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. Der Gebrauch von LNA-modifizierten Primern führte zu einer erhöhten Sensitivität und Effizienz der RT-PCR Reaktionen und ermöglichte somit eine Detektion von nifH Transkripten, die bei ausschließlicher Nutzung von DNA-Primern scheiterte. RT-PCR wurde verwendet, um aktive Diazotrophe in Zuckerrohrwurzelproben aus Afrika und Amerika zu untersuchen. Die Profile der denaturierenden Gradientengel- Elektrophorese (denaturing gradient gel electrophoresis, DGGE) aller untersuchten Proben zeigten eine geringe Diversität, unabhängig vom Gebiet, aus dem die Proben stammten. Ein nifH-Hauptphylotyp mit hoher DNA-Sequenzidentität (93.9–99.6%) zu Teilen der nifH-Sequenz von Rhizobium rosettiformans wurde entdeckt, welches einer Gattung angehört, die typischerweise in Leguminosen vorkommt. Die Prävalenz dieses global verbreiteten nifH-Phylotyps deutet auf eine enge Interaktion zwischen den entsprechenden stickstofffixierenden Bakterien und ihrem Wirt hin. Des Weiteren wurden in Mopane-Proben keine Wurzelknöllchen entdeckt. Jedoch zeigten die Seitenwurzeln einiger Proben eine auswuchsartige Protuberanz. Ihre wurzelassoziierten Bakterienisolate gehörten zu den Actinobacteria, den Firmicutes
4 Zusammenfassung
und den Proteobacteria. Die gefundenen nifH-Phylotypen waren verwandt mit den Rhizobiales, den Spirochaetes, den Firmicutes, den Bacteroidetes und den - Proteobacteria. Sie unterschieden sich von den Phylotypen, die mittels 16S rRNA- Analyse gefunden wurden, welche vor allem von Actinobacteria-ähnlichen Sequenzen dominiert wurden. Des Weiteren wurden vermeintlich nutzbringende Bakterien traditioneller Anbaupflanzen (Sorghum, Perlhirse und Mais) untersucht, die von selbstversorgenden Bauern in der Kavango-Region Namibias angebaut wurden. Gram-positive Bakterien (Firmicutes und Actinobacteria) wiesen eine höhere Diversität als gram-negative Bakterien auf (Proteobacteria). Pflanzenwuchsfördernde Charakteristika wurden in vitro getestet und ließen vielversprechende Kandidaten mit vielfältigen nutzbringenden Eigenschaften erkennen. Daher hat diese Sammlung nativer Mikroorganismen das Potential zur Anwendung als an regionale Bedingungen angepasstes Inokulum. Zusammenfassend zeigt diese Doktorarbeit, dass die Anwendung eines nifH-mRNA basierten Ansatzes Einblicke in die aktiven diazotrophen Gemeinschaften von Zuckerrohr und Mopane, welche erstmalig mittels molekularer Ansätze erforscht wurden, erlaubt. Es konnte festgestellt werden, dass die biologische Stickstofffixierung in Zuckerrohrpflanzen hauptsächlich auf Rhizobien zurückzuführen ist, was das Wirtspflanzenspektrum dieser typischen stickstofffixierenden Wurzelknöllchenbakterien um diese Süßgraspflanze erweitert. Im Gegensatz dazu waren die Diazotrophen, welche mit Mopane assoziiert waren, neben den Rhizobiales auch mit anderen Bakterien verwandt, was auf ein breiteres Spektrum an Stickstofffixierern in dieser Leguminose hinweist. Weitere Studien werden notwendig sein, um diese Bakterien zu isolieren und auf ihre Stickstoffixierungskapazität in planta zu überprüfen. Insbesondere für Zuckerrohr wird es interessant sein zu wissen, ob Stickstofffixierung lediglich in der Wurzel und/oder in dem Pflanzenspross stattfindet und was jeweils der relative Beitrag zur Gesamtmenge an fixiertem Stickstoff ist.
5 Introduction
Introduction
The diversity of life
Our vision about the basic organization of life changed drastically since the pioneer work carried out by Carl Woese in the 1970s. We realized that the ancient notion that all living things should be either classified as plant or animal in nature was too reductive (Woese et al. 1990). Furthermore, the five-kingdom scheme (Animalia, Plantae, Fungi, Protista and Monera) proposed by Whittaker and Margulis (1978) was neither phylogenetically correct nor a natural system (Woese et al. 1990). Instead of using the classical phenotypic criteria, the new classification was based on molecular data. In order to determine the relationships between all extant living species, it was necessary to use a molecule of broad distribution such as the ribosomal RNA (rRNA). This molecule is a component of all self-replicating systems and its sequence changes slowly throughout time. In this way the changes occurring in the different species could be detected (Woese 1977). This powerful approach to determine evolutionary relationships led to the current view on the diversity of life in this planet, where there are three domains: Bacteria, Archaea and Eucarya (Woese et al. 1977, 1990). The root of the tree separates Bacteria from the other two primary groups, showing a common history between Archaea and Eucarya making them specific –but distant– relatives (Woese et al. 1990, Pace 1997).
Microbial diversity
Two out of the three domains of the rRNA phylogenetic tree of life belong to the microbial world. Our knowledge about this diversity was limited because microorganisms are tiny, individually invisible to the eye and their morphology is not enough to distinguish between them (Pace 1997). Standard cultivation methods can select for certain organisms, leading to a restriction and potential biases (Amann et al. 1995, Pace 1997). As a result, more than 99% of the microorganisms observable in nature typically defy cultivation and we are just able to isolate a small portion of the entire microbial world. In order to understand the ecology of different environments (surface waters,
6 Introduction
oceans, and soils) and also the global elemental cycles on which the biosphere depends, it is indispensable to study their microbial component and its activities. A molecular approach that bypassed the necessity to cultivate a microorganism to determine its 16S rRNA gene was developed by Pace and colleagues (1985). Basically, bulk nucleic acids are directly extracted from the environmental samples, 16S rDNAs are amplified typically by PCR, cloned and compared with known sequences (Figure 1). These environmental cloned sequences can be assigned to a phylogenetic tree and then be used as markers to target the organism from which they are coming from (Hugenholtz 2002). The approach can include the design of nucleic acid probes specific for the organisms of interest. This allows the visualization and quantification of the target group in the environment using techniques like fluorescence in situ hybridization (FISH) (Amann et al. 1995).
Figure 1. Full-cycle rRNA approach to characterize microorganisms by a culture-independent approach (modified from Hugenholtz 2002).
Using the rRNA approach mentioned above, the occurrence of phylogenetic types of organisms, “phylotypes”, and their distribution in natural communities can be surveyed
7 Introduction
directly from the environment. Phylotypes that are less abundant but contribute significantly to a certain place can be detected (Pace 1997). Since 1987 where only about 12 phyla of Bacteria were recognized (Woese et al. 1987), the number of phyla continues to increase due to culture activities and especially environmental rRNA gene surveys. Currently the public databases identify 70 phyla of bacteria (Pace 2009). Sequencing rRNA genes is then the method of choice for phylogenetic reconstruction, nucleic acid-based detection and quantification of microbial diversity. We can consider it as the “gold-standard” methodology. The exponential increase of public available rRNA sequences made the necessity to have specialized databases for their storage (Amaral-Zettler et al. 2008). The three main database projects that provide access to the dataset and alignments are Ribosomal Database Project II (RDPII, http://rdp.cme.msu.edu/), Greengenes (http://greengenes.lbl.gov/) and the SILVA system (http://www.arb-silva.de/). At the moment we are approaching almost 2.0 million of sequences that are available to the public in the last released from the SILVA database in this year 2011.
Functional microbial diversity
To evaluate functional microbial diversity 16S rRNA is mostly not a suitable gene because the biochemical, physiological or ecological capabilities of bacteria and their taxonomic affiliation are often not correlated. The use of protein-coding genes allows surveying for particular capabilities or processes and can also be helpful for identification (Hurek and Reinhold-Hurek 2005). Depending on the function studied several genes have been chosen. For example, people interested in the diversity of methane oxidizing bacteria use the pmoA gene, which encodes the PmoA subunit of the membrane-associated form of the methane monooxygenase. It has been shown that this protein is evolutionarily highly conserved among methanotrophs (Holmes et al. 1995, Henckel et al. 1999, Hoshino et al. 2001, Pester et al. 2004, Cunliffe et al. 2008, Tchawa et al. 2010, Lücke et al. 2010), therefore, it is a gene that can be used for diversity analysis. Another case is the study of nitrifying bacterial diversity where researchers have used the gene amoA that encodes the active site of ammonia monooxygenase. The phylogenies based on this gene and the 16S rRNA are congruent, a fact that has been taking into account to use extensively this gene as a molecular marker to explore and characterize ammonia-
8 Introduction
oxidizing bacterial communities (Sinigalliano et al. 1995, Purkhold et al. 2003, Moin et al. 2009, Dang et al. 2010). When the focus of the study has been the diversity of nitrogen fixing bacteria, the nifH gene that encodes for the dinitrogenase reductase subunit of the nitrogenase has been extensively used (Ueda et al. 1995, Zehr et al. 1998, 2003, Hamelin et al. 2002, Tan et al. 2003, Langlois et al. 2005, Deslippe & Egger 2006, Severin et al. 2010, Burbano et al. 2011) (see below).
Biological nitrogen fixation
Nitrogen is an essential element for all living organisms because it is the basic component of proteins, nucleic acids, enzymes and other cellular elements. The most common form found in nature is nitrogen gas that makes up for 80% of all gases in the atmosphere (McArthur 2006). The majority of organisms is unable to use atmospheric nitrogen and therefore N2 could be a limiting nutrient for their growth and reproduction. The process by which atmospheric nitrogen enters into the nitrogen cycle and becomes accessible to all living organisms is called biological nitrogen fixation (BNF), a process by which the nitrogen gas (N2) is converted to ammonia (NH3). This energetically costly process is exclusively carried out by a limited, but diverse, group of microorganisms (bacteria and Archaea) known as diazotrophs that possess the oxygen sensitive enzyme nitrogenase (Zehr et al. 2003, Young 2005, Zehr and Paerl, 2008). BNF is estimated to contribute globally between 200-300 million tons of fixed-nitrogen per year: terrestrial systems accounting for 90-130 million tons fixed N2 per year
(Galloway et al. 1995) ad marine systems for 100-200 million tons fixed N2 per year (Karl et al. 2002). Nitrogenase, the enzyme that catalyzes the process above mentioned is a complex composed of two subunits: the iron containing dinitrogenase reductase (or Fe protein) encoded by the nifH gene and the dinitrogenase (or Mo-Fe protein) which generally contains molybdenum and is composed of alpha and beta subunits encoded by nifD and nifK genes respectively (Burris 1991, Dean & Jacobson 1992, Rees et al., 2005). The mechanism of BNF can be summarized in five fundamental steps: (i) reduction of the Fe protein by electron donors such as ferredoxin and flavodoxin, (ii) association of two MgATP to the Fe protein, (iii) association of the Fe and MoFe proteins to form a complex, (iv) electron transfer (one for each cycle) coupled to the MgATP hydrolysis and (v) dissociation of the complex. This cycle is repeated until the Fe-Mo protein
9 Introduction
accumulates eight electrons necessary for the reduction of N2 (Dixon & Cahn 2004).
Per one molecule of N2 reduced, two molecules of NH3 are produced, and hydrogen is also generated as a by-product of this reaction (Simpson & Burris 1984). Sixteen molecules of ATP are hydrolyzed during a complete reaction cycle and eight electrons have to be transferred from the Fe protein to the Mo-Fe protein. Therefore, for every transferred electron, two ATP molecules are consumed (Rees & Howard 1999, Halbleib & Ludden 2000). Nitrogen fixing organisms can exist in a wide variety of habitats. They can be found as free-living organisms in soils and water, they can have close association such as the one with members of the grass family, or they can form symbiotic associations: inside termite guts, actinorhizal associations with woody plants, cyanobacterial symbioses with various plants and root–nodule symbioses with legumes (Dixon & Cahn 2004). One of the best-studied symbiotic interactions involves plant legumes and bacteria collectively known as rhizobia. More than 18.000 legume species are nodulated by members of the - and -Protebacteria. The nitrogen fixed by this interaction accounts for a fourth of all the nitrogen fixed annually on earth (Sprent 2008, Masson-Boivin et al. 2009). In this case, the bacteria fix nitrogen as endosymbionts inside root nodules in a nutrient-rich, oxygen-controlled microenvironment (Reinhold-Hurek & Hurek 1998). On the other hand, members of the grass family also benefit from BNF but specialized symbiotic structures are not detectable (Hurek et al. 2002). Brazilian sugarcane varieties can obtain a substantial part of plant nitrogen from BNF (Lima et al. 1987, Boddey et al. 1995). Likewise, significant nitrogen fixation has also been demonstrated for rice grown under waterlogged conditions (Boddey et al. 1995, Wu et al. 1995).
nifH gene as a functional marker
Although root-associated diazotrophs have been reported in culture dependent studies (e.g. Gillis et al. 1989, Hurek et al. 1994, James 1994, Baldani et al. 1996), it is also known that these bacteria may be recalcitrant to isolate in pure culture (Hurek et al. 2002). This is the case for the endophytic diazotroph Azoarcus sp. BH72 found in Kallar grass, which can colonize field-grown non-diseased plants in high numbers and become the most actively nitrogen-fixing bacterium there (Reinhold et al. 1986, Hurek et al. 2002). However, these bacteria are usually not culturable in planta (Hurek et al. 2002). Culture-independent methods have the capability to reveal a wide range of
10 Introduction
mostly uncultured bacteria that occur in natural habitats including gramineous plants (Zehr et al. 2003, Hurek and Reinhold-Hurek 2005). For example, in a survey of the activity of diazotrophs in the rhizosphere of the smooth cordgrass Spartina alterniflora, none of the phylotypes could be ascribed to a cultivated bacterium (Brown et al. 2003). To monitor diazotrophs the most reliable molecular marker has been the iron-protein gene (nifH, anfH or vnfH, together called “nifH”) of the evolutionary conserved nitrogenase protein complex (Zehr and MecReynolds 1989, Howard and Rees 1996, Hurek and Reinhold-Hurek 2005). There are three types of related nitrogenases: a molybdenum-based (nif), an iron-only based (anf), and a vanadium-based (vnf) nitrogenase (Hurek and Reinhold-Hurek 1995). The majority of the N2-fixing prokaryotes carry only nif genes, however, some organisms like Azotobacter vinelandii, are able to synthesize any of the three nitrogenases, depending on the growth conditions (Bishop and Premakumar 1992). As mentioned before, the nifH gene has been extensively used as a molecular marker to assess the diversity of nitrogen-fixing bacteria from different environments. The database for the nifH gene has become one of the largest non-ribosomal gene datasets of uncultivated microorganisms (Zehr et al. 2003). There are relatively few nifD and nifK sequences available with respect to the number of nifH sequences. Therefore, the use of these genes as phylogenetic markers is still limited (Ueda et al. 1995). Phylogenetic analysis of each of the components of the nitrogenase protein together with their known homologs, revealed that they separate into distinct, topologically consistent groups (Raymond et al. 2004): Group I is formed by the typical Mo-Fe nitrogenases, predominantly composed of proteobacterial and cyanobacterial phyla; group II is constituted by Mo-Fe nitrogenases from a wide range of predominantly anaerobic organisms (clostridia, acetogenic bacteria, and several methanogens); the alternative nitrogenases, including the Mo-independent anf and vnf genes are part of the group III; uncharacterized nif homologs detected only in methanogens and some anoxygenic photosynthetic bacteria form the group IV; and finally the group V is composed by bacteriochlorophyll and chlorophyll biosynthesis genes common to all phototrophs. A phylogenetic tree constructed from concatenated homologs of the NifH and NifD proteins shows the main groups described above (Figure 2).
