The role of NtrC in the adaptation of Herbaspirillum seropedicae SmR1 to nitrogen limitation and to nitrate.

The RNA-Seq profiling of Herbaspirillum seropedicae SmR1 wild-type and ntrC mutant was performed under aerobic and three nitrogen conditions (ammonium limitation, ammonium shock, and nitrate shock) to identify the major metabolic pathways modulated by these nitrogen sources and those dependent on NtrC. Under ammonium limitation, H. seropedicae scavenges nitrogen compounds by activating transporter systems and metabolic pathways to utilize different nitrogen sources and by increasing proteolysis, along with genes involved in carbon storage, cell protection, and redox balance, while downregulating those involved in energy metabolism and protein synthesis. Growth on nitrate depends on the narKnirBDHsero_2899nasA operon responding to nitrate and NtrC. Ammonium shock resulted in a higher number of genes differently expressed when compared to nitrate. Our results showed that NtrC activates a network of transcriptional regulators to prepare the cell for nitrogen starvation, and also synchronizes nitrogen metabolism with carbon and redox balance pathways.

A mesocosm study on bacteria-kelp interactions: Importance of nitrogen availability and kelp genetics.

Macroalgal holobiont studies involve understanding interactions between the host, its microbiota, and the environment. We analyzed the effect of bacteria-kelp interactions on phenotypic responses of two genetically distinct populations of giant kelp, Macrocystis pyrifera (north and south), exposed to different nitrogen (N) concentrations. In co-culture experiments with different N concentration treatments, we evaluated kelp growth responses and changes in three specific molecular markers associated with the N cycle, both in epiphytic bacteria (relative abundance of nrfA-gene: cytochrome c nitrite reductase) and macroalgae (expression of NR-gene: nitrate reductase; GluSyn-gene: glutamate synthase). Both kelp populations responded differently to N limitation, with M. pyrifera-south sporophytes having a lower specific growth rate (SGR) under N-limiting conditions than the northern population; M. pyrifera-north sporophytes showed no significant differences in SGR when exposed to low-N and high-N concentrations. This corresponded to a higher GluSyn-gene expression in the M. pyrifera-north sporophytes and the co-occurrence of specific nrfA bacterial taxa. These bacteria may increase ammonium availability under low-N concentrations, allowing M. pyrifera-north to optimize nutrient assimilation by increasing the expression of GluSyn. We conclude that bacteria-kelp interactions are important in enhancing kelp growth rates under low N availability, although this effect may be regulated by the genetic background of kelp populations.

Biochemical metabolism of young plants of Ucuúba (Virola surinamensis) in the presence of cadmium.

Virola surinamensis is a forest species widely distributed in the estuaries of the Amazon. These ecosystems are susceptible to contamination by Cadmium (Cd), indicating that the plant has strategies for tolerating this metal. The aim of this study was to assess the nitrogen and carbon metabolism of young plants of Ucuúba (Virola surinamensis) in the presence of cadmium with the perspective of the phytoremediation of contaminated environments. The used experimental design was a completely randomized design with five Cd concentrations (0, 15, 30, 45, and 60 mg L- 1), for 60 days. In general, Cd did not affect nitrate concentration in the root but had a positive effect on leaves. The reduction of nitrate reductase (NR) in plants exposed to Cd was followed by a decrease in ammonia, total soluble amino acids (TSA), and total soluble proteins (TSP). Cd promoted an increase in the concentration of total soluble carbohydrates (TSC), proline, sucrose, and reducing sugars in the plants. The increase in TSC, sucrose and proline, suggests a metabolic regulatory mechanism of V. surinamensis against Cd stress.

Aplicação de piraclostrobina em genótipos de milho cultivados em segunda safra / Pyraclostrobin application in different corn genotypes cultivated off season

Objetivou-se com esse estudo avaliar os efeitos fisiológicos, o controle de doenças e os efeitos nos componentes de produção, ocasionados pela aplicação do fungicida piraclostrobina, em diferentes estádios fenológicos de genótipos de milho cultivado em segunda safra, no município de Jataí-GO. Utilizou-se o delineamento de blocos ao acaso, no esquema fatorial 3 x 4, com quatro repetições. Os tratamentos consistiram de três genótipos de milho e quatro épocas de aplicação de piraclostrobina. Não houve interação entre genótipos e aplicações para as variáveis avaliadas. A aplicação de piraclostrobina não interfere na fisiologia, desenvolvimento e produtividade de grãos das plantas de milho cultivados em segunda safra e não houve diferença significativa no controle das doenças ferrugem polissora, mancha de cercospora, mancha de bipolares e mancha branca pela aplicação de piraclostrobina nos genótipos estudados.(AU)

