ROLE OF ACC DEAMINASE PRODUCING PLANT GROWTH PROMOTING RHIZOBACTERIA IN AMELIORATING THE SALINITY STRESS CONDITIONS: A REVIEW

Salinity in agricultural soil is a severe problem that affects the growth and production in numerous crops all over the world. The country's salt?affected land is estimated to be 6.74 million hectares. According to estimates, approximately 10% more land is becoming salinized each year, and by 2050, nearly half of all arable land will be contaminated by salt. Plants may have bacterial companions that shield them from the negative consequences of salt stress (SS). Plant growth?promoting bacteria (PGPR) can minimize the usage of agrochemicals while also improving plant production, nutrition, and biotic–abiotic stress tolerance. The enzyme 1? aminocyclopropane?1?carboxylic acid deaminase (ACCD) is found in certain bacteria and works by degrading ACC (ethylene precursor in higher plants) into ??ketobutyrate and ammonia (NH ), thereby reducing the ACC levels, thus, inhibits excessive biosynthesis of3 ethylene under numerous stress circumstances. This is one of the most effective methods for inducing plant tolerance to SS. The current review highlighted the recent works of ACCD under SS environment. Further, the relevance of reducing the negative effect of ROS and increasing plant development under SS were also discussed. We propose a path for the community to employ beneficial microorganisms to boost agricultural yield and achieve sustainable development by highlighting plant?microbe interactions in this review.

Estimación de la sustentabilidad de fincas productoras de arveja en el municipio de Ipiales, Nariño-Colombia / Estimation of the sustainability of farm producers of vegetables in the municipality of Ipiales, Nariño-Colombia

RESUMEN Los agroecosistemas muestran una gran heterogeneidad en cuanto a las prácticas y características, debido a que dependen de las lógicas de la agricultura familiar, convirtiéndose en un recurso potencialmente valioso, cuando se estudia el uso y el soporte del medio productivo. Es por ello, que esta investigación evaluó el sistema de arveja (Pisum sativum L.) en el municipio de Ipiales, a través de una metodología multidimensional, usando 26 indicadores, agrupados en cuatro dimensiones de sustentabilidad agroecológica. La estimación, se realizó sobre 30 fincas, que representaron tres tipos de campesinos, previamente descritos, como C1, C2 y C3; los resultados permitieron identificar los Puntos Críticos de Sustentabilidad (PCS) y el Índice General de Sustentabilidad (IGS) del sistema, estableciendo que las diferentes situaciones sociales, ambientales, económicas y técnico-productivas determinan la existencia de PCS específicos. El IGS tuvo una calificación de 2,65, para C1; 2,90, para C2 y 2,62, para C3, evidenciando medidas de monitoreo y de manejo, que se deberían discutir en la región, para disminuir las externalidades generadas por el modelo productivo.

ABSTRACT Agroecosystems present great heterogeneity in terms of practices and characteristics, since they depend on the logic of family farming, which makes them a potentially valuable resource when studying the use and support of the productive environment. For this reason, this research evaluated the pea system (Pisum sativum L.) in the municipality of Ipiales, through a multidimensional methodology that uses 26 indicators grouped into four dimensions of agroecological sustainability. The estimation was made on 30 farms that represented three types of farmers previously described as C1, C2 and C3; the results allowed identifying the Critical Points of Sustainability (CPS) and the General Index of Sustainability (GIS) of the system. It was established that the different social, environmental, economic and technical-productive situations determine the existence of specific CPS, the GIS had a rating of 2.65 for C1, 2.90 for C2 and 2.62 for C3, this evidenced monitoring and management measures that should be discussed in the region to reduce the externalities generated by the production model.