11 Introduction
Figure 2. Phylogenetic tree of concatenated NifH and NifD homologs found in complete genomes where the five phylogenetic groups are shown (Raymond et al. 2004).
Study of active diazotrophic bacteria
The characterization of diazotrophic communities based on the amplification of nif DNA has the drawback that the detected sequences do not necessarily represent diazotrophs that are presently active (Röling and Head 2005). Moreover, the nif-DNA surveys can only be used to identify the presence or absence of nitrogenase genes, as a catalog of the diazotrophic diversity in a particular environment. In contrary, if we use nif-mRNA surveys, we can know the actual activity of the microbes and evaluate whether the bacteria studied are of any importance for BNF in natural environments (Hurek & Reinhold-Hurek 2005). There is a tight relationship between nitrogenase activity and nifH transcription in diazotrophs. Expression of the nif genes is regulated in response to the cellular nitrogen status and oxygen concentration (Merrick and Edwards 1995). If nitrogen fixation is repressed because of combined nitrogen is
12 Introduction
present or there are unfavorable O2 concentrations, the nif genes are not transcribed + (Merrick 1992). For example, in the presence of 10 mM of NH4 , no nifH transcripts from the grass endophyte Azoarcus sp. BH72 could be detected in northern blots (Egener et al. 2001). In agreement with this result, no nifH reverse transcription- polymerase chain reaction (RT-PCR) products were obtained using RNA preparations + from cultures of the same bacteria grown in a complex medium with 10 mM of NH4 (Hurek, unpublished). The use of RT-PCR in the study of expression of nitrogenase genes, allows not only the evaluation of the active diazotrophs but also the identification of the primary diazotrophic bacteria by a comparison of sequences retrieved from the environment with sequences from cultivated organisms (Hurek et al. 2002, Hurek & Reinhold-Hurek 2005). To capture the majority of nifH sequences, specific primers targeting a sequence common to all nifH genes must be degenerate. Various sets of universal nifH primers are available (Zehr and McReynolds 1989, Ueda et al. 1995, Okhuma et al. 1996, Piceno et al. 1999, Widmer et al. 1999, Poly et al. 2001, Zani et al. 2001), which amplify effectively fragments of distantly related diazotrophs but only those of Zehr and McReynolds (1989) have been shown to amplify highly diverged nifH genes with equal efficiencies (Tan et al. 2003, Demba Diallo et al. 2008). Despite the fundamental importance of fixed-nitrogen for essential ecological and biogeochemical processes, the appropriate identification of the full diversity of diazotrophs in many environments is still very limited (Zehr 2003, Hurek and Reinhold- Hurek 2005, Ward et al. 2007). Although PCR based approaches targeting nifH genes have significantly expanded our understanding of these microbial communities (Zehr et al. 2001), there are some limitations to this approach and some improvements that have to be done to fully characterize the community of nitrogen-fixing bacteria within a system.
13 Objectives
Objectives
One of the most widely utilized fertilizers in agriculture is fixed-nitrogen and its use continues to increase globally. Its availability often limits plant growth in natural ecosystems and impacts productivity. BNF provides the majority of fixed-nitrogen and counterbalances nitrogen losses (Reinhold-Hurek and Hurek 2007). The main goal of this PhD work was the study of active diazotrophic bacteria associated to plants. This is the first step to be able in the future to further improve soil fertility for a more sustainable land use. To get a closer insight into the active diversity of plant-associated diazotrophic bacterial community and even more important, to be able to capture the majority of its individuals in environmental samples, strategies to improve culture-independent methodologies were the first objective of this thesis, specifically, to progress on the nifH gene detection by implementing the use of locked nucleic acids (LNAs) modified primers (Chapter 1). The most important crops worldwide (wheat, maize, rice, sugarcane) belong to the Poaceae family, which do not form naturally specialized symbiotic structures. However, it has been shown that some sugarcane cultivars may benefit substantially from BNF (Boddey et al. 1995) and several diazotrophic bacteria have been isolated from stems and roots parts (Baldani et al. 2002, Ando et al. 2005). Nevertheless, their role in BNF with sugarcane is largely unknown. The second purpose of this work was to identify the key active nitrogen fixing diazotrophic bacteria associated to roots of sugarcane plants from Africa and America by culture independent studies (Chapter 2). The legume plant mopane (Colophospermum mopane) is an indigenous tree from Southern Africa that is widely distributed and is used for several domestic purposes, being of economical importance for the region. Mopane belongs to the subfamily Caesalpinioideae and the tribe Detarieae. Nodulation in the subfamily is uncommon and many members lack the ability to produce root nodules (Allen & Allen 1981, Sprent et al. 2007). Nevertheless, the occurrence of nodulation in this subfamily is still not clear. Studies that investigated the production of root nodules in mopane pot cultures showed that this plant does not produce them (Basak and Goyal 1980, de Faria et al. 1989). However, roots collected in South Africa showed some structures that might represent primitive nodules (Jordaan et al. 2000). To understand and learn more about its ecology and how mopane has been able to grow in the nitrogen-poor soils of the
14 Objectives
region, the third goal of the thesis was to study the possible nodulation in mopane roots and the analysis of the root-associated bacteria, to assess its active diazotrophic diversity and total microbial diversity by culture dependent and independent techniques (Chapter 3). In the Kavango region of Namibia, subsistence farmers cultivate several crops (pearl millet, maize, cowpea, sorghum) with low input of nutrients ending up with very low yields. Microbial populations may contribute to improve yields by increasing plant tolerance to stress, biocontrol of pathogens and direct plant growth-promotion. The last objective of the project was the isolation of root-associated native bacteria of pearl millet, maize and sorghum, important traditional agricultural crops from the region. Bacteria were taxonomically characterized and their putative plant-growth-promoting traits studied. These isolates can be considering potential candidates for application in sustainable agriculture in the region (Chapter 4).
15 General discussion
General discussion
Nitrogen fixation and photosynthesis are the basis of all life on earth. Nitrogen fixation provides the basic component, fixed nitrogen as ammonia, of two major groups of macromolecules, nucleic acids and proteins (Newton 2004). Despite the great ecological and practical significance of the process, the key players of the process, the diazotrophic bacteria and its diversity in natural communities, are still poorly understood. New powerful molecular biology techniques based on cultivation independent approaches allows a detailed analysis of microbial populations in the environment (Felske and Osborn 2005). In this manner, we have started to explore their full diversity and to understand the complex interaction between these bacteria and their environment.
Refinement of methods for detection of bacterial mRNA
In this thesis the measurement of gene expression via detection of mRNA after RT- PCR was the method of choice to analyze functional diazotrophic diversity. Since prokaryotic gene expression is a finely regulated process (Grunberg-Manago 1999), detection of transcripts for a given gene, constitutes significant evidence of the occurrence of a given biological process within the environment (Nogales 2005). RNA is synthesized only by actively growing cells and degrades relatively rapidly once produced. It arises from the functioning members of environmental microbial communities and can thus identify them (Hirsh et al. 2010). Working with RNA brings intrinsically some problems that have to be considered. Prokaryotic mRNA is a labile molecule with a short half-life (Grunberg-Manago 1999). Because of that, substantial care has to be taken to avoid RNA degradation during its isolation and the subsequent preparation for the RT-PCR (Nogales 2005). In order to isolate mRNA, total RNA extracts were performed. In addition to this fraction, also the more abundant ribosomal RNA (rRNA) and the transfer RNA (tRNA) fractions were obtained. The methodologies used in this thesis (Chang et al. 1993, TRIzol-Invitrogen) in the majority of the cases allowed us to have a RNA that could be used for the following steps. Nevertheless, depending on the root sample used, the RT-PCR reaction was sometimes impeded and did not give the desired amplification product. Interference might be caused by PCR inhibition or low template concentrations. The occurrence of possible false negative results is one of the drawbacks that can occur
16 General discussion
when PCR-based approaches are used on complex environmental samples. In all the reactions a positive control was used to distinguish between possible errors in the preparation of the reaction and samples-related problems. We spiked the sample with positive-control DNA to establish if the absence of PCR product was caused by the presence of inhibitory material in the PCR reaction. Dilution of the nucleic acid sample was another method, which we used to reduce the levels of the putative inhibitory contaminant to levels lower than the one at which the PCR is inhibited. In general inhibitors act at one or more of three fundamental points in the reaction: (i) interference with the cell lysis in the nucleic acid extraction, (ii) interference by nucleic acid degradation or capture, (iii) inhibition of polymerase activity for amplification of the target molecule (Wilson 1997). The clearest source of PCR inhibitors in endogenous contamination is compounds present in insufficiently purified target DNA. Inhibition of PCR by phenolic substances, polysaccharides or humic acids has often been reported in association with nucleic acid extraction from plant tissues (Demeke et al. 1992, John 1992, Tsai et al. 1992, Jacobsen et al. 1992). Phenolic compounds from the sample or carried over from organic nucleic acid purification procedures can inhibit the reaction by binding to or denaturing the lytic enzymes, and failing to expose the DNA (Jacobsen et al. 1992, Young et al. 1993, Simon et al. 1996). The phenolic groups of humic compounds can denature biological molecules by bonding to amides or by oxidizying to form a quinone, which covalently bonds to DNA or proteins (Wilson 1997). The addition of polyvinylpolypyrrolidone (PVPP) or polyvinylpyrrolidone (PVP) can overcome the inhibition and allow the separation of humic compounds from DNA during agarose gel electrophoresis. Dimethyl sulfoxide (DMSO) has been shown to improve reaction yield during RT-PCR (Sidhu et al. 1996). This compound may enhance PCR by eliminating nonspecific amplification, altering the thermal activity of the polymerase, or may improve the annealing efficiencies of primers by destabilizing secondary structures within the template (Wilson 1997). Another important consideration that has to be taken into account while working with RNA is to assure that the amplification products are not derived from contaminating DNA. Usually a DNAse treatment is performed to ensure that only RNA is amplified. A reaction without a previous RT reaction is used as a control. All experiments done in this project had this necessary type of control, fundamental to then speak about active microorganisms. Additionally, to check for possible contaminations we included a negative control to which no template was added.
17 General discussion
The above-mentioned difficulties led us to develop strategies to improve RT detection by increasing sensitivity and efficiency of PCR reactions. Locked nucleic acids (LNAs) are DNA analogues where the furanose ring in the sugar phosphate backbone is chemically locked (Yamada et al. 2008). The modification provides the LNA bases with stronger binding strength for complementary sequences (Petersen et al. 2000, Jensen et al. 2001). These bases can be incorporated into DNA or RNA oligonucleotides inducing a conformational change in the local helix (Kaur et al. 2006). LNAs have been used in several applications such as real-time PCR probes (Tolstrup et al. 2003, Mouritzen et al. 2004), antisense oligonucleotides (Wahlestedt et al. 2000), detection of single nucleotide polymorphism (SNP) analyses (Ørum et al., 1999, Simeonov and Nikiforov 2002), microarray probes (Castoldi et al. 2008) and PCR primers (Latorra et al. 2003, Ballantyne et al. 2008, Yamada et al. 2008). However, they have not yet been widely used in molecular microbial ecology. Therefore, we studied the use of LNA-substituted primers on the detection of nifH transcripts (see Chapter 1). A fundamental aspect in the analysis of microbial communities is the retrieval of a uniform picture of the whole microbial population. For that reason PCR amplification should proceed without major bias. In the case of diazotrophic diversity, specific primers targeting a sequence common to all nifH genes must be degenerate (Demba Diallo et al. 2008). However, using this type of primers could lead to low amplification efficiencies, sensitivity and specificity (Martin et al. 1985, Watkins and Santa Lucia 2005). Various sets of nifH PCR primers have been designed (Zehr & McReynolds 1989, Ueda et al. 1995, Widmer et al. 1999, Poly et al. 2001) but just the primers designed by Zehr & McReynolds (1989) have been shown to amplify very different nifH genes with equal efficiencies (Tan et al. 2003, Zhang et al. 2007, Demba Diallo et al. 2008) and to cover the greater part of nifH genes without mismatches in the primer target region (Demba Diallo et al. 2008). Because of this, we took the sequence of these primers and designed their LNA-modified primers counterparts. We studied the effect of these substitutions on the detection of nifH transcripts by RT-PCR in roots of rice and sugarcane. Previous reports have shown that LNA modifications increased the specificity of PCR amplification, improved sequencing read quality, and reduced the amount of template required (Ballantyne et al. 2008). In our case the use of the LNA-modified RT primer increased at least 25-fold more the sensitivity of the RT-PCR. This primer could have improved the RT step by enhancing hybridization to the target RNA and/or by
18 General discussion
efficient primer extension through RT as has been shown before (Fratczak et al. 2009). Similar results have been found in studies of microorganisms from digestive tracts of horses and termites (Yamada et al. 2008). When LNA primers were used in rolling circle amplification, they increased the overall sensitivity of inverse PCR from 10 to 1000 times, detecting as many as 10 copies of the target DNA in a sample. Additionally, the use of the RT LNA primer allowed the amplification of RT-PCR products in sugarcane samples that were not possible to obtain when the non-modified RT primer was employed. The reduction of the false negative rate achieved by our methodology is an important result, specifically when the key diazotrophic bacteria under study are unknown as in sugarcane (see Chapter 2). In some root samples of sugarcane it was not possible to get a direct RT-PCR product because of that and in order to analyze all the plant root samples in the same manner, a nested PCR approach developed by Zani et al. (2000) was applied. Additionally to the Zehr & McReynolds primers, this group designed a pair of primers called nifH4 and nifH3 based on conserved sequences outside of the ZehrF and ZehrR primers. In the case of the mopane root samples the only way to get an amplification product was using this nested approach but with all four primers carrying LNA- modifications (see Chapter 3). The nested PCR approach we applied has been widely used for the assessment of complex nitrogen-fixing communities and has been shown to amplify nifH sequences from all major lineages of nitrogenases involved in nitrogen fixation (Langlois et al. 2005, Moisander et al. 2008). The target sites of the four primers used are conserved throughout nifH genes in all the lineages (Zani et al. 2000) and consequently are highly suitable for broad-range nifH specific PCR amplification. As mentioned before, high coverage of divergent nifH genes and amplification with equal efficiencies are well documented for the inner primers (Zehr & McReynolds 1989) that were also used here for PCR followed by denaturant-gradient gel electrophoresis (DGGE) (Tan et al. 2003, Zhang et al. 2007, Demba Diallo et al. 2008, Burbano et al. 2010). To study the structure of the diazotrophic communities, different culture independent techniques were used: clone libraries, DGGE and terminal restriction fragment length polymorphism (T-RFLP). Moreover, we used bioinformatic tools to evaluate the diversity of the nifH phylotypes found. A phylogenetic analysis complemented all the studies (see below).