Objective with this study evaluate the physiological effects, disease control and effects on the production components of pyraclostrobin fungicide, applied at different phenological stages of of second crop corn genotypes, in Jataí city. In randomized blocks in a factorial design 3 x 4 with four replications. The treatments consisted in three corn genotypes and four periods of pyraclostrobin application. There was no interaction between genotype and applications for the variables evaluated. For the majority of the variables the only difference were between genotypes. The application of pyraclostrobin does not interfere in the physiology, development and grain production of maize plants grown in second crop and there was no significant difference in the control of polissora blight,cercospora spot, bipolaris spot stain and white stain by the application of pyraclostrobin in the genotypes studied.(AU)

Aplicação de piraclostrobina em genótipos de milho cultivados em segunda safra / Pyraclostrobin application in different corn genotypes cultivated off season

Objetivou-se com esse estudo avaliar os efeitos fisiológicos, o controle de doenças e os efeitos nos componentes de produção, ocasionados pela aplicação do fungicida piraclostrobina, em diferentes estádios fenológicos de genótipos de milho cultivado em segunda safra, no município de Jataí-GO. Utilizou-se o delineamento de blocos ao acaso, no esquema fatorial 3 x 4, com quatro repetições. Os tratamentos consistiram de três genótipos de milho e quatro épocas de aplicação de piraclostrobina. Não houve interação entre genótipos e aplicações para as variáveis avaliadas. A aplicação de piraclostrobina não interfere na fisiologia, desenvolvimento e produtividade de grãos das plantas de milho cultivados em segunda safra e não houve diferença significativa no controle das doenças ferrugem polissora, mancha de cercospora, mancha de bipolares e mancha branca pela aplicação de piraclostrobina nos genótipos estudados.

Objective with this study evaluate the physiological effects, disease control and effects on the production components of pyraclostrobin fungicide, applied at different phenological stages of of second crop corn genotypes, in Jataí city. In randomized blocks in a factorial design 3 x 4 with four replications. The treatments consisted in three corn genotypes and four periods of pyraclostrobin application. There was no interaction between genotype and applications for the variables evaluated. For the majority of the variables the only difference were between genotypes. The application of pyraclostrobin does not interfere in the physiology, development and grain production of maize plants grown in second crop and there was no significant difference in the control of polissora blight,cercospora spot, bipolaris spot stain and white stain by the application of pyraclostrobin in the genotypes studied.

Enhanced Nitric Oxide Synthesis Through Nitrate Supply Improves Drought Tolerance of Sugarcane Plants.

Nitric oxide (NO) is an important signaling molecule associated with many biochemical and physiological processes in plants under stressful conditions. Nitrate reductase (NR) not only mediates the reduction of NO3 - to NO2 - but also reduces NO2 - to NO, a relevant pathway for NO production in higher plants. Herein, we hypothesized that sugarcane plants supplied with more NO3 - as a source of N would produce more NO under water deficit. Such NO would reduce oxidative damage and favor photosynthetic metabolism and growth under water limiting conditions. Sugarcane plants were grown in nutrient solution and received the same amount of nitrogen, with varying nitrate:ammonium ratios (100:0 and 70:30). Plants were then grown under well-watered or water deficit conditions. Under water deficit, plants exhibited higher root [NO3 -] and [NO2 -] when supplied with 100% NO3 -. Accordingly, the same plants also showed higher root NR activity and root NO production. We also found higher photosynthetic rates and stomatal conductance in plants supplied with more NO3 -, which was associated with increased root growth. ROS accumulation was reduced due to increases in the activity of catalase in leaves and superoxide dismutase and ascorbate peroxidase in roots of plants supplied with 100% NO3 - and facing water deficit. Such positive responses to water deficit were offset when a NO scavenger was supplied to the plants, thus confirming that increases in leaf gas exchange and plant growth were induced by NO. Concluding, NO3 - supply is an interesting strategy for alleviating the negative effects of water deficit on sugarcane plants, increasing drought tolerance through enhanced NO production. Our data also provide insights on how plant nutrition could improve crop tolerance against abiotic stresses, such as drought.

