Molecular analysis of root-associated diazotrophs in important plants from Southern Africa and South America Claudia Sofía Burbano Roa Dissertation submitted in partial fulfillment of the requirements for the degree Dr. rer. nat. Bremen, April 2011 The experiments of the presented work have been carried out from May 2007 until October 2010 at the Department of Biology/Chemistry of Bremen University, Germany, under the guidance of Prof. Dr. Barbara Reinhold-Hurek and Dr. Thomas Hurek. Die Untersuchungen zur folgenden Arbeit wurden von Mai 2007 bis Oktober 2010 am Fachbereich Biologie/Chemie der Universität Bremen unter der Leitung von Prof. Dr. Barbara Reinhold-Hurek und Dr. Thomas Hurek durchgeführt. Vom Fachbereich Biologie/Chemie der Universität Bremen als Dissertation angenommen am: Datum der Disputation: First reviewer: Prof. Dr. Barbara Reinhold-Hurek Second reviewer: Prof. Dr. Michael Friedrich Dedicated to the memory of my grandfather Ignacio Parts of the presented results have been written as manuscript, submitted to a journal, or are already published: Burbano CS, Reinhold-Hurek B, Hurek T. LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples. Environ. Microbiol. Rep. 2010 (2): 251-257. Burbano CS, Liu Y, Rösner K, Reis V, Caballero-Mellado J, Reinhold-Hurek B, Hurek T. Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field grown sugarcane plants and in Norway spruce. Environ. Microbiol. Rep. doi:10.1111/j.1758-2229.2010.00238.x Burbano CS, Grönemeyer JL, Hurek T, Reinhold-Hurek B. Study of the microbial community structure and functional diazotrophic diversity in Collophospermum mopane. In preparation. Grönemeyer JL, Burbano CS, Hurek T, Reinhold-Hurek B. Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia. Submitted to Plant & Soil. In revision. Table of contents Table of contents ABBREVIATIONS 1 SUMMARY 2 ZUSAMMENFASSUNG 4 INTRODUCTION 6 The diversity of life 6 Microbial diversity 6 Functional microbial diversity 8 Biological nitrogen fixation 9 nifH gene as a functional marker 10 Study of active diazotrophic bacteria 12 OBJECTIVES 14 GENERAL DISCUSSION 16 Refinement of methods for detection of bacterial mRNA 16 Searching for the primary active diazotrophic bacteria in sugarcane 20 A non-nodulated legume tree with active diazotrophic bacteria associated to it 22 Discovering the diversity of beneficial bacteria using the classical approach 23 Active diazotrophic bacterial communities 24 CONCLUSIONS AND OUTLOOK 31 REFERENCES 33 Appendices CHAPTER 1. LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples. Table of contents CHAPTER 2. Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field grown sugarcane plants and in Norway spruce. CHAPTER 3. Study of the microbial community structure and functional diazotrophic diversity in Collophospermum mopane. CHAPTER 4. Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia. ACKNOWLEDGMENTS DECLARATION Abbreviations Abbreviations amoA Ammonium monooxygenase anf Genes encoding for nitrogen-fixation, alternative Fe-only nitrogenase ATP Adenosine triphosphate BNF Biological nitrogen fixation DGGE Denaturing gradient gel electrophoresis DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid HGT Horizontal gene transfer LNAs Locked nucleic acids mRNA Messenger ribonucleic acid nif Genes encoding for nitrogen-fixation, typical Mo nitrogenase nifD Gene encoding for the dinitrogenase alpha subunit nifH Gene encoding for the dinitrogenase reductase nifK Gene encoding for the dinitrogenase beta subunit PCR Polymerase chain reaction pmoA Methane monooxygenase PVP Polyvinylpyrrolidone PVPP Polyvinylpolypyrrolidone rDNA Ribosomal deoxyribonucleic acid RNA Ribonucleic acid rRNA Ribosomal ribonucleic acid RT Reverse transcriptase tRNA Transfer ribonucleic acid T-RFLP Terminal restriction fragment length polymorphism SNP Single nucleotide polymorphism SRB Sulfate reducing bacteria vnf Genes encoding for nitrogen-fixation, alternative Va-based nitrogenase 1 Summary Summary Biological nitrogen fixation (BNF) is a process exclusively carried out by a group of prokaryotes known as diazotrophs that possess the enzyme nitrogenase. This enzyme converts Nitrogen (N2) to ammonia (NH3), which can then be assimilated by plants for growth. The study of active diazotrophic bacterial communities in natural environments has been carried out using both culture-dependent and culture-independent techniques. This thesis focuses on the active nitrogen-fixing bacteria associated with the roots of non-legume and legume plants, specifically sugarcane (a principal crop for sugar and biofuel production) and the South African legume tree, mopane (used