19 General discussion
Searching for the primary active diazotrophic bacteria in sugarcane
Sugarcane is a non-leguminous field crop that is cultivated on a wide diversity of soil types (Malavolta 1994) and in large areas of many tropical countries around the world (Renouf 2010). Apart from its most common uses (sugar and alcohol production), a diverse range of other products such as electricity, organic chemicals and paper can also be derived from it or its products (Paturau 1989, Manohar Rao 1997). BNF has been identified as the principal nitrogen contributor in some sugarcane varieties (Urquiaga et al. 1992, Boddey et al. 1995), and diazotrophic bacteria have been detected, isolated and reported as endophytes of sugarcane cultivars (Baldani et al. 2002, Loiret et al. 2004, Ando et al. 2005). Nevertheless, these studies have not answered yet the question of which microorganisms are responsible for the nitrogen- fixing activity detected under natural conditions (Baldani et al. 2002). The opportunity to have sugarcane root samples from three different continents (Africa, North and South America) and the possibility to use culture-independent techniques, allowed us to elucidate the major nitrogen-fixing bacteria associated to this plant and to find out how similar or different the diazotrophic populations might be depending of the site of sampling (see Chapter 2). We found that a major and almost unique nifH phylotype highly related to the order Rhizobiales was found in all the samples studied regardless of the site from where they were taken. An aspect covered and extensively described in Chapter 2 of this thesis. For the other nifH phylotypes found the closest cultivated bacteria they were related to, were also determined. In general, all the sequences obtained were related to the phyla Proteobacteria and Cyanobacteria. All the Brazilian nifH phylotypes were related only to members of the Rhizobiales, while the Mexican and Namibian samples showed nifH phylotypes related to other bacteria. With respect to the Mexican samples, the minority of the sequences clustered with nifH sequences from Methylococcus capsulatus, from which mRNA transcripts have also been found in stems of rice (Elbeltagy et al. 2008). The most diverse sample was from the Namibian sugarcane, having nifH phylotypes related not only to Rhizobiales but also to cyanobacteria, - and -Proteobacteria. The majority of the sequences clustered together with nifH sequences from sulfate reducing bacteria (SRB) Desulfovibrio gigas. Recently, however, it has been shown that there has been horizontal gene transfer from members of the family Desulfovibrionaceae, most likely
20 General discussion
D. vulgaris or D. gigas to the cyanobacteria Microcoleus chthonoplastes (Bolhuis et al. 2010). This bacterium contains a functional nif-gene cluster, which is not typical cyanobacterial. Phylogenetic analysis showed that the genes of the nifHDKENB cluster grouped with -proteobacteria rather than with cyanobacteria. In our case, the nifH sequences had ~85% and ~83% sequence similarity to D. magneticus and D. gigas, respectively. Our nifH sequences had 69.9% - 71.8% sequence identity to the nifH of M. chthonoplastes, values lower than the similarity found to the Desulfovibrio species. Sequences related to this genus have been found in rice roots and rhizosphere of some grasses (Ueda et al. 1995, Hamelin et al. 2002, Brown et al. 2003, Gamble et al. 2009), bringing the idea that N fixation by SRB may be important not only in marine environments as found before (Bertics et al. 2010) but also in non-legume plants. Some other sequences clustered together with cyanobacteria (Anabaena sp. and Lyngbya sp.) known to be predominant in aquatic environments (Zehr et al. 2003) but also able to form symbiosis with plants such as Azolla and Gunnera fixing N2 (Silvester and Smith 1969, Newton and Herman 1979). Moreover, Anabaena species have been isolated from rice fields in China and Taiwan (Ley et al. 1959, Chen 1984) and recently sequences similar to this phylum have also been found in sweet potato stems (Terakado-Toonoka et al. 2008) and as the dominant diazotroph (87%) in red soils in China (Teng et al. 2009). A unique sequence clustered together with Ideonella sp. Long 7 nifH, a bacterium previously isolated by T. Hurek from roots of the wild rice Oryza longistaminata. This genus has also been found in roots and stems from maize (Roesch et al. 2008) and sorghum rhizosphere (Cohelo et al. 2008). Additionally, we also studied diazotrophic bacteria in rhizospheric soil from plants of Mexican sugarcane and shoots from Brazilian sugarcane. We analyzed RT-PCR products by direct sequencing and clone library approach. We found that the sequences from rhizospheric soil and shoot were related to the partial nifH sequences from the -proteobacterium Sulfurospirillum multivorans and the -proteobacterium Bradyrhizobium elkanii, respectively. Based on these results and the ones presented later in Chapter 2, it is important to note that no nifH transcripts were found from all the previously isolated bacteria (from shoot or root of sugarcane). This opens the possibility that other bacteria could be important for the N2 fixation in sugarcane and that more studies will be required to isolate the bacteria and test their N2 fixation capacity in planta.
21 General discussion
A non-nodulated legume tree with active diazotrophic bacteria associated to it
Another important part of this thesis was the study of possible nodulation and microbial communities associated with the roots of the mopane legume tree (see Chapter 3). Mopane has been studied in the context of plant-herbivore interactions (Ferwerda et al. 2005, Oppong et al. 2009, Kohi et al. 2010) because its leaves are the principal food source for animals in the region, specially elephants and insects like the caterpillar of the mopane emperor moth (Imbrasia belina), widely harvested and consumed as a high protein food source by the local people there (Illgner and Nel 2000). An interesting aspect about the microorganisms associated with mopane, was that either in the bacterial isolates spectrum or the 16S rRNA sequences recovered, the majority of the bacteria and phylotypes found were related to the phylum Actinobacteria (Figure 3). In the context of nitrogen fixation, Frankia has been the most studied species from this phylum, because is able to grow as a microsymbiont in the root nodules of several woody dicotyledonous plants (Benson et al. 1993). Nonetheless, other diazotrophic species (Arthrobacter, Nocardia and Rhodococcus strains) have been found in humus of Norway spruce (Elo et al. 2000), and even more interesting for us, in other legumes like in surface sterilized roots of Casuarina equisetifolia where two nitrogen-fixing bacteria were isolated and clustered together with the families Thermomonosporaceae and Micromonosporaceae (Valdés et al. 2005). Recently, a high number of actinomycete colonies was isolated from surface-sterilized root nodules of Lupinus angustifolius that belong to genus Micromonospora. Some of the isolates were able to fix nitrogen and their nifH sequences were 99% similar with nifH gene from Frankia alni, suggesting that this genus is a natural inhabitant of nitrogen-fixing root nodules (Trujillo et al. 2010). In our case, the presence of the nifH gene in the actinobacteria isolates is still not clear because we have obtained contradictory results, and a good quality sequence is still not available to do further analysis. It is well known that in rhizobia, the nif genes are on either plasmids or genomic islands (Galibert et al. 2001, Kaneko et al. 2000; 2002), which are parts of the genome that are prone to transfer at least between related bacteria (Young 2005). A possible explanation might be that the structural genes for nitrogen fixation are also located on plasmids and they might be lost in some of the isolates we recovered. However, this hypothesis has to be corroborated. With
22 General discussion
respect to the 16S rRNA sequences, just 10 out of the 195 Actinobateria-like sequences were related to the genera (Frankia, Arthrobacter, Nocardia and Rhodococcus) known to be nitrogen-fixers in plants. However, the actinomycetes associated with the roots of mopane might be playing also other roles. They might promote plant-growth by phytohormones and siderophores production or protect the plant against pathogens by producing antibiotics or extracellular enzymes (Clegg and Murray 2002, Bailey et al. 2006, Nimnoi et al. 2010). Regarding the possible diazotrophic diversity associated with mopane roots, it is important to emphasize that we found active nifH phylotypes in all the mopane roots analyzed with diversity higher than the one expected (see Chapter 3 for further explanation).
Figure 3. Major bacterial groups found associated with root of mopane trees by culture dependent (A) and independent (B) techniques.
Discovering the diversity of beneficial bacteria using the classical approach
Apart from the use of molecular tools to explore and understand diazotrophic communities associated with legume and non-legume plants, the last part of the thesis was the isolation and characterization of bacteria from crops grown in the Kavango region of Namibia. A screening of possible plant-growth promoting characteristics was also performed on the isolates (see Chapter 4). We obtained 44 bacterial isolates that were coming from maize, sorghum and pearl millet, crops that are cultivated primarily for subsistence in Namibia. The majority of the isolates (67%) belonged to the gram-
23 General discussion
positive bacteria (Firmicutes and Actinobacteria) and the rest to the gram-negative (Proteobacteria). These results were in agreement with the ones we obtained from the isolate spectrum in mopane samples where 64% of the bacteria belonged to gram- positives (Chapter 3, Table 1). The high number of gram-positive bacteria isolated from all the plants we sampled in Namibia, may indicate that these microorganisms are able to survive in the nutrient poor soils of the region. Plant-bacteria interactions have been widely studied in gram-negative bacteria but have been less documented in gram- positive ones (Francis et al. 2010). It has been documented that gram-positives can be used as biocontrol bacteria (El-Tarabily & Sivasithamparam, 2006) and they can also promote plant growth (Tsavkelova et al. 2006, Spaepen et al. 2007, Sziderics et al.2007, Jorquera et al. 2008). In this part it is important to mention that the use of the classical cultivation approach is still needed to be able to understand characteristics and properties of bacteria. We can learn how they contribute to the environment they are coming from and expand the knowledge of the existing microbial diversity (Hugenholtz et al. 1998, Rodríguez-Valera 2002, Schleifer 2004, Pontes et al. 2007). In our case, the combination of conventional and molecular methodologies allowed us for the first time to describe isolates in the Kavango region with potential use as growth-promoting bacteria (see further details in Chapter 4).
Active diazotrophic bacterial communities
This PhD work focused on the study of active diazotrophic bacteria associated with roots of legume and non-legume plants. Two of the plants studied are monocotyledons and belong to the Poaceae family (rice and sugarcane) while the third one (mopane), is a dicotyledon and is included in the Fabaceae family. Rice is one of the most important sources of energy and protein of the diet in humans. After wheat, this cereal occupies the second place as the world’s most important staple food grain and is cultivated on 10% of the earth’s arable land (Vaughan & Geissler 2009). On the other hand, sugarcane is a tropical crop that not only generates the 86% of all the world’s sugar (Vaughan & Geissler 2009) but also together with corn produces 40% of the feedstock needed for biofuel production in form of ethanol (Sims et al. 2008). Finally, the importance of mopane resides in its multiple uses for domestical purposes -especially timber- and also as a food plant for animals in
24 General discussion
the region (Ferwerda et al. 2005). We detected nifH transcripts in all of the root samples used and further analyzed the diversity of the diazotrophic community associated with each of the plants above mentioned. The possibility to survey nif-mRNA let us determined the community members involved in nitrogenase gene expression because of the tight relationship between nitrogenase activity and nif-mRNA transcription (Greer et al. 2001). A total of 352 nifH transcripts were obtained from which 93 came from rice, 144 from sugarcane and 115 from mopane. All these sequences were included in a phylogenetic analysis to have an overview of these diazotrophic communities, and to compare them depending from which plants they were obtained from (Figure 4). None of the sequences belonged to Fe nitrogenases (group III). Rice sequences were obtained from roots of the wild species Oryza longistaminata, a plant that grows in Namibia and shows consistent nifH transcription levels (Demba Diallo et al., 2008). The nifH transcripts we obtained were distributed over the whole tree. This suggested that the community of active nitrogen-fixing bacteria associated with roots of this wild rice species was diverse and predominated by Proteobacteria and few other phyla, a result that is consistent with a previous study where O. longistaminata root samples from the natural habitat were compared with microcosm grown plants (Demba Diallo et al. 2008). In the case of sugarcane nifH transcripts, the diazotrophic diversity was lower. The majority of the sequences were related to nifH fragments from cultured bacteria affiliated with the order Rhizobiales, mostly to Rhizobium rosettiformans, a species recently described and isolated from a dump site in India (Kaur et al. 2010). In this respect it has been shown that other species of Rhizobium are also able to colonize roots of plants different from legumes. For example, R. leguminosarum bv. trifolii efficiently colonizes roots of several monocotyledons, such as wheat, maize and rice (Schloter et al. 1997, Yani et al. 1997, Dazzo & Yanni 2006) and R. etli has been found as an endophyte of maize (Gutierrez-Zamora & Martínez-Romero 2001). Our results expand the spectrum of colonization of the Rhizobium genus and particularly of R. rosettiformans to other non-legume plants such as sugarcane. A recent study on Japanese sugarcane cv. NiF8 also showed that bacteria related to rhizobia express nitrogenase genes in shoots and roots but with much higher diversity than our findings. They found a large number of sequences related to Bradyrhizobium sp. and also other sequences were associated to Azorhizobium caulinodans and Sinorhizobium fredii (Thaweenut et al. 2011). With respect to mopane, the third plant analyzed, it is
25 General discussion
important to highlight that this is the first study showing that active nitrogen-fixing bacteria are associated with its roots. The nifH transcripts we obtained were not just related to rhizobia but also to other phyla such as Spirochaetes and other proteobacterial classes, opening new ideas about diazotrophic associations with legume plants. To estimate how similar or different the diazotrophic communities were between the three plants studied, we used the measurement of phylogenetic distances. This type of measurements takes into account the degree of divergence between different sequences (Lozupone & Knight 2005). The Unifrac significance test and the P test were carried out to compare the three environments. The first test, measures the similarity between communities as the fraction of branch length in the tree that is unique for an environment, while the second uses the number of changes from one environment to another in a branch that is required to explain the distribution of sequences between environments in the tree (Lozupone et al. 2006). Both results showed that there were significant differences between all of the environments (P 0.03). However, a cluster analysis revealed that the rice community was the most distant, while the mopane and sugarcane communities clustered together. The PCoA (Principal Coordinate Analysis) also revealed this pattern between the samples, the first principal coordinate, which explained 70.44% of the variation in the data separated mopane and sugarcane from rice diazotrophic communities (Figure 5). A possible explanation for this result is that diazotrophic communities in rice were more diverse (several phylotypes were found along the phylogenetic tree), while the diazotrophs found in mopane and sugarcane where mainly related to Proteobacteria and specifically to the Rhizobiales (Figure 4). Therefore, the diazotrophic communities associated to these two plants are more similar to the scenario found in nodulated host plants, where the diversity of the nitrogen-fixing Rhizobium and Frankia symbionts in the symbiotic tissue is typically low. Nevertheless, a low diversity of root-associated nitrogen-fixing bacteria in other angiosperms seems to be rare. One exception is Kallar grass, where Azoarcus sp. is the most active nitrogen-fixing bacterium in roots of field-grown plants (Hurek et al. 2002).
