Defense responses in plants of Eucalyptus elicited by Streptomyces and challenged with Botrytis cinerea.

MAIN CONCLUSION: Elicitation of E. grandis plants with Streptomyces PM9 reduced the gray-mold disease, through increasing the levels of enzymes directly related to the induction of plant defense responses, and accumulation of specific phenolic compounds. Members of Eucalyptus are economically important woody species, especially as a raw material in many industrial sectors. Species of this genus are susceptible to pathogens such as Botrytis cinerea (gray mold). Biological control of plant diseases using rhizobacteria is one alternative to reduce the use of pesticides and pathogen attack. This study evaluated the metabolic and phenotypic responses of Eucalyptus grandis and E. globulus plants treated with Streptomyces sp. PM9 and challenged with the pathogenic fungus B. cinerea. Metabolic responses were evaluated by assessing the activities of the enzymes polyphenol oxidase and peroxidase as well as the levels of phenolic compounds and flavonoids. The incidence and progression of the fungal disease in PM9-treated plants and challenged with B. cinerea were evaluated. Treatment with Streptomyces sp. PM9 and challenge with B. cinerea led to changes in the activities of polyphenol oxidase and peroxidase as well as in the levels of phenolic compounds in the plants at different time points. Alterations in enzymes of PM9-treated plants were related to early defense responses in E. grandis. Gallic and chlorogenic acids were on average more abundant, although caffeic acid, benzoic acid and catechin were induced at specific time points during the culture period. Treatment with Streptomyces sp. PM9 significantly delayed the establishment of gray mold in E. grandis plants. These results demonstrate the action of Streptomyces sp. PM9 in inducing plant responses against B. cinerea, making this organism a potential candidate for biological control in Eucalyptus.

Baccharis anomala DC. extract reduces inflammation and attenuates hepatic fibrosis in vivo by decreasing NF-kB and extracellular matrix compounds.

Baccharis anomala DC. (BA) is a plant species found in the tropical regions of South America and is widely used for its hepatoprotective effects, as well as for the treatment of gastrointestinal diseases. Studies have recently reported its antioxidant and anti-inflammatory potential. BA extract can reverse the activated phenotype of hepatic stellate cells (HSC), which plays a central role in extracellular matrix (ECM) deposition in the development of liver fibrosis. Thus, this study aimed to evaluate the effects of the treatment with BA extract on liver fibrosis in a CCl4-induced liver fibrosis model in BALB/c mice. Methanolic extract was obtained from BA leaves, a gas chromatography/mass spectrometry (GC/MS) to detect the compounds present was performed, and then administered by intraperitoneal injection in Balb/C mice at a concentration of 50 and 100 mg/kg together with the administration of CCl4 for inducing liver fibrosis. After 10 weeks, blood analysis, histopathology, oxidative stress, as well as protein and gene expression in the hepatic tissue were performed. Treatment with BA extract was able to reduce profibrotic markers by reducing the expression of α-SMA and Col-1 proteins, as well as reducing the formation of free radicals and lipid peroxidation. (BA extract showed anti-inflammatory effects in the liver by suppressing NF-kB activation and reducing gene expression of signaling targets (IL-6 and iNOS). The data obtained showed that BA extract has antifibrotic and anti-inflammatory effects.

Coumaric acid from M. polymorphum extracts reverses the activated state of hepatic stellate cells (GRX) and inhibits their proliferation by decreasing the p53/p21 pathway.

Coumaric acid is a phenolic compound found in medicinal plants. Its use has been reported in the treatment of inflammatory diseases, prevention of alterations induced by oxidative stress, as well as acetaminophen-induced hepatotoxicity. Thus, this study evaluated coumaric acid as a potential treatment for liver fibrosis. Cell proliferation was assessed by the trypan blue exclusion technique and the cytotoxicity of coumaric acid was performed using an LDH assay. Mechanisms of cell apoptosis were evaluated by flow cytometry. The expression of genes associated with apoptosis, cell cycle control, and fibrosis was assessed by qPCR. The production of lipid droplets was quantified by oil red staining. The experiments performed showed that the treatment with coumaric acid was able to reduce cell proliferation without causing cell cytotoxicity or apoptosis. Coumaric acid was able to inhibit the expression of cyclin D1 and CDK's (CDK2, CDK4, and CDK6), increasing p53 and p21, which could lead to cell cycle arrest. Treatment with coumaric acid was also able to revert the activated phenotype of GRX cells to their quiescent state. Thus, our results suggest that coumaric acid has a potential therapeutic effect against liver fibrosis.