Seed characterization and early nitrogen metabolism performance of seedlings from Altiplano and coastal ecotypes of Quinoa.

BACKGROUND: Early seed germination and a functional root system development during establishment are crucial attributes contributing to nutrient competence under marginal nutrient soil conditions. Chenopodium quinoa Willd (Chenopodiaceae) is a rustic crop, able to grow in marginal areas. Altiplano and Coastal/Lowlands are two representative zones of quinoa cultivation in South America with contrasting soil fertility and edaphoclimatic conditions. In the present work, we hypothesize that the ecotypes of Quinoa from Altiplano (landrace Socaire) and from Coastal/Lowland (landrace Faro) have developed differential adaptive responses in order to survive under conditions of low availability of N in their respective climatic zones of Altiplano and Lowlands. In order to understand intrinsic differences for N competence between landraces, seed metabolite profile and germinative capacity were studied. Additionally, in order to elucidate the mechanisms of N uptake and assimilation at limiting N conditions during establishment, germinated seeds of both landraces were grown at either sufficient nitrate (HN) or low nitrate (LN) supply. We studied the photosynthetic performance, protein storage, root morphometrical parameters, activity and expression of N-assimilating enzymes, and the expression of nitrate transporters of roots in plants submitted to the different treatments. RESULTS: Seeds from Socaire landrace presented higher content of free N-related metabolites and faster seed germination rate compared to Faro landrace. Seedlings of both ecotypes presented similar physiological performance at HN supply, but at LN supply their differences were exalted. At LN, Socaire plants showed an increased root biomass (including a higher number and total length of lateral roots), a differential regulation of a nitrate transporter (a NPF6.3-like homologue) belonging to the Low Affinity Transport System (LATS), and an upregulation of a nitrate transporter (a NRT2.1-like homologue) belonging to the High Affinity nitrate Transport System (HATS) compared to Faro. These responses as a whole could be linked to a higher amount of stored proteins in leaves, associated to an enhanced photochemical performance in Altiplano plants, in comparison to Lowland quinoa plants. CONCLUSIONS: These differential characteristics of Socaire over Faro plants could involve an adaptation to enhanced nitrate uptake under the brutal unfavorable climate conditions of Altiplano.

The activity-based cost of drug-susceptibility test of Mycobacterium tuberculosis through Kit SIRE Nitratase® Plastlabor.

Background: Drug-resistant tuberculosis (TB) is an ongoing health threat, and the greatest challenge to adequate control of TB in many countries lies in the lack of proper laboratory drug-susceptibility test. The aim of this study was to evaluate the activity-based costs (ABC) of Kit SIRE Nitratase® (Kit SIRE) and compare its values with the conventional drug-susceptibility test. Methods: The ABC was calculated for three different approaches: Kit SIRE (clinical samples and cultures), proportion methods in Lowenstein Jensen (PM-LJ), and the Bactec™ MGIT™ 960 system based on Mycobacterial Research Laboratory's routine. Results: The ABC of Kit SIRE from cultures was US$ 148.54, while from clinical samples was US$ 136.12. In the case of conventional tests, the ABC of Bactec™ MGIT™ 960 was US$ 227.63 and of the PM-LJ was US$ 132.64. The Kit SIRE has a lower ABC when clinical samples are used instead of cultures. Compared to conventional tests, the ABC is similar to the PM-LJ and lower the Bactec™ MGIT™ 960. Conclusion: The Kit SIRE should be used as a screening method in clinical specimens and in culture for laboratories that do not have Bactec™ MGIT™ 960. Therefore, it can be incorporated into the routine of laboratories in countries with low resources and a high burden of TB and drug-resistant TB.

Do nitrogen sources and molybdenum affect the nutritional quality and nitrate concentrations of hydroponic baby leaf lettuce?