26 General discussion
Figure 4. Minimum-evolution NifH protein tree. The tree shows the phylogenetic affiliation of 352 nifH cDNA fragments recovered from roots of rice, sugarcane and mopane root samples. Numbers at branches represent internal branch test (IBT) confidence values >50% from 500 replicates. The scale bars show the number of nucleotide substitutions per site. The parenthesis next to the name of the samples represents the number of identical sequences found.
27 General discussion
Figure 5. Clustering analysis (A) and Principal Coordinate Analysis -PcoA- (B) of active diazotrophic communities associated to roots of rice, mopane and sugarcane plants.
Additionally, these results are interesting because although rice and sugarcane belong to the same family, their respective root-associated diazotrophic communities were different, and despite that, mopane clustered together with sugarcane. Even though mopane is part of the legume family, nodules have not been found yet. Only in few of our samples a thickening of roots or protuberance was observed. Previously, an anatomically study from the roots of mopane showed that in lateral roots there is a formation of a coralloid-like outgrowths that “seems to be induced by bacteria” and it was proposed that the root clusters can be regarded as primitive root nodules (Jordaan et al. 2000). However, another possibility is that the thickening of the roots might be caused by secondary growth. Several examples of secondary woodiness have been demonstrated in at least eight orders spread throughout the eudicots (Spicer & Groover 2010). A deep anatomical analysis together with the chemical study of the compound that gives to the roots the pink color will clarify more aspects about the possible nodulation in this plant. Another important aspect in all these type of studies is that we based our analysis on the proper description of sequences deposited in databases available to the public. It is important to be aware that databases cannot be entirely reliable, and that there are some elements such as correct strain identities and nomenclature that may create unnecessary confusions (Tindall 1994). An appropriate phylogenetic analysis is very helpful because it can show the evolutionary relationships between the sequences found. We can also use it for comparative genomics, functional predictions, the identification of new microorganisms and the detection of horizontal gene transfer -
28 General discussion
HGT- (Dereeper et al. 2008). In our case, some sequences from mopane roots that were related to a Bacillus sp. by Blast analysis, clustered together with members of the genus Bradyrhizobium after the phylogenetic analysis. In this case there is the open question to know if the Bacillus sequence deposited in the database is really coming from this bacteria or for the contrary it can by a case of HGT (see below). Unfortunately there is no more information available on the bacteria. As mentioned before, an issue that one has to pay attention when studying diazotrophic communities is HGT. This process can obscure the identity of the organisms from which the nifH sequences are coming from. There have been some examples where there is strong evidence for lateral gene transfer. Two cases are known for members of the phylum Proteobacteria: in the first one nifH sequences from two species of the genus Azoarcus (-Proteobacteria) clustered together with nifH sequences from -Proteobacteria (Hurek et al. 1997); in the second example, there is evidence for HGT between bacteria within the same class, the -Proteobacteria. In the NifH phylogeny, strains of Rhodopseudomonas palustris were associated with Rhodobacter spp. and other phototrophic purple non-sulfur bacteria, and not with its taxonomically close relative Bradyrhizobium japonicum and the phototrophic rhizobia as was deduced from comparative sequence analysis of the 16S rRNA (Cantera et al. 2004). More recently, by studying the genome of the cyanobacteria Microcoleus chthonoplastes it was possible to illustrate that this bacterium contains a functional nif- gene cluster that is not typical cyanobacterial and that most likely was acquired by HGT from a member of the -Proteobacteria (Bolhuis et al. 2010). From the sequences we retrieved, there were some nifH sequences from sugarcane roots related to Methylococcus capsulatus (-Proteobacteria) that in the phylogeny were together with strains of -Proteobacteria. This could be explained by the assumption that lateral transfer of this subclass of nif gene-sequence types might have distributed effective nitrogen-fixation capabilities among diverse microorganisms (Boulygina et al. 2002, Dedysh et al. 2004), like it was shown for other cases above mentioned. As was pointed out, the importance of HGT for the evolution of nitrogen fixation will become clearer with more bacterial genomes sequenced and including sequences from other nitrogenase structural genes into phylogenetic studies (Zehr et al. 2003, Young 2005).
29 General discussion
Furthermore, the nifH sequences from the bacteria that have transferred the gene (at least the cases mentioned above) have only a low percentage DNA sequence identity to dinitrogenase reductase genes of other genera. For example, in the case of Microcoleus chthonoplastes (accession no. NZ_DS989843) the nifH from another genus with the highest percentage of DNA sequence identity is from the verrucomicrobium Coraliomargarita akajimensis (accession no. CP001998) with 75% DNA sequence identity. This suggests that lateral transfer had occurred early in the evolution of nitrogenase (Zehr et al. 2003). In the case of nifH fragments with near perfect DNA sequence matches to nifH sequence from identified bacteria (Hurek and Reinhold-Hurek, 2005) and if the databases contain reliable data, the identification of new environmental nifH phylotypes by Blast analysis together with the phylogenetic analysis, can still be used for nifH-based taxonomic identification of diazotrophic communities associated with roots of plants.
30 Conclusions and outlook
Conclusions and outlook
It is important to study BNF due to its role in our planet’s biogeochemical processes. Nitrogen is a nutrient that is required in high amounts and its availability is a major factor limiting plant growth in natural as well as agriculture environments. Studies based on nitrogenase gene sequences and particularly nifH sequences continue helping us to discover the diversity of diazotrophic bacteria in nature and also let us identify and classify uncultivated microorganisms. This PhD focused on active diazotrophic diversity associated to roots of legume and non-legume plants. Culture-independent techniques were successfully used and in some cases refined (LNAs-modified primers) to be able to study samples from different plants. Particularly, these techniques made possible for the first time to carry out the molecular detection of nitrogen-fixing bacteria in sugarcane. The use of these techniques is also expected to improve RT-PCR detection of various genes in different environmental samples. In general, the active diazotrophic diversity varied depending on the type of plant studied. In some cases it was lower than expected, for example for sugarcane, where a predominant phylotype affiliated to the genus Rhizobium was found. But in others the diversity was high, i.e. rice, where phylotypes related to several bacteria (group I and II of nitrogenases) were found. Moreover, active diazotrophs associated to mopane roots were found for first time. The findings in sugarcane open the possibility that other bacteria –in particular R. rosettiformas-, which are different from previously isolated microorganisms, could be the most active nitrogen-fixing bacteria in sugarcane fields. It is mandatory the isolation of these bacteria to test their N2 fixation capacity in planta. It is also important to study if the nitrogen fixation occurs in roots or shoots and discover the relative contribution of each part of the plant to the total N2 fixed. The studies performed in this thesis pave the way for future studies of plant-microbe interactions in sugarcane, which can then be used to improve agricultural practices i.e. lowering the use of chemical fertilizers. A metagenome and metatranscriptome analysis of roots and shoot endophytes from sugarcane varieties that have shown to derive considerable quantities of N2 from BNF, will further increase the knowledge about functions and features of this group of bacteria. With respect to mopane there are still many open questions that will require more studies. It will be interesting to know which type of compound is the one that gives the roots their pink color. Electron microscopy analysis will also help to answer if the
31 Conclusions and outlook
bacteria associated to mopane roots are true endophytes. More detailed anatomical studies will also resolve if the outgrowths found in the lateral roots can be considered as “primitive nodules”. Efforts to continue to isolate the diazotrophic bacteria inhabiting these structures will be needed. The isolation and characterization of bacteria should continue. Native microorganisms can be use for improving the growth of plants with human and agricultural importance. It was possible in this thesis to have the first characterization of native microorganisms isolated from important crops for the Kavango region in Namibia. This characterization is the base for future studies on the impact of these bacteria in its natural environment. Plant-growth promotion features will have to be tested first at pot and later at field scale in the region. It is important to be aware of the limitations of the cultivation-independent studies. These approaches enable insights into the immense diversity of bacteria. Nonetheless, fundamental conclusions on the genetic and metabolic versatility are not possible. Only the combination of methodologies, including classical cultivation techniques and culture independent techniques gives a comprehensive insight into the bacterial diversity in natural habitats. Next generation sequencing will be valuable to cover the full diversity in the interactions plant-microbe. In order to study activity measurements, the application of stable isotope probing on mRNA will be for example a method to identify the bacteria assimilating specific substrates.
32 References
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40 Chapter 1
CHAPTER 1
LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples
Claudia Sofía Burbano, Barbara Reinhold-Hurek and Thomas Hurek
Status of the manuscript: Published in Environmental Microbiology Reports
Own contribution: - Execution of all experiments - Writing of the manuscript
Environmental Microbiology Reports (2010) 2(2), 251–257 doi:10.1111/j.1758-2229.2009.00107.x
LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in
environmental samplesemi4_107 251..257
Claudia Sofía Burbano, Barbara Reinhold-Hurek and terminal restriction fragment length polymorphism and Thomas Hurek* (T-RFLP) generally employ polymerase chain reaction Laboratory of General Microbiology, Center for (PCR) to amplify genetic markers. While for the Biomolecular Interactions Bremen (CBIB), University of cultivation-independent identification of bacteria the 16S Bremen, D-28359 Bremen, Germany. rRNA approach is most widely used, the molecular char- acterization of microbial communities with specialized physiological capabilities generally relies on the compara- Summary tive sequence analysis of protein encoding genes. In order to study the active diazotrophic bacterial To take account of the redundancy of the genetic code, community and to capture the majority of its individu- primers targeting a sequence common to all homologues als in environmental samples, strategies improving of a particular gene are usually degenerate. However, gene detection by increasing sensitivity and effi- because of an imperfect hybridization of primers with ciency of PCR reactions are highly desirable. Since mixed bases to the target sequence, amplification efficien- LNA (locked nucleic acids) modifications might alle- cies are typically low, and sensitivity and specificity are viate a low sensitivity and specificity often limiting often lacking when highly degenerate primers are used in PCR reactions utilizing degenerate primers, the effect PCR reactions (Martin et al., 1985; Watkins and SantaLu- of LNA substituted primers on the detection of nifH cia, 2005). These problems become even more serious transcripts in roots of rice and sugar cane by direct when template concentrations are low and inhibitors are reverse transcription polymerase chain reaction (RT- present, a situation often encountered when complex PCR) was studied. The LNA substitution of the RT environmental samples, e.g. from plants or soil are primer increased the sensitivity of the RT-PCR up to analysed (Poussier et al., 2002). In order to avoid a low 26-fold, whereas LNA substitution of the PCR primers PCR sensitivity and specificity, nested PCR assays are decreased specificity. Terminal restriction fragment frequently used, but this approach might introduce length polymorphism (T-RFLP) analysis of RT-PCR Taq-polymerase reading errors (Tindall and Kunkel, 1988; products showed that LNA substitutions in the Barnes, 1992) because of higher PCR cycle numbers RT-primer did not change the pattern of nifH cDNA than direct PCR-based methods. A promising new strat- phylotypes. The use of the LNA-substituted RT-primer egy could be to use locked nucleic acid (LNA) substituted allowed the detection of nifH transcripts in sugar degenerate primers in direct PCR assays. cane, where DNA primers alone failed to produce Locked nucleic acids are DNA analogues in which the RT-PCR products. These results suggest that similar furanose ring in the sugar phosphate backbone is chemi- improvements to PCR detection of nucleic acids can cally locked (Yamada et al., 2008). The LNA bases can be be expected for other environmental samples and incorporated into DNA or RNA oligonucleotides inducing genes likewise, when LNA-substituted primers are a conformational change in the local helix (Kaur et al., used. 2006). This modification provides the LNA bases with stronger binding strength for complementary sequences (Petersen et al., 2000; Jensen et al., 2001). LNA has been Introduction used in many applications like the detection of single Culture-independent molecular methods have been nucleotide polymorphism (SNP) analyses(Ørum et al., widely used to characterize microbial communities. Using 1999; Simeonov and Nikiforov, 2002), real-time PCR DNA as a signature molecule, fingerprinting methods probes (Tolstrup et al., 2003; Mouritzen et al., 2004), such as denaturing gradient gel electrophoresis (DGGE) antisense oligonucleotides (Wahlestedt et al., 2000), microarray probes (Castoldi et al., 2008) and PCR Received 27 June, 2009; accepted 21 October, 2009. *For correspon- dence. E-mail [email protected]; Tel. (+49) 421 218 2370; primers (Latorra et al., 2003; Ballantyne et al., 2008; Fax (+49) 421 218 9058. Yamada et al., 2008). LNA modifications have been
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd 252 C. S. Burbano, B. Reinhold-Hurek and T. Hurek
Table 1. Oligonucleotides used in this study.
