Biotechnological potential of Bacillus sp. S26 for alleviation of abiotic and biotic stresses in vine.

Grapevine (Vitis spp.) is one of the most cultivated fruit plants in the world. Vineyard growers apply copper-based products in these crops to prevent fungal diseases, generating worries about Cu contamination in soils and food. In this context, this study identifies prokaryotic communities associated with grapevine plants grown under different levels of Cu-contaminated soils. Moreover, the study isolates new bacteria to improve Cu resistance in plants. Soil Cu content correlated inversely with operational taxonomic units (OTUs) belonging to the groups Acidobacteria (SubGroup 2), Latescibacteria, Pedosphaeraceae, and Candidatus Udaeobacter. A total of 14 new bacterial isolates were obtained from copper-contaminated soils. These isolates produced Indolic Compounds (IC) in a range of 25 to 96 µg mL- 1, highlighting bacterial strains S20 and S26 as the highest producers. These new bacteria also produced siderophores, highlighting strains S19 and S26, which removed 58 and 59% of Fe ions from the CAS complex, respectively. From the in vitro antagonistic activity against Colletotrichum spp. strains, the authors identified some bacterial strains that inhibited phytopathogen growth. Bacterial strain Bacillus sp. S26 was chosen for inoculation experiments in grapevine plants. This bacterial isolate improved the growth of grapevine plants in Cu-contaminated soils. However, growth promotion did not occur in unstressed plants. More studies are necessary for developing a new bioinoculant containing S26 cells aiming to reduce biotic and abiotic stresses in grapevine.

Bacillus subtilis strain F62 against Fusarium oxysporum and promoting plant growth in the grapevine rootstock SO4.

Fusarium wilt is a fungal disease that causes economic losses to viticulture, whose causal agent Fusarium sp. has been associated with the decline and death of young vines. This work had the objective of evaluating the antagonistic potential of Bacillus subtilis F62 against F. oxysporum in vitro and in vivo, as well as the growth promotion in the grapevine rootstock SO4. In the in vitro assay, the antagonism by diffusible and volatile compounds of B. subtilis F62 and the inhibition of conidial germination of four Fusarium sp. isolates were evaluated. In the in vivo assay, cuttings and micropropagated plants of SO4 were submitted to four treatments: control, Bac (B. subtilis F62 inoculation), Fus (F. oxysporum inoculation) and Bac + Fus. We observed that inhibition of mycelial growth occurred mainly by diffusible compounds. B. subtilis F62 had a positive effect on the growth promotion and in the biocontrol of F. oxysporum, reducing the frequency of pathogen re-isolation in cuttings (18.1%) and in micropropagated plants (52.4%). These results demonstrate the ability of B. subtilis F62 to upgrade plant development and assist in controlling of the Fusarium wilt in the grapevine rootstock SO4.

Antifungal activity of essential oil from Eucalyptus staigeriana against Alternaria alternata causing of leaf spot and black rot in table grapes.

Alternaria alternata causes leaf spot and black rot diseases in leaves and grapes of grapevines, respectively, and leads to huge economic losses in table grapes production. As natural antifungal agents, essential oils (EOs), which are generally recognized as safe substances, shows strong antifungal activity against fungal phytopathogens. The aim of this study was to determine the chemical composition of Eucalyptus staigeriana EO and its in vitro and in vivo effects against A. alternata. The major compounds of E. staigeriana EO were citral (34.32%, of which 21.83% geranial and 12.49% neral), limonene (20.60%) and 1,8-cineole (12.33%). E. staigeriana EO exhibited the highest inhibitory activity on mycelial growth and conidial germination at 1 µL mL-1. Moreover, the EO was able to reduce the incidence and severity of leaf spot disease in leaves and black rot disease in table grapes caused by A. alternata. These results represent a possible alternative to reduce the use of synthetic molecules for the control of diseases in postharvest of table grapes and in vineyard.

Oryza sativa cv. Nipponbare and Oryza barthii as Unexpected Tolerance and Susceptibility Sources Against Schizotetranychus oryzae (Acari: Tetranychidae) Mite Infestation.