Lettuce is one of the most popular vegetables, and the market niche of the baby leaf variety is expanding. The manner in which nitrogen (N) is supplied and the available concentration of the micronutrient molybdenum (Mo) affect N metabolism, with reflects on the nutritional quality of that vegetable. Here, two Mo concentrations (0.06 and 0.12 mg/L) and four proportions (%) of nitrate (NO3 - )/ammonium (NH4 + )(100/0, 75/25, 50/50, and 25/75) were supplied to hydroponic baby leaf lettuce floating type to evaluate their effects on plant growth and leaf mineral and NO3 - compositions. Shoot dry mass did not differ among the different treatments, although fresh mass was lower in treatments with larger proportions of NH4 + . Higher leaf concentrations of NO3 - were observed in plants treated with 100% N-NO3 - , but they were still below tolerable limits for human health. The enzyme nitrate reductase was not found to be sensitive to the nitrogen sources or to Mo concentrations. N proportions and Mo concentrations differently affected macro- (C, N, P, K, Ca, Mg, and S) and micronutrients (Cu, Fe, Mn, Mo, and Zn) leaf concentrations. Although treatment with 100% N-NO3 - favored higher mineral concentrations in lettuce leaves, the addition of 25% N-NH4 + allowed fresh mass production with the lowest NO3 - concentrations. As such, and considering the healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity, the 75/25 NO3 - /NH4 + proportion is recommended for the hydroponic cultivation of baby leaf var. Mimosa lettuce. PRACTICAL APPLICATION: We demonstrate a direct link between the constitution of nutrient solution with nitrate accumulation by hydroponic lettuce and indicate the best source of N as well as the concentration of Mn to healthy reduction of NO3 - consumption by humans and the maintenance of plant productivity.

Does nitrogen source influence cadmium distribution in Arabidopsis plants?

The purpose of the present work was to study the effect of the nitrogen source (NO3- vs NH4+) on cadmium (Cd) uptake, translocation and partition and its associated toxicity in hydroponically-grown Arabidopsis plants. After a short growth period on a complete Hoagland nutrient solution, Arabidopsis seedlings continued in the same growth medium (NA) or were switched to NO3- (N) or NH4+ (A) as sole N sources and supplied with 2.5 µM Cd. Unrelated to the nitrogen source, Cd reached higher levels in roots than in leaves. However, when ammonium was the source of nitrogen, Cd accumulation in roots was lower than in N or NA medium and the metal translocation to the aerial part was restricted, reaching values 25%-35% below the levels observed in plants grown with N or NA. Cadmium negatively affected chlorophyll content and PSII quantum yield, independently of the nitrogen source, with the highest decrease (35%) under NA treatment. Proline content increased, either with NA, N or A supplied in the presence of Cd, whereas a rise in total anthocyanin content was clearly favored when ammonium was the source of nitrogen, with or without Cd. In leaves, while NIA1 and NIA2 expression was markedly reduced by Cd in the presence of N or NA, ammonium source slightly reduced NIA1 expression but greatly upregulated NIA2 expression upon Cd exposure. The decay in NR activity was independent of the nitrogen source when Cd was applied and this decay was accompanied by a great increase in NH4+ levels either with nitrates or ammonium in the medium in the presence of Cd. Only NIA1 was detected in roots and its expression, together with NR activity and nitrates levels, was the highest in N medium devoid of Cd. The possibility of reducing Cd health risks through nitrogen fertilization practices is discussed.

Detection of Drug Resistant Mycobacterium Tuberculosis Strains Using Kit SIRE Nitratase®: a Multicenter Study

Abstract (1) Background: The Commercial Kit SIRE Nitratase® PlastLabor, is a drug susceptibility test kit used to detect Mycobacterium tuberculosis resistance to first-line TB treatment drugs. The present study aimed at evaluating its performance in a multicenter study. (2) Methods: To determine its accuracy, the proportion methods in Lowenstein Jensen medium or the BACTECTMMGITTM960 system was used as a gold standard. (3) Results: The study revealed that the respective accuracies of the kit with 190 M. tuberculosis clinical isolates, using the proportion methods in Lowenstein Jensen medium or BACTECTMMGITTM960 system as a gold standard, were 93.9% and 94.6%, 96.9% and 94.6%, 98.0% and 97.8%, and 98.0% and 98.9%, for streptomycin, isoniazid, rifampicin, and ethambutol, respectively. (4) Conclusion: Thus, the kit can rapidly screen resistance to streptomycin, isoniazid, rifampicin, and ethambutol. Additionally, it does not require sophisticated equipment; hence, it can be easily used in the laboratories of low and middle income countries.

Identification and Unusual Properties of the Master Regulator FNR in the Extreme Acidophile Acidithiobacillus ferrooxidans.