Name Sequence (5′-3′) Reference
TH580-DNA GTTRYADATVAKVCCVCCVAG Hurek et al. (2002) TH580-LNA GTTRYADATVAKVCCVCCVAG This study Zehr-F-DNA TGYGAYCCNAARGCNGA Zehr and McReynolds (1989) Zehr-F-LNA TGYGAYCCNAARGCNGA This study Zehr-R-DNA ADNGCCATCATYTCNCC Zehr and McReynolds (1989) Zehr-R-LNA ADNGCCATCATYTCNCC This study
LNA residues are depicted in boldface and are underlined. shown to increase the specificity of PCR amplification, substitutions near the 3′ end and to use LNA pyrimidine to improve sequencing read quality, and to reduce the instead of purine substitutions because they provide more amount of template required (Ballantyne et al., 2008). stabilization. Finally, the substitution of adenine bases by Therefore, LNA modifications might also alleviate a low LNA is advocated since adenine substitutions have the PCR sensitivity and specificity often limiting PCR utilizing lowest binding strength among LNA substituted bases degenerate primers. (C > T > G >> A) and since a high binding strength may Biological nitrogen fixation is a natural source for reac- be detrimental in PCR amplifications (McTigue et al., tive nitrogen on our planet and is exclusively carried out 2004; Levin et al., 2006; Ballantyne et al., 2008). Follow- by prokaryotic cells. In order to study the active diaz- ing these guidelines our primers were designed to have otrophic bacterial community and to capture the majority three LNA substitutions that were placed towards the 5′ of its individuals in the environmental sample, PCR-based end of the sequence. However, because of the contextual surveys of transcribed nitrogenase (nifH) genes with sequences at the 5′ end of the primers in this study, we degenerate primers have been widely used. One of the could not strictly substitute only adenine bases by LNA drawbacks with PCR-based approaches is the occur- (Table 1). rence of false negatives as a result of PCR inhibition or In order to test the performance of the LNA-modified low template concentrations when complex environmen- primers, comparisons between these with their standard tal samples are surveyed. Therefore, strategies improving DNA primers counterparts were done. Both LNA and DNA PCR detection by increasing sensitivity and efficiency primers were used for RT of total RNA extracted from of PCR reactions are highly desirable. N2-fixing pure cultures of Azoarcus sp. BH72, and for PCR Here we studied the effect of LNA substitution on the detection of nifH transcripts by direct reverse trans- cription (RT)-PCR in roots of rice and sugarcane. For PCR we used the highly degenerate, nifH-specific primers developed by Zehr and McReynolds (1989), which are 128- and 96-fold degenerate at 5 and 4 positions respec- tively. For RT primer TH580 was used, which is 1944-fold degenerate at eight positions. These primers have been shown to amplify highly divergent nifH genes with equal efficiencies (Tan et al., 2003; Zhang et al., 2007; Demba
Diallo et al., 2008) and to cover the majority of nifH genes Fig. 1. Comparison of amplified products of nifH-mRNA targeted without mismatches in the primer target region (Demba RT-PCR, using as template RNA extracted from pure culture Diallo et al., 2008). Azoarcus sp. BH72 or from Oryza longistaminata roots respectively. 1: RT-PCR with DNA primers; 2: RT with DNA primer, PCR with LNA primers; 3: RT with LNA primer, PCR with DNA primers; 4: Results and discussion RT-PCR with LNA primers; C: negative control without template. Controls for presence of DNA by heat inactivation of reverse Influence of LNA-modified primers on nifH gene transcriptase followed by PCR-reactions were negative (not RT-PCR amplification shown). Total RNA was extracted following a protocol of Chang and colleagues (Chang et al., 1993) with slight modifications as The LNA-substituted primers were designed taking described previously (Hurek et al., 2002; Knauth et al., 2005). For RT-PCR the protocol from Knauth and colleagues (2005) was account of the guidelines given by Latorra and colleagues followed. Different accessions of O. longistaminata (accessions (2003) and Levin and colleagues (2006). These authors BP and G1) were grown in microcosms in the phytotron. recommend to use no more than three LNA substitutions Representative results of triplicate experiments are shown. For Azoarcus sp. BH72 0.4 mg, for BP 0.5 mg and for G1 1.5 mg total in the primers, which should be preferentially located RNA were used as template. For plant samples, total RNA was near the 5′ end. Furthermore, it is suggested to avoid LNA extracted from 500 mg root fresh weight throughout this analysis.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 LNA substitutions for nifH RT-PCR 253
et al., 2008). Since this was the case for all three LNA primers, and because of the results obtained, we contin- ued our study applying condition (iii) with the LNA modi- fied primers used for the RT step, only. In order to obtain a quantitative estimate of LNA primer performance, a semi-quantitative nifH RT-PCR approach was carried out using 0.4 mg of total RNA from Azoarcus sp. BH72. Aliquots of RT-PCR reactions were taken at different cycle numbers in order to avoid saturation of the PCR amplification. Band intensities of the approximately 360 bp nifH fragments were quantified for three repeti- tions and for each cycle indicated in Fig. 2. Estimation of band intensities revealed that 2.4- and 2.3-fold more RT-PCR products accumulated after 34 and 40 cycles, respectively, when LNA instead of DNA primers were used (Fig. 2C). These data suggest that the use of the LNA primer in the RT step probably improved RT, a result that is consistent with Fratczak and colleagues (2009), which have shown that LNA-substituted primers may improve RT by enhancing hybridization to the target RNA, followed by efficient primer extension through reverse transcriptase.
Fig. 2. Expression of the dinitrogenase reductase gene (nifH) from Azoarcus sp. strain BH72. Ethidium bromide-stained RT-PCR products after 21, 24, 28, 34 and 40 cycles using TH580-DNA (A) and TH580-LNA (B) primers. Controls for presence of DNA by heat inactivation of reverse transcriptase followed by PCR reactions were negative (not shown). (C) Quantification of RT-PCR products as relative fluorescence units with standard deviations are given for three independent experiments, each. The nifH-mRNA levels were quantified as described previously (Reinhold-Hurek et al., 2006). Means are shown with error bars indicating standard deviation (SD). amplification of nifH cDNA. The conditions tested were as follows: (i) RT-PCR with only DNA primers; (ii) RT with DNA primers and PCR with LNA primers; (iii) RT with LNA primers and PCR with DNA primers; and (iv) RT-PCR with only LNA primers. Figure 1 shows RT-PCR amplification products of the appropriate size (360 base pairs; bp) with the four differ- ent conditions. The use of only one LNA primer in the RT step was the condition that showed the best performance Fig. 3. Amplification products of nifH-mRNA targeted RT-PCR using as template RNA extracted from different root samples. C, (lane 3), whereas the use of 2 LNA primers in the PCR negative control without template. Controls for presence of DNA by step (lane 2), and 3 LNA primers in RT and PCR steps heat inactivation of reverse transcriptase followed by PCR-reactions (lane 4) yielded unspecific products. The same four com- were negative (not shown). Representative results of triplicate experiments are shown. For S45 0.15 mg, for Y3 1.5 mg and for parisons were performed using total RNA extracts of sugarcane 0.5 mg total RNA were used as template. two different samples of wild rice (Oryza longistaminata) A. RT-PCR products after 40 cycles were compared using roots (accessions BP and G1) (Fig. 1). In both cases the TH580-DNA (lane 1) and TH580-LNA (lane 2) primers. Azoarcus sp. strain BH72 RNA was used as a positive control, and different number of unspecific products increased in PCR and accessions of O. longistaminata (Rice S45, G1 and Y3) grown in RT-PCR reactions when LNA-modified primers were microcosms in the phytotron, or sugar cane sampled in Namibia used. These results may be explained by a detrimental at the Okavango river. B. RT-PCR products after 40 cycles using 10-fold serial dilutions effect of a too high binding strength when bases in PCR of RNA extracted from O. longistaminata (accession BP) roots to primers other than A are substituted by LNA (Ballantyne compare the TH580-DNA and TH580-LNA primers.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 254 C. S. Burbano, B. Reinhold-Hurek and T. Hurek
Efficiency and sensitivity of the RT LNA primer produced an amplification product was scored. NifH in plant root samples amplification products were detected in almost all the dilutions tested (2 out of 3) when the LNA modified primer To test the performance of LNA primers in environmental was used, whereas in the case of the DNA primer no samples, different root samples from wild rice (O. longis- bands were obtained from the most diluted sample (10-5- taminata) and sugarcane plants were used to extract total fold diluted). From this analysis the minimum concen- RNA, and nifH RT-PCR was performed. In all cases the tration required for obtaining the DNA fragment of the use of the LNA primer improved the intensity of the ampli- appropriate size with the LNA primer was estimated to be fied product. More importantly, for samples such as sug- 4.5 pg (CI 95%: 21 pg, 0.5 pg), in contrast to the DNA arcane, only with the LNA primer a PCR product could be primer, where 117 ng (CI 95%: 216 pg, 21 pg) total RNA obtained (Fig. 3A). These results were corroborated by a was necessary. One of the 3 replicates of the MPN Southern blot hybridization using a digoxygenin-labelled RT-PCR is shown in Fig. 3B. nifH probe (not shown). These results showed that at least 25-fold more product In order to compare the relative template requirements was formed when the RT-primer was substituted by LNA, of the DNA and LNA primers, the RNA extracted from one indicating that the use of this primer drastically increased of the wild rice roots samples was subjected to serial RT-PCR sensitivity. Similar results have been shown dilutions, and to most-probable-number (MPN) RT-PCR when LNA primers were used in rolling circle amplification analysis as described previously (Hurek et al., 2002). increasing the overall sensitivity of inverse PCR from 10 Three RNA extractions were subjected to RT-PCR reac- to 1000 times in samples of the digestive tracts from tions with a starting template concentration of 0.5 mg total horses and termites detecting as many as 10 copies of the RNA, and the number of positive and negative tubes that target DNA in a sample (Yamada et al., 2008).
Fig. 4. Comparison of expressed nifH gene pools associated with roots of O. longistaminata (accession BP) using TH580-DNA (1) and TH580-LNA (2) primers. T-RFLP patterns obtained from RT-PCR products were compared after digestion with two different enzymes, BmgBI and BstUI (Fermentas), as described previously (Tan et al., 2003; Knauth et al., 2005). Numbers at peaks indicate the sizes of predicted T-RFs and the number of clones represented in T-RFLP analyses in parentheses. Marker given in nucleotides.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 LNA substitutions for nifH RT-PCR 255
Effect of LNA substitution on mRNA-based profiling of pools associated with roots of O. longistaminata plants nitrogenase (nifH) genes (Demba Diallo et al., 2008), nifH transcript sequences appeared to be randomly distributed over several stati- The T-RFLP analysis was used to examine mRNA-based stically highly supported clades in the NifH protein tree profiles of nitrogenase (nifH) genes obtained with DNA harbouring nifH homologues from well known N -fixing and LNA RT-primers. For this purpose T-RFLP patterns of 2 bacteria (Fig. S1). Statistical comparison of both clone RT-PCR products from roots of an individual wild rice libraries with ∫-LibShuff (Schloss et al., 2004) and Unifrac plant (accession BP) were compared. Results showed (Lozupone et al., 2006) confirmed that the two libraries that the patterns obtained were largely congruent for LNA are not significantly different from each other at P = 0.63 or DNA primers used in the RT reaction (Fig. 4). and P = 0.38, respectively, supporting our conclusion that Profiles were highly reproducible with respect to size LNA substitution of the RT-primer is unlikely to introduce and relative abundance of T-RFs (terminal restriction a significant PCR bias. Analyses were conducted using fragments) (Fig. 4, Table 2). Repeated analysis of the the default settings of ∫-LibShuff version 1.22 and of the two samples gave equivalent results. Besides one small UniFrac web server (http://bmf2.colorado.edu/unifrac/). 187 bp peak in the case of amplicons treated with the The 93 nifH gene cDNA sequences were deposited in restriction enzyme BmgBI, no variation in the presence of the EMBL nucleotide database, accession numbers T-RFs among the two samples was found (Table 2). FN555007–FN555099. Terminal restriction fragments from experimental runs In conclusion, our results show that LNA substitution of were compared with predicted T-RFs from 46 and 47 nifH the RT-primer increased the sensitivity of the RT-PCR gene cDNA clones obtained by RT-PCR with DNA and without introducing a significant PCR bias. This allowed the LNA substituted RT-primers respectively. Estimated T-RF detection of diazotrophic bacteria in samples where DNA sizes showed reasonable agreement (Osborn et al., primers alone failed to produce PCR products and made 2000) with the predicted T-RF sizes and allowed the sugarcane for the first time accessible for cultivation- assignment of all major T-RFs with a relative abundance independent detection of nitrogen fixing bacteria, which > 5% to nifH gene fragments (Fig. 4 and Table 2). Also a are likely to be involved in nitrogen fixation with this plant. comparative sequence analysis of the 93 cDNA clones Similar improvements to RT-PCR detection of RNA can provided no evidence that the representation of nifH gene be expected for other samples and genes likewise. transcript sequences in community analyses might be affected differently by DNA than by LNA substituted RT primers; sequences from both libraries clustered indivi- Acknowledgements dually and not according to the type of RT primer used. This research was supported by grant 01LC0021 from the Consistent with previous work on expressed nifH gene Bundesministerium für Bildung und Forschung to B.R.-H. and
Table 2. Fragment lengths and relative abundances of T-RFs from amplified nifH gene transcripts using DNA and LNA RT primers.a
Fragment length (nt; mean Ϯ SDb) Relative abundance of T-RFs T-RFLP analysis In silico analysis (%; mean Ϯ SD)
Restriction enzyme DNA LNA DNA LNA DNA LNA
BstUI 69 Ϯ 169Ϯ 170704Ϯ 0.5 4 Ϯ 0.5 73 Ϯ 173Ϯ 1ND7412Ϯ 3.0 13 Ϯ 0.5 79 Ϯ 180Ϯ 180ND2Ϯ 0.5 2 Ϯ 0.5 179 Ϯ 1 180 Ϯ 1 181 181 3 Ϯ 1.0 4 Ϯ 0.5 184 Ϯ 1 184 Ϯ 1 182 ND 35 Ϯ 5.0 36 Ϯ 1.5 187 Ϯ 1 186 Ϯ 1 188 188 21 Ϯ 2.0 20 Ϯ 6.0 256 Ϯ 1 255 Ϯ 1 253 253 23 Ϯ 9.0 21 Ϯ 4.0 BmgBI 90 Ϯ 190Ϯ 190904Ϯ 0.5 4 Ϯ 1.0 109 Ϯ 1 109 Ϯ 1 111 111 21 Ϯ 1.5 19 Ϯ 2.0 182 Ϯ 1 183 Ϯ 1NDND3Ϯ 2.0 2 Ϯ 1.0 187 Ϯ 1NDNDND2Ϯ 1.0 ND 196 Ϯ 1 196 Ϯ 1 ND 194 31 Ϯ 1.0 40 Ϯ 11.0 263 Ϯ 1 263 Ϯ 1 264 264 23 Ϯ 6.0 22 Ϯ 4.0 279 Ϯ 1 278 Ϯ 1 276 276 16 Ϯ 1.0 13 Ϯ 4.0 a. NifH gene cDNAs were obtained by RT-PCR from roots of O. longistaminata (Rice BP) as described in the legend of Fig. 4. A baseline threshold of 50 fluorescence units was used to distinguish ‘true peaks’ from background noise. The relative abundance of a T-RF represents the peak height of a T-RF relative to the total peak height of all T-RFs in a sample. b. Mean Ϯ SD; mean with standard deviation from three replicates. ND, not detected.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 256 C. S. Burbano, B. Reinhold-Hurek and T. Hurek
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(2001) A comparison of the solution structures of labeled locked nucleic acid (LNA) probes and fluorescence an LNA:DNA duplex and the unmodified DNA:DNA duplex. polarization detection. Nucleic Acids Res 30: e91. J Chem Soc, Perkin Trans 2 1224–1232. Tan, Z., Hurek, T., and Reinhold-Hurek, B. (2003) Effect of Kaur, H., Arora, A., Wengel, J., and Maiti, S. (2006) Ther- N-fertilization, plant genotype and environmental condi- modynamic, counterion, and hydration effects for the in- tions on nifH gene pools in roots of rice. Environ Microbiol corporation of locked nucleic acid nucleotides into DNA 5: 1009–1015. duplexes. Biochemistry 45: 7347–7355. Tindall, K.R., and Kunkel, T.A. (1988) Fidelity of DNA syn- Knauth, S., Hurek, T., Brar, D., and Reinhold-Hurek, B. thesis by the Thermus aquaticus DNA polymerase. (2005) Influence of different Oryza cultivars on expression Biochemistry 27: 6008–6013. of nifH gene pools in roots of rice. Environ Microbiol 7: Tolstrup, N., Nielsen, P.S., Kolberg, J.G., Frankel, A.M., 1725–1733. Vissing, H., and Kauppinen, S. (2003) OligoDesign: optimal Latorra, D., Arar, K., and Hurley, J.M. (2003) Design con- design of LNA (locked nucleic acid) oligonucleotide capture siderations and effects of LNA in PCR primers. Mol Cell probes for gene expression profiling. Nucleic Acids Res Probes 17: 253–259. 31: 3758–3762. Levin, J.D., Fiala, D., Samala, M.F., Kahn, J.D., and Peter- Wahlestedt, C., Salmi, P., Good, L., Kela, J., Johnsson, T., son, R.J. (2006) Position-dependent effects of locked Hökfelt, T., et al. (2000) Potent and nontoxic antisense nucleic acid (LNA) on DNA sequencing and PCR primers. oligonucleotides containing locked nucleic acids. Proc Natl Nucleic Acids Res 34: e142. Acad Sci USA 97: 5633–5638. Lozupone, C., Hamady, M., and Knight, R. (2006) UniFrac – Watkins, N.E., Jr, and SantaLucia, J., Jr (2005) Nearest- an online tool for comparing microbial community diversity neighbor thermodynamics of deoxyinosine pairs in DNA in a phylogenetic context. BMC Bioinform 7: 371. duplexes. Nucleic Acids Res 33: 6258–6267. McTigue, P.M., Peterson, R.J., and Kahn, J.D. (2004) Yamada, K., Terahara, T., Kurata, S., Yokomaku, T., Tsuneda, Sequence-dependent thermodynamic parameters for S., and Harayama, S. (2008) Retrieval of entire genes from
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 LNA substitutions for nifH RT-PCR 257
environmental DNA by inverse PCR with pre-amplification in bold. The boxed text at the nodes of these clades indicates of target genes using primers containing locked nucleic how many transcript sequences within these clades were acids. Environ Microbiol 10: 978–987. obtained with DNA and LNA RT primers. All other clades were Zehr, J.P., and McReynolds, L.A. (1989) Use of degenerate condensed for clarity. The three major groups harbouring
oligonucleotides for amplification of the nifH gene from NifH homologues from established N2-fixing bacteria the marine cyanobacterium Trichodesmium thiebautii. Appl (Raymond et al., 2004) were labelled as outlined in Demba Environ Microbiol 55: 2522–2526. Diallo and colleagues (2008). Bars show the number of Zhang, L., Hurek, T., and Reinhold-Hurek, B. (2007) A nifH- amino acid substitutions per site. A total of 272 translated based oligonucleotide microarray for functional diagnostics partial nifH sequences (including 179 sequences from
of nitrogen-fixing microorganisms. Microb Ecol 53: 456– cultivated N2-fixing bacteria) were aligned with MAFFT 470. (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) using the G-INS-i setting at default (Katoh et al., 2002). The result- Supporting information ing alignment consisted of 111 sites. The phylogenetic tree was constructed with MEGA 4 (Tamura et al., 2007) using a Additional Supporting Information may be found in the online minimum evolution analysis with a Jones-Taylor-Thornton version of this article: (JTT) amino acid substitution model (Jones et al., 1992), and the pairwise deletion option. The 93 nifH gene cDNA Fig. S1. Minimum-evolution NifH protein tree showing the sequences were deposited in the EMBL nucleotide database, phylogenetic affiliation of 46 and 47 nifH cDNA fragments accession numbers FN555007–FN555099. recovered with DNA and LNA RT primers, respectively, from roots of Oryza longistaminata (accession BP). Numbers at Please note: Wiley-Blackwell are not responsible for the branches represent internal branch test (IBT) confidence content or functionality of any supporting materials supplied values > 50% from 500 replicates. Clades with nifH transcript by the authors. Any queries (other than missing material) sequences and with IBT confidence values Ն 95% are drawn should be directed to the corresponding author for the article.