Cultivated rice (Oryza sativa L.) is frequently exposed to multiple stresses, including Schizotetranychus oryzae mite infestation. Rice domestication has narrowed the genetic diversity of the species, leading to a wide susceptibility. This work aimed to analyze the response of two African rice species (Oryza barthii and Oryza glaberrima), weedy rice (O. sativa f. spontanea), and O. sativa cv. Nipponbare to S. oryzae infestation. Surprisingly, leaf damage, histochemistry, and chlorophyll concentration/fluorescence indicated that the African species present a higher level of leaf damage, increased accumulation of H2O2, and lower photosynthetic capacity when compared to O. sativa plants under infested conditions. Infestation decreased tiller number, except in Nipponbare, and caused the death of O. barthii and O. glaberrima plants during the reproductive stage. While infestation did not affect the weight of 1,000 grains in both O. sativa, the number of panicles per plant was affected only in O. sativa f. spontanea, and the percentage of full seeds per panicle and seed length were increased only in Nipponbare. Using proteomic analysis, we identified 195 differentially abundant proteins when comparing susceptible (O. barthii) and tolerant (Nipponbare) plants under control and infested conditions. O. barthii presents a less abundant antioxidant arsenal and is unable to modulate proteins involved in general metabolism and energy production under infested condition. Nipponbare presents high abundance of detoxification-related proteins, general metabolic processes, and energy production, suggesting that the primary metabolism is maintained more active compared to O. barthii under infested condition. Also, under infested conditions, Nipponbare presents higher levels of proline and a greater abundance of defense-related proteins, such as osmotin, ricin B-like lectin, and protease inhibitors (PIs). These differentially abundant proteins can be used as biotechnological tools in breeding programs aiming at increased tolerance to mite infestation.

Essential oil as sustainable alternative for diseases management of grapes in postharvest and in vineyard and its influence on wine.

This study aimed to determine the chemical composition of Eucalyptus staigeriana essential oil (EO) and its effect in vivo against Botrytis cinerea and Colletotrichum acutatum in postharvest of grapes and in a vineyard. Moreover, grapes collected from de field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. The major compounds of E. staigeriana EO were citral (30.91%), 1,8-cineole (24.59%), and limonene (19.47%). In the postharvest experiment, EO was efficient, reducing the incidence and severity of disease caused by B. cinerea and the incidence of disease caused by C. acutatum, both in preventive and curative treatment. Moreover, this EO reduced the incidence and severity of gray rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum in the field. The alternative control did not significantly influence the alcoholic fermentation and volatile composition of wines, except for the residual presence of 1,8-cineole that can contribute to the aroma complexity of 'Isabella' wine. These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative to control fungal rots on grapes.

Alternative control of grape rots by essential oils of two Eucalyptus species.

BACKGROUND: Essential oils (EOs) are volatile natural compounds produced by plant secondary metabolism, and some of them exhibit antimicrobial activity. The objective of the present study was to determine the chemical composition the EOs of Eucalyptus staigeriana and Eucalyptus globulus, and their effect in vitro and in vivo against Botrytis cinerea and Colletotrichum acutatum, the most important fungal rot diseases of grapes. Moreover, grapes collected from field experiments were used to evaluate the impact of the alternative control on the alcoholic fermentation and wine composition. RESULTS: The major compound of E. staigeriana EO were citral 30.91% (19.74% geranial, 11.17% neral), 1.8-cineole (24.59%) and limonene (19.47%), while 1.8-cineole represented 68.26% of E. globulus EO. The two EOs showed in vitro antifungal activity against both pathogens. Eucalyptus staigeriana EO exhibited the highest activity inhibiting mycelial growth (MG) and conidial germination at 0.5 µL mL-1 . Moreover, this EO was able to reduce the incidence and severity of grey rot caused by B. cinerea and the severity of ripe rot caused by C. acutatum The alternative control did not significantly influence alcoholic fermentation, the physicochemical characteristics, and the volatile composition of wines. CONCLUSION: These results are promising and indicate that E. staigeriana EO might be further investigated as a natural alternative for the control of fungal rots on wine grapes. © 2019 Society of Chemical Industry.

Inhibition of Alternaria stem canker on tomato by essential oils from Baccharis species.

