Abstract. Resumen. Roger Guillermo Cañón-Cortázar 1,4, Lizeth Manuela Avellaneda-Torres 2,4, and Esperanza Torres- Rojas 3,4* - PDF

Associated microorganisms to the nitrogen cycle in soils under three systems of use: potato crop, livestock and páramo, in Los Nevados National Natural Park, Colombia Microorganismos asociados al ciclo

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Associated microorganisms to the nitrogen cycle in soils under three systems of use: potato crop, livestock and páramo, in Los Nevados National Natural Park, Colombia Microorganismos asociados al ciclo del nitrógeno en suelos bajo tres sistemas de uso: cultivo de papa, ganaderia y páramo, en el Parque Los Nevados, Colombia Roger Guillermo Cañón-Cortázar 1,4, Lizeth Manuela Avellaneda-Torres 2,4, and Esperanza Torres- Rojas 3,4* 1 Agronomist Engineer. 2 Chemist, M.Sc.. 3 Biologist, Ph.D., Associated profesor, exclusive dedication. 4 Faculty of Agronomy, Universidad Nacional de Colombia and Colombian Center of Genomics and Bioinformatics in Extreme environments GEBIX, Bogot[a, Colombia. *corresponding author: Rec.: Acept.: Abstract This study estimated the population density of microorganisms associated with the metabolism of nitrogen (N) in soils with three uses: potato crop, livestock and low intervention zones (high plains or páramo) in El Bosque district of Los Nevados National Natural Park in Colombia. The following microorganisms were indirectly quantified: amonifiers (AMO), proteolytic (PRO), ammonium oxidizing (BOA), nitrite oxidizing (BON) and denitrifiers (DEN) in soils of three farms located at different altitudes and under two climatic seasons. The non-parametric Kruskal-Wallis paired test with a significance level of P 0.05 was used to detect statistically significant differences among abundances of functional groups of microorganisms. Then a principal component analysis (PCA) was performed to evaluate the relationships among different variables. The results showed differences among all the groups, with páramo soils having the lowest density of microorganisms in the soil. The PRO microorganisms were the most abundant in all soils, followed by DEN, with a higher density in the páramo ecosystem. The microbial population density was not significantly affected by altitude but showed some differences that did not have a unique behavior pattern. Additionally, differences were seen in the seasons, being higher in the dry season for the PRO, AMO, BOA and BON groups; and in the rainy season for DEN. This study provided knowledge of the relationships between land use and the presence of microorganisms associated with the nitrogen cycle. Key words: Ammonium oxidizing, amonifiers, denitrifiers, nitrite oxidizing, proteolytic. Resumen En la vereda El Bosque del Parque Nacional Natural de Los Nevados (PNNN), Colombia, se estimó la densidad poblacional de microorganismos asociados con el metabolismo de nitrógeno (N) en suelos bajo tres sistemas de uso: cultivo de papa, ganadería y zonas poco intervenidas (páramo). Indirectamente se cuantificaron microorganismos amonificantes (AMO), proteolíticos (PRO), oxidantes del amonio (BOA), oxidantes del nitrito (BON) y denitrificantes (DEN) en suelos de tres fincas ubicadas a diferente altitud y en épocas seca y lluviosa. Se realizó la prueba no-paramétrica de Kruskal-Wallis por pares, con un nivel de significancia P 0.05, para detectar diferencias estadísticamente significativas entre las diferentes abundancias de grupos funcionales de microorganismos. Luego, se realizó análisis de componentes 339 ASSOCIATED MICROORGANISMS TO THE NITROGEN CYCLE IN SOILS UNDER THREE SYSTEMS OF USE: POTATO CROP, LIVESTOCK AND PÁRAMO, IN LOS NEVADOS NATIONAL NATURAL PARK, COLOMBIA principales (ACP) para evaluar las relaciones entre las diferentes variables estudiadas. Los resultados mostraron diferencias para los grupos evaluados, siendo menor la densidad de microorganismos en suelos de páramo. Los PRO fueron más abundantes en todos los tipos de suelos, seguido de los DEN con mayor densidad en el ecosistema páramo. La densidad poblacional microbiana no fue afectada por la altitud, sin embargo se presentaron algunas diferencias que no mostraron un patrón único de comportamiento. Se observaron diferencias como resultado de la época climática, siendo mayor en la época seca para los grupos PRO, AMO, BOA y BON, y en época de lluvias para los DEN. Este estudio contribuye al conocimiento de las relaciones que existen entre el uso del suelo y la presencia de los microorganismos asociados con el ciclo de nitrógeno. Palabras clave: Amonificantes, denitrificantes, oxidantes del amonio, oxidantes del nitrito, proteolíticos. Introduction The microbe community associated to the nitrogen (N) cycle represents an evolutive advantage by fixing atmospheric N 2 to convert it in forms assimilable for other organisms. The N is present in several forms, which are transformed during the cycle by the action of amonifiers (AMO), proteolytic (PRO), ammonium oxidizing (BOA), nitrite oxidizing (BON) and denitrifiers (DEN) microorganisms, among others (Loomis and Connor, 2002). Knowledge on the diversity and metabolic capacity of the soil microorganisms associated to the N cycle is scarce. However, there are different methods to analyze the crop dependent and independent microbe communities (Nannipieri et al., 2003). Among the first ones is the most-probable-number (MPN) technique which indirectly estimates the population density without doing a real counting of the microorganisms or colonies. Despite that the generated information id restricted only to communities that can be cultured and is sensitive to the inoculum density (Kirk et al., 2004), this is a fast and cheap technique that generates useful information to estimate the abundance of the microbe community in the soil. The type of soil is considered as a factor that directly or indirectly controls the structure of the microbe community in the soil (Han et al., 2011). Microbe biomass is one of the indicators that better reflects responses to environmental changes, even better than physical and chemical parameters (Kaschuk et al., 2011), and therefore, is fundamental for the evaluation of soil quality (Acosta-Martínez et al., 2008). Páramo is located between 3200 and 4500 MASL and constitutes a fragile ecosystem of great importance for water storage and hydric regulation (Hofstede et al., 2003). In Colombia this ecosystem corresponds to an area of 30,000 km 2, being the country with the largest area of páramos (Ramírez, 2011) and in Los Nevados National Natural Park (PNNN) is the most representative ecosystem with an estimated area of 38,600 ha (66.21%) (PNN de Colombia, 2011). In the Colombian Páramos potato is cultivated and livestock activities are developed, however, it is unknown the effect of these practices on soil properties and microbe communities. Therefore, the objective of this study was to estimate the population density of microorganisms associated to N metabolism in soils of the farm El Bosque in the PNNN, used for potato crop and livestock, compared to páramo soils with native vegetation. It is expected the establishment of possible relationships between soil use and presence of microorganisms that belong to different functional groups associated to N metabolism. Materials and methods Location and sample collection Samples from rhizosferic soils for this study were taken in the farms Buenos Aires (3769 MASL), El Edén (3590 MASL) and La Secreta (3432 MASL) in the location El Bosque, Pereira, Risaralda. In each location were evaluated soil uses: páramo forest, potato (Solanum tuberosum) crop and livestock in the dry and 340 ACTA AGRONÓMICA. 61 (4) 2012, p rainy season. Livestock is double-purpose (milk and meat production), with emphasis on milk production given the zone characteristics. Grasses sowed in the zone are Orchoro (Dactylis glomerata), Raygrass (Lolium sp.) and Plegadera (Lachemilla sp.). Páramo has predominantly vegetation of Cortaderia selloana, Pernettya prostrata, Buddleja sp., Lupinus albus, Dendropanax sp., Chusquea sp. in each one of the soi use types three samplings were performed, composed of 10 subsamples, for a total of 54 samples. In each sample was determined the abundance of five microorganism functional groups associated with N metabolism: PRO, AMO, BOA, BON and DEN. Microorganism abundance and culture media Microorganism abundance for the different functional groups was estimated by the MPN method (Cochran, 1950). Different culture media and carbon and N sources were used according to the functional group to study. For PRO and BOA the culture media was prepared using as basis the Winogradsky saline solution (Pochon, 1954) at a rate of 50 ml/l supplemented with a trace element solution (1 ml/l) (Balch et al., 1977). For the PRO microorganisms it was used gelatin (30 g/l) as only carbon and N source for an incubation time of 15 days. For BOA microorganisms it was used calcium carbonate (1 g/l) and ammonium sulfate (0.5 g/l), as carbon and N sources respectively, and an incubation time of 30 days (Flórez-Zapata and Uribe-Vélez, 2011). For AMO microorganisms it was used as culture media and only carbon and N source bacteriological peptone (2 g/l) and an incubation time of 24 h (IGAC, 2006). To isolate BON it was used as culture media potassium nitrite (0.006 g/l), dipotassium phosphate (1 g/l), iron sulfate heptahydrate (0.03 g/l), sodium (0.3 g/l), magnesium sulfate heptahydrate (0.1 g/l), calcium carbonate (1 g/l) and calcium chloride (0.3 g/l); incubation time was 96 h (IGAc, 2006). Finally, for DEN microorganisms the culture media used was a mix of two solutions, A and B, with the following composition, solution A: potassium nitrate (2 g/l), asparagine (2 g/l), bromothymol blue 1% (10 g/l); solution B: sodium citrate (17 g/l), monopotassium phosphate (2 g/l), magnesium sulfate (2 g/l), calcium chloride hexahydrate (0.4 g/l), ferric chloride hexahydrate (0.1 g/l); incubation time was 72 h (IGAC, 2006) at 28 C. Microorganisms count Presence or absence of organisms was determined using as characteristics the liquefaction