© 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports, 2, 251–257 Supporting Information Burbano et al. 2010
Fig. S1. Minimum-evolution NifH protein tree showing the phylogenetic affiliation of 46 and 47 nifH cDNA fragments recovered with DNA and LNA RT primers, respectively, from roots of Oryza longistaminata (accession BP). Numbers at branches represent internal branch test (IBT) confidence values >50% from 500 replicates. Clades with nifH transcript sequences and with IBT confidence values >90% are drawn in bold. The boxed text at the nodes of these clades indicates how many transcript sequences within these clades were obtained with DNA and LNA RT primers. All other clades were condensed for clarity. The three major groups harboring NifH homologues from established N2-fixing bacteria (Raymond et al., 2004) were labeled as outlined in Demba Diallo et al. (2008). Bars show the number of amino acid substitutions per site. 272 translated partial nifH sequences (including 179 sequences from cultivated N2-fixing bacteria) were aligned with MAFFT (http://align.bmr.kyushu-u.ac.jp/mafft/online/server/) using the G-INS-i setting at default (Katoh et al., 2002). The resulting alignment consisted of 111 sites. The phylogenetic tree was constructed with MEGA 4 (Tamura et al., 2007) using a minimum evolution analysis with a Jones-Taylor-Thornton (JTT) amino acid substitution model (Jones et al., 1992), and the pairwise deletion option. The 93 nifH gene cDNA sequences were deposited in the EMBL nucleotide database, Accession Nos FN555007-FN555099.
Supporting Information Burbano et al. 2010
References
Demba Diallo, M., Reinhold-Hurek, B., and Hurek, T. (2008) Evaluation of PCR primers for universal nifH gene targeting and for assessment of transcribed nifH pools in roots of Oryza longistaminata with and without low nitrogen input. FEMS Microbiol. Ecol. 65: 220- 228.
Jones, D. T., Taylor, W. R., and Thornton, J. M. (1992) The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8: 275-282.
Katoh, K., Misawa, K., Kuma, K., and Miyata, T. (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30:3059- 3066.
Raymond, J., Siefert, J. L., Staples, C. R., and Blankenship, R. E. (2004) The natural history of nitrogen fixation. Mol. Biol. Evol. 21: 541–554.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596-1599.
Experimental procedures Burbano et al. 2010
Appendix S1: Experimental procedures
Preparation of RNA
Total RNA from cells of Azoarcus sp. strain BH72, growing on N2 under controlled oxygen concentrations (2μM dissolved O2) in a bioreactor, and total RNA from root samples was extracted following a protocol of Chang and colleagues (1993) with slightly modifications as described previously (Hurek et al., 2002; Knauth et al.,
2005). For each extraction, equal root mass (fresh weight) was used for normalization. In order to remove the DNA from RNA preparations, DNase I (Roche,
Mannheim, Germany) treatment was followed by one chloroform-phenol- isoamylalcohol extraction before precipitation. RNA was finally resuspended in 1x
RNA-secure (Ambion, Austin, TX, U.S.A.).
Oligonucleotide primers
A universal primer for the nitrogenase Fe protein gene (nifH) (Hurek et al. 2002)
NIFHR-580-DNA was used for the reverse transcription (RT). A locked nucleic acid- containing primer was also design (NIFHR-580-LNA), where three bases were modified and also used for the RT. For PCR amplification, universal primers (Zehr-F and Zehr-R) for nifH were used (Zehr and McREynolds, 1989). LNA primers were also design. For T-RFLPs the forward primer was 5 -labelled with Cy5. All the LNA primers were designed as suggested by Levin et al. (2006).
RT-PCR analysis of nifH
The Ready-to-Go RT-PCR beads were used for RT-PCR according to the manufacturer’s instructions (Amersham Biosciences). To synthesize the cDNA, 0.5μl of total RNA was used as template. The RT step was done for 30 min at 47°C followed by inactivation for 5 min at 95°C and by cycling for 1 min at 94°C, for 2 min Experimental procedures Burbano et al. 2010 at 57°C, and for 2 min at 72°C, followed by a 10 min extension at 72°C as described by Knauth and colleagues (2005). The absence of DNA template was controlled by inactivation of the reverse transcriptase for 15 min at 95°C prior to the reaction. The nifH amplification products were about 362 bp long and were visualized on 1.8% agarose gels with ethidium bromide. Intensities of signals were measured using a
Typhoon 8600 optical scanner (Molecular Dynamics, Sunnyvale) using
IMAGEQUANT software (Molecular Dynamics).
T-RFLPs analysis
The nifH amplification products were purified by electroelution, digested over night with two enzymes separately (BmgBI and BstUI), using 1 μg of DNA and 2.5 U of restriction enzyme (Fermentas), and separated on an ALFexpress automated sequencer using High Resolution polyacrylamide gel (Amersham Biosciences) as described previously (Tan et al. 2003, Knauth et al. 2005). The sizes of the terminal restriction fragments were determined by using ALFexpress sizer 50-500.
References
Chang S, Puryear J, Cairney J. (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113–116.
Hurek T, Handley LL, Reinhold-Hurek B, Piché Y. (2002) Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 15: 233-242.
Knauth S, Hurek T, Brar D, Reinhold-Hurek B. (2005) Influence of different Oryza cultivars on expression of nifH gene pools in roots of rice. Environ Microbiol 7: 1725-1733.
Levin JD, Fiala D, Samala MF, Kahn JD, and Peterson RJ (2006) Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers. Nucleic Acids Res 34: e142.
Tan Z, Hurek T, Reinhold-Hurek B. (2003) Effect of N-fertilization, plant genotype and environmental conditions on nifH gene pools in roots of rice. Environ Microbiol 5: 1009-1015.
Zehr JP, McReynolds LA. (1989) Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55: 2522–2526.
Chapter 2
CHAPTER 2
Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field grown sugarcane plants and in Norway spruce
Claudia Sofía Burbano, Yuan Liu, Kim Rösner, Veronica Reis, Jesus Caballero- Mellado, Barbara Reinhold-Hurek and Thomas Hurek
Status of the manuscript: Published in Environmental Microbiology Reports
Own contribution: - Majority of sugarcane experiments - Writing of the manuscript
Contribution Y. Liu: - Norway spruce experiments
Contribution K. Rösner: - Screening of sugarcane nifH clone libraries by DGGE
Contribution V. Reis: - Cultivation of sugarcane and sample collection
Contribution J. Caballero-Mellado: - Cultivation of sugarcane and sample collection Environmental Microbiology Reports (2011) doi:10.1111/j.1758-2229.2010.00238.x
Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field-grown sugarcane
plants and in Norway spruceemi4_238 1..7
Claudia Sofía Burbano,1 Yuan Liu,1 Introduction Kim Leonie Rösner,1 Veronica Massena Reis,2 Sugarcane is the only known non-leguminous field crop Jesus Caballero-Mellado,3† Barbara Reinhold-Hurek1 that can obtain up to 80% of total plant N from atmo- and Thomas Hurek1* spheric N through plant-associated biological nitrogen 1Lab of General Microbiology, Center for Biomolecular 2 fixation (BNF) (Boddey et al., 1995). BNF is a highly spe- Interactions Bremen (CBIB), University of Bremen, cialized prokaryotic process which is poorly understood in D-28359 Bremen, Germany. sugarcane. Cultivated sugarcane is a hybrid of Saccha- 2Embrapa Agrobiologia, km 447, Estrada Antiga Rio-São rum officinarum L. and other species (Gaut, 2002) and is Paulo, Seropédica, 23890-000, Rio de Janeiro, Brazil. with corn the most important crop for biofuel production 3Centro de Ciencias Genomicas, UNAM, Apdo. Postal today, where it provides the feedstock for about 40% of all No. 565-A, Cuernavaca, Mor., México. fuel ethanol (Sims et al., 2008). Only few sugarcane lines are known to derive large quantities of N from BNF Summary (Boddey et al., 1995), and in contrast to the well-known Rhizobium–legume symbiosis, symbiotic structures har- Although some sugarcane cultivars may benefit sub- bouring bacterial endosymbionts are not formed (James stantially from biological nitrogen fixation (BNF), the and Olivares, 1998). For a better understanding of this responsible bacteria have been not identified yet. nitrogen-fixing system as well as for its application and Here, we examined the active diazotrophic bacterial optimization, it is instrumental to identify the active diaz- community in sugarcane roots from Africa and otrophs providing nitrogen to the host. America by reverse transcription (RT)-PCR using Various nitrogen-fixing bacteria have been detected broad-range nifH-specific primers. Denaturing gradi- (Ando et al., 2005) and isolated from this plant (Baldani ent gel electrophoresis (DGGE) profiles obtained from et al., 2002), such as Gluconacetobacter diazotrophicus, sugarcane showed a low diversity at all sample loca- Herbaspirillum seropedicae or H. rubrisubalbicans. tions with one phylotype amounting up to 100% of the However, their role in BNF with sugarcane is largely nifH transcripts. This major phylotype has 93.9–99.6% unknown. Although in gnotobiotic experiments G. diaz- DNA identity to the partial nifH sequence from a strain otrophicus provided fixed nitrogen to the host plant, the affiliated with Rhizobium rosettiformans. In addition, gains did not explain the observed contributions in nature nifH transcripts of this phylotype were also detected in (Sevilla et al., 2001). Up to now the most active nitrogen- spruce roots sampled in Germany, where they made fixing bacteria in sugarcane fields have not been identified up 91% of nifH transcripts detected. In contrast, yet. Identifying these bacteria is methodologically chal- in control soil or shoot samples two distinct nifH lenging, because they are difficult to access for transcript sequences distantly related to nifH from cultivation-independent methods (Burbano et al., 2010) Sulfurospirillum multivorans or Bradyrhizobium and may be recalcitrant to isolation in pure culture, as elkanii, respectively, were predominant. These results reported for Azoarcus sp. BH72, nitrogen-fixing endo- suggest that R. rosettiformans is involved in phytes in Kallar grass (Reinhold et al., 1986; Hurek et al., root-associated nitrogen fixation with sugarcane 2002). Azoarcus sp. BH72 is an endophytic diazotroph and spruce, plants that do not form root–nodule that can colonize field-grown non-diseased plants in symbioses. remarkably high numbers (Reinhold et al., 1986) and become the most actively nitrogen-fixing bacterium there (Hurek et al., 2002). These bacteria are usually not cul- turable in planta, in the field (Hurek et al., 2002). Yet, Received 11 August, 2010; accepted 3 December, 2010. *For corre- spondence. E-mail [email protected]; Tel. (+49) 421 218 62868; Azoarcus sp. BH72 can grow vigorously on regular media. Fax (+49) 421 218 62873. †Deceased. Therefore, it is reasonable to assume that, apart from
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd 2 C. S. Burbano et al.
Azoarcus sp. BH72, the culturability of many other active nitrogen-fixing bacteria in planta may be lower than in pure culture, as well. Low culturability of these bacteria in planta makes their isolation into pure culture challenging, which might explain why the most active nitrogen-fixing bacteria in sugarcane fields have not been obtained yet by cultivation-dependent methods. Spruce represents another plant which may obtain nitrogen from plant-associated nitrogen-fixing bacteria. Conifers like spruce are of great ecological and commer- cial importance in the northern hemisphere temperate forests and in similar climates all over the world. It has been known for long that forest ecosystems may accumu- late significant, albeit low amounts of nitrogen without symbiotic nitrogen-fixing bacteria and without forming any symbiotic structures (Bormann et al., 1977; Pérez et al., 2003), but not much is known on the composition of plant-associated nitrogen-fixing communities in this environment. Here, we report on the active diazotrophic bacterial communities in roots of sugarcane (from Namibia, Mexico and Brazil) and spruce (from Germany) which are surpris- ingly similar and have a low diversity.