The toxicity of four essential oils extracted from Baccharis articulata, Baccharis ochracea, Baccharis psiadioides and Baccharis trimera was tested against the phytopathogen Alternaria alternata, which causes Alternaria stem canker on tomatoes. Diseases caused by Alternaria fungi are responsible for great economic losses in terms of production and are controlled by synthetic fungicides; however, essential oils offer an alternative, since they have been proven to be effective for controlling against various plant pathogens. In this way, the antifungal activity of Baccharis essential oils was tested using potato dextrose agar medium with concentrations ranging from 0.1 to 20.0 µL mL-1. Baccharis trimera and Baccharis ochracea essential oils presented 100% mycelial growth inhibition of A. alternata and were also able to control Alternaria stem canker disease under greenhouse conditions. Tomato plants treated with these essential oils exhibited area under the disease progress curve (AUDPC) values of 230.10 and 241.42, differing from the control condition, which showed an AUDPC value of 268.92. The essential oils of B. trimera and B. ochracea can be an alternative for controlling Alternaria stem canker disease of tomatoes and should be formulated as a potential fungicide against the A. alternata pathogen.

Antimicrobial and antibiofilm activity of the essential oil from dried leaves of Eucalyptus staigeriana / Atividade antimicrobiana e antibiofilme do óleo essencial de folhas secas de Eucalyptus staigeriana

In recent years, compounds with biological properties produced by plants have received attention as an alternative to control microorganisms. Essential oils extracted from green leaves of Eucalyptus sp. have been demonstrated to have antimicrobial activities, but so far there are no reports of antimicrobial activity of essential oils extracted from dried leaves of Eucalyptus staigeriana. So, the objectives of this study were to determine the chemical composition of the essential oils obtained from dried leaves of E. staigeriana (EOdlES) and to evaluate in vitro antimicrobial and antibiofilm activities of EOdlES against gram-positive and gram-negative, resistance and multiresistant Enterococcus faecalis isolated from food and clinical samples. The characterization of EOdlES was performed by gas chromatography-mass spectrometry (GC/MS). For this study, 26 bacterial strains were used, which included 11 reference strains and 15 antibiotic resistant and multiresistant E. faecalis strains. Antimicrobial activities of EOdlES against gram-positive and gram-negative were determined using the disc diffusion method. The minimum inhibitory concentration (MIC) value was evaluated by a microbroth dilution technique. The antibiofilm effects were assessed by microtiter plate method. As a result, 21 compounds were identified, being oxygenated monoterpenes (69.58%) the major chemical family. EOdlES showed only antimicrobial activity against gram-positive strains. E. faecalis resistant and multiresistant strains show the lowest MIC (3.12 to 6.25%), when compared with reference E. faecalis strain. EOdlES has the ability to inhibit the biofilm formation, but little or none ability to inhibit the preformed biofilm. This study demonstrates that EOdlES is a promising alternative to control important foodborne and clinic gram-positive resistant bacteria.(AU)

Nos últimos anos, compostos com propriedades biológicas produzidas por plantas têm recebido atenção como alternativa de controle de micro-organismos. Óleos essenciais extraídos de folhas verdes de Eucalyptus sp. têm demonstrado atividades antimicrobianas. No entanto, até o momento não há nenhum relato de atividade antimicrobiana de óleos essenciais extraídos de folhas secas de Eucalyptus staigeriana. O objetivo deste estudo foi determinar a composição química dos óleos essenciais obtidos de folhas secas de E. staigeriana e avaliar in vitro a sua atividade antimicrobiana e de antibiofilme contra gram-positivas e gram-negativas e também resistentes e multirresistentes de Enterococcus faecalis isolados de amostras de alimentos e clínicas. A caracterização de E. staigeriana foi realizada por CG-EM. Para este estudo foram utilizadas 26 cepas bacterianas, que incluíram 11 cepas referência e 15 cepas de E. faecalis resistentes a antibióticos. A atividade antimicrobiana de E. staigeriana contra gram-positivas e gram-negativas foi determinada utilizando o método de disco-difusão. Os valores da concentração inibitória mínima foram avaliados pela técnica de microdiluição. Os efeitos de antibiofilme foram avaliados pelo método de placa de microtitulação. Como resultado, 21 compostos foram identificados, sendo monoterpenos oxigenados (69,58%) a grande família química. E. staigeriana mostrou apenas atividade antimicrobiana contra cepas gram-positivas. Cepas de E. faecalis resistentes e multirresistentes mostraram a menor concentração inibitória mínima (3,12 para 6,25%) quando comparado com a cepa referência de E. faecalis. E. staigeriana apresentou a capacidade de inibir a formação de biofilme, mas pouca ou nenhuma capacidade de inibir o biofilme pré-formado. Este estudo demonstra que o óleo essencial obtido de folhas secas de E. staigeriana é uma alternativa promissora para controle importante de bactérias gram-positivas resistentes de origem alimentar e clínicas.(AU)

