<p>Kao jedna od najstarijih grupa fotoautotrofnih mikroorganizama, cijanobakterije predstavljaju &scaron;iroko rasprostranjene prokariote sa raznovrsnim metaboličkim strategijama u cilju preživljavanja i adaptacije na različite uslove životne sredine. Upravo zbog toga, cijanobakterije su značajne kao producenti različitih biolo&scaron;ki aktivnih metabolita. Međutim, većina studija o cijanobakterijama uglavnom je vezana za cijanobakterije vodenih ekosistema. Predmet istraživanja ovog rada je<br />utvrđivanje diverziteta autohtonih cijanobakterija &scaron;umskih ekosistema i karakterizacija odabranih terestričnih cijanobakterijskih predstavnika planinskih područja Srbije izolovanih tokom trogodi&scaron;njeg monitoringa kroz utvrđivanje njihovih osnovnih ekofiziolo&scaron;kih, biohemijskih i genetičkih karakteristika. Rezultati dobijeni u ovom radu su ukazali na diverzitet zemlji&scaron;nih cijanobakterija &scaron;umskih stani&scaron;ta ispitivanih planinskih područja kao i na njihov metabolički diverzitet, odnosno potencijal produkcije različitih bioaktivnih jedinjenja. Na osnovu taksonomski važnih odlika&nbsp;identifikovano je i okarakterisano 20 cijanobakterijskih sojeva za koje je utvrđeno da pripadaju sledećim rodovima:&nbsp;<em>Nostoc, Anabaena, Tolypothrix, Calothrix</em>, <em>Cylindrospermum, Lyngbya, Oscillatoria, Phormidium</em>. Identifikacija izolovanih cijanobakterija primenom molekularanog markera 16S rRNK u većini slučajeva (90%) je potvrdila preliminarnu identifikaciju rodova na osnovu morfolo&scaron;kih kriterijuma. U pogledu produkcije biomase dobijeni rezultati su pokazali da je&nbsp;produkcija biomase kod odabranih testiranih cijanobakterijskih sojeva zavisila od primenjenih uslova kultivacije. Utvrđeno je da su azot, glukoza i saharoza delovali u pravcu stimulacije produkcije biomase kod velikog broja sojeva. Najveća produkcija biomase detektovana je kod soja&nbsp;<em>Calothrix&nbsp;</em>M2 u prisustvu azota u medijumu. Kod soja&nbsp;<em>Nostoc&nbsp;</em>T18 zabeleženo je najveće povećanje produkcije biomase u prisustvu glukoze i saharoze u medijumu. Takođe, sadržaj fikobiliproteina bio je povećan kod većine testiranih sojeva u prisustvu glukoze i saharoze u medijumu. Ispitivanjem sadržaja ugljenih hidrata (glukoze, fruktoze i ksiloze) konstatovano je prisustvo sva tri monosaharida kod svih sojeva pri čemu je svaki soj imao specifičan ugljeno-hidratni profil. Sadržaj monosaharida kod svih testiranih sojeva opadao je u sledećem redosledu glukoza ˃fruktoza ˃ ksiloza. Izuzetnu sposobnost produkcije heksoza i pentoza ispoljila su tri soja<em>&nbsp;Nostoc&nbsp;</em>M1<em>, Phormidium&nbsp;</em>T11 i&nbsp;<em>Calothrix&nbsp;</em>M2. Antibakterijska aktivnost intracelularnih ekstrakata registrovana je kod 16 testiranih&nbsp;cijanobakterijskih sojeva i zavisila je od kombinacije cijanobakterijski-bakterijski soj i tipa primenjenog ekstrakta. U odnosu na heksanske ekstrakte, metanolni ekstrakti su pokazali veću efikasnost, ukazujući na prirodu bioaktivnih jedinjenja sa antibakterijskim delovanjem. Najefikasnijim su se pokazali 75% MeOH ekstrakti cijanobakterijskih sojeva<em>&nbsp;Oscillatoria</em>&nbsp;M2, <span id="cke_bm_336S" style="display: none;">&nbsp;</span><em>Calothrix</em><span id="cke_bm_336E" style="display: none;">&nbsp;</span> M2,<em>&nbsp;Lyngbya</em>&nbsp;T7 i&nbsp;<em>Cylindr<span id="cke_bm_337E" style="display: none;">&nbsp;</span>ospermum</em>&nbsp;K1 koji su ispoljili antibakterijsku aktivnost na 4 testirane bakterije. Hemijskom analizom masno kiselinskog sastava utvrđeno je da je masno kiselinski sadržaj cijanobakterijskih sojeva varirao u zavisnosti od soja. Najznačajniji konstituenti testiranih cijanobakterijskih sojeva bile su 18-to i 16-to ugljenične masne kiseline poput linolne kiseline i &alpha;-linoleinske. Najveći sadržaj linolne kiseline detektovan je kod sojeva <span id="cke_bm_344S" style="display: none;">&nbsp;</span><em>Phormidium</em><span id="cke_bm_344E" style="display: none;">&nbsp;</span> T11 i <em>Tolypothrix</em> K11 &scaron;to ukazuje na sojeve kao potencijalno značajne izvore esencijalnih masnih kiselina. Antiradikalska aktivnost detektovana je kod svih testiranih cijanobakterijskih sojeva. U DPPH eseju, etanolni ekstrakti soja<em> Calothrix</em> M2 ispoljili su najefikasniju sposobnost &bdquo;hvatanja&ldquo; DPPH&middot;radikala dok je u slučaju FRAP metode najveću redukujuću moć imao etanolni ekstrakt soj <em>Cylindrospermum</em> K1. Hemijskom analizom fenolnog sastava kod analiziranih cijanobakterijskih sojeva identifikovano je i kvantifikovano ukupno 21 fenolno jedinjenje. Fenolni sastav je varirao u zavisnosti od soja, a najče&scaron;će detektovana fenolna jedinjenja bila su luteolin-7-O-glukozid, bajkalin i kemferol. Soj sa najznačajnijom sposobno&scaron;ću