as animal feed and for domestic purposes in the region). The presence of the nifH gene, which encodes for the iron protein of the nitrogenase complex, has been widely used as a functional and phylogenetic marker to indicate nitrogen fixation in the environment. In order to improve nifH gene detection, the effect of locked nucleic acids (LNAs) substituted primers in RNA extracts from roots of rice and sugarcane was studied by direct reverse transcription polymerase chain reaction (RT-PCR). It was found that the use of an LNA modified RT primer increases the sensitivity and efficiency of RT-PCR reactions and therefore allowed the detection of nifH transcripts where DNA primers alone failed to produce RT-PCR products. RT-PCR was then used to detect active diazotrophs in sugarcane root samples from Africa and America. Denaturing gradient gel electrophoresis profiles showed a low diversity of diazotrophs in all samples locations. A major nifH phylotype was found to have a high DNA sequence identity (93.9–99.6%) with the partial nifH sequence from Rhizobium rosettiformans, which belongs to a genus typically found in legume plants. The prevalence of this globally distributed nifH phylotype indicates a tight interaction between the corresponding nitrogen fixing bacteria and their host. In mopane samples, root nodules were not detected. However, in some samples the lateral roots showed an outgrowth-like protuberance. Their root-associated bacterial isolates belonged to Actinobacteria, Firmicutes and Proteobacteria. The nifH phylotypes found are related to Rhizobiales, Spirochaetes, Firmicutes, Bacteroidetes and -Proteobacteria. These were different from the phylotypes found by the 16S rRNA analysis, which are mainly dominated by Actinobacteria-like sequences. Additionally, the isolation and characterization of putatively beneficial bacteria from traditional crops (sorghum, pearl millet and maize) grown by subsistence farmers in the 2 Summary Kavango region of Namibia was performed. Gram-positive bacteria (Firmicutes and Actinobacteria) showed a higher diversity than gram-negative bacteria (Proteobacteria). Plant-growth promoting characteristics were tested in vitro and revealed promising candidates with multiple beneficial properties. This collection of native microorganisms has the potential for application as inoculants adapted to regional conditions. In conclusion, this PhD work shows that the application of a nifH-mRNA based approach can provide insights into the active diazotrophic communities of mopane and sugarcane, plants that were studied for the first time by molecular approaches. It was found that sugarcane plants are mainly associated with one diazotroph, extending the typically root-nodule-associated rhizobia to this graminaceous plant. On the contrary, the diazotrophs associated with mopane are related not only to Rhizobiales, increasing the spectrum of nitrogen fixers in this legume plant. Further studies are required to isolate these bacteria and test their N2 fixation capacity in planta. For sugarcane in particular, it is important to determine whether the N2 fixation occurs only in the root and/or the shoot and to quantify the relative contribution of each part to the total amount of N2 fixed. 3 Zusammenfassung Zusammenfassung Die biologische Stickstofffixierung ist ein Prozess, der ausschließlich durch eine als diazotroph bezeichnete Gruppe Prokaryoten durchgeführt wird, die das Enzym Nitrogenase besitzen. Dieses Enzym konvertiert atmosphärischen Stickstoff (N2) zu Ammoniak (NH3), welcher dann von Pflanzen zum Wachstum genutzt werden kann. Aktive diazotrophe Bakteriengemeinschaften aus natürlichen Lebensräumen wurden sowohl in Kultur, als auch mittels kultivierungsunabhängiger Methoden untersucht. Diese Dissertation fokussiert auf aktive stickstofffixierende Bakterien, die mit Wurzeln leguminoser sowie nicht-leguminoser Pflanzen assoziiert sind - insbesondere des Zuckerrohres (eine Hauptanbaupflanze für Zucker- und Biokraftstoffproduktion) und des südafrikanischen Leguminosenbaums Mopane (genutzt als Futterpflanze für Tiere und für den regionalen Hausgebrauch). Das Vorhandensein des nifH Gens, welches für das Eisenprotein des Nitrogenasekomplexes kodiert, ist ein weitverbreiteter funktionaler und phylogenetischer Marker für potentielle Stickstofffixierung in der Umwelt. Zur Verbesserung der nifH Gendetektion in RNA-Extrakten von Reis- und Zuckerrohrwurzeln wurde der Effekt von Primern mit verbrückten Nukleinsäuren (locked nucleic acids (LNAs)) in einer direkten reversen Polymerase-Kettenreaktion (reverse transcription polymerase chain reaction (RT-PCR)) untersucht.