Author
Abstract: This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied.1. IntroductionThe Amazon forest area represents 50% of the world’s remaining rainforests [1]. This biome spreads across Brazil, Bolivia, Peru, Ecuador, Colombia, Venezuela, Republic of Guyana, and French Guyana. The Amazon is the largest Brazilian biome and occupies an area of 4,196,943 km2, corresponding to 67% of the Brazilian territory [2]. The Amazon forest provides important ecosystem services such as hydrological cycles and carbon sequestration and storage. More importantly, it hosts over 20% of all plant and animal species in the world [3], indicating its high species diversity.Amazon’s biodiversity encompasses not only macroflora and macrofauna but also its microorganisms, which are often neglected. Mineral materials and organic compounds present in soil create distinct microhabitats populated by different microbial communities. Microorganisms are crucial to the balance of ecosystems, with soil microbial communities playing important roles in soil fertility, plant health, and essential biogeochemical processes such as nitrification, ammonia oxidation, and methanogenesis [4–7].As of 2010, oil palm was cultivated on 112,500 hectares of land in Brazil [8], primarily in the Amazon region. Oil palm (Elaeis guineensis Jacq.) is a highly productive perennial crop, yielding 2,000–8,000 kg oil/ha [8]. The oil, which is extracted from the fruit, has diverse applications in the food and cosmetic industries and can also be used for biodiesel production [9].Although the Amazon is one of the most species-rich biomes on Earth, little is known about its archaeal diversity. To date only one published microbial ecology study has focused on Amazon soil archaeal diversity using 16S rRNA gene sequencing [10], and only one soil type (i.e., Amazonian dark earth, also called Terra Preta) was studied.The archaeal taxonomy is a matter of constant change since its proposal, in 1977. Initially two phyla were recognized: Crenarchaeota and Euryarchaeota, but in the subsequent years, many new phyla were proposed. One example is the Thaumarchaeota phylum [11], composed predominantly of mesophilic members; it encompasses the ammonia-oxidizing archaea. The phylum Korarchaeota was proposed in 1996, after the identification of DNA sequences from the Obsidian Pool, in Yellowstone National Park [12], composed of one candidate thermophilic species, whose genome was completely sequenced [13]. Recent works have described putative new phyla, such as Nanoarchaeota, Aigarchaeota, Aenigmarchaeota, Parvarchaeota, and Lokiarchaeota, but these phyla are not widely accepted yet, due to the low number of specimens or DNA sequences available. In addition, further analyses have positioned sequences belonging to these phyla in already described phyla, such as Euryarchaeota or Thaumarchaeota [14–17]. There are some archaeal groups, such as MCG (Miscellaneous Crenarchaeotic Group), which are poorly characterized in terms of phylogenetic affiliation; recent data revealed that this group is probably more closely related to the phylum Thaumarchaeota than to the Crenarchaeota.Mesophilic archaea seem to play important roles in the cycling of important nutrients such as nitrogen and carbon. The importance of ammonia-oxidizing archaea (AOAs) has been well documented in different ecosystems, such as soils, marine, and freshwater environments, where they sometimes can be found in higher abundance than the ammonia-oxidizing bacteria (AOBs) [18–20]. On the other hand, their real role in nitrification is not yet well understood due to the scarce number of cultured AOAs and the few physiological studies available for this group. The methanogens are among the nonextremophilic archaea, which are widely distributed in anaerobic environments, such as flooded soils, or marine soils and vents. Methanogens are also found in the gut of termites, in rumen of cows, or even in the mouth and intestine of humans. These organisms play important roles in the carbon cycle, transforming small compounds such as acetate and propionate into methane, and removing the hydrogen, which is potentially hazardous to some bacterial cells. On the other hand, methane production is one of the major gases involved in the planet’s greenhouse effect (reviewed by [21]).There are several studies associating land use with changes in the structure and abundance of soil microbial communities, such as the influence of the land use over the diversity of AOA and AOB in grassland soils [22] and the impacts of edaphic factors on those archaea in tropical soils [23]. Therefore, the conversion of native forest into palm tree culture can be another example of a potential impact