Unraveling Rice Tolerance Mechanisms Against Schizotetranychus oryzae Mite Infestation.

Rice is the staple food for over half of the world's population. Infestation of Schizotetranychus oryzae (Acari: Tetranychidae) causes great losses in rice productivity. To search for rice genotypes that could better tolerate S. oryzae infestation, we evaluated morphological and production parameters in Brazilian cultivars, and identified two cultivars with contrasting responses. Leaf damage during infestation was similar for all cultivars. However, infestation in Puitá INTA-CL resulted in reduction in the number of seeds per plant, percentage of full seeds, weight of 1,000 seeds, and seed length, whereas infestation in IRGA 423 increased weight of 1,000 seeds and seed length. Reduction in seed weight per plant caused by infestation was clearly higher in Puitá INTA-CL (62%) compared to IRGA 423 (no reduction detected), thus Puitá INTA-CL was established as susceptible, and IRGA 423 as tolerant to S. oryzae infestation. Photosynthetic parameters were less affected by infestation in IRGA 423 than in Puitá INTA-CL, evidencing higher efficiency of energy absorption and use. S. oryzae infestation also caused accumulation of H2O2, decreased cell membrane integrity (indicative of cell death), and accelerated senescence in leaves of Puitá INTA-CL, while leaves of IRGA 423 presented higher levels of total phenolics compounds. We performed proteomics analysis of Puitá INTA-CL and IRGA 423 leaves after 7 days of infestation, and identified 60 differentially abundant proteins (28 more abundant in leaves of Puitá INTA-CL and 32 in IRGA 423). Proteins related to plant defense, such as jasmonate synthesis, and related to other mechanisms of tolerance such as oxidative stress, photosynthesis, and DNA structure maintenance, together with energy production and general metabolic processes, were more abundant in IRGA 423. We also detected higher levels of silicon (as amorphous silica cells) in leaves of infested IRGA 423 plants compared to Puitá INTA-CL, an element previously linked to plant defense, indicating that it could be involved in tolerance mechanisms. Taken together, our data show that IRGA 423 presents tolerance to S. oryzae infestation, and that multiple mechanisms might be employed by this cultivar. These findings could be used in biotechnological approaches aiming to increase rice tolerance to mite infestation.

High infestation levels of Schizotetranychus oryzae severely affects rice metabolism.

High levels of Schizotetranychus oryzae phytophagous mite infestation on rice leaves can severely affect productivity. Physiological characterization showed that S. oryzae promotes a decrease in chlorophyll concentration and the establishment of a senescence process in rice leaves. Late-infested leaves also present high levels of superoxide radical and hydrogen peroxide accumulation, along with high levels of membrane integrity loss, which is indicative of cell death. To better understand the rice molecular responses to high levels of mite infestation, we employed the Multidimensional Protein Identification Technology (MudPIT) approach to identify differentially expressed proteins. We identified 83 and 88 proteins uniquely present in control and late-infested leaves, respectively, along with 11 and one proteins more abundant in control and late-infested leaves, respectively. S. oryzae infestation induces a decreased abundance of proteins related to translation, protease inhibition, and photosynthesis. On the other hand, infestation caused increased abundance of proteins involved in protein modification and degradation. Our results also suggest that S. oryzae infestation interferes with intracellular transport, DNA structure maintenance, and amino acid and lipid metabolism in rice leaves. Proteomic data were positively correlated with enzymatic assays and RT-qPCR analysis. Our findings describe the protein expression patterns of late-infested rice leaves and suggest several targets which could be tested in future biotechnological approaches aiming to avoid the population increase of phytophagous mite in rice plants.