Moquiniastrum polymorphum subsp. polymorphum extract inhibits the proliferation of an activated hepatic stellate cell line (GRX) by regulating the p27 pathway to generate cell cycle arrest.

ETHNOPHARMACOLOGICAL RELEVANCE: The chosen plant and its extracts have been an alternative in the treatment of several inflammatory and oxidant diseases, and is therefore a viable option for the treatment of hepatic fibrosis. AIM OF THE STUDY: This study aimed to use Moquiniastrum polymorphum subsp. polymorphum, mainly the ethanolic extract and fractions, in the treatment of hepatic fibrosis. MATERIALS AND METHODS: Extracts were prepared from dried leaves in 100% ethanol (ET) and fractionated with an increased polarity solvent (dichloromethane to methanol). The quantification of compounds in the extracts was characterized by GCMS. The decrease in cell proliferation and the cytotoxicity of the extracts were evaluated together with the mechanisms of apoptosis and autophagy. The expression of genes associated with decreased fibrosis and cell cycle control was assessed and the production of lipid droplets was quantified by Oil Red O staining. RESULTS: The experiments showed that treatment with ET and fraction 1 (F1) inhibited the expression of CDKIs (CCDN1, CDK2, CDK4 and CDK6) through an increase in p27, related to an increase in autophagic vesicles. The extract and F1 were able to decrease proliferation and revert the activated state of GRX cells to their quiescent state. CONCLUSION: Our results suggest that extracts obtained from Moquiniastrum polymorphum subsp. polymorphum have a potential therapeutic effect against liver fibrosis.

Streptomyces spp. enhance vegetative growth of maize plants under saline stress.

Saline stress is one of the abiotic stresses that most compromises the yield of crops and can be mitigated by plant growth-promoting rhizobacteria (PGPR). This work characterized rhizobacteria isolates from the genus Streptomyces as PGPR and evaluated their role on growth and alleviation of the effects caused by saline stress in maize (Zea mays L.). Production of indolic compounds (IC), siderophores, ACC deaminase, phenazines, and promotion of plant growth were determined to characterize bacterial isolates. Salinity tolerance was accessed by culturing the Streptomyces isolates under NaCl increasing concentrations (0-300 mM). Four Streptomyces isolates exhibiting PGPR traits and salinity tolerance were selected and their effect on tolerance of maize plants to saline stress was evaluated. Plants obtained from bacterized seeds and submitted to 100 and 300 mM NaCl were used. All Streptomyces spp. produced IC and siderophores, CLV178 being the best producer of these two compounds. ACC deaminase was detected in six of the 10 isolates (CLV95, CLV97, CLV127, CLV179, CLV193, and CLV205), while phenazines were found only in CLV186 and CLV194. All isolates were tolerant to salinity, growing at concentrations up to 300 mM NaCl, with exception of CLV188. Increased concentrations of IC were detected in most of the isolates exposed to salinity. CLV97 and CLV179 significantly promoted growth of roots and leaves of maize plants and attenuated the negative effects of salinity on plant growth. Root colonization by Streptomyces spp. was confirmed in plants cultivated 20 days under saline stress.

Methoxyeugenol deactivates hepatic stellate cells and attenuates liver fibrosis and inflammation through a PPAR-É£ and NF-kB mechanism.