The ability to conserve energy in the presence or absence of oxygen provides a metabolic versatility that confers an advantage in natural ecosystems. The switch between alternative electron transport systems is controlled by the fumarate nitrate reduction transcription factor (FNR) that senses oxygen via an oxygen-sensitive [4Fe-4S]2+ iron-sulfur cluster. Under O2 limiting conditions, FNR plays a key role in allowing bacteria to transition from aerobic to anaerobic lifestyles. This is thought to occur via transcriptional activation of genes involved in anaerobic respiratory pathways and by repression of genes involved in aerobic energy production. The Proteobacterium Acidithiobacillus ferrooxidans is a model species for extremely acidophilic microorganisms that are capable of aerobic and anaerobic growth on elemental sulfur coupled to oxygen and ferric iron reduction, respectively. In this study, an FNR-like protein (FNRAF) was discovered in At. ferrooxidans that exhibits a primary amino acid sequence and major motifs and domains characteristic of the FNR family of proteins, including an effector binding domain with at least three of the four cysteines known to coordinate an [4Fe-4S]2+ center, a dimerization domain, and a DNA binding domain. Western blotting with antibodies against Escherichia coli FNR (FNREC) recognized FNRAF. FNRAF was able to drive expression from the FNR-responsive E. coli promoter PnarG, suggesting that it is functionally active as an FNR-like protein. Upon air exposure, FNRAF demonstrated an unusual lack of sensitivity to oxygen compared to the archetypal FNREC. Comparison of the primary amino acid sequence of FNRAF with that of other natural and mutated FNRs, including FNREC, coupled with an analysis of the predicted tertiary structure of FNRAF using the crystal structure of the related FNR from Aliivibrio fisheri as a template revealed a number of amino acid changes that could potentially stabilize FNRAF in the presence of oxygen. These include a truncated N terminus and amino acid changes both around the putative Fe-S cluster coordinating cysteines and also in the dimer interface. Increased O2 stability could allow At. ferrooxidans to survive in environments with fluctuating O2 concentrations, providing an evolutionary advantage in natural, and engineered environments where oxygen gradients shape the bacterial community.

Nitrate reductase mediates nitric oxide-dependent gravitropic response in Arabidopsis thaliana roots.

Plant roots respond positively to gravity force and orientate it growth providing anchorage to the soil and gathering water and nutrient sources. The gravitropic response is a complex process wherein nitric oxide (NO) participates as a key signaling molecule. Here, we used genetically impaired genotypes to demonstrate the role of the nitrate reductase (NR) enzyme as a possible source of endogenous NO during gravitropic response in Arabidopsis thaliana (A. thaliana) roots. A. thaliana has two NR genes, NIA1 and NIA2. The single mutants nia1 and nia2, and the double mutant nia1/nia2 showed perturbed gravitropism. Complementation with the exogenous NO donor, S-nitroso-L-cysteine, partially rescued the wild-type phenotype in nia2 and nia1/nia2 but not in the nia1 mutant. Our findings showed that each NR gene differentially contributes to reaching the optimum level of NO during the gravitropic response, suggesting that NIA1 and NIA2 isoforms are not equivalent and have potential regulatory feedback to each other during the gravitropic response in A. thaliana roots.

Nitrate reductase activity in leaves as a plant physiological indicator of in vivo biological nitrification inhibition by Brachiaria humidicola.

The tropical forage grass Brachiaria humidicola (Bh) controls soil microbial nitrification via biological nitrification inhibition (BNI). The aim of our study was to verify if nitrate reductase activity (NRA) in Bh roots or leaves reflects in vivo performance of BNI in soils. NRA was measured in roots and leaves of contrasting accessions and apomictic hybrids of Bh grown under controlled greenhouse and natural field conditions. Nitrate (NO3-) contents were measured in soil solution and in Bh stem sap to validate NRA data. Potential soil nitrification rates (NRs) and leaf δ15N values were used to verify in vivo BNI by the NRA assay in the field study. NRA was detected in Bh leaves rather than roots, regardless of NO3- availability. NRA correlated with NO3- contents in soils and stem sap of contrasting Bh genotypes substantiating its reflectance of in vivo BNI performance. Additionally, leaf NRA data from the field study significantly correlated with simultaneously collected NRs and leaf δ15N data. The leaf NRA assay facilitated a rapid screening of contrasting Bh genotypes for their differences in in vivo performance of BNI under field and greenhouse conditions, but inconsistency of the BNI potential by Bh germplasm was observed. Among Bh genotypes tested, leaf NRA was closely linked with nitrification activity, and consequently with actual BNI performance. It was concluded that NRA in leaves of Bh can serve as an indicator of in vivo BNI activity when complemented with established BNI methodologies (δ15N, NRs) under greenhouse and field conditions.