Cambios fisiológicos de dos variedades de arveja almacenada en diferentes tipos de empaque y temperatura / Physiological changes of two varieties of pea stored in different types of packing and temperature

RESUMEN La arveja, una fuente económica para pequeños y medianos agricultores de la zona andina, evidencia un incipiente manejo pos-cosecha y un nulo nivel de transformación. Tras la cosecha y debido a su naturaleza temporal y perecedera, la arveja acelera su metabolismo, disminuyendo su calidad y vida útil. Una opción económica y que esté disponible para los productores y LOS comercializadores para conservar sus características es el uso de empaques y la refrigeración. El objetivo fue evaluar tipos de empaques (bolsa de polietileno de baja densidad -PE-LD-, envase plástico termoformado perforado -PE-HD- y bandeja de poliestireno -PS-) y temperaturas de almacenamiento (0°C, 4°C y 18°C) en dos variedades de arveja, Sureña y Obonuco Andina. Se utilizó un diseño irrestrictamente al azar con arreglo factorial 3x3x2, teniendo como factores temperatura, empaque y variedad y como variables de estudio, las constantes cinéticas (K) de porcentaje de brotación, firmeza, pérdida de peso e índice de color. Los resultados mostraron que, para la brotación, se presentó el valor más bajo de K en Sureña, en PE-LD, a 0°C. La temperatura afectó la firmeza de los granos, conservándose a 0°C. La menor pérdida de peso se evidenció en PE-HD, a 0°C, independiente de la variedad. Para el índice de color, la K más baja se encontró en PS, a 0°C, en Obonuco Andina. Con la ecuación de Arrhenius en la variable índice de color, se estimó tiempos de vida útil de 13; 7; 5 y 2 días a temperaturas de 0°C, 6°C, 10°C y 24°C, respectivamente.

ABSTRACT The pea, an economic source for small and medium farmers in the Andes zone, evidences an incipient postharvest handling and a null level of transformation. After the harvest and due to its temporary and perishable nature, the pea accelerates its metabolism, decreasing its quality and shelf life. An economic option that is available to producers and marketers to preserve their characteristics is the use of packaging and refrigeration. The objective was to evaluate types of packaging (low density polyethylene bag (PE-LD), perforated thermoformed plastic container (PE-HD) and polystyrene tray (PS)) and storage temperatures (0°C, 4°C and 18°C) in two pea varieties (Sureña and Obonuco Andina). An unrestricted random design with a 3x3x2 factorial arrangement was used, taking as factors temperature, packing and variety; and as study variables the kinetic constants (K) of percentage of sprouting, firmness, weight loss and color index. The results showed that, for the sprouting, the lowest value of K was shown in Sureña, in PE-LD at 0°C. The temperature affected the firmness of the grains, remaining at 0°C. The lowest weight loss was evidenced in PE-HD at 0°C, regardless of the variety. For the color index, the lowest K was found in PS at 0°C, in Obonuco Andina. With the Arrhenius equation in the variable color index, useful life times of 13; 7; 5 and 2 days were estimated at temperatures of 0°C, 6°C, 10°C and 24°C respectively.

Adequacy of methodology for conducting germination test in forage pea seeds / Adequação da metodologia para condução do teste de germinação em sementes de ervilha forrageira

Seeds presents variable germination performance at different temperatures and substrates, which are basic components of germination test. Within this context, this work aimed to adapt the methodology for the seeds germination's test of P. sativum subsp. arvense. The experiment required four portions of seeds, five temperature degrees (15, 20, 25, 20-30 and 15-25 °C), and two substrata (roll of paper and in sand). Seeds evaluation considered the following: moisture degree and germination test. The effect of seed portion, substrata and temperature in germination was assessed on a daily basis. It was calculated the rate of germination, of anormal plants, of dormant and dead seeds, and of speed of germination index; initial, final and average times, and synchrony. The experimental design consisted of random blocks with four replications, while conjoint analysis was the statistical procedure adopted. The germination test for forage pea seeds should be conducted at the constant temperature 20 °C in roll of paper substratewith the first count and final count on the 4th and 7th day, respectively.

As sementes apresentam desempenho germinativo variável em diferentes temperaturas e substratos, que são componentes básicos do teste de germinação. Dentro deste contexto, este trabalho teve como objetivo adequar a metodologia para o teste de germinação de sementes de P. sativum subsp. arvense. Para tanto, foram utilizados quatro lotes de sementes, cinco temperaturas (15, 20, 25, 20-30 e 15-25 °C) e dois substratos (rolo de papel e entre areia). O teor de água das sementes foi determinado antes das avaliações e o efeito dos lotes, substratos e temperaturas sobre a germinação foi avaliado diariamente. Calculou-se a porcentagem de germinação, de plântulas anormais, de sementes dormentes e mortas, o índice de velocidade de germinação, os tempos inicial, final e médio e sincronia. O delineamento adotado foi o de blocos casualizados, com quatro repetições e o procedimento estatístico adotado foi análise conjunta. O teste de germinação deve ser conduzido na temperatura constante de 20 °C, em substrato rolo de papel com primeira contagem e contagem final ao 4° e 7° dia, respectivamente.

