Rizobacterias promotoras de crecimiento en Guarianthe skinneri (Orchidaceae) / Growth-promoting rhizobacteria from Guarianthe skinneri (Orchidaceae)

Resumen La orquídea Guarianthe skinneri está incluida en la norma NOM-059-ECOL-2010 de México como una especie amenazada. Con el fin de estudiar las BPCV (bacterias promotoras del crecimiento vegetal) en esta orquídea, se recolectaron 10 raíces de diferentes plantas para aislar bacterias asociadas a las raíces, que se analizaron mediante pruebas in vitro como: producción de AIA, fijación de nitrógeno, interacción con el hongo micorrízico Thanatephorus sp. cepa RG26 y solubilización de fosfato. De los 71 aislados bacterianos se caracterizaron 10 cepas mediante secuenciación con el marcador 16s rADN y se identificaron seis cepas: Sphingomonas sp., Sinorhizobium sp., Bacillus sp., Nocardia cerradoensis, Bacillus megaterium y Burkholderia phytofirmans. Se observó que la bacteria Sinorhizobium sp. produjo mayor cantidad de AIA (69.189 µg/ml) y Bacillus sp. presentó mayor reducción de acetileno (10.251 nmol cultivo/96 h). En las interacciones de las bacterias y el hongo RG26 se presentaron cuatro categorías (sumamente positivo, positivo, antagonismo 50-50 e inhibición). En relación a la solubilización de fosfato, la bacteria Burkholderia phytofirmans presentó mayor IS a las 48 y 96 hr con IS de 3.11 y 3.48, respectivamente. Los resultados indican que Bacillus sp. pudiera tener las mejores características para promover el desarrollo de la orquídea G. skinneri mediante la inoculación de semillas y plántulas.

Abstract The Guarianthe skinneri orchid is included in NOM-059-ECOL-2010, Mexico standard as an endangered species. In order to study PGPR (promoting growth plant rhizobacteria) from this orchid, 10 roots were collected from different plants to isolate bacteria associated with the roots, which were analyzed by in vitro tests such as: production of AIA, nitrogen fixation, interaction with the mycorrhizal fungus Thanatephorus sp. strain RG26 and phosphate solubilization. We obtain 71 bacterial isolates, 10 strains of them were characterized by sequencing with the 16d rDNA marker identifying six bacteria: Sphingomonas sp. Sinorhizobium sp. Bacillus sp. Nocardia cerradoensis, Bacillus megaterium and Burkholderia phytofirmans. We observed that the bacterium Sinorhizobium sp. produced a greater amount of AIA (69.189 μg/ml) and Bacillus sp. performed greater acetylene reduction (10.251 nmol cultivo/96h). In the interactions of the bacteria and the fungus RG26, four categories were presented (extremely positive, positive, antagonism 50-50 and inhibition). In relation to the solubilization of phosphate, Burkholderia phytofirmans presented higher IS after 48 and 96 hr with an IS of 3.11 and 3.48, respectively. The results indicate that Bacillus sp. it could have the best characteristics to promote the development of the G. skinneri orchid by inoculating seeds and seedlings. Rev. Biol. Trop. 66(3): 953-968. Epub 2018 September 01.

Aislados de Bacillus provenientes de la rizósfera de cactus incrementan la germinación y la floración en Mammillaria spp. (Cactaceae) / Bacillus isolates from rhizosphere of cacti improve germination and bloom in Mammillaria spp. (Cactaceae)

Las cactáceas son la vegetación característica de las zonas áridas en México, donde las lluvias son escasas, la evapotranspiración es elevada y la fertilidad de los suelos es baja. Las plantas han desarrollado estrategias fisiológicas como la asociación con microorganismos en la zona de la rizósfera para incrementar la captación de nutrientes. En el presente trabajo se obtuvieron 4 aislados bacterianos de la rizósfera de Mammillaria magnimamma y Coryphantha radians, los que fueron nombrados como QAP3, QAP19, QAP22 y QAP24 e identificados genéticamente como pertenecientes al género Bacillus. Estos aislados exhibieron in vitro propiedades bioquímicas como solubilización de fosfatos, producción de ácido indolacético y actividad ACC deaminasa, que se relacionan con la promoción del crecimiento de las plantas. Dicha promoción fue ensayada inoculando semillas de M. magnimamma y evaluando luego algunos parámetros. Se encontró que todos los aislados incrementaron la germinación desde un 17% hasta un 34,3% (con respecto a las semillas testigo sin inocular); el aislado QAP24 fue el que presentó el mayor efecto en este sentido y permitió la germinación de todas las semillas viables (84,7%) 3 días antes que en el testigo. La inoculación de este aislado en plantas de Mammillaria zeilmanniana mostró un efecto positivo sobre la floración: en 2 meses dentro del período de un año se detectó un incremento en el número de plantas en floración con respecto a las plantas testigo, de hasta el 31,0% en uno de ellos. Se concluye que los aislados de Bacillus spp. caracterizados poseen potencial para ser empleados en programas de conservación de especies vegetales de zonas áridas.