of gelatin (PRO) and nitrite production, measured as Griess Ilosvay reactive (BOA) (Flórez-Zapata and Uribe-Vélez, 2011). The nitrite consumption was determined with the Griess Ilosvay reactive (BON), ammonium production by the Nessler reactive (AMO) and gas production using Durham bells and changes in blue coloration (DEN) (IGAC, 2006). Countings were expressed as colony forming units/g of dry soil (CFU/g). Statistical analysis Data analysis on the different abundances of the microorganisms functional groups was performed by Anova, however, due to the fact that the data do not support the normality principle, the non-parametric Kruskal-Wallis (KW) test by pairs with a significance level P 0.05 was done, in order to detect statistical significant differences (Kruskal and Wallis, 1952). The above stated was done using the statistical software SAS 9.0 (009) and STATA 11.1( Then, a principal component analysis (PCA) was performed to evaluate the relations between the different variables evaluated. Results and discussion Differences in the abundance of the different functional groups (P = ) were found (Table 1). In general, the microbe abundance was lower in páramo soil compared with potato crop and livestock, which did not show differences within them (P = 0.289). According to Roldán et al. (2009) this is possibly due to the fact that in páramo soils the densities of the functional groups associated with N cycle are lower because this use has high plant diversity, offering substrates that favor microorganisms richness, but not diversity, which is different from the intensive management uses. However, the counting strategy by microorganisms culture could be a limiting factor for 341 ASSOCIATED MICROORGANISMS TO THE NITROGEN CYCLE IN SOILS UNDER THREE SYSTEMS OF USE: POTATO CROP, LIVESTOCK AND PÁRAMO, IN LOS NEVADOS NATIONAL NATURAL PARK, COLOMBIA Table 1. Density (CFU/g) of functional groups microorganisms associated with soil metabolism of N. the values are averages of replicates for: soil use (18), altitude (farm) (18) and season of the year (27). Factors Microorganisms density (CFU/g) PRO AMO BOA BON DEN Soil use Páramo 35,059 a* 73 a 20 a 117 a 428 a Potato crop 213,052 b 213 b 1917 b 1297 b 1062 b Livestock 194,416 b 142 b 2001 b 741 b 2447 b Altitude 3769 (Buenos Aires) 175,927 a 130 a 329 a 597 a 722 a 3590 (El Edén) 142,235 a 107 a 923 ab 397 a 2032 b 3432 (La secreta) 127,394 a 191 a 6959 b 891 a 1184 ab Season Rainy 77,392 a 76 a -665 a 372 a 2020 a Dry 219,646 b 210 b -395 a 1064 a 605 b PRO:proteolitics, AMO: amonifiers, BOA: ammonium oxidizing bacteria, BON: nitrite oxidizing bacteria and DEN: denitrifiers. Values on the same column and factor followed by same letters do not significantly differ (P 0.05) possible recuperation and counting of those that are not cultured, which could be acting on a determined role within the N cycle in the soil, although they are not detected. Páramo soils showed lower PRO densities compared with soils used for potato crop and livestock (Table 1) due, possibly, to the increment of nutrients that are easily available in these uses because of application of chemical synthesis fertilizers (N, P, K), as well as the mechanical labors that are done in these soils which increases their mineralization processes and, therefore, nutrient availability for microorganism growth. However, livestock soils show lower microorganism densities when compared to potato crop, as they are part of a rotation system in which potato is sowed for two years and then they are used for livestock (Ramírez, 2011). PRO density did not change (P 0.05) for the altitude effect, but for the effect of the season (P = ), being the abundance higher in the dry season (Table 1). Possibly, this is due to the fact that in the rainy season the water affects soil temperature, humidity and ph, factors that affect microbial activity (Roldán et al., 2009). AMO density changed for the soil use effect (P = 0.001) with lower density in the páramo ecosystem (Table 1). Calvo-Vélez et al., (2008) state that in tropical soils the ammonification rate is affected by soil ph; thus, a ph between 5.5 and 6.0 is ideal for a maximum microorganism activity, but in more acidic conditions the activity is reduced. In this study there were no differences found in ph due to soil use, therefore this factor might not be a limiting condition for ammonification in the páramo agroecosystem. One of the factors that can condition low AMO density in páramo is the low amount of substrate coming from PRO. Differences in the AMO densities in the altitudes of this study were not significant (P 0.05). On the other hand, in the dry season it was found a higher density than in the rainy season. This behavior of the AMO and PRO microorganisms is consistent as in soils with less PRO there were also less AMO, which is coherent because the PRO products are potential substrates for AMO. However, the later ones can be aerobic or anaerobic and in some cases they are able to form resistant reproductive structures that are viable during adverse periods when there is low humidity, low nutrient availability or flooding (Ponzuelo, 1991). BOA concentrations were different (P ) with respect to the soil use, being the livestock soils the ones with the highest densities (Table 1), but it was not the same in the potato crop (P = 0.526); similarly to the previous groups, BOA density was lower in the páramo ecosystem. Flórez-Zapata (2010) found 1.5x10 2 propagules/g of BOA in soil cultivated with potato, and Philippot and Germon (2005) consider that densities between 10 5 and 10 7 CFU bacterial/g are co- 342 ACTA AGRONÓMICA. 