Characteristics of root-cultivable endophytic fungi from Rhododendron ovatum Planch.

Ericoid mycorrhiza can improve the competitiveness of their host plants at the ecosystem level. The ability of ericoid mycorrhizal fungi to thrive under harsh environmental conditions suggests that they are capable of decomposing plant organic matter. This study aims to characterize 2 strains of root-cultivable endophytic fungi, RooDK1 and RooDK6, from Rhododendron ovatum Planch using colony and hyphal morphology, molecular analysis, observations of mycorrhiza, and investigations of adaptation to different sources of organic matter. Nitrogen utilization was also investigated by assessing protease production and growth on different nitrogen sources. Morphological studies indicated that both species are ericoid mycorrhizal fungi; our molecular studies confirmed RooDK1 as Oidiodendron maius and classified RooDK6 as Pezicula ericae. We observed that only RooDK1 can assist in host plant survival by degrading organic matter. This species also secretes protease and has the highest nitrate reductase activity of these 2 endophytes. Thus, RooDK1 has a greater ability to help the host plants thrive in a harsh habitat.

Exploitation of new endophytic bacteria and their ability to promote sugarcane growth and nitrogen nutrition.

Few studies have evaluated endophytic bacteria in relation to plant growth promotion, nitrogen uptake and biological control. The aim of this study was to molecularly and physiologically characterize thirteen endophytic bacteria strains, evaluate their biological control properties and their ability to promote plant growth and plant N nutrition. All the strains produced indole acetic acid and promoted increase of plant biomass, N accumulative amount and N-use efficiency index. None of the strains carries the nifH gene. Four strains stimulated plant nitrate reductase activity, four solubilized phosphate, nine produced siderophores and none produced HCN. Seven strains inhibited Bipolaris sacchari growth and one was antagonistic to Ceratocystis paradoxa. The pathogens were inhibited by the production of diffusible and volatile metabolites by the bacterial strains. Moreover, this is the first study to demonstrate the effect of Delftia acidovorans on sugarcane plant growth, nitrogen metabolism improvement and antagonism to B. sacchari. The most efficient strains in promoting plant growth and exhibiting antagonistic activities towards fungal pathogens were Herbaspirillum frinsingense (IAC-BECa-152) and three Pantoea dispersa strains (IAC-BECa-128, IAC-BECa-129, and IAC-BECa-132). These bacteria show potential to be used as inoculants for sustainable agricultural management, mainly at the seedling production phase.

NO3-/NH4+ ratios affect nutritional homeostasis and production of Tanzania guinea grass under Cu toxicity.

Nitrogen (N) can alleviate metal toxicity. However, as of yet, there have been no studies showing the efficacy of NO3-/NH4+ in mitigating Cu toxicity. The objective of this study was to evaluate the Cu toxicity on the nutritional and productive attributes of Panicum maximum cv. Tanzania as well as the role of NO3- and NH4+ ratios in nutritional homeostasis. The experiment was conducted using 3 × 4 factorial treatments arranged in a randomized complete block design with three replicates. The treatments were three NO3-/NH4+ ratios (100/0, 70/30, and 50/50) and four Cu rates (0.3, 250, 500, and 1000 µmol L-1) in nutrient solution. Copper concentrations in the diagnostic leaves (DL) were highest in plants grown under 70/30 NO3-/NH4+ ratios and a Cu rate of 1000 µmol L-1. In this combination, it was observed that DL had higher concentrations of NH4+, greater glutamine synthetase activity, lower chlorophyll concentration (SPAD value), and lower shoot dry mass, suggesting high disorders of nutritional homeostasis. Plants receiving N in the form of NO3- and 1000 Cu µmol L-1 showed that DL had lower concentrations of Cu, higher concentration of chlorophyll, higher NO3- concentration, higher nitrate reductase activity, and higher NO3- accumulation in the roots, suggesting a reduction in disorders of nutritional homeostasis. The disorders on mineral uptake, N assimilation, and biomass production caused by Cu toxicity are shown to be affected by NO3-/NH4+ ratios, and N supply via NO3- allowed for better homeostasis of the forage grass.