Chemical Characterization and Cytotoxic Activity of Blueberry Extracts (cv. Misty) Cultivated in Brazil.

Vaccinium corymbosum (L.) varieties cultivation is relatively recent in Brazil, but its production has been intensified given its good adaptability to the Southern Brazil climate. Blueberries are a rich source of phenolic compounds and contain significant levels of anthocyanins, flavonols, chlorogenic acids, and procyanidins, which lead to different biological activities. Chemical identification of skin and whole hydroalcoholic blueberry extracts (ExtSB and ExtWB) revealed the presence of anthocyanins concentrated in the skin and others chemicals compounds as quercetin glycosides, proanthocyanins dimers, citric, and chlorogenic acid in the pulp. Selectivity for tumor cell lines (Hep-2, HeLa, HT-29) using ExtSB and ExtWB extracts was observed through MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay after 24 h of treatment when compared to nontumor cells (MRC-5). Morphological changes and late stages of apoptotic and necrosis process were seen in HT-29 cell line after ExtWB treatment, compared to nontumor cell line MRC-5. These results are in agreement with other studies that indicate the activity of compounds such as anthocyanins and other molecules found in Southern Highbush blueberry variety, attributed to promote beneficial effects on health that may respond as cytotoxic natural agent and contribute to cancer treatment.

In vitro antifungal activity of four chemotypes of Lippia alba (Verbenaceae) essential oils against Alternaria solani (Pleosporeaceae) isolates.

Several volatile natural compounds produced by plant secondary metabolism have been proven to present antimicrobial action, enabling their use in phytopathogen control. They also present low environmental impact when compared to conventional pesticides. Essential oils contain these compounds and can be found in several plant species, such as Lippia alba (Mill.) N.E. Brown (Verbenaceae). Essential oils of four chemotypes of L. alba, characterized by their major compounds, namely camphor, citral, linalool and camphor/1,8-cineole, were tested against the phytopathogen Alternaria solani Sorauer (Pleosporaceae), which causes early blight on tomatoes and is responsible for great economic losses regarding production. Essential oils antifungal action was tested in vitro using potato dextrose agar medium with essential oil concentrations at 0.1, 0.5, 1.0, 1.5 and 2.0 µL mL-1. The chemotype that had the best performance was citral, showing significant inhibition compared to the others, starting at the 0.5 µL mL-1 concentration. The essential oil belonging to the linalool chemotype was efficient starting at the 1.5 µL mL-1 concentration. Conversely, the camphor chemotype did not show any action against the phytopathogen. Moreover, the essential oils had no remarkable effect on tomato germination and growth. In conclusion, these essential oils presented fungicidal action against A. solani.

Identification of new adventitious rooting mutants amongst suppressors of the Arabidopsis thaliana superroot2 mutation.

The plant hormone auxin plays a central role in adventitious rooting and is routinely used with many economically important, vegetatively propagated plant species to promote adventitious root initiation and development on cuttings. Nevertheless the molecular mechanisms through which it acts are only starting to emerge. The Arabidopsis superroot2-1 (sur2-1) mutant overproduces auxin and, as a consequence, develops excessive adventitious roots in the hypocotyl. In order to increase the knowledge of adventitious rooting and of auxin signalling pathways and crosstalk, this study performed a screen for suppressors of superroot2-1 phenotype. These suppressors provide a new resource for discovery of genetic players involved in auxin signalling pathways or at the crosstalk of auxin and other hormones or environmental signals. This study reports the identification and characterization of 26 sur2-1 suppressor mutants, several of which were identified as mutations in candidate genes involved in either auxin biosynthesis or signalling. In addition to confirming the role of auxin as a central regulator of adventitious rooting, superroot2 suppressors indicated possible crosstalk with ethylene signalling in this process.

When stress and development go hand in hand: main hormonal controls of adventitious rooting in cuttings.