61 (4) 2012, p mmon in agricultural soils BOA densities in livestock soils could be associated with the increase in organic residues by cattle manure and animal urine, which are an ureic substrate that promotes microorganism growth (Orozco-Patio, 1999). The increase of microorganisms in cropping soils compared with páramo can be associated with fertilizers use, which increases the N availability that can be used as energy source by chemolithotrophs (BOA and BON) microorganisms (Roldán et al., 2009). Additionally, the total biomass of the microorganisms involved to the N cycle increases with the intensity of soil use in grasses (Berner et al., 2011). However, an amount of NH 4 + larger than 800 mg/kg inhibits the development of autotrophic nitrifying microflora (Ponzuelo, 1991), which makes possible the use of the functional group (BOA) as indicator of changes in soil use (Roldán et al., 2009). BOA had an increase in population density as the altitude was reduced, this could be associated with the increase in temperature happening as altitude is reduced, approximately 0.57 C each 100 m (Guevara-Díaz, 2003). BON had low population density in páramo ecosystem (Table 1), which can be associated with low substrate coming for the oxidation of NH + 4 that is done by BOA in this ecosystem. Ponzuelo (1991) consider that the NO - 2 is a form easily unmovable due to its capacity to react with the soil organic matter (SOM), in special with phenolic compounds. These reactions happen in all type of soils and are favored by acidity and high SOM contents, as the ones found in páramo soils that range from 9.05 and 18.81% (data not shown). BON neither show differences within altitudes of the farms in study nor with the period effect, however higher density was observed for the dry season (Table 1). DEN revealed lower density in páramo and higher in livestock (P = 0.006). Denitrification is a heterotrophic process that occurs in anoxia or hypoxia conditions (Francis et al., 2007) and is related to humid and high bulk density environments, as it is the case in the livestock soil in its first 20 cm that are compacted, favoring high DEN densities (Ramírez, 2011). This also carries a reduction in soil microbe biomass (Li et al., 2004) and an increase of the DEN microorganisms activity, which is close to 5% of the soil microbe biomass (Hai, 2009), that means, that both microorganisms are found in high densities in soils compared to AMO, BOA and BON. Flórez-Zapata (2010) considers that DEN respond to mineral fertilizer applications, because they obtain energy from these compounds. In this study, DEN were affected by the season of the year (P = 0.001), being its density higher in rainy seasons (Table 1). However, in páramo and livestock in the El Edén farm it was observed a higher activity in dry season, possibly due to low variation in soil humidity between seasons (data not published). In the multivariate analysis were observed differences in microorganisms concentrations due to the soil use (Figure 1), where the páramo soil presents a different behavior compared to the potato and livestock soils; the later ones were correlated (0.8 and 0.73, respectively) to the axis 1, while in páramo the correlation to the axis 1 was negative (-0.48). a similar trend between the values of the livestock/potato crop systems could be due to the rotation between them that can cause fragmentation of the humus-allophanes complexes that favor SOM decomposition and increments in densities of microorganisms associated to these processes (Orozco-Patio, 1999). AMO presented a higher density in soils used for livestock, this could be due to the fact that the cattle manner has compounds like methylamine which inhibits the AMO function (Orozco-Patio, 1999). In the same way happens in the crops because of the urea addition during fertilization, as it is expected, chemical fertilizers additions increase the nutrient availability (Roldán et al., 2009). On the other hand, it was observed a lower density of microorganisms in the páramo ecosystem (Table 2 and Figure 1) which can be associated with the SOM stability, because humic substances predominate, allophane is present and do not allow decomposition of this matter due to their stable structure. It was observed that PRO were more abundant in the páramo, which is important 343 ASSOCIATED MICROORGANISMS TO THE NITROGEN CYCLE IN SOILS UNDER THREE SYSTEMS OF USE: POTATO
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