Cacti are the most representative vegetation of arid zones in Mexico where rainfall is scarce, evapotranspiration is high and soil fertility is low. Plants have developed physiological strategies such as the association with microorganisms in the rhizosphere zone to increase nutrient uptake. In the present work, four bacterial isolates from the rhizosphere of Mammillaria magnimamma and Coryphantha radians were obtained and named as QAP3, QAP19, QAP22 and QAP24, and were genetically identified as belonging to the genus Bacillus, exhibiting in vitro biochemical properties such as phosphate solubilization, indoleacetic acid production and ACC deaminase activity related to plant growth promotion, which was tested by inoculating M. magnimamma seeds. It was found that all isolates increased germination from 17 to 34.3% with respect to the uninoculated control seeds, being QAP24 the one having the greatest effect, accomplishing the germination of viable seeds (84.7%) three days before the control seeds. Subsequently, the inoculation of Mammillari zeilmanniana plants with this isolate showed a positive effect on bloom, registering during two months from a one year period, an increase of up to 31.0% in the number of flowering plants compared to control plants. The characterized Bacillus spp. isolates have potential to be used in conservation programs of plant species from arid zones.

Caracterización de rizobacterias promotoras de crecimiento en plántulas de Eucalyptus nitens / Characterization of growth-promoting rhizobacteria in Eucalyptus nitens seedlings

Se aislaron bacterias rizosféricas y endófitas a partir de rizósfera y tejidos de raíz de árboles de Eucalyptus nitens con el objetivo de evaluar su capacidad de promover el crecimiento en plántulas de la misma especie en condiciones de invernadero. Los aislamientos que incrementaron el crecimiento de las plántulas fueron identificados y caracterizados por su capacidad de producir ácido indolacético (AIA), solubilizar fosfato y expresar la 1-aminociclopropano-1-carboxilato (ACC) desaminasa. Los 105 aislamientos obtenidos fueron morfológicamente diferentes y solo 15 promovieron significativamente el crecimiento de plántulas de E. nitens. Los máximos incrementos observados fueron en el peso seco aéreo (142 %) y de la raíz (135 %); también aumentaron la altura de las plantas (50 %) y el largo de raíces (45 %) de las mismas. Las rizobacterias pertenecieron a los géneros Arthrobacter, Lysinibacillus, Rahnella y Bacillus. Los aislados identificados como A. phenanthrenivorans 21 y B. cereus 113 incrementaron la emergencia de E. nitens a los 12 días en un valor promedio de 3,15 veces con relación al control. R. aquatilis aislado 78 presentó la mayor producción de AIA (97,5 ± 2,87 μg/ml) en presencia de triptófano y el mayor índice de solubilización de fósforo (2,4). B. amyloliquefaciens aislado 60 fue positivo para la actividad ACC desaminasa. Los resultados obtenidos indican el potencial de las rizobacterias estudiadas como promotoras de emergencia y crecimiento de plántulas de E. nitens y su posible uso como inoculantes, ya que presentan más de un mecanismo de acción asociado a la promoción del crecimiento.

