence is sufficient and necessary for correct vacuolar sorting - the plant specific insert (PSI). Even though both PSI domains from cardosin A (PSI A) and cardosin B (PSI B) present high similarity between them, their sorting mediated routes are very different: PSI A is able to mediate a Golgi bypass route, directly delivering proteins from the Endoplasmic Reticulum (ER) to the Vacuole. In contrast, PSI B mediated sorting route follows the conventional vacuolar sorting pathway, ER - Golgi apparatus - Pre-Vacuolar Compartment - Vacuole. The main goal of this study is to identify intermediate playersin such trafficking pathways and involved in PSIs sorting processes We expressed, purified and isolated from bacterial cultures both PSIs and several endomembrane reporters involved in specific events of protein transport and will study their interaction through pulldown assays. Furthermore, purified PSIs were also used as bait for co-Immunoprecipitation in Tobacco and Arabidopsis extracts. Preliminary results using yeast-two hybrid assays already showed positive interactions between sorting related proteins and the PSIs, that need to be confirmed. The data retrieved from this work will set the basis of a broader objective aiming at mapping the PSIs network of interactions, that will help the characterization and define intermediate players of unconventional trafficking.

Soil contamination is particularly serious. One of the main factors contributing to this is the accelerated development of agriculture, which has led to a strong increase in the use of herbicides. Studies show that herbicides formulated on the basis of glyphosate, have a carcinogenic potential in laboratory animals, being classified by the WHO as a probable carcinogen for humans. In this context, our research works in the area of ​​decontamination of agricultural soils, using native microorganisms, where the pollutants are transformed into by-products that are less harmful to the environment. A sampling of a wine farm in Douro was collected, followed by isolation of bacterial by means of selective cultures and their respective pure culture. Subsequently, bacterial tests were performed in vitro, with normal and in duplicate concentrations of glyphosate. It the end was isolated 379 bacteria. The results were as expected, with greater bacterial growth occurring in low soil dilutions of around 26%, as well as in recommended concentrations. On the other hand, growth at high dilutions and concentrations was found to be lower with a percentage of 12%. After bacterial tests on microplates, it was found that only 4.80% of the bacteria have the potential for glyphosate degradation. Through this study it is concluded that in soils where the use of herbicides is recurrent, there are promising bacteria for their degradation.

This work is part of the development and enhancement of natural substances and the search for effective, non-polluting and beneficial control methods for human health and for the environment against insect pests of stored cereals. For this purpose, the essential oil extracted by hydro-distillation using Clevenger of aromatic plants Mentha spicata L. and Salvia officinalis L. of the Lamiaceae family, have been tested for a possible effect bio-insecticides, by the method of direct contact against two different species of insect pests of stored cereals belonging to the order Coleoptera: wheat weevil Sytophilus granarius Linnaeus, 1758 (Curculionidae) and confused flour beetle Tribolium confusum Jacquelin du Val, 1863 (Tenebrionidae). This method involves the use of three solutions of essential oils (1ml, 2ml, 10ml of essential oil). 1ml of each solution was applied uniformly to a filter paper disc placed in a petri dish. A batch of adult insect individuals was introduced into each petri dish which was immediately closed and placed in their respective oven. Evaluation of the effectiveness of the product tested: this involves counting the dead individuals daily in the first hours after launching the tests. The test results show that the aerial part of Sage and Spearmint have a toxic effect on adults of Weevil (more than 60% mortality for Spearmint and more than 90% for Sage) and Tribolium (with more than 70% mortality for spearmint and more than 90% for sage). The dose (D3) is the most effective concentration. The insecticidal activity of the essential oil of Spearmint and Sage officinale more effective with respect to both species. In conclusion, these two aromatic and medicinal plants are naturally occurring substances that act as insecticides to control the stored cereals insect pest species.

The olive tree (Olea europaea subsp. europaea L.) is a typical plant of the Mediterranean vegetation, well adapted to drought and poor soils being also tolerant to high solar irradiance. The phyllosphere microbiota associated with these plants is likely to play a role in their tolerance to such harsh environmental conditions. Here, we have characterized the endophytic and epiphytic fungal community present in leaves of olive trees, for potential application of these insights to climate change adaptation. Leave samples were collected in two rainfed olive orchards near Mirandela (NE Portugal), a Mediterranean type climate region with typical short, hot, dry, and mostly clear summers and cold to very cold, wet winters. Fungi were isolated from the surface and inner tissues of leaves. The isolates obtained were identified by ITS rRNA gene sequencing and their phylogenetic diversity was then analyzed. Strains were phylogenetically placed all across the fungal tree of life, which make evident the high diversity of fungi associated with olive tree leaves. At higher taxonomic ranks, Ascomycota was the more abundant phylum either for endophytes (82.6%) or epiphytes (95.6%), while Dothideomycetes (41.1% and 53.2%, respectively) was the most abundant class which includes the order Pleosporales (19.3% and 25.4%). A Celerioriella-like species and two unassigned species belonging to Phaeomoniellaceae and Pleosporineae were the most abundant taxa within 23 species (out of 161) found in both epiphytic and endophytic subsamples. These strains are good candidates to be studied for their resilience to climate changes in order to be applied as "tolerance inducers" in olive crops from this Mediterranean area.