ETHNOPHARMACOLOGICAL RELEVANCE: Studies have shown interest in nutraceuticals for the prevention of liver diseases. Methoxyeugenol, is a molecule found in foods, such as nutmeg (Myristica fragrans Houtt.) and Brazilian red propolis. These two sources of methoxyeugenol, propolis and nutmeg, are used in folk medicine for the treatment of hepatic and gastrointestinal disorders, although little is known about their effects on the prevention of liver fibrosis. Natural PPAR (Peroxisome proliferator-activated receptor) agonists would represent unique molecules for therapy, considering the lack of therapeutics to treat liver fibrosis in chronic liver disease. Thus, investigation on new alternatives are necessary, including the search for natural compounds from renewable and sustainable sources. Liver fibrosis is a pathological process characterized by an exacerbated cicatricial response in the hepatic tissue, which compromises liver function. Therefore, inhibition of HSC (hepatic stellate cell) activation and hepatocyte damage are considered major strategies for the development of new anti-fibrotic treatments. AIM OF THE STUDY: This study aimed to investigate the effects of methoxyeugenol treatment on HSC phenotype modulation in human and murine cells, hepatocyte damage prevention, and protective effects in vivo, in order to evaluate its therapeutic potential for liver fibrosis prevention. METHODS: We investigated the effects of methoxyeugenol in (i) in vitro models using human and murine HSC and hepatocytes, and (ii) in vivo models of CCl4 (carbon tetrachloride) -induced liver fibrosis in mice. RESULTS: We herein report that methoxyeugenol decreases HSC activation through the activation of PPAR-É£, ultimately inducing a quiescent phenotype highlighted by an increase in lipid droplets, loss of contraction ability, and a decrease in the proliferative rate and mRNA expression of fibroblast markers. In addition, methoxyeugenol prevented hepatocytes from oxidative stress damage. Moreover, in mice submitted to chronic liver disease through CCl4 administration, methoxyeugenol decreased the inflammatory profile, liver fibrosis, mRNA expression of fibrotic genes, and the inflammatory pathway signaled by NF-kB (Nuclear factor kappa B). CONCLUSION: We propose methoxyeugenol as a novel and potential therapeutic approach to treat chronic liver disease and fibrosis.

Therapeutic effect of uridine phosphorylase 1 (UPP1) inhibitor on liver fibrosis in vitro and in vivo.

Potassium 5-cyano-4-methyl-6-oxo-1,6-dihydropyridine-2-olate (CPBMF65) is a potent inhibitor of the uridine phosphorylase 1 (UPP1) enzyme. Its non-ionized analog has already demonstrated biological properties by reducing adverse effects caused by the chemotherapeutic 5-fluorouracil (5-FU). In addition, it has been demonstrated that uridine inhibits inflammation and fibrosis in bleomycin lung injury, decreasing collagen production. The purpose of this study was to investigate the in vitro and in vivo effects of CPBMF65 on activated hepatic stellate cells (HSC) and on carbon tetrachloride-induced liver fibrosis in mice. After incubation with CPBMF65, decreased cell proliferation and phenotype reversion were observed in vitro. In addition, CPBMF65 promoted a protective effect on tetrachloride-induced liver fibrosis in mice, demonstrated by its antifibrotic and anti-inflammatory actions. The results of the present study indicate that the UPP1 inhibitor (CPBMF65) may have potential as a novel therapeutic agent for the treatment of liver fibrosis.

Streptomyces sp. CLV45 from Fabaceae rhizosphere benefits growth of soybean plants.

Plant growth-promoting bacteria such as Streptomyces are an attractive alternative for increasing the sustainability of agricultural systems. In this study, Streptomyces isolates obtained from rhizosphere soil of plants in the family Fabaceae were characterized for their plant growth-promoting traits, including the production of siderophores, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA), and phenazines. Soybean seeds were bacterized with selected isolates to test growth promotion. All isolates produced IAA, and the isolate CLV45 was the most efficient, reaching 398.53 mg of IAA per gram of cells. CLV41, CLV45, and CLV46 showed high activity for ACC deaminase whereas CLV42, CLV44, and CLV46 were efficient in siderophore production. Pyocyanin was detected in all isolates; CLV41, CLV43, and CLV45 produced phenazine-carboxylic acid as well. Selected for IAA and ACC deaminase production combined with production of siderophores and phenazines, CLV42, CLV44, and CLV45 were tested for their growth promotion potential. Seed bacterization with CLV45 resulted in plants with increased shoot growth (36.63%) and dry mass (17.97%) compared to control plants. Results suggest that moderate or high levels of auxin and ACC deaminase production by the isolate CLV45 positively affected the growth of soybean plants, making it a strong candidate for further studies on biofertilizer formulation.

Therapeutic effect of Baccharis anomala DC. extracts on activated hepatic stellate cells.