produkcije fenolnih jedinjenja bio je&nbsp;<em>Phormidium&nbsp;</em>M1 kod koga je identifikovano prisustvo 11 fenolnih jedinjenja. Testirajem toksičnosti intracelularnih ekstrakata u biotestovima <span id="cke_bm_355S" style="display: none;">&nbsp;</span><span id="cke_bm_353S" style="display: none;">&nbsp;</span><em>A. salina</em><span id="cke_bm_355E" style="display: none;">&nbsp;</span><span id="cke_bm_353E" style="display: none;">&nbsp;</span>, <span id="cke_bm_356S" style="display: none;">&nbsp;</span><em>D. magna</em><span id="cke_bm_356E" style="display: none;">&nbsp;</span> i <em>D. rerio</em> ukupno 40<span id="cke_bm_357E" style="display: none;">&nbsp;</span>% testiranih sojeva ispoljilo je toksičan efekat. Najtoksičnijim sojem se pokazao soj&nbsp;<em>Nostoc</em> T7 koji je ispoljio tok<span id="cke_bm_354E" style="display: none;">&nbsp;</span>sičnost u sva tri testa. U slučaju biotesta&nbsp;<em>A. salina&nbsp;</em>najtoksičnijim sojevima pokazali su se&nbsp;<em>Nostoc</em><span id="cke_bm_368E" style="display: none;">&nbsp;</span><span id="cke_bm_366E" style="display: none;">&nbsp;</span>&nbsp;T7,<em>&nbsp;Oscillatoria&nbsp;</em>M2, <em>Oscillatoria&nbsp;</em>T18 i&nbsp;<em>Nostoc</em>&nbsp;K15. Cijanobakterijski sojevi koji su ispoljili najpotentniju aktivnost u biotestu&nbsp;<em>D. magna</em> bili su <span id="cke_bm_382S" style="display: none;">&nbsp;</span><span id="cke_bm_380S" style="display: none;">&nbsp;</span><em>Tolypothrix</em><span id="cke_bm_382E" style="display: none;">&nbsp;</span><span id="cke_bm_380E" style="display: none;">&nbsp;</span> K15, <span id="cke_bm_383S" style="display: none;">&nbsp;</span><em>Nostoc</em><span id="cke_bm_383E" style="display: none;">&nbsp;</span> T7 i <em>Calothrix</em> M2. <span id="cke_bm_384E" style="display: none;">&nbsp;</span><span id="cke_bm_381E" style="display: none;">&nbsp;</span>U biotestu sa embrionima zebrica, soj sa najznačajnijim teratogenim efektom bio je&nbsp; <em>Cylindrospermum</em>&nbsp;K1. U pogledu uticaja ekstrakta testiranog soja na ekspresiju gena&nbsp; kod model organizma&nbsp;<span id="cke_bm_392S" style="display: none;">&nbsp;</span><em>D. rerio</em><span id="cke_bm_392E" style="display: none;">&nbsp;</span>,&nbsp;<em>Cylindrospermum</em>&nbsp;K1 je ispoljio sposobnost modulacije biolo&scaron;kih procesa poput cirkadijalnog ritma kao i sposobnost p<span id="cke_bm_393E" style="display: none;">&nbsp;</span>rodukcije&nbsp;<br />jedinjenja sa estrogenim efektom. Rezultati analize toksigeničnosti testiranih cijanobakterijskih sojeva su pokazali da geni&nbsp;<em>mcyB</em>&nbsp;i&nbsp;<em>mcyE</em>&nbsp;koji su uključeni u&nbsp; produkciju cijanotoksina mikrocistina nisu detektovani ni u jednom od testiranih sojeva. Odsustvo dva gena <span id="cke_bm_399E" style="display: none;">&nbsp;</span>iz mcy genskog klastera ukazuje na to da su druga jedinjenja odgovorna za uočen toksični efekat u primenjenim biotestovima. Dobijeni<br />rezultati ukazuju na značaj ispitivanja zemlji&scaron;nih cijanobakterija, s obzirom na to da su rezultati ovog rada&nbsp; ukazali na velik metabolički diverzitet ispitivanih sojeva i izražen potencijal produkcije različitih bioaktivnih jedinjenja.</p> / <p>As one of the oldest groups of photoautotrophic microorganisms, cyanobacteria represent widespread prokaryotes&nbsp; with diverse metabolic strategies in order to survive and adapt to different environmental conditions. For this reason, cyanobacteria are significant as producers of various biologically active metabolites. However, most of the studies are mainly related to cyanobacteria of aquatic ecosystems. The subject of the research of this dissertation&nbsp; is to determine the diversity of autochthonous cyanobacteria of forest ecosystems and to characterize selected terrestrial cyanobacterial representatives of mountainous areas of Serbia isolated during three year monitoring by determining their basic ecophysiological, biochemical and genetic characteristics. The results obtained in this dissertation&nbsp; have show&nbsp; the diversity of soil cyanobacteria of forest habitats of the investigated mountain areas as well&nbsp; as their metabolic diversity and potential to produce&nbsp; various&nbsp; bioactive compounds. Based on taxonomically important features, 20 cyanobacterial strains have been identified&nbsp; to belong to the following genera:&nbsp; <em>Nostoc, Anabaena</em>, <em>Tolypothrix, Calothrix, Cylindrospermum, Lyngbya, Oscillatoria, Phormidium</em>. Identification of&nbsp; the&nbsp; isolated cyanobacteria using the molecular marker 16S rRNA in most cases (90%) confirmed the preliminary identification of genera based on&nbsp; morphological criteria. In terms of biomass production, the obtained results showed that the production of biomass in the selected tested cyanobacterial strains&nbsp; hepended on the applied cultivation conditions. It was found that nitrogen, glucose and sucrose acted towards the stimulation of biomass production in a large number of strains. The largest biomass production was detected in strain <em>Calothrix</em> M2 in the presence of nitrogen in the medium. In the presence of glucose and sucrose in the medium&nbsp; the highest increase in biomass production was recorded&nbsp; in&nbsp; cyanobacterial strain&nbsp;<em> Nostoc&nbsp;</em> T18. Also, the content of phycobiliproteins&nbsp; has been increased in most&nbsp; of the&nbsp; tested strains&nbsp; in the presence of glucose and sucrose in the medium.