of the oil palm cultivation in the archaeal communities of Amazonian soils.This work aimed to improve our understanding of how the soil archaeal community is impacted by oil palm cultivation. To this end, microbial DNA was extracted from soil samples from native forest and an adjacent area under oil palm cultivation. The archaeal 16S ribosomal RNA (rRNA) gene was amplified and sequenced using high-throughput methods for comparative analysis. Here we show for the first time that soil archaeal diversity is reduced in soil under oil palm cultivation compared to native forest soil.2. Materials and Methods2.1. Site Description, Sampling, and ProcessingSoil samples were collected in the State of Pará, Brazil, in an oil palm-cultivated area and an adjacent area of Amazon native forest near the city of Moju (Figure 1). The tropical forest in this region is dense, with trees that are 25–35 m tall [24]. The climate is equatorial, hot, and humid (Ami type according to the Köppen climate classification). Annual temperatures range from 25°C to 27°C, and rainfall is 2,000–3,000 mm per year being irregularly distributed [25]. The soil is predominantly “Latossolo Amarelo” (a type of Oxisol) [26].Figure 1: (a) Native forest and (b) oil palm-cultivated sites.The oil palm cultivation in the sampled farm is not as controlled as other crops (Figure 1(b)). There is no irrigation regime; natural precipitation of the rainforest is the only way these plants are irrigated. In the Amazon, the soil is very moist, due to the high precipitation levels during the year. In the studied area, the annual period of flooding is from February to April. Furthermore, the soil around the palm trees is not fertilized in a homogeneous fashion, since only one side of the plants is directly fertilized.In October 2010, after plant litter was removed, a soil borer was used to obtain four 10 cm deep soil samples from three points in the oil palm-cultivated area (S02°00′28.9′′/W048°37′57.4′′, S02°00′29.2′′/W048°37′56.6′′, and S02°00′31.3′′/W048°37′54.3′′) (Figure 1(b)) and four samples from the native Amazon forest area (S02°00′27.2′′/W048°35′53.0′′) (Figure 1(a)). The samples collected in each area were mixed, ground, and sieved to remove larger particles, yielding one composite sample for each area, with approximately 1 kg each. The samples were stored in plastic bags on dry ice during transportation. A subsample was sent to physicochemical analysis at SoloQuímica Análises de Solo Ltda. (Brasília, DF, Brazil). The rest of the samples were then stored at −80°C until DNA extraction. Initially, the physicochemical characteristics of the soils samples were evaluated individually and a high variation among replicas was observed. This result was due to the heterogeneous fertilization of the palm trees in the cultivation fields in Amazon; therefore, composite samples were necessary to describe the microbiota in oil palm soil.2.2. DNA Extraction, PCR, and Pyrosequencing AnalysisTotal DNA was extracted according to the protocol of Smalla et al. [27], using 2 g soil per sample. To minimize DNA extraction bias, this procedure was performed in quadruplicate. PCR reactions were performed using the following primers specific for Archaea: 340F (5′-CCC TAY GGG GYG CAS CAG-3′) and 1000R (5′-GGC CAT GCA CYW CYT CTC-3′) [28]. Archaea 16S rRNA genes were amplified, yielding 660 bp amplicons. Adapters used as priming sites for both amplification and sequencing (454 Life Sciences, Branford, CT, USA) were ligated to the 5′ end of the primer sequences. Each 20 μL PCR reaction contained 10–30 ng total DNA, 1x reaction buffer, 4 μM dNTP, 10 μM of each primer, 200 μg/mL bovine serum albumin, 0.5 U KAPA2G Robust HotStart polymerase, and Milli-Q water. Amplification was performed in an Applied Biosystems GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using the following program: 2 min at 98°C; followed by 30 cycles of 30 seconds at 95°C, 30 seco
For further details log on website :
http://www.scilit.net/article/10.1155/2016/3762159
Archaea, Volume 2016, pp 1-14; doi:10.1155/2016/3762159