The therapeutic potential of Baccharis anomala DC. extracts was evaluated through its cytotoxic and antiproliferative effect and their phenotypic reversion property in activated hepatic stellate cells (HSCs). Baccharis anomala is distributed in Brazil (southeastern and south regions) and used for diuretic effect in folk medicine. Four fractions were obtained from the fractionation of the methanolic extract. Fractions III and IV decreased cell proliferation without increasing cell necrosis markers levels and induced cell cycle arrest in G1 phase. Fraction III induced phenotypic reversion through PPAR-γ activation pathway, while fraction IV did not alter PPAR-α/γ expression levels, suggesting that there is an independent PPAR-α/γ pathway involved. Hydroxybenzoic, chlorogenic and coumaric acids were identified. Fractions III and IV showed antiproliferative effect and ability to induce reversion of activated phenotype of HSCs.

Plant growth and resistance promoted by Streptomyces spp. in tomato.

Plant Growth Promoting Rhizobacteria (PGPR) represent an alternative to improve plant growth and yield as well as to act as agents of biocontrol. This study characterized isolates of Streptomyces spp. (Stm) as PGPR, determined the antagonism of these isolates against Pectobacterium carotovorum subsp. brasiliensis (Pcb), evaluated the ability of Stm on promoting growth and modulating the defense-related metabolism of tomato plants, and the potential of Stm isolates on reducing soft rot disease in this species. The VOC profile of Stm was also verified. Promotion of plant growth was assessed indirectly through VOC emission and by direct interaction with Stm isolates in the roots. Evaluation of soft rot disease was performed in vitro on plants treated with Stm and challenged with Pcb. Enzymes related to plant defense were then analyzed in plants treated with three selected isolates of Stm, and PM1 was chosen for further Pcb-challenging experiment. Streptomyces spp. isolates displayed characteristics of PGPR. PM3 was the isolate with efficient antagonism against Pcb by dual-culture. Most of the isolates promoted growth of root and shoot of tomato plants by VOC, and PM5 was the isolate that most promoted growth by direct interaction with Stm. Soft rot disease and mortality of plants were significantly reduced when plants were treated with StmPM1. Modulation of secondary metabolism was observed with Stm treatment, and fast response of polyphenoloxidases was detected in plants pretreated with StmPM1 and challenged with Pcb. Peroxidase was significantly activated three days after infection with Pcb in plants pretreated with StmPM1. Results suggest that Streptomyces sp. PM1 and PM5 have the potential to act as PGPR.

Streptomyces rhizobacteria modulate the secondary metabolism of Eucalyptus plants.

The genus Eucalyptus comprises economically important species, such as Eucalyptus grandis and Eucalyptus globulus, used especially as a raw material in many industrial sectors. Species of Eucalyptus are very susceptible to pathogens, mainly fungi, which leads to mortality of plant cuttings in rooting phase. One alternative to promote plant health and development is the potential use of microorganisms that act as agents for biological control, such as plant growth-promoting rhizobacteria (PGPR). Rhizobacteria Streptomyces spp have been considered as PGPR. This study aimed at selecting strains of Streptomyces with ability to promote plant growth and modulate secondary metabolism of E. grandis and E. globulus in vitro plants. The experiments assessed the development of plants (root number and length), changes in key enzymes in plant defense (polyphenol oxidase and peroxidase) and induction of secondary compounds(total phenolic and quercetinic flavonoid fraction). The isolate Streptomyces PM9 showed highest production of indol-3-acetic acid and the best potential for root induction. Treatment of Eucalyptus roots with Streptomyces PM9 caused alterations in enzymes activities during the period of co-cultivation (1-15 days), as well as in the levels of phenolic compounds and flavonoids. Shoots also showed alteration in the secondary metabolism, suggesting induced systemic response. The ability of Streptomyces sp. PM9 on promoting root growth, through production of IAA, and possible role on modulation of secondary metabolism of Eucalyptus plants characterizes this isolate as PGPR and indicates its potential use as a biological control in forestry.

Long-term maintenance of in vitro cultures affects growth and secondary metabolism of St. John's Wort / A manutenção prolongada de culturas in vitro afeta o crescimento e o metabolismo secundário de hipérico