&nbsp; Examination of the carbohydrate content (glucose, fructose and xylose)&nbsp; showed that all three monosaccharides were present in all strains and that each strain had a specific carbohydrate profile&nbsp; whereby the content of monosaccharides in&nbsp; all tested strains declined in the following order: glucose ˃ fructose ˃ xylose. Among the tested strains, three strains namely <span id="cke_bm_329S" style="display: none;">&nbsp;</span><em>Nostoc </em><span id="cke_bm_329E" style="display: none;">&nbsp;</span>M1, <em>Phormidium </em>T11 and&nbsp; <em>Calothrix</em> M2 showed the exceptional ability to produce hexose and pentose. The antibacterial activity of intracellular extracts was recorded in 16 tested cyanobacterial strains and depended on the combination of cyanobacterial-bacterial strain and the type of applied extract. Compared to hexane extracts, methanol extracts showed greater efficiency, indicating on the nature of bioactive compounds with antibacterial activity. The most effective were 75% MeOH extracts of cyanobacterial strains&nbsp; <span id="cke_bm_338S" style="display: none;">&nbsp;</span><em>Oscillatoria</em><span id="cke_bm_338E" style="display: none;">&nbsp;</span>&nbsp; M2,&nbsp; <span id="cke_bm_339S" style="display: none;">&nbsp;</span><em>C<span id="cke_bm_330E" style="display: none;">&nbsp;</span>alothrix</em><span id="cke_bm_339E" style="display: none;">&nbsp;</span>&nbsp; M2,&nbsp; <em>Lyngby</em>a&nbsp; T7 an<span id="cke_bm_340E" style="display: none;">&nbsp;</span>d <em>Cylindrospermum</em>&nbsp; K1 which exhibited antibacterial activity on 4 tested bacteria.&nbsp; Results of the analysis of the fatty&nbsp; acid composition showed that the fatty acid content of tested cyanobacterial strains varied depending on the strain. The most significant constituents of the tested&nbsp; cyanobacterial strains were 18 and 16 carbonic fatty acids such as linoleic acid, &alpha;-linoleic. The highest content of linoleic acid was detected in two strains, <span id="cke_bm_350S" style="display: none;">&nbsp;</span><span id="cke_bm_348S" style="display: none;">&nbsp;</span><em>Phormidium</em><span id="cke_bm_350E" style="display: none;">&nbsp;</span><span id="cke_bm_348E" style="display: none;">&nbsp;</span> T11 and <span id="cke_bm_351S" style="display: none;">&nbsp;</span><em>Tolypot<span id="cke_bm_351E" style="display: none;">&nbsp;</span>hri</em>x K11, indicatin<span id="cke_bm_352E" style="display: none;">&nbsp;</span>g that these strains can be potentially significant sources of essential fatty acids.&nbsp; Results of antioxidant&nbsp; tests showed that all tested strains had antiradical activity. In the case of DPPH assay, ethanolic extracts&nbsp; of <em>Calothrix </em>M2 exhibited the most effective ability to scavenge DPPH &bull;radical while in the case of the FRAP method,&nbsp; ethanolic extract of&nbsp;<em> Cylindrospermum</em>&nbsp; K1 had&nbsp; the greatest reduction power. Accordi<span id="cke_bm_349E" style="display: none;">&nbsp;</span>ng to data obtained from&nbsp; chemical analysis of&nbsp; the phenolic composition of the analyzed cyanobacterial strains, a total of 21 phenol compounds were identified and quantified. The phenolic composition varied depending on the strain, and the most frequently detected phenolic compounds were luteolin-7-O-glucoside, baicalin and kaempferol. The strain with the&nbsp; most&nbsp; asignificant ability to produce phenolic compounds was<em> Phormidium</em> M1, in which the presence of 11 phenolic compounds was identified.&nbsp; The results of the toxicity of intracellular extracts obtained in <em>A. salina, D. magna and D. rerio&nbsp; </em>biotests, showed&nbsp; that a total of 40% of&nbsp; the&nbsp; tested strains exhibited a toxic effect.