Rhizospheric and endophytic bacteria were isolated from the rizosphere and root tissue of Eucalyptus nitens. The objective of this work was to evaluate their capacity to promote growth in seedlings of the same species under greenhouse conditions. The isolates that improved seedling growth were identified and characterized by their capacity to produce indoleacetic acid (IAA), solubilize phosphates and increase 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. One hundred and five morphologically different strains were isolated, 15 of which promoted E. nitens seedling growth, significantly increasing the height (50%), root length (45%) as well as the aerial and root dry weight (142% and 135% respectively) of the plants. Bacteria belonged to the genus Arthrobacter, Lysinibacillus, Rahnella and Bacillus. Isolates A. phenanthrenivorans 21 and B. cereus 113 improved 3.15 times the emergence of E. nitens after 12 days, compared to control samples. Among isolated R. aquatilis, 78 showed the highest production of IAA (97.5±2.87 μg/ml) in the presence of tryptophan and the highest solubilizer index (2.4) for phosphorus, while B. amyloliquefaciens 60 isolate was positive for ACC deaminase activity. Our results reveal the potential of the studied rhizobacteria as promoters of emergence and seedling growth of E. nitens, and their possible use as PGPR inoculants, since they have more than one mechanism associated with plant growth promotion.

Influência da inoculação de rizóbios sobre a germinação e o vigor de plântulas de alface / Influence of rhizobial inoculation on seedling vigor and germination of lettuce

Os rizóbios, conhecidos por sua capacidade de fixar N2 em associação com leguminosas, também se mostram capazes de promover o crescimento de não-leguminosas, especialmente pela produção de ácido indol-acético (AIA). Neste trabalho, objetivou-se selecionar rizóbios produtores de AIA e avaliar o efeito de diferentes concentrações deste fitormônio sobre a germinação e o desenvolvimento inicial de plântulas de alface. Foram selecionados quatro isolados de Bradyrhizobium sp. e um isolado de Rhizobium leguminosarum biovar trifolii, os quais foram crescidos por quatro dias em meio levedura-manitol enriquecido com triptofano. Após esse período, avaliou-se a produção de AIA e procedeu-se à inoculação de sementes de alface com os isolados. O isolado TV-13, de R. leguminosarum biovar trifolii produziu 171,1µg mL-1 de AIA, causando prejuízos para o desenvolvimento das plântulas de alface. Por outro lado, os isolados de Bradyrhizobium sp. produziram entre 1,2 e 3,3µg mL-1 de AIA e aumentaram o vigor das plântulas em relação ao tratamento sem inoculação com rizóbios. Para verificar se essas diferenças foram decorrentes das concentrações de AIA, foram realizados mais dois experimentos, nos quais as sementes foram embebidas em culturas de TV-13 com ou sem a presença de triptofano ou em doses crescentes de AIA sintético. O isolado TV-13 crescido na presença de triptofano causou danos progressivos sobre o desenvolvimento das plântulas de alface, o que não ocorreu na ausência de triptofano. Também foi verificado um retardo na germinação das sementes quando submetidas a altas concentrações de AIA sintético. Os resultados indicam a influência do AIA sobre os parâmetros de germinação, de modo que a inoculação de sementes de alface com rizóbios que produzem baixas quantidades de AIA é uma prática recomendável.

Rhizobia are known by their ability to fix nitrogen in symbiosis with legumes, but they are also capable of promote the growth of non-legume, mainly due to indoleacetic acid production (IAA). In this research, it was aimed to select rhizobia producers of IAA and evaluate the effect of different levels of this hormone over the germination and initial development of lettuce seedlings. Four isolates of Bradyrhizobiumsp. and one isolate of Rhizobium leguminosarum biovar trifolii were grown during four days in yeast manitol medium enriched with tryptophan. After that period, the production of IAA was evaluated and the isolates were inoculated in lettuce seeds. The isolate TV-13, of R. leguminosarum biovar trifolii, produced 171.1µg mL-1 of IAA, causing damages to lettuce seedlings. On other hand, Bradyrhizobium sp. isolates produced between 1.2 and 3.3µg mL-1 of IAA and improved seedlings vigor. In order to verify if these results were due to IAA concentrations, other two assays were carried out, in which lettuce seeds were imbibed in TV-13 cultures with or without tryptophan or in increasing dosages of synthetic IAA. The isolate TV-13 grown in the presence of tryptophan caused progressive damages to lettuce seedlings development, fact that did not occur in the absence of tryptophan. It was also verified a delay in germination of seeds exposed to high levels of synthetic IAA. The results show the influence of IAA on germination parameters, so that the inoculation of lettuce seeds with rhizobia that produce low amounts of IAA is a recommended practice.