Macroalgae as Soil Conditioners or Growth Promoters of Pisum sativum (L)

Mexico has long littorals that receive great amounts of marine algae that are wasted despite being potentially useful as growth promoters without the inconveniences of agrochemicals. Some macroalgae have been used with excellent results as soil conditioners and fertilisers in agriculture. In this study nine macroalgae from the coasts of Veracruz (Gulf of Mexico) and one from Guerrero (Mexican Pacific), Mexico, were tested. They were added to the soil in the form of fragments and of silage in liquid around Pisum sativum plants. A two-way random design with four replicas was followed. Two controls, hormones and water, were also used. Growth was recorded every third day. Results showed that four algae promoted growth (p<0.05) Ulva fasciata (ensiling treatment) and Ulva lactuca (ensiling treatment) and Gracilaria caudata (fragment treatment) and Palisada perforata (fragment treatment), compared with the hormones and water controls. The activity of Ulva fasciata and Palisada perforata is recorded for the first time. Since the control plants treated with hormones grew much less than those treated with the four algae, we conclude that the release of all algae compounds was responsible for the growth, and not the hormone mimetics.

Phytotoxicity of glyphosate in the germination of Pisum sativum and its effect on germinated seedlings

The present study evaluated the effects of glyphosate on Pisum sativum germination as well as its effect on the physiology and biochemistry of germinated seedlings. Different physico-chemical biomarkers, viz., chlorophyll, root and shoot length, total protein and soluble sugar, along with sodium and potassium concentration, were investigated in germinated seedlings at different glyphosate concentrations. This study reports the influence of different concentrations of glyphosate on pea seeds and seedlings. Physicochemical biomarkers were significantly changed by glyphosate exposure after 15 days. The germination of seedlings under control conditions (0 mg/L) was 100% after 3 days of treatment but at 3 and 4 mg/L glyphosate, germination was reduced to 55 and 40%, respectively. Physiological parameters like root and shoot length decreased monotonically with increasing glyphosate concentration, at 14 days of observation. Average root and shoot length (n=30 in three replicates) were reduced to 14.7 and 17.6%, respectively, at 4 mg/L glyphosate. Leaf chlorophyll content also decreased, with a similar trend to root and shoot length, but the protein content initially decreased and then increased with an increase in glyphosate concentration to 3 mg/L. The study suggests that glyphosate reduces the soluble sugar content significantly, by 21.6% (v/v). But internal sodium and potassium tissue concentrations were significantly altered by glyphosate exposure with increasing concentrations of glyphosate. Biochemical and physiological analysis also supports the inhibitory effect of glyphosate on seed germination and biochemical effects on seedlings.

The morphology and bioactivity of the rice field cyanobacterium Leptolyngbya / Morfología y bioactividad de la cianobacteria Leptolyngbya en los campos de cultivo de arroz

The genus Leptolyngbya comprises filamentous cyanobacteria that are important in rice fields. In the rhizosphere, cyanobacteria produce a variety of secondary metabolites such as auxins that are important in agriculture soil performance. To assess this, Leptolyngbya strain MMG-1, was isolated from the rhizosphere of rice plants and described. For this, the morphology of this strain was studied by light microscopy as well as by confocal laser scanning microscopy. Besides, the ability of this strain to synthesize an auxin-like bioactive compound was demonstrated under various culture conditions (different amounts of tryptophan; pH; different alternating light:dark periods; duration of the incubation). The auxin-like compound was extracted from the culture of Leptolyngbya strain MMG-1 and identified as indole-3-acetic acid (IAA) by thin layer chromatography (TLC) as well as by high performance liquid chromatography (HPLC). Our results showed that the strain required the precursor L-tryptophan for the synthesis of IAA. Leptolyngbya strain MMG-1 accumulated IAA intracellularly. The IAA secreted by Leptolyngbya strain MMG-1 was significantly correlated with the initial concentration of L-tryptophan in the medium, as well as with the duration of the incubation. The bioactivity of the secreted IAA was determined by its effect on the rooting pattern of Pisum sativum seedlings. The culture supernatant of Leptolyngbya strain MMG-1 stimulated the seedling lateral rooting, while it decreased root length. Hence, rhizospheric Leptolyngbya produced auxin under different conditions and affected the plants rooting pattern. Rev. Biol. Trop. 62 (3): 1251-1260. Epub 2014 September 01.

