Julieta Orlando

Diversity and Activity of Denitrifiers of Chilean Arid Soil Ecosystems

The Chilean sclerophyllous matorral is a Mediterranean semiarid ecosystem affected by erosion, with low soil fertility, and limited by nitrogen. However, limitation of resources is even more severe for desert soils such as from the Atacama Desert, one of the most extreme arid deserts on Earth. Topsoil organic matter, nitrogen and moisture content were significantly higher in the semiarid soil compared to the desert soil. Although the most significant loss of biologically preferred nitrogen from terrestrial ecosystems occurs via denitrification, virtually nothing is known on the activity and composition of denitrifier communities thriving in arid soils. In this study we explored denitrifier communities from two soils with profoundly distinct edaphic factors. While denitrification activity in the desert soil was below detection limit, the semiarid soil sustained denitrification activity. To elucidate the genetic potential of the soils to sustain denitrification processes we performed community analysis of denitrifiers based on nitrite reductase (nirK and nirS) genes as functional marker genes for this physiological group. Presence of nirK-type denitrifiers in both soils was demonstrated but failure to amplify nirS from the desert soil suggests very low abundance of nirS-type denitrifiers shedding light on the lack of denitrification activity. Phylogenetic analysis showed a very low diversity of nirK with only three distinct genotypes in the desert soil which conditions presumably exert a high selection pressure. While nirK diversity was also limited to only few, albeit distinct genotypes, the semiarid matorral soil showed a surprisingly broad genetic variability of the nirS gene. The Chilean matorral is a shrub land plant community which form vegetational patches stabilizing the soil and increasing its nitrogen and carbon content. These islands of fertility may sustain the development and activity of the overall microbial community and of denitrifiers in particular.

Phylogenetic Diversity of Peltigera Cyanolichens and Their Photobionts in Southern Chile and Antarctica

The lichen genus Peltigera has been mainly revised in the Northern Hemisphere, with most species being recorded in Europe and North America. This study assessed the phylogenetic diversity of the mycobionts and cyanobionts of Peltigera cyanolichens collected in Southern Chile and Antarctica, areas in which lichens are extremely diverse but poorly studied. The operational taxonomic units (OTUs) of each symbiont were defined by analyzing the genetic diversity of the LSU and SSU rDNA of the mycobionts and cyanobionts, respectively, and a phylogenetic approach was used to relate these OTUs with sequences previously reported for Peltigera and Nostoc. Among the 186 samples collected, 8 Peltigera and 15 Nostoc OTUs were recognized, corresponding to sections Peltigera, Horizontales, and Polydactylon, in the case of the mycobionts, and to the Nostoc clade II, in the case of the cyanobionts. Since some of the OTUs recognized in this study had not previously been described in these areas, our results suggest that the diversity of Peltigera reported to date in the regions studied using traditional morphological surveys has underestimated the true diversity present; therefore, further explorations of these areas are recommended.

Genetic diversity of terricolous Peltigera cyanolichen communities in different conservation states of native forest from southern Chile.

Decreasing quality of forest habitats is among the major factors leading to a loss of epiphytic lichen diversity. However, there is little information about how this factor influences the diversity of terricolous lichens, which do not grow over living trees and could be less susceptible to such disturbances. In this work we describe the genetic diversity of Peltigera terricolous cyanolichens and their cyanobiont (Nostoc) from three habitats at the Karukinka Natural Park (Tierra del Fuego, southern Chile), which represent different conservation states: native mature-forest (low disturbance intensity), native young-forest (medium disturbance intensity) and grassland (high disturbance intensity). In both forest contexts, a higher diversity and a higher number of unique OTUs (operational taxonomic units) were found. In contrast, in the grassland, the diversity was lower and the Peltigera species were mostly cosmopolitan. The presence of unique OTUs and the higher diversity of lichens in native forest areas highlight the importance of their preservation, indicating that decreasing forest quality also has a negative impact on terricolous lichens diversity.

Comparison of water availability effect on ammonia-oxidizing bacteria and archaea in microcosms of a Chilean semiarid soil

Water availability is the main limiting factor in arid soils; however, few studies have examined the effects of drying and rewetting on nitrifiers from these environments. The effect of water availability on the diversity of ammonia-oxidizing bacteria (AOB) and archaea (AOA) from a semiarid soil of the Chilean sclerophyllous matorral was determined by microcosm assays. The addition of water every 14 days to reach 60% of the WHC significantly increased nitrate content in rewetted soil microcosms (p < 0.001). This stimulation of net nitrification by water addition was inhibited by acetylene addition at 100 Pa. The composition of AOA and AOB assemblages from the soils microcosms was determined by clone sequencing of amoA genes (A-amoA and B-amoA, respectively), and the 16S rRNA genes specific for β-proteobacteria (beta-amo). Sequencing of beta-amo genes has revealed representatives of Nitrosomonas and Nitrosospira while B-amoA clones consisted only of Nitrosospira sequences. Furthermore, all clones from the archaeal amoA gene library (A-amoA) were related to “mesophilic Crenarchaeota” sequences (actually, reclassified as the phylum Thaumarchaeota). The effect of water availability on both microbial assemblages structure was determined by T-RFLP profiles using the genetic markers amoA for archaea, and beta-amo for bacteria. While AOA showed fluctuations in some T-RFs, AOB structure remained unchanged by water pulses. The relative abundance of AOA and AOB was estimated by the Most Probable Number coupled to Polymerase Chain Reaction (MPN-PCR) assay. AOB was the predominant guild in this soil and higher soil water content did not affect their abundance, in contrast to AOA, which slightly increased under these conditions. Therefore, these results suggest that water addition to these semiarid soil microcosms could favor archaeal contribution to ammonium oxidation.