&nbsp; The most toxic strain was&nbsp;<em> Nostoc&nbsp;</em> T7 due to the fact that it showed toxicity in all three tests. In the case of&nbsp; <em>A. salina </em>biotest, the most potent strains were <span id="cke_bm_365S" style="display: none;">&nbsp;</span><em>Nostoc</em><span id="cke_bm_365E" style="display: none;">&nbsp;</span> T7, <em>Oscillatoria</em> M2,&nbsp; <em>Oscillatoria&nbsp;</em> T18 and <em>Nostoc</em> K15. Cyano<span id="cke_bm_366E" style="display: none;">&nbsp;</span>bacterial strains that exhibited the most prominent activity&nbsp; in&nbsp; <em>D. magna</em>&nbsp; biotest were <span id="cke_bm_378S" style="display: none;">&nbsp;</span><span id="cke_bm_376S" style="display: none;">&nbsp;</span><em>Tolypothrix </em><span id="cke_bm_378E" style="display: none;">&nbsp;</span><span id="cke_bm_376E" style="display: none;">&nbsp;</span>K15,<span id="cke_bm_379S" style="display: none;">&nbsp;</span><em> Nostoc</em><span id="cke_bm_379E" style="display: none;">&nbsp;</span> T7 and <em>Calothrix</em> M2. In bi<span id="cke_bm_380E" style="display: none;">&nbsp;</span><span id="cke_bm_377E" style="display: none;">&nbsp;</span>otest with zebrafish embryos, the strain with the most significant teratogenic effect was <em>Cylindrospermum</em> K1. Regarding the effect of cyanobacterialextract on gene expression in model organism<span id="cke_bm_388S" style="display: none;">&nbsp;</span><em> D. rerio,</em><span id="cke_bm_388E" style="display: none;">&nbsp;</span> cyanobacterial strain&nbsp; <em>Cylindrospermum</em>&nbsp; K1&nbsp; exhibited the ability to modulate biological processes such as circadian rhythm as well as <span id="cke_bm_389E" style="display: none;">&nbsp;</span>the ability to produce compounds with an estrogenic effect. The results of the&nbsp; toxicogenetic analysis of the tested cyanobacterial strains have shown that the genes&nbsp;<span id="cke_bm_394S" style="display: none;">&nbsp;</span><em> mcyB</em><span id="cke_bm_394E" style="display: none;">&nbsp;</span>&nbsp; and&nbsp; <em>mcyE&nbsp;</em> involved in the production&nbsp; of cyanotoxin microcystins have not been detected in any of the tested strains. The absence of two genes from the&nbsp; mcy&nbsp; gene cluster indicates<span id="cke_bm_395E" style="display: none;">&nbsp;</span> that other compounds are responsible for the observed toxic effect in applied biotests. The obtained results point&nbsp; out on the importance of the study of soil cyanobacteria, since the obtained results have indicated that the&nbsp; tested strains possess a large metabolic&nbsp; diversity and potential to produce various bioactive compounds.</p>

Soil microorganisms play important roles in maintaining soil quality and ecosystem health. Development of effective methods for studying the composition, diversity, and behavior of microorganisms in soil habitats is essential for a broader understanding of soil quality. Forest management strategies and practices are of vital significance for sustainable forest production. How the different forest management measures will influence soil microbial communities is a widespread concern of forest industry and scientific communities. Only a small proportion (~0.1%) of the bacteria from natural habitats can be cultured on laboratory growth media. Direct extraction of whole-community DNA from soil, followed by polymerase chain reaction (PCR) and other analysis circumvents the problems of the culture-dependent methods and may shed light on a broader range of microbial communities in the soil. DNA-based molecular methods rely on high quality soil microbial DNA as template, and thus extraction of good quality DNA from soil samples has been a challenge because of the complex and heterogeneous nature of the soil matrix. The objectives of this research were to establish a set of DNA-based molecular methods and to apply them to investigate forest soil microbial composition and diversity. Soil samples were collected from different forest ecosystems, i.e., the natural forest (YNF) and the first rotation (~ 50 years) (Y1R) and the second rotation (~ 1 year) (Y2R) of hoop pine plantations at Yarraman, and from different forest residue management practices (the experiments had established 6.4 years before the samples were collected) at Gympie, two long-term experimental sites of the Queensland Department of Primary Industry-Forestry in subtropical Queensland, Australia. Some DNA-based molecular techniques, including DNA extraction and purification, PCR amplification, DNA screening, cloning, sequencing and phylogenetic analyses, were explored using Yarraman soil samples, which were high in organic matter, clay and iron oxide contents. A set of methods was assembled based on the recommendations of the method development experiments and applied to the investigations of the microbial composition and diversity of the Yarraman and Gympie soil samples. Four soil DNA extraction methods, including the Zhou method (Zhou et al., 1996), the Holben method (Holben, 1994), the UltraClean (Mo Bio) and FastDNA (Bio 101) soil DNA extraction kits, were explored. It was necessary