Results and discussion
Analysis of the active diazotrophic populations by reverse transcription (RT)-PCR Fig. 1. nifH transcription of Rhizobium rosettiformans-cluster- affiliated phylotypes in roots of sugarcane. We sampled four cultivated sugarcane varieties and one A. Map showing sampling sites of plants and the country (in black), from where R. rosettiformans was isolated. wild sugarcane (Saccharum spontaneum cv. Krakatau) B. DGGE fingerprint analysis of expressed nitrogenase genes from three different continents (Fig. 1A). The wild sugar- in sugarcane roots from three continents: Brazil (Krakatau, cane is one of the few sugarcane lines, for which a sub- RB-72454), Mexico (69–290) and Namibia (local race). Marker: molecular weight marker (Demba Diallo et al., 2004); control: RNA stantial nitrogen input from N2 fixation had been shown extract of Azoarcus sp. BH72 grown on N2. Bands representing R. (Boddey et al., 1995). Using RNA extracts of roots, we rosettiformans-cluster-affiliated phylotypes are marked by an arrow. surveyed the expression of nifH in these sugarcane vari- Representative results from triplicate experiments are shown. C. Reproducibility of DGGE profiles from four different plant eties. An optimized reverse transcription (RT)-PCR strat- samples (58, 60, 65 and 72) of the cultivar Krakatau sampled in egy with broad-range nifH-specific primers (Zani et al., Brazil. See Appendix S1 for sample details and experimental 2000) was applied to amplify nifH transcripts. This procedure. approach is widely used for the detection of nitrogen- fixing microorganisms in natural environments: There is a et al., 2002). The RT-PCR yielded amplification products tight relationship between nitrogenase activity and of the appropriate size (364 bp) from all sugarcane root expression of nifH (encoding the iron protein of nitroge- samples analysed. The amplified products hybridized to nase) (Merrick and Edwards, 1995; Egener et al., 1999), nifH in Southern blot hybridization (data not shown). Addi- and because of this, communities that are actively tran- tionally, the PCR products were directly sequenced and scribing nifH are also likely to fix nitrogen. Moreover, the showed that nifH was retrieved in all cases. All control degenerate primers used here have been shown to PCR reactions to test for DNA contamination of the RNA amplify different concentrations of phylogenetically differ- extracts were negative. ent nifH genes without a bias towards a 1:1 ratio of prod- Denaturing gradient gel electrophoresis (DGGE) was ucts in artificial mixtures of up to six different templates used for profiling of nifH sequences. The resulting DGGE (Tan et al., 2003; Zhang et al., 2007). Employing this profiles of nifH transcripts showed surprisingly little diver- approach, the nitrogen-fixing endophytes that supply sity in each sample and had one band in common, rep- nitrogen to their host had previously been identified resenting up to 100% of the nifH transcripts detected among several clades present at the DNA level (Hurek (Fig. 1B). The patterns were reproducible between repli-
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports Transcription of nifH in sugarcane and spruce 3 cates and repetitions, and plant-to-plant variation was lowed by cyanobacteria (23%), Rhizobium sp. (13%) and very low, as illustrated in the example cv. Krakatau finally one sequence related to Ideonella. Melting profiles (Fig. 1C). Thus nifH-mRNA fragments of only few or of predominant clones from libraries matched to the even one phylotype were dominating, suggesting that one DGGE fragments retrieved from root samples (data not major active diazotrophic bacterium was present in roots shown). of sugarcane. Surprisingly none of the abundant sequences was related to the cultivated diazotrophs suspected to interact with sugarcane, such as H. seropedicae or G. diazotrophi- nifH phylogenetic diversity cus. In partial accordance with our findings on Rhizobiales A clone library approach was carried out independently to in roots is the study by Ando and colleagues (2005) who corroborate our DGGE results. Four clone libraries were used a cultivation-independent approach and detected generated for RT-PCR products retrieved from sugarcane nifH DNA sequences in sugarcane stems (cv. NiF8, cv. roots. A total of 144 clones were analysed, and the identity NCo310 and cv. KF92-93) collected in different locations of the nucleotide sequences compared with database of Japan, primarily related to Bradyrhizobium (45%), Kleb- entries revealed that all clones were nifH sequences siella (37%) and Serratia (13%). (Table 1). In general, the nifH clone libraries from all of the Estimations of species coverage, richness, evenness samples yielded also low levels of diversity. All sequences and diversity were calculated for all the libraries analysed from Brazilian libraries (cv. RB-72454 and cv. Krakatau) and also to the combined data set (Table 2). Good’s cov- showed a sequence similarity of 98–100% between each erage was greater than 97% in all the cases, which indi- other. In the case of the Mexican library (cv. 69–290) the cates that the nifH sequences identified in these samples majority of the sequences (78%) were also similar among represent the majority of the root-associated bacteria each other (97.5–100% identity). They were all related to sequences present in sugarcane. The Chao1 estimator of Rhizobium sp. In the case of the Namibian library (local species richness conformed to the number of phylotypes race), the phylogenetic distribution was different as to be found in the Brazilian and Mexican libraries, while the expected from the DGGE profiles. The majority of the library from Namibian sugarcane was not saturated yet. sequences were related to uncultured bacteria isolated Conventional diversity indices were higher for the from marine and soil environments, with the closest culti- Mexican and Namibian libraries where more than one vated organisms belonging to Desulfovibrio (63%), fol- phylotype was found in the active diazotrophic community
Table 1. Phylotypes found in the nifH transcript clone libraries of sugarcane and spruce root samples.
nifH transcript sequence Closest cultivated neighbour present in public database
Sequence No. Clone librarya Assigned accession No. similarity, % clones Accession No. Description
Local raceN FN665973–FN665979 99.0–99.6 7 GQ241353.1 Rhizobium rosettiformans W3 Krakatau72B FN665866–FN665896 98.8–99.4 31 isolated from hexachloro- 69–290M FN665897–FN665917 98.8–99.4 21 cyclohexane dumpsite in India RB-72454B FN665805–FN665834 98.5–99.4 29 FN665834 97.9 1 Spruce FN665981–FN666015 98.1–99.0 35 FN665980 93.9 7 69–290M FN665918, FN665920, FN665922 92.9–93.5 3 GQ289565.1 Bradyrhizobium japonicum clone from reddish paddy soil 69–290M FN665919, FN665921, FN665923 91.8–92.1 3 AE017282.2 Methylococcus capsulatus nifH
Local raceN FN665924–FN665939 84.5–85.4 34 AP010904.1 Desulfovibrio magneticus nifH FN665941–FN665958 FN665940 82.9 1 Local raceN FN665959–FN665960 97.5–98.1 5 EU381369.1 Anabaena sp. LG2 clone from a FN665967–FN665968 subtropical estuary FN665971 Local raceN FN665961–FN665966 97.8–98.7 8 EF392710.1 Lyngbia sp. CCAP 1446/10 clone FN665969–FN665970 Spruce FN666016–FN666019 98.7 4 AM110707.1 Burkholderia vietnamiensis isolated from spruce EcM Local raceN FN665972 94.0 1 AY231580.o Ideonella sp. isolated from roots of Oryza longistaminata a. See Appendix S1 for description of experimental procedures. BBrazil; MMexico; NNamibia.
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports 4 C. S. Burbano et al.
Table 2. nifH transcript sequence diversity and library coverage estimations for sugarcane and spruce root samples.
Measurementsa RB-72454B KrakatauB Mex 69–290M Local raceN Combined Spruce
No. of clones 30 31 27 56 144 46 Rhizobium rosettiformans W3-related clones, % 100 97 78 13 62 91 Phylotypes 1 2 2 7 12 2 Singletons 0 1 0 3 4 0 Chao1 estimator of species richness (OTUs) 1 2.47 2.00 11.43 19.55 2 Shannon’s index for diversity 0 0.142 0.529 1.245 1.953 0.184 Simpson’s index for diversity 0 0.062 0.345 0.589 0.827 0.086 Evenness 1 0.576 0.849 0.496 0.587 0.601 Good’s estimator of coverage, % 100 97 100 98 97 100 a. See Appendix S1 for description of experimental procedures. BBrazil; MMexico; NNamibia. in contrary to the Brazilian ones. With respect to the the RT-PCR products amplified from the corresponding Evenness index, the lowest value was for the Namibian rhizosphere soil samples, unambiguously readable iden- library, which is in accordance with the number of phylo- tical sequences of high quality were obtained (Accession types recorded. No. FR720331) which had the closest sequence match (91% DNA sequence identity) with the partial nifH sequence from the epsilonproteobacterium Sulfurospiril- Predominant nifH transcript phylotypes related to lum multivorans (Accession No. DQ337206), and only a Rhizobium rosettiformans in non-Fabales low similarity with the nifH sequence from R. rosettifor- The predominant rhizobial nifH fragment detected in all mans (< 65% DNA sequence identity). A clone library samples (arrow Fig. 1B) corresponded to a phylotype approach, which was carried out in parallel, showed that that is most closely related (93.9–99.6% DNA identity) to all sequenced 40 clones (Accession No. FR720332-71) the partial nifH sequence from a cultivated bacterium, had a DNA identity Ն 98.3% to the direct sequence named strain W3 (Tables 1 and 2). This isolate was (Accession No. FR720331). Since nifH transcripts from R. obtained from a dump site in India and was recently rosettiformans could not be detected in rhizospheric soil assigned to the genus Rhizobium as the new species attached to the roots where nifH transcripts from this Rhizobium rosettiformans (Kaur et al., 2010). Surpris- bacterium were predominant, nifH transcription of these ingly, nifH transcripts of this phylotype were also bacteria with Mexican sugarcane cv. CP 72-2086 and detected in Norway spruce roots sampled from a forest probably also with the other plants studied is specific to ecosystem in Germany (Fig. 1A), where they made up roots and not soil-borne. To find out whether nifH tran- 91% of the nifH transcripts detected (Tables 1 and 2). A scription from R. rosettiformans is abundant in shoots, phylogenetic analysis showed that 69% of the 190 too, we also analysed RT-PCR products amplified from partial nifH cDNA sequences from sugarcane and sugarcane shoots by a direct sequencing and a clone spruce formed a distinct cluster together with the nifH library approach. We used for this investigation shoots sequence from R. rosettiformans strain W3 (Fig. 2). nifH from three plants of the Brazilian sugarcane variety cDNA sequences within this cluster were at least 8% Krakatau for which we had shown that nifH transcripts different from the sequence of the nearest described from R. rosettiformans were predominant in their roots bacterium, Ideonella sp. (Fig. 1). From direct sequencing unambiguously readable To find out whether nifH transcription of R. rosettifor- identical sequences of high quality were obtained (Acces- mans is confined to roots, we analysed the active diaz- sion No. FR720372) which had the closest sequence otrophic bacterial community in rhizosphere soil from two match (91.3% DNA sequence identity) with the partial nifH plants of Mexican sugarcane cv. CP 72-2086 and in sequence from the alphaproteobacterium Bradyrhizobium shoots from three plants of the wild S. spontaneum variety elkanii (Accession No. AM110714) and only 84.6% DNA cv. Krakatau. DGGE fingerprint analysis of expressed sequence identity with the nifH sequence from R. rosetti- nitrogenase genes confirmed the presence of the unique formans. Also in this case a clone library approach carried band representing the R. rosettiformans-cluster-affiliated out in parallel did not allow the detection of nifH transcripts phylotype in roots of Mexican sugarcane cv. CP 72-2086 from R. rosettiformans in the pool of less abundant nifH (data not shown). Direct sequencing of this fragment mRNAs. From the 40 clones (Accession Nos. FR720373– revealed that its sequence (Accession No. FR727038) 412) examined by comparative sequence analysis, none matched with the nifH sequence from R. rosettiformans had a DNA sequence identity > 85% to this clade. Con- (Accession No. GQ241353). From direct sequencing of sistent with this, the shoot nifH transcript sequences did
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports Transcription of nifH in sugarcane and spruce 5
Fig. 2. Minimum-evolution nifH DNA tree. The tree shows the phylogenetic affiliation of 231 nifH cDNA fragments recovered from roots of four sugarcane cultivars (144 sequences), of Norway spruce (46 sequences) and of shoots from sugarcane cv. Krakatau for comparison (41 sequences). Numbers at branches represent internal branch test (IBT) confidence values > 50% from 500 replicates. The scale bars show the number of nucleotide substitutions per site. Maximum parsimony and maximum likelihood analyses gave similar topologies and supported the Rhizobium rosettiformans clade with bootstrap values > 95% (not shown). See Appendix S1 for experimental procedure. not fall into the R. rosettiformans clade (Fig. 2). Therefore, bacteria express nifH, which raises the question of nifH transcription by R. rosettiformans is probably con- the relative contribution of root and shoot residing fined to roots in the plants studied. Our results further- nitrogen-fixing populations in providing fixed nitrogen to more suggest that in shoots different, so far unknown sugarcane.
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports 6 C. S. Burbano et al.