Abstract: This study compared soil archaeal communities of the Amazon forest with that of an adjacent area under oil palm cultivation by 16S ribosomal RNA gene pyrosequencing. Species richness and diversity were greater in native forest soil than in the oil palm-cultivated area, and 130 OTUs (13.7%) were shared between these areas. Among the classified sequences, Thaumarchaeota were predominant in the native forest, whereas Euryarchaeota were predominant in the oil palm-cultivated area. Archaeal species diversity was 1.7 times higher in the native forest soil, according to the Simpson diversity index, and the Chao1 index showed that richness was five times higher in the native forest soil. A phylogenetic tree of unclassified Thaumarchaeota sequences showed that most of the OTUs belong to Miscellaneous Crenarchaeotic Group. Several archaeal genera involved in nutrient cycling (e.g., methanogens and ammonia oxidizers) were identified in both areas, but significant differences were found in the relative abundances of Candidatus Nitrososphaera and unclassified Soil Crenarchaeotic Group (prevalent in the native forest) and Candidatus Nitrosotalea and unclassified Terrestrial Group (prevalent in the oil palm-cultivated area). More studies are needed to culture some of these Archaea in the laboratory so that their metabolism and physiology can be studied.1. IntroductionThe Amazon forest area represents 50% of the world’s remaining rainforests [1]. This biome spreads across Brazil, Bolivia, Peru, Ecuador, Colombia, Venezuela, Republic of Guyana, and French Guyana. The Amazon is the largest Brazilian biome and occupies an area of 4,196,943 km2, corresponding to 67% of the Brazilian territory [2]. The Amazon forest provides important ecosystem services such as hydrological cycles and carbon sequestration and storage. More importantly, it hosts over 20% of all plant and animal species in the world [3], indicating its high species diversity.Amazon’s biodiversity encompasses not only macroflora and macrofauna but also its microorganisms, which are often neglected. Mineral materials and organic compounds present in soil create distinct microhabitats populated by different microbial communities. Microorganisms are crucial to the balance of ecosystems, with soil microbial communities playing important roles in soil fertility, plant health, and essential biogeochemical processes such as nitrification, ammonia oxidation, and methanogenesis [4–7].As of 2010, oil palm was cultivated on 112,500 hectares of land in Brazil [8], primarily in the Amazon region. Oil palm (Elaeis guineensis Jacq.) is a highly productive perennial crop, yielding 2,000–8,000 kg oil/ha [8]. The oil, which is extracted from the fruit, has diverse applications in the food and cosmetic industries and can also be used for biodiesel production [9].Although the Amazon is one of the most species-rich biomes on Earth, little is known about its archaeal diversity. To date only one published microbial ecology study has focused on Amazon soil archaeal diversity using 16S rRNA gene sequencing [10], and only one soil type (i.e., Amazonian dark earth, also called Terra Preta) was studied.The archaeal taxonomy is a matter of constant change since its proposal, in 1977. Initially two phyla were recognized: Crenarchaeota and Euryarchaeota, but in the subsequent years, many new phyla were proposed. One example is the Thaumarchaeota phylum [11], composed predominantly of mesophilic members; it encompasses the ammonia-oxidizing archaea. The phylum Korarchaeota was proposed in 1996, after the identification of DNA sequences from the Obsidian Pool, in Yellowstone National Park [12], composed of one candidate thermophilic species, whose genome was completely sequenced [13]. Recent works have described putative new phyla, such as Nanoarchaeota, Aigarchaeota, Aenigmarchaeota, Parvarchaeota, and Lokiarchaeota, but these phyla are not widely accepted yet, due to the low number of specimens or DNA sequences available. In addition, further analyses have positioned sequences belonging to these phyla in already described phyla, such as Euryarchaeota or Thaumarchaeota [14–17]. There are some archaeal groups, such as MCG (Miscellaneous Crenarchaeotic Group), which are poorly characterized in terms of phylogenetic affiliation; recent data revealed that this group is probably more closely related to the phylum Thaumarchaeota than to the Crenarchaeota.Mesophilic archaea seem to play important roles in the cycling of important nutrients such as nitrogen and carbon. The importance of ammonia-oxidizing archaea (AOAs) has been well documented in different ecosystems, such as soils, marine, and freshwater environments, where they sometimes can be found in higher abundance than the ammonia-oxidizing bacteria (AOBs) [18–20]. On the other hand, their real role in nitrification is not yet well understood due to the scarce number of cultured AOAs and the few physiological studies available for this group. The methanogens are among the nonextremophilic archaea, which are widely distributed in anaerobic environments, such as flooded soils, or marine soils and vents. Methanogens are also found in the gut of termites, in rumen of cows, or even in the mouth and intestine of humans. These organisms play important roles in the carbon cycle, transforming small compounds such as acetate