Acknowledgments: This work is supported by FEDER funds through the COMPETE (Operational Programme for Competitiveness Factors) and by National funds through the FCT (Foundation for Science and Technology) within the POCI-01-0145-FEDER-031133 (MicOlives) project.

The “citrus greening disease” or huanglongbing is caused by the Gram-negative bacterium Candidatus liberibacter spp. One vector of HLB, the African citrus psyllid Trioza erytreae (del Guercio, 1918), was recorded for the first time in 2014 in mainland Spain and since then it spread throughout the Iberian Peninsula. Measures against T. erytreae include chemical treatments and quarantine although eradication programmes have been of limited success or even failed. The biological control of T. erytreae has been little developed yet. Among natural enemies, spiders may encompass a promising group of predators. The objective of this work was to assess the potential of spiders as natural enemies of T. erytreae. A Citrus limon orchard was selected in northwestern Portugal in the area colonized by T. erytreae. A total of 100 webs (50 above and 50 below 1.5m) were visually inspected throughout the orchard in August 2020. The family of the web-builder, web size, number of adults of T. erytreae captured by each web, and the temperature and humidity were registered. Temperature and humidity did not significantly differ between the two vertical strata. Web size was significantly higher in the lower strata whereas the number of captured adults of T. erytreae was significantly higher in the upper strata. The dominant family in the upper strata was Theridiidae corresponding with the most successful webs capturing adults of T. erytreae. Our results suggest that web architecture was the only factor driving the number of captured adults of T. erytreae. Thus, space web builders could be the most successful natural enemies of T. erytreae among aerial web builders.

The authors are grateful to the Foundation for Science and Technology (FCT, Portugal), for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020) and to the project PRE-HLB—Preventing HLB epidemics for ensuring citrus survival in Europe (H2020-SFS-2018-2 Topic SFS-05-2018-2019-2020, proj. No. 817526).

Olive anthracnose, caused by Colletotrichum spp., is one of the most damaging fruit diseases in olive crops worldwide. Their control is very difficult and relies mostly on the use of copper-based fungicides. The plant-associate fungal community has been increasingly recognized for playing an important role in plant health. Thus, in this work, the olive associated fungi of cultivars with different levels of resistance to anthracnose were characterized to identify potential protecting fungi for a sustainable strategy of biocontrol. A culture-dependent approach was used to assess both epiphytic and endophytic fungal communities of leaves of olive cultivars Madural (susceptible to anthracnose) and Cobrançosa (moderately tolerant), and the isolates obtained were identified by rRNA sequencing.

Overall, Ascomycota phylum and Aureobasidium genus were the most dominant, being epiphytes significantly more diverse and abundant than endophytes. The host plant (at cultivar level) had a negligible effect on the composition of fungal communities of the leaves. However, the host plant effect was found to be greater on epiphytic than on endophytic community structure. Among the genera identified in the most-resistant cultivar, Aspergillus and Pseudocercospora were the most frequently isolated within epiphytic and endophytic fungal communities, respectively; whereas in the susceptible cultivar, Aureobasidium and Didymocyrtis, were the most frequently isolated within epiphytic and endophytic fungal communities, respectively. Determination of indicator value indices has shown significant preference/specificity of a set of fungal species with the most-resistant cultivar. Future research needs to decipher these complex interactions between plant-fungi and assess their role in plant health.

Acknowledgments: This work is supported by FEDER funds through the COMPETE (Operational Programme for Competitiveness Factors) and by National Funds through the FCT (Foundation for Science and Technology) within the POCI-01-0145-FEDER-031133 (MicOlives) project.

Arginine (Arg) being one of the most important amino acids of plants involved in biosynthesis and induction of vital amino acid derivatives, enzymes, phytohormones and signaling molecules. The effect of exogenous Arg (3.0 mM) was investigated in salt (120 and 150 mM NaCl) treated lentil (Lens culinaris cv. BARI Mashur-2). Seven-day-old lentil seedlings were subjected to salt stress alone and in combination with Arg for 48 h. Salt stress highly raised the hydrogen peroxide (H₂O₂) content and increased membrane lipid peroxidation as indicated by increased malondialdehyde (MDA). Reduced ascorbate (AsA), increased glutathione disulfide (GSSG) content, decreased glutathione (GSH):GSSG together with altered activities of antioxidant enzymes caused the oxidative stress in salt affected plants. Salt stress decreased leaf relative water content (RWC) but increased proline (Pro) content. Chlorophyll (chl) a, b and total chl contents decreased under salt stress. Arginine co-treatment with salt stress decreased the oxidative stress through improving the AsA and GSH pool and activities of catalase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase. Decreased Pro level, improved RWC, chl content, growth parameters like plant height, root length and seedling dry weight in Arg supplemented salt affected plants are the indication of Arg-induced relaxation of salt stress and improved salt tolerance in lentil plants.