Adventitious rooting (AR) is a multifactorial response leading to new roots at the base of stem cuttings, and the establishment of a complete and autonomous plant. AR has two main phases: (a) induction, with a requirement for higher auxin concentration; (b) formation, inhibited by high auxin and in which anatomical changes take place. The first stages of this process in severed organs necessarily include wounding and water stress responses which may trigger hormonal changes that contribute to reprogram target cells that are competent to respond to rooting stimuli. At severance, the roles of jasmonate and abscisic acid are critical for wound response and perhaps sink strength establishment, although their negative roles on the cell cycle may inhibit root induction. Strigolactones may also inhibit AR. A reduced concentration of cytokinins in cuttings results from the separation of the root system, whose tips are a relevant source of these root induction inhibitors. The combined increased accumulation of basipetally transported auxins from the shoot apex at the cutting base is often sufficient for AR in easy-to-root species. The role of peroxidases and phenolic compounds in auxin catabolism may be critical at these early stages right after wounding. The events leading to AR strongly depend on mother plant nutritional status, both in terms of minerals and carbohydrates, as well as on sink establishment at cutting bases. Auxins play a central role in AR. Auxin transporters control auxin canalization to target cells. There, auxins act primarily through selective proteolysis and cell wall loosening, via their receptor proteins TIR1 (transport inhibitor response 1) and ABP1 (Auxin-Binding Protein 1). A complex microRNA circuitry is involved in the control of auxin response factors essential for gene expression in AR. After root establishment, new hormonal controls take place, with auxins being required at lower concentrations for root meristem maintenance and cytokinins needed for root tissue differentiation.

Genus Mikania: chemical composition and phytotherapeutical activity

The genus Mikania ranks high in the list of best-selling natural products in the world. Its main distribution is in South America, but some species are found in Asia, North America and Africa. It is used for treating fever, rheumatism, colds and respiratory diseases, as well as snake bites and scorpion stings, due to its broad spectrum of action. There are approximately 430 species of this genus and only 12% have been studied, highlighting their chemical and pharmacological diversity. The main chemical groups are: coumarins and derivatives, sesquiterpenes, sesquiterpenes lactones, diterpenes, phytosterols/terpenoids and flavonoids. This review aims to supply useful references for scientists interested in natural products and the search for new compounds, from over the 300 already described for the genus.

Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of AUXIN RESPONSE FACTOR transcripts and microRNA abundance.

The development of shoot-borne roots, or adventitious roots, is indispensable for mass propagation of elite genotypes. It is a complex genetic trait with a high phenotypic plasticity due to multiple endogenous and environmental regulatory factors. We demonstrate here that a subtle balance of activator and repressor AUXIN RESPONSE FACTOR (ARF) transcripts controls adventitious root initiation. Moreover, microRNA activity appears to be required for fine-tuning of this process. Thus, ARF17, a target of miR160, is a negative regulator, and ARF6 and ARF8, targets of miR167, are positive regulators of adventitious rooting. The three ARFs display overlapping expression domains, interact genetically, and regulate each other's expression at both transcriptional and posttranscriptional levels by modulating miR160 and miR167 availability. This complex regulatory network includes an unexpected feedback regulation of microRNA homeostasis by direct and nondirect target transcription factors. These results provide evidence of microRNA control of phenotypic variability and are a significant step forward in understanding the molecular mechanisms regulating adventitious rooting.

Mineral nutrition and adventitious rooting in microcuttings of Eucalyptus globulus.

We characterized the adventitious rooting response of Eucalyptus globulus Labill. to various concentrations of calcium, nitrogen, phosphorus, iron, manganese, zinc, boron and copper. The parameters analyzed were percent rooting, root number, root length and mean rooting time. Root number and root length were significantly affected by mineral nutrition, whereas mean rooting time and rooting percentage seemed to be closely related to auxin availability. Root number was affected by calcium, nitrogen source and zinc, whereas root length was influenced by concentrations of phosphorus, iron and manganese, and by nitrogen source. Based on these results, we evaluated various combinations of several concentrations of these minerals in each rooting phase. Cuttings that were rooted in an optimized mineral nutrient medium and acclimatized to ex-vitro conditions for two months showed significantly higher survival after transplanting and drought stress than cuttings rooted in basal medium and treated in the same way.