Hypericum perforatum is a traditional medicinal plant with wound healing and antidepressant properties. Efficiency of micropropagation is often related to the long term maintenance of tissues in culture, which may alter the secondary metabolism of plants. The objective of this study was to evaluate growth and secondary metabolism of in vitro shoots of H. perforatum on short and long term maintenance of cultures (30 and 100 days). The effect of BA and NAA supplementation was evaluated during 30 days of culture. Adventitious shoots were cultivated on MS medium supplemented with 4.4mM BA alone or in combination with 0.05mM NAA for 30 days. A hormone-free medium was used as control. Shoots cultivated for 100 days were maintained in presence of 4.4mM BA. Biomass, multiplication of shoots, contents of phenolic compounds, flavonoids and hypericin were evaluated. No difference between BA and BA+NAA was observed on growth, multiplication of shoots and levels of flavonoids at the end of 30 days of culture. Production of phenolic compounds was promoted by addition of BA+NAA to the medium, whereas hypericin was increased by the presence of BA. The time of culture (30 and 100 days) affected all the parameters analyzed, except the levels of flavonoids in the short term experiment.

Hypericum perforatum é uma planta medicinal que apresenta propriedades cicatrizante e antidepressiva. Frequentemente, a eficiência da micropropagação está relacionada à manutenção dos tecidos em cultura por longos períodos, o que pode alterar o metabolismo secundário das plantas. O objetivo deste trabalho foi avaliar o crescimento e o metabolismo secundário de brotações adventícias de H. perforatum mantidas por diferentes tempos de cultivo (30 e 100 dias). O efeito da adição de BA e ANA foi avaliado no período de 30 dias. As brotações foram cultivadas em meio MS suplementado com 4,4mM BA como único regulador ou em combinação com 0,05mM ANA, por 30 dias. Um meio de cultivo sem adição de reguladores foi utilizado como controle. As brotações cultivadas por 100 dias foram mantidas em presença de 4,4mM BA. A biomassa, a multiplicação dos brotos, as concentrações de compostos fenólicos, flavonoides e hipericina foram os parâmetros avaliados. Nenhuma diferença entre a adição de BA ou BA+ANA foi observada quanto ao crescimento, ao número de brotos e aos níveis de flavonoides ao final de 30 dias de cultivo. Diferenças neste período foram detectadas nos níveis de compostos fenólicos e de hipericina quando os brotos foram cultivados em presença de BA+NAA e de BA, respectivamente. O tempo de cultivo (30 e 100 dias) afetou todos os parâmetros avaliados, com exceção dos níveis de flavonoides no período de 30 dias.

Plant regeneration through somatic embryogenesis of yacón [smallanthus sonchifolius (poepp. and endl.) H. Robinson]

Smallanthus sonchifolius has tuberous roots containing large amounts of fructo-oligosaccharides and its medicinal use has increased due to the hypoglycemic properties reported for this species. An efficient system for propagation via somatic embryogenesis is reported using petiole segments cultivated on MS medium supplemented with combinations of BA, kinetin and 2,4-D, under light and darkness conditions. Embryogenic callus was formed in most of the treatments; however, somatic embryogenesis was promoted by the presence of light. Clusters of somatic embryos appeared on callus surface after 50 days of culture. The highest number of embryos was produced on 0.45 µM BA and 4.5 µM 2,4-D. Embryogenic calli were maintained on MS medium containing 4.5 µM BA and 0.045 µM 2,4-D. Embryos converted on hormone-free half-strength MS medium with 2 g.L-1 activated charcoal and plantlets were transferred to non-sterile conditions for acclimatization, showing 100 percent of survival.

Smallanthus sonchifolius apresenta raízes tuberosas, que contêm grandes quantidades de frutoligosacarídeos e seu uso medicinal tem aumentado devido à propriedade hipoglicemiante relatada para esta espécie. Um sistema eficiente para propagação via embriogênese somática é descrito, utilizando segmentos peciolares cultivados em meio MS suplementado com diversas combinações de BA, cinetina e 2,4-D, sob condições de luz ou escuro. A maioria dos tratamentos resultou na formação de calos embriogênicos; no entanto, a embriogênese somática foi promovida em presença de luz e agregados de embriões somáticos foram observados na superfície dos calos após 50 dias de cultivo. O maior número de embriões foi obtido em presença de 0,45 µM BA e 4,5 µM 2,4-D. Os calos embriogênicos foram mantidos em meio MS com adição de 4,5 µM BA e 0,045 µM 2,4-D. A conversão dos embriões somáticos foi obtida em meio MS, com a concentração de sais reduzida à metade, 2 g.L-1 de carvão ativado, sem reguladores de crescimento. As plantas regeneradas foram aclimatadas em condições ambientais, com sobrevivência de 100 por cento.