to modify these methods for Yarraman soil. I designed and introduced a pre-lysis buffer washing step, to partially remove soil humic substances and promote soil dispersion. This modification greatly improved the quality of the extracted DNA, decreasing co-extracted humic substances by 31% and increasing DNA yield by 24%. The improved Holben method was recommended for fungal community studies, and the improved Zhou method for bacterial community studies. The extracted DNA was good in quality, with a consistent size of ~20 kb and a yield of 48-87 g g-1 soil, and could be successfully used for 16S (Zhou method) and 18S (Holben method) rDNA amplifications. For less difficult environmental samples, UltraClean kits could be a good option, because they are simple and fast and the extracted DNA are also of good quality. Screening of the DNA PCR products using TGGE, Heteroduplex-TGGE and SSCP was also explored. These methods were not so effective for the screening of the soil DNA PCR products, owing to the difficulty in interpretation of the results. Cloning was a necessary step to obtain a single sequence at species level in soil microbial community studies. The screening of the clone library by TGGE, Heteroduplex-TGGE and SSCP could only separate the clones into several major bands, although SSCP gave better separation. Sequencing of selected clones directly from the clone library obtained ultimate results of microbial taxonomic composition and diversity through well-established sequence analysis software packages and the databases. It was recommended that, in this project with the target of microbial community composition and diversity, soil DNA PCR products were directly cloned to construct clone libraries and a sample of clones were sequenced to achieve an estimate of the taxonomic composition of the soil. Fungal communities of the Yarraman soil samples under the natural forest (YNF) and the hoop pine plantations (YHP) were investigated using 18S rDNA based cloning and sequencing approaches. Twenty-eight clone sequences were obtained and analysed. Three fungal orders, i.e., Zygomycota, Ascomycota and Basidiomycota were detected from the YNF and YHP samples. By contrast, culture-based analyses of fungi in the literature were mostly Ascomycetes. YNF appeared to have more Ascomycota but less Zygomycota than YHP, and within the Zygomycota order, YHP had more unidentified species than YNF. Bacterial communities of Yarraman soil samples of YNF, Y1R and Y2R were investigated using 16S rDNA-based cloning and sequencing approaches. 305 16S rDNA clone sequences were analysed and showed an overall bacterial community composition of Unclassified bacteria (34.4%), Proteobacteria (22.0%), Verrucomicrobia (15.7%), Acidobacteria (10.2%), Chloroflexi (6.9%), Gemmatimonadetes (5.6%), and Actinobacteria (5.2%). There was a significant difference among YNF, Y1R and Y2R in the taxonomic group composition. YNF had a greater proportion of Acidobacteria (18.0%), Verrucomicrobia (23.0%) and Chloroflexi (9.0%) than Y1R and Y2R (corresponding to 6.3%, 12.1% and 5.9%, respectively), while Y1R and Y2R had a higher percentage of the Unclassified group (38.5% for Y1R and 46.5% for Y2R) than YNF (18.0%). For the Proteobacteria group, YNF had more Alpha-subdivision but Y1R and Y2R had more Delta-subdivision. From YNF to Y1R to Y2R, the clone sequence variable site ratios, 5% and 10% OTU numbers and Shannon's diversity index H' values tended to decrease, indicating the soil bacterial diversity decreased from the natural forest to the first and the second rotation hoop pine plantations. The large amount of unclassified clone sequences could imply a novel group of bacteria in the soil, particularly in the hoop pine soil samples. Alternatively they may result from artefacts during the PCR process. Bacterial communities of the Gympie soil under different residue management practices, i.e., residue (litter plus logging residue) removed (G0R), residue retained (G1R), and residue doubled (G2R), were also investigated using the 16S rDNA-based cloning and sequencing approaches. Acidobacteria (37.6%) and Proteobacteria (35.6%, including Alpha-subdivision of 29.9% and Gamma-subdivision of 5.7%) were dominant components of the communities, followed by Actinobacteria (14.7%), Verrucomicrobia (7.3%) and Unclassified bacteria. There was no significant difference among G0R, G1R and G2R in the bacterial community compositions and diversity. These findings provided an in-depth vision of the soil microbial communities under different forest management practices. Their combination with other soil analysis results, such as physical and chemical properties, and forest production data, could provide an improved understanding of sustainable forest management strategies.