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase / 药物分析学报

Objective To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P＜0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

Study of the mechanism on the apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase / 西安交通大学学报·英文版

Objective: To investigate the mechanisms of apoptosis induced in Human leukemia cell line K562 by the combination of indole-3-acetic acid and horseradish peroxidase. Methods: Human leukemia cell line K562 were exposed to indole-3-acetic acid (IAA) at 20, 40, 60, 80 or 100 mol/L and horseradish peroxidase(HRP) at 1.2 g/mL for varying times. MTT assay was applied to detect the cell proliferation. Flow cytometry was performed to detect the arrest of cell cycle. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to measure apoptosis. 2, 7-dichlorofluorescin diacetate (DCFH-DA) uptake was measured to determine free radical by confocal microscope. Content of malondiadehyde (MDA) and activity of superoxide dismutase (SOD) were measured by biochemical methods. Results: IAA/HRP initiated growth inhibition of K562 cells in a dose- and time-dependent manner. Flow cytometry revealed that cell cycle arrested at G1/G0 after 24 hours treatment. After 72 hours treatment, apoptotic rate of 100 mol/L IAA group increased to 43.9%, which was 5 times that of control(P<0.01). Content of MDA and activity of SOD increased respectively in treatments compared with control. Meanwhile, IAA/HRP stimulated the formation of free radical, which was increased by IAA concentration-dependently. Conclusion: The combination of IAA and HRP can inhibit the growth of Human leukemia cell line K562 in vitro by inducing apoptosis which is associated with the increase of free radical. The combination of IAA and HRP might be a promising chemopreventive and chemotherapeutic agent against human leukemia.

La plaqueta como marcador biológico periférico de la función serotoninérgica neuronal