El género Leptolyngbya comprende cianobacterias filamentosas que son importantes en los campos de cultivo de arroz. En la rizosfera, las cianobacterias producen una variedad de metabolitos secundarios, tales como auxinas, que son importantes en el rendimiento de la agricultura del suelo. La cepa Leptolyngbya MMG-1, fue aislada de la rizosfera de plantas de arroz y se describe en este trabajo. La morfología de esta cepa se estudió por microscopía de luz, así como por microscopía confocal de barrido láser. Además, se estimó la capacidad de esta cepa para sintetizar el compuesto bioactivo auxina como se demostró en diversas condiciones de cultivo (diferentes cantidades de triptófano; pH; diferente luz alterna: períodos oscuros; duración de la incubación). La auxina se extrajo a partir del cultivo de la cepa Leptolyngbya MMG-1 y se identificó como ácido indol-3-acético (AA) por cromatografía de capa fina (TLC), así como por cromatografía líquida de alta resolución (HPLC). Nuestros resultados mostraron que la cepa requiere el precursor de L-triptófano para la síntesis de IAA. La cepa Leptolyngbya MMG-1 acumula intracelularmente IAA. El IAA secretada por la cepa Leptolyngbya MMG-1 se correlacionó significativamente con la concentración inicial de L-triptófano en el medio, así como con la duración de la incubación. La bioactividad de la IAA secretada se determinó por su efecto sobre el patrón de enraizamiento de plantas de semillero de Pisum sativum. El sobrenadante del cultivo de la cepa Leptolyngbya MMG-1 estimuló el enraizamiento lateral en la plántula, mientras que se redujo la longitud de la raíz. Por lo tanto, la producción de auxina por Leptolyngbya rizosférica afectó el crecimiento de las plantas.

Character Association Studies in M2 Generation of Fieldpea (Pisum sativum var. arvense L.).

An experiment was carried out with an objective to study the association among characters of fieldpea in M2 generation. The parent material, seeds of PUSA212 variety were irradiated with 10kR, 15kR, 20kR, 25kR and 30kR doses of gamma rays at NBRI, Lucknow. Next day after treatment, the seeds along with control were space planted for raising M1 generation. Each M1 plant was harvested separately. Desirable ten M1 individual plant progenies from each treatment were bulked and laid in RBD for rising M2 generation. Induced mutations delivered fairly good amount of genotypic correlation and phenotypic correlation. Grain yield per plant shown significant positive correlation with number of pods per plant, days to flowering, days to pod setting harvest index, seed protein content and negatively significant correlation with days to maturity as well as plant height at phenotypic level. Path coefficient analysis revealed that most of the characters under study exhibited positive direct effect on grain yield per plant at both genotypic and phenotypic level. However days to pod setting shown negative direct effect on grain yield per plant yield at both genotypic and phenotypic level. Thus pods per plant, harvest index determining grain yield indicating scope for improving fieldpea yield by selection. The mutants with small pods, tall and increased number of pods per plant were isolated in M2 generation.

Phytotoxic effect of leachates of industrial solid waste on the growth of Pisum sativum.

The present investigation deals with the study on the phytotoxicity of leachates obtained from Eveready Industries India Ltd., Flashlight Lucknow. Analysis of heavy metals in leachates and effluent showed that leachates contained heavy metals in the following order Ni>Zn>Cr>Cu, whereas effluent mainly contained Hg in high (0.22 mg ml-1) concentration. Phytotoxicity of leachates on different parameters of P. sativum was studied in pot experiment. The results revealed that leachate induced concentration dependent decline in the germination percentageand protein content. However, lower concentration of leachate (5-20%) showed stimulatory effect on the chlorophyll and carotenoid content. A continuous dumping of such solid waste in the vicinity of populated area will ultimately cause surface and ground water pollution due to leaching of the waste.