Environmental context shapes the bacterial community structure associated to Peltigera cyanolichens growing in Tierra del Fuego, Chile

The structure of the associated bacterial community of bipartite cyanolichens of the genus Peltigera from three different environmental contexts in the Karukinka Natural Park, Tierra del Fuego, Chile, was assessed. The sampling sites represent different habitat contexts: mature native forest, young native forest and grassland. Recently it has been determined that the bacterial community associated to lichens could be highly structured according to the mycobiont or photobiont identities, to the environmental context and/or to the geographic scale. However, there are some inconsistencies in defining which of these factors would be the most significant on determining the structure of the microbial communities associated with lichens, mainly because most studies compare the bacterial communities between different lichen species and/or with different photobiont types (algae vs. cyanobacteria). In this work bipartite lichens belonging to the same genus (Peltigera) symbiotically associated with cyanobacteria (Nostoc) were analyzed by TRFLP to determine the structure of the bacterial community intimately associated with the lichen thalli and the one present in the substrate where they grow. The results indicate that the bacterial community intimately associated differs from the one of the substrate, being the former more influenced by the environmental context where the lichen grows.

Substrates of "Peltigera" Lichens as a Potential Source of Cyanobionts

Photobiont availability is one of the main factors determining the success of the lichenization process. Although multiple sources of photobionts have been proposed, there is no substantial evidence confirming that the substrates on which lichens grow are one of them. In this work, we obtained cyanobacterial 16S ribosomal RNA gene sequences from the substrates underlying 186 terricolous Peltigera cyanolichens from localities in Southern Chile and maritime Antarctica and compared them with the sequences of the cyanobionts of these lichens, in order to determine if cyanobacteria potentially available for lichenization were present in the substrates. A phylogenetic analysis of the sequences showed that Nostoc phylotypes dominated the cyanobacterial communities of the substrates in all sites. Among them, an overlap was observed between the phylotypes of the lichen cyanobionts and those of the cyanobacteria present in their substrates, suggesting that they could be a possible source of lichen photobionts. Also, in most cases, higher Nostoc diversity was observed in the lichens than in the substrates from each site. A better understanding of cyanobacterial diversity in lichen substrates and their relatives in the lichens would bring insights into mycobiont selection and the distribution patterns of lichens, providing a background for hypothesis testing and theory development for future studies of the lichenization process.

Intrinsic factors of Peltigera lichens influence the structure of the associated soil bacterial microbiota

Definition of lichens has evolved from bi(tri)partite associations to multi-species symbioses, where bacteria would play essential roles. Besides, although soil bacterial communities are known to be affected by edaphic factors, when lichens grow upon them these could become less preponderant. We hypothesized that the structure of both the lichen microbiota and the microbiota in the soil underneath lichens is shaped by lichen intrinsic and extrinsic factors. In this work, intrinsic factors corresponded to mycobiont and cyanobiont identities of Peltigera lichens, metabolite diversity and phenoloxidase activity and extrinsic factors involved the site of the forest where lichens grow. Likewise, the genetic and metabolic structure of the lichen and soil bacterial communities were analyzed by fingerprinting. Among the results, metabolite diversity was inversely related to the genetic structure of bacterial communities of lichens and soils, highlighting the far-reaching effect of these substances; while phenoloxidase activity was inversely related to the metabolic structure only of the lichen bacterial microbiota, presuming a more limited effect of the products of these enzymes. Soil bacterial microbiota was different depending on the site and, strikingly, according to the cyanobiont present in the lichen over them, which could indicate an influence of the photobiont metabolism on the availability of soil nutrients.

Designing a SCAR molecular marker for monitoring Trichoderma cf. harzianum in experimental communities