Human activities such as fossil fuel combustion and deforestation have increased atmospheric concentrations of carbon dioxide, reactive nitrogen, and other greenhouse gases. As a result, Earth's surface has warmed by 0.85 °C since the pre-industrial era and will continue to warm. Many northern latitude temperate forest ecosystems mitigate the effects of both elevated carbon dioxide and atmospheric nitrogen deposition through retention of carbon and nitrogen in plants and soils. However, the continued ability of these ecosystems to store carbon and nitrogen will be altered with continued climate change. Warmer winters will lead to reduced depth and duration of snowpack, which insulates soils from cold winter air. Climate change over the next century will therefore affect soil temperatures in northern temperate forests in opposing directions across seasons, with warmer soils in the growing season and colder, more variable soil temperatures in winter. Warmer growing seasons generally increase ecosystem uptake and storage of carbon and nitrogen, whereas a smaller snowpack and colder soils in winter reduce rates of ecosystem nutrient cycling and plant growth. My dissertation aims to understand how climate change in the growing season and winter interact to affect function and nitrogen cycling in northern hardwood forest ecosystems. I accomplished this goal through formal literature review and two climate change manipulation experiments at Hubbard Brook Experimental Forest, NH. I found that although 67% of climate change experiments were conducted in seasonally snow covered ecosystems, only 14% take into account the effects of distinct climate changes in winter. By simulating climate change across seasons, I demonstrated that changes in nitrogen cycling caused by increased soil freezing in winter are not offset by warming in the growing season. Moreover, shifts in plant function due to winter climate change are mediated through a combination of changes in snow depth, soil temperature, and plant-herbivore interactions that differentially affect above- and belowground plant components. These results would not be evident from examining climate change in either the growing season or winter alone and demonstrate the need for considering seasonally distinct climate change to determine how nitrogen and carbon cycling will change in the future.

A herbivoria por grandes mamíferos é um fator decisivo no direcionamento de comunidades vegetais, influenciando o crescimento, sobrevivência e reprodução das plantas. Os ungulados, quando exóticos, podem impactar os ecossistemas diferentemente de herbívoros nativos, gerando maiores efeitos nas comunidades de plantas. Os objetivos deste estudo foram avaliar se a presença de búfalos (densidade de 1 animal/ha) impacta as assembléias de plântulas e de arbustos numa floresta estacional semidecídua no sul do Brasil, além de inferir consequências para a manutenção da comunidade vegetal. Para isso realizamos duas abordagens no município de Sentinela do Sul: na primeira, estabelecemos um experimento de exclusão de bubalinos (duração de 14 meses) e na segunda, comparamos três áreas com presença e protegidas desses herbívoros por três e dez anos. Em ambas as abordagens, registramos as seguintes variáveis ecológicas: riqueza, altura média de plantas, cobertura vegetal, diversidade (Shannon), e biomassa. No experimento de exclusão, nenhuma das variáveis apresentou diferenças significativas perante presença e ausência de búfalos (após 14 meses). Na comparação das três áreas as comunidades vegetais mostraram diferenças qualitativas (composição de espécies) no contraste presença versus dez anos de exclusão de búfalos, e as variáveis riqueza e altura média sofreram, respectivamente, significativos incremento e decréscimo. A diferença observada para o incremento de diversidade de zero a dez anos de exclusão foi marginalmente significativa e a compactação do solo apresentou decréscimo em função do tempo de exclusão do gado. Portanto , os resultados do presente trrabalho indicam que as manchas florestais não respondem à remoção de búfalos em baixas densidades no curto prazo e, na ausência de grandes herbívoros, a floresta tende a incrementar sua complexidade via aumento de riqueza e diversidade. / Herbivory by large mammals is a key factor in plant communities, and has effects on growth, survival and reproduction of plants. Ungulates, when exotic, impact ecosystems in a different way compared to the native ones, and may have a more pronounced effect on plant communities. In this study we evaluated buffalo (1 animal/ha density) impact on the seedling and shrub assemblages in a semideciduous seasonal forest in southern Brazil and checked if such impact would affect plant community maintainability. To address these aims we conducted two studies in Sentinela do Sul County: 1. we established a buffalo exclusion experiment (during 14 months) and 2. compared three areas with presence and protected against these herbivores for three and ten years. In both studies, we measured the following ecological variables: plant cover, richness, diversity (Shannon), biomass and average height. In the exclusion experiment, any ecological variable showed significant differences between buffalo presence and absence plots. When the three areas were compared, the communities showed qualitative differences (species composition) in the contrast presence versus ten years of buffalo exclusion, and richness and average height showed, respectively, increase and decrease. The difference observed for an increase of diversity between zero and ten years exclusion was marginally significant, and soil compactation showed retraction in relation to livestock exclusion time. We conclude that forest spots don’t answer in short-term to low densities buffalo removal and, in the absence of large herbivores, the forest tends to develop its complexity by richness and diversity increase, in a long-term basis.

No description available.