Comparative analysis of the antibacterial activity of some phytolectins.

The aim of this work was to analyse the comparative effects of the antibacterial properties of partially purified lectins from the seeds of Artocarpus heterophyllus (jack fruit), Canavalia ensiformis (jack bean), Lens culinaris (lentil) and Pisum sativum (pea) against the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The lectins were isolated by partial purification using ammonium sulphate precipitation and dialysis. The antimicrobial activity was studied using agar well diffusion method. The results showed that the Jack fruit lectin had a potent anti-bacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa whereas Pea and jack bean lectin were found to be effective bacteriostatic agents which reduced the growth of bacteria and lentil lectin showed the least antibacterial activity. A comparison of the antibacterial activity of phytolectins with conventional antibiotics namely ampicillin and tetracycline was also carried out. Studies revealed that the antibacterial activities of the conventional antibiotics are higher than that of the plant extracts at the same concentration in accordance to literature.

Increasing the rate of drying reduces metabolic imbalance, lipid peroxidation and critical water content in radicles of garden pea (Pisum sativum L.)

Orthodox seeds become desiccation-sensitive as they undergo germination. As a result, germinating seeds serve as a model to study desiccation sensitivity in plant tissues. The effects of the rate of drying on the viability, respiratory metabolism and free radical processes were thus studied during dehydration and wet storage of radicles of Pisum sativum. For both drying regimes desiccation could be described by exponential and inverse modified functions. Viability, as assessed by germination capacity and tetrazolium staining, remained at 100% during rapid (< 24 h) desiccation. However, it declined sharply at c. 0.26 g g¹ dm following slow (c. 5 days) drying. Increasing the rate of dehydration thus lowered the critical water content for survival. Rapid desiccation was also associated with higher activities and levels of malate dehydrogenase and the oxidized form of nicotinamide adenine dinucleotide. It was also accompanied by lower hydroperoxide levels and membrane damage. In addition, the activitiy of glutathione reductase was greater during rapid drying. Ageing may have contributed to increased damage during slow dehydration, since viability declined even in wet storage after two weeks. The results presented are consistent with rapid desiccation reducing the accumulation of damage resulting from desiccation-induced aqueous-based deleterious reactions. In addition, they show that radicles are a useful model to study desiccation sensitivity in plant tissues.

Comparative analysis of the antibacterial activity of some phytolectins.

The aim of this work was to analyse the comparative effects of the antibacterial properties of partially purified lectins from the seeds of Artocarpus heterophyllus (jack fruit), Canavalia ensiformis (jack bean), Lens culinaris (lentil) and Pisum sativum (pea) against the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa. The lectins were isolated by partial purification using ammonium sulphate precipitation and dialysis. The antimicrobial activity was studied using agar well diffusion method. The results showed that the Jack fruit lectin had a potent anti-bacterial activity against S. aureus, B. subtilis, E. coli and P. aeruginosa whereas Pea and jack bean lectin were found to be effective bacteriostatic agents which reduced the growth of bacteria and lentil lectin showed the least antibacterial activity. A comparison of the antibacterial activity of phytolectins with conventional antibiotics namely ampicillin and tetracycline was also carried out. Studies revealed that the antibacterial activities of the conventional antibiotics are higher than that of the plant extracts at the same concentration in accordance to literature.

Impact of dyeing industry effluent on germination and growth of pea (Pisum sativum).

Dye industry effluent was analyzed for physico-chemical characteristics and its impact on germination and growth behaviour of Pea (Pisum sativum). The 100% effluent showed high pH (10.3) and TDS (1088 mg l-1). The germination parameters included percent germination, delay index, speed of germination, peak value and germination period while growth parameters comprised of root and shoot length, root and shoot weight, root-shoot ratio and number of stipules. The study showed the maximum values of positive germination parameters viz. speed of germination (7.85), peak value (3.28), germination index (123.87) and all growth parameters at 20% effluent concentration while the values of negative germination parameters viz. delay index (-0.14) and percent inhibition (-8.34) were found to be minimum at 20% effluent concentration. The study demonstrated that at lower concentrations the dyeing industry effluent caused a positive impact on germination and growth of Pisum sativum.

Studies on antioxidative enzymes induced by cadmium in pea plants (Pisum sativum).