Several species of the fungal genus Trichoderma establish biological interactions with various micro- and macro-organisms. Some of these interactions are relevant in ecological terms and in biotechnological applications, such as biocontrol, where Trichoderma could be considered as an invasive species that colonizes a recipient community. The success of this invasion depends on multiple factors, which can be assayed using experimental communities as study models. Therefore, the aim of this work is to develop a species-specific sequence-characterized amplified region (SCAR) marker to monitor the colonization and growth of T. cf. harzianum when it invades experimental communities. For this study, 16 randomly amplified polymorphic DNA (RAPD) primers of 10-mer were used to generate polymorphic patterns, one of which generated a band present only in strains of T. cf. harzianum. This band was cloned, sequenced, and five primers of 20–23 mer were designed. Primer pairs 2F2/2R2 and 2F2/2R3 successfully and specifically amplified fragments of 278 and 448 bp from the T. cf. harzianum BpT10a strain DNA, respectively. Both primer pairs were also tested against the DNA from 14 strains of T. cf. harzianum and several strains of different fungal genera as specificity controls. Only the DNA from the strains of T. cf. harzianum was successfully amplified. Moreover, primer pair 2F2/2R2 was assessed by quantitative real-time polymerase chain reaction (PCR) using fungal DNA mixtures and DNA extracted from fungal experimental communities as templates. T. cf. harzianum was detectable even when as few as 100 copies of the SCAR marker were available or even when its population represented only 0.1% of the whole community.概要研究目的开发具有种属特异性序列特征性扩增区域(SCAR)标记物来监测哈茨木霉在其入侵的试验菌群中的定殖和生长, 为哈茨木霉应用于生物防治等生态和生物技术中提供支撑。创新要点多种木霉属真菌能与各种微观和宏观的生物有机体建立相互作用。 利用这些相互作用, 木霉可做为原生种群的入侵物种而用于生物防治。 本文通过使用试验菌群为研究模型, 利用随机扩增多态性DNA(RAPD)技术和序列特征性扩增区域(SCAR)标记物来监测菌群中哈茨木霉的生长状态。研究方法利用随机扩增多态性DNA(RAPD)技术, 从16 个10 聚体引物进行多态性筛选, 其中1个引物扩增出对应哈茨木霉的条带。 对该条带进行克隆测序, 并设计5 个20-23 聚体引物。 成功利用引物对2F2/2R2 和2F2/2R3278 分别特异性地扩增出哈茨木霉BpT10a 菌株278 bp和448 bp 的DNA 片段。 同时, 用这两个引物对14 个哈茨木霉菌株和几种不同的真菌菌株进行特异性对照试验, 也只成功扩增出哈茨木霉菌株。 此外, 使用真菌DNA 混合物和试验真菌群的DNA 为模板, 采用实时聚合酶链式反应(PCR)对引物对2F2/2R2 进行评估。 当仅使用100 份SCAR 标记物或哈茨木霉仅占整个菌群的0.1%时, 仍能检测出哈茨木霉。重要结论本研究所建立的SCAR 分子标记能有效监测菌群中的哈茨木霉的定殖和生长, 具有较高特异性、 灵敏度和准确度。

Carbon Consumption Patterns of Microbial Communities Associated with Peltigera Lichens from a Chilean Temperate Forest

Lichens are a symbiotic association between a fungus and a green alga or a cyanobacterium, or both. They can grow in practically any terrestrial environment and play crucial roles in ecosystems, such as assisting in soil formation and degrading soil organic matter. In their thalli, they can host a wide diversity of non-photoautotrophic microorganisms, including bacteria, which play important functions and are considered key components of the lichens. In this work, using the BioLog® EcoPlate system, we studied the consumption kinetics of different carbon-sources by microbial communities associated with the thallus and the substrate of Peltigera lichens growing in a Chilean temperate rain forest dominated by Nothofagus pumilio. Based on the similarity of the consumption of 31 carbon-sources, three groups were formed. Among them, one group clustered the microbial metabolic profiles of almost all the substrates from one of the sampling sites, which exhibited the highest levels of consumption of the carbon-sources, and another group gathered the microbial metabolic profiles from the lichen thalli with the most abundant mycobiont haplotypes. These results suggest that the lichen thallus has a higher impact on the metabolism of its microbiome than on the microbial community of its substrate, with the latter being more diverse in terms of the metabolized sources and whose activity level is probably related to the availability of soil nutrients. However, although significant differences were detected in the microbial consumption of several carbon-sources when comparing the lichen thallus and the underlying substrate, d-mannitol, l-asparagine, and l-serine were intensively metabolized by both communities, suggesting that they share some microbial groups. Likewise, some communities showed high consumption of 2-hydroxybenzoic acid, d-galacturonic acid, and itaconic acid; these could serve as suitable sources of microorganisms as bioresources of novel bioactive compounds with biotechnological applications.

Fungal communities as an experimental approach to Darwin's naturalization hypothesis.

Darwins naturalization hypothesis suggests that the success of an invasive species will be lower when colonizing communities are formed by phylogenetically related rather than unrelated species due to increased competition. Although microbial invasions are involved in both natural and anthropogenic processes, factors affecting the success of microbial invaders are unknown. A biological invasion assay was designed using Trichoderma cf. harzianum as the invader and two types of recipient communities assembled in microcosm assays: communities phylogenetically related to the invader, and communities phylogenetically unrelated to it. Both types of communities were invaded by T. cf. harzianum, and the success of colonization was monitored by qPCR; its effect on the genetic structure of recipient fungal communities was then assessed by DGGE profiles. T. cf. harzianum established itself in both communities, reaching 1000-10,000 times higher copy numbers in the non-related communities. However, invader establishment does not affect the structure of the invaded communities. These results suggest that the composition of recipient communities and their phylogenetic relationship to the invader affect the success of colonization by T. cf. harzianum. While this approach represents a very simplified assay, these microcosms enable an experimental test of Darwins hypothesis in order to understand the biological invasion process in microbial communities.