and propionate into methane, and removing the hydrogen, which is potentially hazardous to some bacterial cells. On the other hand, methane production is one of the major gases involved in the planet’s greenhouse effect (reviewed by [21]).There are several studies associating land use with changes in the structure and abundance of soil microbial communities, such as the influence of the land use over the diversity of AOA and AOB in grassland soils [22] and the impacts of edaphic factors on those archaea in tropical soils [23]. Therefore, the conversion of native forest into palm tree culture can be another example of a potential impact of the oil palm cultivation in the archaeal communities of Amazonian soils.This work aimed to improve our understanding of how the soil archaeal community is impacted by oil palm cultivation. To this end, microbial DNA was extracted from soil samples from native forest and an adjacent area under oil palm cultivation. The archaeal 16S ribosomal RNA (rRNA) gene was amplified and sequenced using high-throughput methods for comparative analysis. Here we show for the first time that soil archaeal diversity is reduced in soil under oil palm cultivation compared to native forest soil.2. Materials and Methods2.1. Site Description, Sampling, and ProcessingSoil samples were collected in the State of Pará, Brazil, in an oil palm-cultivated area and an adjacent area of Amazon native forest near the city of Moju (Figure 1). The tropical forest in this region is dense, with trees that are 25–35 m tall [24]. The climate is equatorial, hot, and humid (Ami type according to the Köppen climate classification). Annual temperatures range from 25°C to 27°C, and rainfall is 2,000–3,000 mm per year being irregularly distributed [25]. The soil is predominantly “Latossolo Amarelo” (a type of Oxisol) [26].Figure 1: (a) Native forest and (b) oil palm-cultivated sites.The oil palm cultivation in the sampled farm is not as controlled as other crops (Figure 1(b)). There is no irrigation regime; natural precipitation of the rainforest is the only way these plants are irrigated. In the Amazon, the soil is very moist, due to the high precipitation levels during the year. In the studied area, the annual period of flooding is from February to April. Furthermore, the soil around the palm trees is not fertilized in a homogeneous fashion, since only one side of the plants is directly fertilized.In October 2010, after plant litter was removed, a soil borer was used to obtain four 10 cm deep soil samples from three points in the oil palm-cultivated area (S02°00′28.9′′/W048°37′57.4′′, S02°00′29.2′′/W048°37′56.6′′, and S02°00′31.3′′/W048°37′54.3′′) (Figure 1(b)) and four samples from the native Amazon forest area (S02°00′27.2′′/W048°35′53.0′′) (Figure 1(a)). The samples collected in each area were mixed, ground, and sieved to remove larger particles, yielding one composite sample for each area, with approximately 1 kg each. The samples were stored in plastic bags on dry ice during transportation. A subsample was sent to physicochemical analysis at SoloQuímica Análises de Solo Ltda. (Brasília, DF, Brazil). The rest of the samples were then stored at −80°C until DNA extraction. Initially, the physicochemical characteristics of the soils samples were evaluated individually and a high variation among replicas was observed. This result was due to the heterogeneous fertilization of the palm trees in the cultivation fields in Amazon; therefore, composite samples were necessary to describe the microbiota in oil palm soil.2.2. DNA Extraction, PCR, and Pyrosequencing AnalysisTotal DNA was extracted according to the protocol of Smalla et al. [27], using 2 g soil per sample. To minimize DNA extraction bias, this procedure was performed in quadruplicate. PCR reactions were performed using the following primers specific for Archaea: 340F (5′-CCC TAY GGG GYG CAS CAG-3′) and 1000R (5′-GGC CAT GCA CYW CYT CTC-3′) [28]. Archaea 16S rRNA genes were amplified, yielding 660 bp amplicons. Adapters used as priming sites for both amplification and sequencing (454 Life Sciences, Branford, CT, USA) were ligated to the 5′ end of the primer sequences. Each 20 μL PCR reaction contained 10–30 ng total DNA, 1x reaction buffer, 4 μM dNTP, 10 μM of each primer, 200 μg/mL bovine serum albumin, 0.5 U KAPA2G Robust HotStart polymerase, and Milli-Q water. Amplification was performed in an Applied Biosystems GeneAmp® PCR System 9700 Thermal Cycler (Applied Biosystems, Foster City, CA, USA) using the following program: 2 min at 98°C; followed by 30 cycles of 30 seconds at 95°C, 30 seco
For further details log on website :
http://www.scilit.net/article/10.1155/2016/3762159
No comments:
Post a Comment