Fast detection of Mycobacterium tuberculosis in culture-positive sputum samples by nitrate reductase activity

ABSTRACT Microscopy and bacterial culture are the main tools in the diagnosis of tuberculosis. Since the slow growth of Mycobacterium tuberculosis impairs rapid diagnosis strategies, especially in countries where the latter are the only available resources, the ongoing development of new and inexpensive tools based on mycobacterial metabolism optimizing growth detection with preliminary identification is greatly welcome. When compared to the other species from the M. tuberculosis complex, M. tuberculosis is a strong nitrate reducer. Current assay compares the nitrate reductase activity of M. tuberculosis from pulmonary specimens cultivated in nitrate-supplemented media. Fifty-five sputum samples were decontaminated and inoculated in conventional (Middlebrook 7H9, Ogawa Kudoh-OK) and in nitrate-supplemented media (Middlebrook 7H9-N, Ogawa Kudoh-N). An aliquot from the media directly reacted with Griess reagent (7H9-N and OK-N) every five days, or transferred to a nitrate substrate solution (7H9, OK). Nitrate to nitrite reduction was considered positive, revealed by the pink color, indicating bacterial growth. As reference method, the Mycobacteria Growth Indicator Tube (MGIT) was used for sensitivity calculations and statistical analysis. 7H9-N and OK-N assays proved to perform better in detecting M. tuberculosis than conventional assays (7H9 and OK). Indeed, broth nitrate-supplemented medium (7H9-N) was comparable to MGIT to detect M. tuberculosis, except in growth detection time. Results show that 7H9-N may be used as an alternative tool particularly in low-income countries since it is a simple and cheap technique, and does not restrict diagnosis to single-source products.

Quadruplex PCR assay for identification of Corynebacterium pseudotuberculosis differentiating biovar Ovis and Equi.

BACKGROUND: Corynebacterium pseudotuberculosis is classified into two biovars, nitrate-negative biovar Ovis which is the etiologic agent of caseous lymphadenitis in small ruminants and nitrate-positive biovar Equi, which causes abscesses and ulcerative lymphangitis in equines. The aim of this study was to develop a quadruplex PCR assay that would allow simultaneous detection and biovar-typing of C. pseudotuberculosis. METHODS: In the present study, genomes of C. pseudotuberculosis strains were used to identify the genes involved in the nitrate reduction pathway to improve a species identification three-primer multiplex PCR assay. The nitrate reductase gene (narG) was included in the PCR assay along with the 16S, rpoB and pld genes to enhance the diagnosis of the multiplex PCR at biovar level. RESULTS: A novel quadruplex PCR assay for C. pseudotuberculosis species and biovar identification was developed. The results of the quadruplex PCR of 348 strains, 346 previously well-characterized clinical isolates of C. pseudotuberculosis from different hosts (goats, sheep, horse, cattle, buffalo, llamas and humans), the vaccine strain 1002 and the type strain ATCC 19410T, were compared to the results of nitrate reductase identification by biochemical test. The McNemar's Chi-squared test used to compare the two methods used for C. pseudotuberculosis biovar identification showed no significant difference (P = 0.75) [95% CI for odds ratio (0.16-6.14)] between the quadruplex PCR and the nitrate biochemical test. Concordant results were observed for 97.13% (338 / 348) of the tested strains and the kappa value was 0.94 [95% CI (0.90-0.98)]. CONCLUSIONS: The ability of the quadruplex assay to discriminate between C. pseudotuberculosis biovar Ovis and Equi strains enhances its usefulness in the clinical microbiology laboratory.

Evaluation of the commercial kit SIRE nitratase for detecting resistant mycobacterium tuberculosis in Brazil

Abstract INTRODUCTION: This study aimed to evaluate a new commercial kit, Kit SIRE Nitratase-PlastLabor, for testing the drug susceptibility of clinical Mycobacterium tuberculosis isolates. METHODS: The accuracy of the Kit SIRE Nitratase was evaluated by examining the susceptibility (streptomycin, isoniazid, rifampicin, and ethambutol) of 40 M. tuberculosis isolates, using the proportion method with Lowenstein-Jensen medium or the BACTEC MGIT 960 system. RESULTS: The detection accuracy for streptomycin, isoniazid, rifampicin, and ethambutol was 95%, 97.5%, 100%, and 80%, respectively. CONCLUSIONS: The exceptional accuracy demonstrated by Kit SIRE Nitratase for isoniazid and rifampicin makes the kit an attractive option for screening M. tuberculosis strain resistance.