A herbivoria por grandes mamíferos é um fator decisivo no direcionamento de comunidades vegetais, influenciando o crescimento, sobrevivência e reprodução das plantas. Os ungulados, quando exóticos, podem impactar os ecossistemas diferentemente de herbívoros nativos, gerando maiores efeitos nas comunidades de plantas. Os objetivos deste estudo foram avaliar se a presença de búfalos (densidade de 1 animal/ha) impacta as assembléias de plântulas e de arbustos numa floresta estacional semidecídua no sul do Brasil, além de inferir consequências para a manutenção da comunidade vegetal. Para isso realizamos duas abordagens no município de Sentinela do Sul: na primeira, estabelecemos um experimento de exclusão de bubalinos (duração de 14 meses) e na segunda, comparamos três áreas com presença e protegidas desses herbívoros por três e dez anos. Em ambas as abordagens, registramos as seguintes variáveis ecológicas: riqueza, altura média de plantas, cobertura vegetal, diversidade (Shannon), e biomassa. No experimento de exclusão, nenhuma das variáveis apresentou diferenças significativas perante presença e ausência de búfalos (após 14 meses). Na comparação das três áreas as comunidades vegetais mostraram diferenças qualitativas (composição de espécies) no contraste presença versus dez anos de exclusão de búfalos, e as variáveis riqueza e altura média sofreram, respectivamente, significativos incremento e decréscimo. A diferença observada para o incremento de diversidade de zero a dez anos de exclusão foi marginalmente significativa e a compactação do solo apresentou decréscimo em função do tempo de exclusão do gado. Portanto , os resultados do presente trrabalho indicam que as manchas florestais não respondem à remoção de búfalos em baixas densidades no curto prazo e, na ausência de grandes herbívoros, a floresta tende a incrementar sua complexidade via aumento de riqueza e diversidade. / Herbivory by large mammals is a key factor in plant communities, and has effects on growth, survival and reproduction of plants. Ungulates, when exotic, impact ecosystems in a different way compared to the native ones, and may have a more pronounced effect on plant communities. In this study we evaluated buffalo (1 animal/ha density) impact on the seedling and shrub assemblages in a semideciduous seasonal forest in southern Brazil and checked if such impact would affect plant community maintainability. To address these aims we conducted two studies in Sentinela do Sul County: 1. we established a buffalo exclusion experiment (during 14 months) and 2. compared three areas with presence and protected against these herbivores for three and ten years. In both studies, we measured the following ecological variables: plant cover, richness, diversity (Shannon), biomass and average height. In the exclusion experiment, any ecological variable showed significant differences between buffalo presence and absence plots. When the three areas were compared, the communities showed qualitative differences (species composition) in the contrast presence versus ten years of buffalo exclusion, and richness and average height showed, respectively, increase and decrease. The difference observed for an increase of diversity between zero and ten years exclusion was marginally significant, and soil compactation showed retraction in relation to livestock exclusion time. We conclude that forest spots don’t answer in short-term to low densities buffalo removal and, in the absence of large herbivores, the forest tends to develop its complexity by richness and diversity increase, in a long-term basis.

A herbivoria por grandes mamíferos é um fator decisivo no direcionamento de comunidades vegetais, influenciando o crescimento, sobrevivência e reprodução das plantas. Os ungulados, quando exóticos, podem impactar os ecossistemas diferentemente de herbívoros nativos, gerando maiores efeitos nas comunidades de plantas. Os objetivos deste estudo foram avaliar se a presença de búfalos (densidade de 1 animal/ha) impacta as assembléias de plântulas e de arbustos numa floresta estacional semidecídua no sul do Brasil, além de inferir consequências para a manutenção da comunidade vegetal. Para isso realizamos duas abordagens no município de Sentinela do Sul: na primeira, estabelecemos um experimento de exclusão de bubalinos (duração de 14 meses) e na segunda, comparamos três áreas com presença e protegidas desses herbívoros por três e dez anos. Em ambas as abordagens, registramos as seguintes variáveis ecológicas: riqueza, altura média de plantas, cobertura vegetal, diversidade (Shannon), e biomassa. No experimento de exclusão, nenhuma das variáveis apresentou diferenças significativas perante presença e ausência de búfalos (após 14 meses). Na comparação das três áreas as comunidades vegetais mostraram diferenças qualitativas (composição de espécies) no contraste presença versus dez anos de exclusão de búfalos, e as variáveis riqueza e altura média sofreram, respectivamente, significativos incremento e decréscimo. A diferença observada para o incremento de diversidade de zero a dez anos de exclusão foi marginalmente significativa e a compactação do solo apresentou decréscimo em função do tempo de exclusão do gado. Portanto , os resultados do presente trrabalho indicam que as manchas florestais não respondem à remoção de búfalos em baixas densidades no curto prazo e, na ausência de grandes herbívoros, a floresta tende a incrementar sua complexidade via aumento de riqueza e diversidade. / Herbivory by large mammals is a key factor in plant communities, and has effects on growth, survival and reproduction of plants. Ungulates, when exotic, impact ecosystems in a different way compared to the native ones, and may have a more pronounced effect on plant communities. In this study we evaluated buffalo (1 animal/ha density) impact on the seedling and shrub assemblages in a semideciduous seasonal forest in southern Brazil and checked if such impact would affect plant community maintainability. To address these aims we conducted two studies in Sentinela do Sul County: 1. we established a buffalo exclusion experiment (during 14 months) and 2. compared three areas with presence and protected against these herbivores for three and ten years. In both studies, we measured the following ecological variables: plant cover, richness, diversity (Shannon), biomass and average height. In the exclusion experiment, any ecological variable showed significant differences between buffalo presence and absence plots. When the three areas were compared, the communities showed qualitative differences (species composition) in the contrast presence versus ten years of buffalo exclusion, and richness and average height showed, respectively, increase and decrease. The difference observed for an increase of diversity between zero and ten years exclusion was marginally significant, and soil compactation showed retraction in relation to livestock exclusion time. We conclude that forest spots don’t answer in short-term to low densities buffalo removal and, in the absence of large herbivores, the forest tends to develop its complexity by richness and diversity increase, in a long-term basis.