Pea plants (Pisum sativum cv. Swati) exposed to different concentration of cadmium (50, 100, 200 9M Cd) under controlled glass house conditions were quantified for different physiological parameters and antioxidative enzymes. In pea plants, Cd produced a significant inhibition of growth and induced chlorosis, marginal yellowing and necrosis in young leaves, the effect being most pronounced at 200 9M Cd supply. An alteration in the activated oxygen metabolism of pea plants were also detected as evidenced by an increase in concentration of H2O2 and TBARS along with decrease in the chlorophyll and carotenoid concentration in leaves. Cadmium toxicity induced an increase in non- protein thiol, ascorbate, proline and cysteine concentration. A significant increment in the activity of SOD, APX and GR, and a decrease in CAT was observed as a result of Cd treatment. The enhanced activity of SOD and inhibition of CAT and POD produces a high build up of H2O2 which appears to be the main cause of oxidative stress due to Cd toxicity in pea plants.

Response of ultraviolet-B and nickel on pigments, metabolites and antioxidants of Pisum sativum L.

Ultraviolet radiation (UV) though harmful but is an important and unavoidable component of terrestrial ecosystem to which plants have been exposed since their migration from aquatic to land habitat. Incoming UV-B radiation and heavy metals abundance in contaminated soils are significant environmental threat affecting metabolic functions of plants through generation of reactive oxygen species. Plants have evolved mechanisms to counteract these reactive radicals and to repair the damage caused by UV-B and heavy metals. This study describes the impact of supplemental UV-B (sUV-B) and nickel (Ni) singly and in combination on photosynthetic pigments, flavonoids, enzymatic and non-enzymatic antioxidants, metabolites and lipid peroxidation of Pisum sativum L. (pea) plants. Compared to the controls, both the stresses individually and in combination led to reductions in photosynthetic pigments, ascorbic acid, protein and catalase (CAT) activity, whereas a reverse trend was observed for flavonoids, phenol, proline and thiol contents, superoxide dismutase (SOD) and peroxidase (POX) activities and lipid peroxidation (LPO). However, flavonoids increased significantly under individual exposure of sUV-B as compared to other treatments. An increase of LPO by 81% indicated the generation of reactive oxygen species under both the stress conditions. sUV-B and Ni in combination acted synergistically with stimulation of CAT activity by 51.6% , additively on SOD activity with increase of 16.4%, while other parameters showed antagonistic action of both the stresses.

Control of Erysiphe pisi Causing Powdery Mildew of Pea (Pisum sativum) by Cashewnut (Anacardium occidentale) Shell Extract

The effect of methanolic extract of cashewnut (Anacardium occidentale) shell extract was seen on conidial germination of Erysiphe pisi and powdery mildew development in pea (Pisum sativum). Maximum conidial germination inhibition of E. pisi on glass slides was observed at 300 ppm. Similar effect on floated pea leaves was observed after 48 h at the same concentration. Conidial germination on intact untreated pea leaves was also assessed on II and IV nodal leaves while IV and II nodal leaves were treated with the extract and vice versa. There was tremendous reduction in conidial germination on all the nodal leaves. The disease intensity of pea powdery mildew was significantly reduced by methanolic extract of cashewnut shells. Maximum reduction was observed with 200 ppm where 39% disease intensity was recorded in comparison to 96.53% in the control. The phenolic acid content of pea leaves following treatments with this extract varied and no definite pattern was observed. Out of several phenolic compounds, namely, gallic, ferulic, chlorogenic, and cinnamic acids, only gallic acid was found to be present consistently in all the treatments with varied amounts.

Recessive monogenic mutation in grain pea (Pisum sativum) that causes pyridoxine requirement for growth and seed production.

A stable pyridoxine-deficient pea mutant was obtained by screening the M2 progeny of azide-treated Pisum sativum cv Pusa Harbhajan. The mutation is visible lethal. The isolation of pyridoxine-deficient mutant demonstrates directly that pea plants synthesize their own pyridoxine and that pyridoxine is an essential growth factor for pea plants. The mutant character is determined by homozygous recessive alleles, designated pdx-1, at a single locus. Pyridoxine-deficient plants are fertile and indistinguishable from the wild type if supplied exogenously with 2 mg of pyridoxine.