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Abstract: Among all neurotransmitters, serotonin or 5-hydroxi-triptamine (5-HT) is probably the most studied in neuropsychopharmacology. Interest in this neurotransmitter is due to cumulative evidences showing that neuronal serotonergic systems are altered in depressed patients, as well as in several behavior dysfunctions like aggressiveness, impulsiveness, and suicide attempts, among others. Also, specific agonists and antagonists have been synthesized, which has enabled the characterization of the serotonergic receptor subtypes. Furthermore, highly selective inhibitors ofserotonin uptake have been developed, and these are capable of working in the synaptic terminals, as well as in other cell systems, such as platelets. This has allowed for the understanding and characterization of the action mechanisms of diverse psychoactive drugs interacting with the serotonergic system. Platelets have been proposed as an outlying model resembling that of serotonergic neurons due to the similarities they present in the uptake, storage, and serotonin release mechanisms, as well as the presence in platelet membranes of serotonin 5-HT2A receptors. The platelets have a serotonergic system consisting of four main components: 1. an uptake mechanism, 2. intracellular storage organelles, 3. serotonergic receptors in the plasmatic membrane, and 4. a mitochondrial enzyme, the monoamine oxidase (MAO), which metabolizes serotonin. All these elements show physiologic similarities with the neuronal serotonergic system. Serotonergic similarities in neurons and platelets In the Central Nervous System (SNC) serotonin acts mainly as an inhibitory neurotransmitter. The precursor for its synthesis is the aminoacid tryptophan. This is taken from the blood to the cerebral interstice, where it is taken up by the nervous terminals and converted into 5-hidroxytryptophan (5-HTP) by the enzyme tryptophan hydroxilase. The conversion to 5-HTP is a key regulatory step in serotonin synthesis, and is converted quickly in 5-HT by the action of the aromatic L-acid descarboxilase. However, platelets do not synthesize 5-HT, since they do not possess tryptophan hydroxilase. Thus they only display uptake, storage, and serotonin release functions. Serotonin actions The neurotransmitter functions of neuronal serotonin, generally inhibitory, depend on the serotonergic receptor characteristics it interacts with. Its action mechanism can be mediated through second messengers (metabotrophic receptors) or through a direct action over ionic channels (ionotrophic receptors). In the platelets, serotonin is stored in a slow replacement depot, where it can be released from by exocythotic mechanisms. Serotonin participates in the platelet activation that allows for their aggregation to each other for blood clotting process. Serotonin uptake To stop the serotonin neurotransmitter function, neuronal serotonin is taken up from the synaptic cleft by transporter proteins. The serotonin neuronal uptake is impelled by a proton gradient that requires ATP. The 5-HT uptake can follow two paths: the 5-HT can be metabolized by the MAO into 5-hydroxy-indolacetic acid, or it can be reintroduced into release vesicles in order to be reutilized as a neurotransmitter. The serotonin uptake by platelets occurs either by passive diffusion or by active transport mechanisms. Under physiological conditions, the active uptake mechanism is the most effective. This uptake is mediated by proteins similar to the ones required for the neuronal serotonin uptake in the brain. It requires energy and the presence of Na+ and Cl-. The platelet uptake system has a relatively high affinity (Kd) for 5-HT, being similar in magnitude from platelets to neurons. The platelet storage of 5-HT is located mainly in the dense bodies and in the storage granules. Serotonin transporters in platelets and synaptic terminals The main form of ending a serotonergic transmission pulse is by taking up 5-HT molecules from the synaptic cleft directed to reduce the serotonin concentration, which then stops the serotonergic neurotransmission. The uptake process involves a molecular recognition of 5-HT by the transporter, its binding, and passing through the membrane to be released within the cellular. Serotonin molecules bound to its transporter protein cross through the membrane using Na+ as a driving force. The return ofthe transporter to its original position requires K+ as the driving force to step this protein toward its original position. When a selective serotonin reuptake inhibitor is administered, the 5-HT concentration increases in the synaptic cleft, which enhances serotonin neurotransmission. This increase induces a down regulation cascade of both: serotonin autoreceptors (presynaptic) and postsynaptic receptors, that may finally reestablish the resting state of the neuron. It has been confirmed that the protein for neuronal as well as platelet serotonin uptake transport are synthesized by the same gene. Experimental evidence has shown that the platelet transporter presents the same functional and pharmacological characteristics than the neuronal transporter. Serotonergicreceptors Seven types of pre and post synaptic serotonin receptors, which have also several subtypes, have been characterized. Pre and post synaptic 5-HT 1 receptors . The 5-HT1 receptors are involved in both pre and post synaptic serotonergic neurotransmission. The presynaptic 5-HT1A receptors are autoreceptors. Due to their localization in the cellular body and in the dendrites, they have been named somatodendritic autoreceptors, which control the serotonin release. The postsynaptic receptors may play a role in hypothalamic thermoregulation. The presynaptic 5-HT1D receptors are autoreceptors that perform a regulation by blocking the 5-HT release. These receptors are not synthesized in platelets. Postsynaptic 5-HT 2 receptors . The 5-HT2 receptor subtypes are 5-HT2A,BandC. When postsynaptic 5-HT2Areceptors are bound to serotonin, they drive the transduction of neuronal impulses through the production of second messengers within the postsynaptic neuron. These second messengers induce the synthesis of intracellular proteins denominated transcription factors, which may regulate the expression of several neuronal genes. Platelet 5-HT2A receptors correspond to the neuronal 5-HT2A metabothropic receptors and induce alterations in platelet density and affinity. 5-HT 3 receptors . These receptors were originally described in the periphery, specifically as part of the enteric nervous system. In the CNS 5-HT3 receptors are densely present in the solitary tract nucleus and in the area postrema. These receptors are the onlymonoaminergic receptors consistingofionic channels operated by aminergic neurotransmitters. The stimulation of 5-HT3 receptors is responsible of several secondary effects of the selective inhibitors of serotonin reuptake (SISR). These effects are not mediated only in the CNS, but also in sites outside the brain, such as the intestine, which possess this type of receptors also. These receptors are not located in the platelets. 5HT 4-7 serotonergic receptors . These receptors are distributed throughout the body, where they stimulate the alimentary tract secretions and facilitate peristaltic reflexes. Their localization in serotonergic areas in the brain and platelets has not been established. Notwhithstanding their limitations, the characteristics reviewed support the conclusion that platelets can be used as partial models to study the neuronal serotonin 5-HT2 binding and uptake functions. As Alfred Pletscher stated: "although the incomplete of the pattern demands care in its application, they could have the advantage of the relative simplicity".

Suppression of auxin stimulated growth of barley coleoptile sections by endosulfan.

Endosulfan, a cyclic sulphurous acid ester commonly used as a broad spectrum insecticide, suppressed the elongation of barley coleoptiles. Indoleacetic acid at optimum concentration overcame the inhibition of growth of coleoptiles treated with 10 ppm endosulfan. However, perfusion of the coleoptile sections with endosulfan and subsequent treatment with indoleacetic acid could not stimulate cell elongation to the extent observed in the control.