Olive fruit fly, Bactrocera oleae (Rossi) (Diptera: Tephritidae) is the most important olive pest and with a major economic importance in the olive production worldwide. Different olive cultivars exhibit different propensities to fruit fly infestation and the causes are still unclear. Here, we want to disclose the potential role of the olive-associated endophytes in conferring such susceptibility differences. Accordingly, the endophytic microbial composition of infested and non-infested fruits from cultivars Madural (susceptible to olive fly) and Cobrançosa (less susceptible) were studied. A culture-dependent approach was used, being the isolates identified by sequencing of their internal transcribed spacer (for fungi) and 16S rRNA gene regions (for bacteria). Overall, there was a larger consortium of bacteria associated to olives than fungi. The bacterial communities were predominantly composed of Proteobacteria and Actinobacteria phyla while the fungal isolates belong to the Ascomycota and Basidiomycota. Both host cultivar and level of fly infestation had a negligible effect on fungal and bacterial community composition. Despite this, it was found a clear positive association of microbial consortia with the resistant cultivar (Kocuria sp., Actinobacterium sp., Rhodococcus sp., Pseudomonas citronellolis, Aspergillus flavus, Cladosporium sp., and Meristemomycetes arctostaphylos) and non-infested fruits(Kocuria sp., Stereum sp., and Vishniacozyma victoria). Their function roles on host cultivar susceptibility/resistance to fruit fly is a topic that requires further studies.

Acknowledgements: This work is supported by FEDER funds through the COMPETE (Operational Programme for Competitiveness Factors) and by National funds through the FCT (Foundation for Science and Technology) within the POCI-01-0145-FEDER-031133 (MicOlives) project.

This work is based on the study of the above- and below-ground tissues collected throughout a year, of the autumn-flowering geophyte Cyclamen graecum Link (Primulaceae). The geophytes are life forms that include underground storage organ reserves that support the initiation of leaf growth and flowering [1, 2, 3]; their buds, resposible for their annual growth, remain protected below the soil surface on an underground perennial organ and their growing and flowering period alternates with a period of dormancy. The purpose of this study is to estimate the seasonal fluctuation of sugars, starch and proline that contribute to the adaptation of C. graecum to the Mediterranean ecosystem. Α considerable amount of substances are allocated to leaves via transfer from root resources, photosynthates and senescing inflorescences. The total sugar content was determined using a modified phenol-sulphuric acid method. Quantitative determination of starch content was accomplished in sugar-free aliquots of dried tissues, which were solubilized in perchloric acid and the starch content was estimated using a modified anthrone method. Proline content was determined with acid-ninhydrin method. The above ground tissues of C. graecum that are exposed to ambient environmental conditions may exhibit seasonal responses to abiotic stresses. Ιt seems likely that monthly fluctuations of proline accumulation, sugars and starch content obtained in the above- and below-ground tissues of C. graecum coincide with the distinct seasonality of the ecosystem. The highest sugar content, among the considered plant parts, was detected in the tubers, whereas the lowest in peduncles and petals in late autumn. Relatively elevated sugar content was detected in the leaves from December to April, probably associated with constraints of the vegetative growth of this species. Throughout the flowering period of C. graecum (September-December), similar trends of sugar content were detected between petals and peduncles. Starch is stored in tubers and leaves of C. graecum and the elevated values from March to April coincide with elevated photosynthetic rates. The accumulation of sugars and proline in petals from September to December may result to a reduction of their osmotic potential, which is expected to maintain their turgor and avoid the harmful effect of the cold period. Additionally, the pronounced proline accumulation in expanded leaves of C. graecum from November to April indicates the protective role of proline in tissues exposed to ambient environmental conditions

References

1. Dafni A., Cohen D., & Noy-Mier I. (1981). Life-cycle variation in geophytes. Annals of the Missouri Botanical Garden, 68(4), 652-660.
2. Negbi M. (1989). Theophrastus on geophytes. Botanical Journal of the Linnean Society 100(1), 15-43.
3. Akita Y., et al. (2010). Comparative analysis of floral pigmentation between wild-type and white-flowered varieties of Cyclamen graecum. The Journal of Horticultural Science and Biotechnology 85(5), 437-443.