The abilities of fungi and bacteria to degrade simple and complex carbon compounds derived from different sources, such as root exudates, litter, soil organic matter or fungal mycelium were studied in this dissertation. Knowledge of functional traits, especially degradation abilities of fungi and bacteria, are important for deciphering the black box of microbial functioning in topsoil and thus aiding in modeling and predicting future directions of microbial communities development in face of global changes. Among fungal cultures form culture collection representing strains with different taxonomy and ecophysiology, the ecophysiology of fungi was more important in manifestation of functional traits than taxonomy. Among bacterial isolates from the litter and soil of spruce forest, Acidobacteria were confirmed to express multiple decomposition enzymes in high rates in vitro and were also abundant and active degraders in acidic spruce forest soil. The expression of degradation capacities of both bacteria and fungi were further studied in situ in spruce forest topsoil, that represents an important environment due to the ubiquity of coniferous forests on the Northern hemisphere. There is an obvious gap of knowledge, when comes to our understanding of seasonal effect on microbial functioning, and this is...

Oomycetes (Stramenopiles, protists) are among the most severe plant pathogens, comprising species with a high economic and ecologic impact on forest ecosystems. Their diversity and community structures are well studied in terrestrial habitats, but tree canopies as huge and diverse habitats have been widely neglected. A recent study highlighted distinct oomycete communities in the canopy stratum compared to the ground region of three temperate deciduous trees (Quercus robur, Tilia cordata, Fraxinus excelsior). While the communities from the two strata were distinct when taking oomycete abundances into account, they were rather similar when only OTU presence/absence was considered. It remains, however, unknown if this homogeneity in the OTU presence also leads to a functional homogenisation among microhabitats within the two strata ground and canopy. In this study, we supplemented functional traits to oomycete communities in the tree microhabitats, which were determined over a time period of 2 years with a metabarcoding approach. Our results showed that even though most oomycetes occurred in all microhabitats, a strong discrepancy between the strata and correspondingly the distribution of oomycete lifestyles could be observed. This pattern was constant over several seasons. Obligate biotrophic species, exclusively feeding on living host tissue, dominated the canopy region, implying tree canopies to be a hitherto neglected reservoir for parasitic protists. OTUs assigned to the genus Hyaloperonospora—parasites highly specialised on hosts that were not sampled—could be determined in high abundances in the canopy and the surrounding air, challenging the strict host dependencies ruled for some oomycetes. Our findings further contribute to the understanding of oomycete ecosystem functioning in forest ecosystems

An understanding of ecosystem-level carbon (C) sequestration, or net ecosystem production (NEP), requires the separation of heterotrophic, microbial respiration (RH) from autotrophic, root-derived respiration (RA) as the components of RS (i.e., NEP = NPP - RH). However, separating these two sources in situ has been problematic since they are closely coupled. This study utilizes two similarly aged Pinus taeda L. stands, 8 and 9 years-old, aimed at quantifying these two respiration components through in-situ root severing. In order to use root-severing treatments to separate RS into RH and RA components, confirmation of carbohydrate depletion coupled to RA decline is crucial. This study evaluated the changes in CO2 flux rates and carbohydrate supply upon root severing in Pinus taeda L. using a controlled laboratory validating a two-part field study. The first field study used root-severing cores to test in-situ if respiration components can be attained based on the depletion of carbohydrate supply. The second field study was aimed at how future changes in climate might affect the ability of forests to store C and how modern forestry practices might affect changes and was conducted over the course of two installations, spring and summer 2012. In this study we examined the effects of fertilization (0 and 100.9 kg N ha-1 ) and throughfall reduction (0 and -30%) on total soil respiration (RS) as well as the heterotrophic contribution to RS, in a fully replicated (n=4), 2x2 factorial design. In the controlled lab experiment RS and RA declined by 86% and 95% respectively by the end of an 86 day trial and NSC carbohydrates declined by 60% for soluble, 29% for insoluble, and 43% for total (soluble + insoluble). The decline of RA was highly correlated to with the decline of NSC’s at 0.90, 0.69 and 0.93 for soluble, insoluble and total, respectively. The companion field study revealed a mean decrease 21±0.5% of over the final three dates when severed root respiration stabilized. In the second study, testing throughfall reduction and fertilization levels there were no fertilization by throughfall reduction interactions on the contribution of RH to RS in either the spring or summer; however, the main effect of throughfall reduction was significant in the spring. During the spring, the mean contribution of RH to RS for ambient throughfall plots was 96±6.4%, while the mean contribution under throughfall reduction was 68±1.9%. During the summer, there were no differences among treatments and the overall contribution of RH to RS was 78±1.6%. Collectively, both of these studies revealed that the severing of roots from their primary energy source and the subsequent depletion of stored NSC that the use of in-situ methods allows for the quantification of soil respiration components RA and RH. Using these estimates to model NEP in the short-term can be variable by season, however, long-term monitoring may simplify future NEP modeling scenarios / Master of Science