Nodular nitrogen-fixing legume/rhizobia and acti- in association with non-Fabales, which extends the host norhizal/frankiae symbioses in plants are restricted to the range of this diazotroph. Some sequences (FN665835– dicot subclass Rosidae within the angiosperms (Soltis FN665856) also clustered together with a sequence et al., 1995). In nodulated host plants, the diversity of the from the metagenome of the endophytic community of nitrogen-fixing Rhizobium and Frankia symbionts in the rice (ADIE01000000 nifH meta46). B. Reinhold-Hurek, P. symbiotic tissue is typically low. However, a low diversity Hardoim, J. Döring, A. Weilharter, A. Krause, T. Woyke of root-associated nitrogen-fixing bacteria in other and colleagues (unpublished) found that the active angiosperms seems to be rare. An exception is Kallar endophytic community was dominated by this alphapro- grass, which may closely associate with Azoarcus sp. as teobacterial clade related to Rhizobium, extending the the most active nitrogen-fixing bacterium in roots of field- typically root–nodule-associated rhizobial nitrogen fixa- grown plants (Hurek et al., 2002). Therefore our observa- tion activity also to another cereal. tion that the composition of the active nitrogen-fixing In conclusion, our results suggest that R. rosettiformans communities in the rhizospheres of Norway spruce and is involved in root-associated nitrogen fixation with sugar- sugarcane is similar and their diversity is low is surprising. cane and spruce. The prevalence of a distinct, globally Even more intriguing is the high sequence similarity of the distributed clade of nifH transcript sequences in sugar- dominant nifH transcripts with the partial sequence of R. cane and spruce indicates a tight interaction between the rosettiformans nifH, since it is generally assumed that corresponding nitrogen-fixing bacteria and their host. The rhizobia express nitrogenase genes only in a successfully preponderance of nifH expression by R. rosettiformans in established symbiosis within their host and rhizobial nifH sugarcane and spruce roots is quite surprising, since transcript sequences from gymnosperms like spruce have within the family Rhizobiales nifH transcription in non-host not been reported yet. We analysed the 2264 environ- environments seems to be rare. Since nodulation genes mental nifH transcript sequences available up to February (nodA, nodC and nodD) could not be detected in R. roset- 2010 in the EBI database to identify how many of them tiformans (Kaur et al., 2010), the interaction between R. were highly similar (95–100%) to cultivated bacteria from rosettiformans and these plants is probably not depen- the order Rhizobiales (Supplementary information, Table dent on nod genes, which is consistent with the absence S1). Less than 1% of all sequences were related to this of symbiotic structures in sugarcane and spruce. group of bacteria. Furthermore, nifH transcript sequences with a > 95% DNA sequence identity to cultivated members of this order represented only 0.88% of the 565 Acknowledgements nifH transcript sequences available from non-leguminous Financial support was provided by Bundesministerium für plants in the EBI database up to February 2010, confirm- Bildung und Forschung (grant 01LC0621A2) in the BIOLOG ing that transcription in a non-host environment seems to framework and by the Deutsche Forschungsgemeinschaft be rare. Five sequences were related to Bradyrhizobium (Grants Hu 579/7-1 and Hu 579/8-1). Root samples of sug- sp. retrieved from sweet potato tubers, rice roots and a arcane from Namibia were collected in the Okavango region fjord, indicating nitrogen fixation with plants other than under the collection permits 1138/2007 and 821/2004, under Fabales. Two sequences from marine environments were Material Transfer Agreements with NBRI (National Botanical- Research Institute), Windhoek, Namibia. We thank Mr 99% similar to R. rosettiformans W3 nifH, suggesting that Paulinho for helping and kindly allowing us to collect sugar- this phylotype fixes nitrogen not only associated with cane from his farm near Rundu, in the Okavango region of plants but even in marine environments. Namibia, and Elvira Prugger for help with analyses of rhizo- Also Bradyrhizobium sp.-related sequences detected sphere soil and shoot samples of sugarcane. as expressed in another sugarcane sample (Krakatau, sample 65, FN665835–FN665856 and FN665857– FN665865) are interesting in this context. Some References phylotypes (FN665835–FN665848) were highly similar Ando, S., Goto, M., Meunchang, S., Thongra-ar, P., Fujiwara, (94–97%) to nitrogenase genes of uncultured bacteria T., Hayashi, H., and Yoneyama, T. (2005) Detection of nifH isolated from soils and Sorghum rhizosphere, as was sequences in sugarcane (Saccharum officinarum L.) and also confirmed by phylogenetic analysis (Fig. 2). pineapple (Ananas comosus (L.) Merr.). Soil Sci Plant Nutr Bradyrhizobium is known to colonize roots or rhizo- 51: 303–308. sphere of non-legumes such as maize (Roesch et al., Baldani, J.I., Reis, V.M., Baldani, V.L.D., and Döbereiner, J. 2008), Sorghum (Coelho et al., 2008) or mangroves (2002) A brief story of nitrogen fixation in sugarcane – reasons for success in Brazil. Funct Plant Biol 29: 417– (Zhang et al., 2008). In pure culture of Bradyrhizobium 423. japonicum, nifH is derepressed when oxygen concentra- Boddey, R.M., De Oliveira, O.C., Urquiaga, S., Reis, V.M., De tions are sufficiently low. This also supports our obser- Olivares, F.L., Baldani, V.L.D., and Doebereiner, J. (1995) vation that Bradyrhizobium sp. appears to express nifH Biological nitrogen fixation associated with sugar cane and
© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports Transcription of nifH in sugarcane and spruce 7
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© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports Supporting Information Burbano et al. 2011
Table S1. Environmental nifH transcript phylotypes related to cultivated bacteria of the order Rhizobiales from 2264 nifH mRNA sequences1 available up to February 2010 in EBI.
Environmental nifH phylotype Cultivated organism
Sequence Accession no. Retrieved from identity, % No. of bases Accession no. Species affiliation
AB208392 Rice 95 349 AB367742 Mesorhizobium loti AB365432 Sweet potato 100 316 DQ485715 Sinorhizobium fredii CPAC 402 AB265689 Sweet potato 96 309 CU234118 Bradyrhizobium sp. ORS278 AB365432 Sweet potato 100 316 EU357923 Bradyrhizobium sp. SR78 EU818925 B. liaoningense LMG 18230 EU707168 B. japonicum CCBAU15477 EU357921 B. japonicum SR102 EU146014 B. liaoningense CCBAU 83789 EU146009 B. liaoningense CCBAU 83689 EU146008 B. liaoningense CCBAU 83672 EU146007 B. liaoningense CCBAU 83669 EU146002 B. japonicum CCBAU 83626 EU146001 B. japonicum CCBAU 83623 AB365434 Sweet potato 98 306 AB470340 Bradyrhizobium sp. AT1 FJ233714 Norwegian fjord 96 344 CU234118 Bradyrhizobium sp. ORS278 342 FJ347434 Bradyrhizobium sp. ORS318 330 FJ347422 Bradyrhizobium sp. ORS294 330 FJ347445 Bradyrhizobium sp. ORS361 328 FJ347443 Bradyrhizobium sp. ORS356 329 FJ347427 Bradyrhizobium sp. ORS303 95 329 CP000494 Bradyrhizobium sp. BTAi1
1 Supporting Information Burbano et al. 2011
340 AB079616 Bradyrhizobium sp. IRBG 228 340 AB079615 Bradyrhizobium sp. IRBG 2 327 FJ347451 Bradyrhizobium sp. ORS400 AB208371 Rice 96 366 CU234118 Bradyrhizobium sp. ORS278 95 359 AB079617 Bradyrhizobium sp. IRBG 230 363 AB079615 Bradyrhizobium sp. IRGB 2 DQ471674 Estuary 99 321 GQ241353.1 Rhizobium rosettiformans W3 GQ442429 Marine mats 99 314 GQ241353.1 Rhizobium rosettiformans W3
1 Sequences sharing 95% DNA sequence identity
2 Supporting Information Burbano et al. 2011
Appendix S1: COMPLETE SAMPLING DETAILS AND EXPERIMENTAL
PROCEDURES
Sample collection
The root samples from Mexican sugarcane cv. Mex 69-290 and cv. CP 72-2086 were collected in August 2006 in Tuxtepec, Oaxaca State (18°0522 N; 96°0827 W), whereas the root samples from the wild Sacharum spontaneum variety cv. Krakatau and from the cultivated sugarcane variety cv. RB-72454, a hybrid of S. officinarum and S. spontaneum, were harvested in February 2007 at EMBRAPA in Seropédica,
Brazil. The African cultivated sugarcane was collected in the Okavango region of
Namibia (local race sample S1) in April 2007 (17º5226.5 S; 19º5422.1 E). The root samples of Norway spruce (Picea abies L. Karst) were collected in July 2009 from the Solling plateau in Central Germany (51°46'N, 9°35'E; elevation 500 m). All plants were collected at least in triplicate and were sampled by excavating the roots to a depth of 25-30 cm. For Mexican sugarcane and the wild S. spontaneum variety cv. Krakatau also the corresponding rhizosphere soil and shoots between the 1st and
3rd node were sampled, respectively. After collection all roots, rhizosphere soil, and shoot samples were stored in liquid nitrogen and transferred to the laboratory for further analysis.
RNA extraction and purification
Total RNA from root samples of sugarcane was extracted following the Trizol method
(Invitrogen, Karlsruhe, Germany) with minor modifications. An additional step of bead-beating at speed 4 for 10 seconds was performed three times, using the Lysing
Matrix D (MP Biomedicals, Esschwege, Germany) and the FastPrep FP120 instrument (BIO 101, La Jolla, CA, USA). Equal root mass (200 mg fresh weight) was used for normalization in all the extractions. DNase I (Roche, Mannheim, Germany) treatment was done as described previously (Hurek et al., 2002). For spruce, RNA
1 Supporting Information Burbano et al. 2011 was extracted according to the method of Chang et al. (1993). RNA was further purified by RNeasy mini-spin columns (Qiagen, Hilden, Germany) according to the manufacturer’s protocol and was resuspended in 40 μl of H2O. RNA concentrations were estimated with the Nanodrop ND-1000 spectrophotometer (peqlab, Erlangen,
Germany) and the RNA quality was verified by agarose gel electrophoresis.
RT- PCR
The Ready-to-Go RT-PCR beads were used for RT and PCR according to the manufacturer’s instructions (GE Health Care, Munich, Germany). For sugarcane samples a nested RT-PCR protocol was used. The RT step was done for 30 min at
42°C using the primer nifH3 (Zani et al., 2000). The PCR amplification was carried out with an initial inactivation at 95°C (5 min), followed by 30 cycles of denaturation at 95°C (1 min), annealing at 55°C (1 min), and extension at 72°C (1 min), and a final extension at 72°C (10 min) using the nifH3 and nifH4 primers as described by Zani and colleagues (2000). The absence of DNA template was controlled by inactivation of the reverse transcriptase at 95°C (15 min) prior to the reaction. The second round of the nested PCR amplification was performed with 1 μl of the first-round product with the primers ZehrF and ZehrR (Zehr and McReynolds, 1989). For spruce, the reverse transcription and PCR amplification were done using LNA substituted primers as described previously (Burbano et al., 2010). The nifH amplification products (approx. 360 bp long) were visualized on 1.8% agarose gels with ethidium bromide. RNA was analyzed from at least two samples each, (except the Namibian sugarcane and spruce that were sampled only once) with three replicates each. All control reactions testing for DNA contamination of the RNA extracts were negative
(data not shown).
2 Supporting Information Burbano et al. 2011
Denaturing gradient gel electrophoresis (DGGE)
One μl of the RT-nested-PCR products was used as a template to reamplify the nifH amplicons with the forward primer ZehrF containing the GC clamp (10 cycles were performed following the conditions of the nested PCR). The DGGE technique was carried out using the D-Code system from Bio-Rad laboratories (Bio-Rad
Laboratories, Hercules, CA, USA). The PCR products were analyzed by DGGE as described previously (Demba Diallo et al., 2004, 2008).
nifH Clone library and sequencing
The nifH RT-nested-PCR amplicons obtained from the sugarcane roots were cloned into the pJET1.2/blunt plasmid vector from the CloneJET PCR cloning kit
(Fermentas, St- Leon-Rot, Germany) following the manufacturer’s instructions and transformed into chemically competent E. coli cells. Clone libraries were screened by colony PCR. Positive clones were additionally screened by spot blot hybridization at medium stringency with a 60 nt long digoxygenin-multilabeled nifH probe. A clone library was created for every root sample for a total of five. The selected clones were sequenced by Qiagen (Hilden, Germany). To identify bands found in DGGE fingerprints (Fig. 1B) selected clones were screened by DGGE to match the melting profile of the band of interest (data not shown). From the nifH RT-PCR fragment obtained from spruce roots a clone library was constructed using the TOPO TA cloning system (Invitrogen, Darmstadt, Germany) according to the manufacturer’s instructions. The nifH RT-PCR product was also directly sequenced by custom sequencing (LGC Genomics, Berlin, Germany) with the Zehr-R-LNA primer (Burbano et al., 2010). The unambiguously readable sequence was deposited in the EMBL nucleotide database (accession no. FN869040).
Phylogenetic assignment
NifH cDNA sequences were compared by Blast analyses (Altschul et al., 1997)
3 Supporting Information Burbano et al. 2011 against Genbank NR, and nifH sequences from cultivated bacteria that showed the closest match to the query sequence were included for phylogenetic analysis. The nucleotide sequences were aligned with MAFFT using the G-INS-i setting at default
(Katoh et al., 2002). A phylogenetic tree was constructed with MEGA 4 (Tamura et al., 2007) using a minimum evolution analysis with a maximum composite likelihood nucleotide model (Tamura et al., 2004), and the pairwise deletion option. Internal branch test (IBT) confidence values were obtained based on 500 replicates.
Maximum parsimony and maximum likelihood analyses were conducted at
Phylogeny.fr (http://www.phylogeny.fr/) using standard settings. Sequences were submitted to EMBL with the accession numbers FN665805 to FN665834 and
FN665866 to FN666269.
Diversity Measurements and Statistical Analysis
The observed number of nifH phylotypes was calculated using the FastGroupII tool
(Yu et al., 2006) with the method “Percent Sequence Identity with gaps of 97% similarity”. Richness estimations and diversity indices were determined using the web interface from ASLO (Kemp and Aller, 2004) and the software PAST (Hammer et al.,
2001), respectively. The percentage of coverage was estimated by Good`s method using the formula [1-(n/N)] x 100, where n is the number of phylotypes appearing only once in a library, and N is the library size (Good, 1953).
References
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Burbano, CS, Reinhold-Hurek B, Hurek T. (2010). LNA-substituted degenerate primers improve detection of nitrogenase gene transcription in environmental samples Environ Micribiol Rep 2: 251–257.
Chang S, Puryear J, Cairney J. (1993). A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Rep 11: 113–116.
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Good IJ. (1953). The population frequencies of species and the estimation of population parameters. Biometrika 40: 237-264.
Demba Diallo M, Willems A, Vloemans N, Cousin S, Vandekerckhove TT, de Lajudie P, Neyra. (2004). Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal. Environ Microbiol 6: 400–415.
Demba Diallo, M, Reinhold-Hurek, B, Hurek, T. (2008). Evaluation of PCR primers for universal nifH gene targeting and for assessment of transcribed nifH pools in roots of Oryza longistaminata with and without low nitrogen input. FEMS Microbiol Ecol 65: 220–228.
Hammer Ø, Harper DAT, P. Ryan PD.(2001). PAST: Paleontological Statistics Software Package for Education and Data Analysis. Palaeontol Electronica 4: art. 4.
Hurek T, Handley L, Reinhold-Hurek B, and Piché Y. (2002). Azoarcus grass endophytes contribute fixed nitrogen to the plant in an unculturable state. Mol Plant Microbe Interact 15: 233–242.
Katoh K, Misawa K, Kuma K, Miyata T. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 15: 3059-3066.
Kemp PF, Aller JY. (2004). Estimating prokaryotic diversity: When are 16S rDNA libraries large enough? Limnol Oceanogr Methods 2: 114-125.
Tamura K, Nei M, Kumar S. (2004). Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Nat Acad Sci (USA). 101:11030-11035.
Tamura K, Dudley J, Nei M, Kumar S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24: 1596-1599
Yu Y, Breitbart M, McNairnie P, Rohwer F. (2006). FastGroupII: A web-based bioinformatics platform for analyses of large 16S rDNA libraries. BMC Bioinformatics 7:57.
Zani S, Mellon MT, Collier JL, Zehr JP. (2000). Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR. Appl Environ Microbiol 66: 3119-3124.
Zehr JP, McReynolds LA. (1989). Use of degenerate oligonucleotides for amplification of the nifH gene from the marine cyanobacterium Trichodesmium thiebautii. Appl Environ Microbiol 55: 2522–2526.
5 Chapter 3
CHAPTER 3
Study of the microbial community structure and functional diazotrophic diversity in Colophospermum mopane
Claudia Sofía Burbano, Jann Lasse Grönemeyer, Barbara Reinhold-Hurek and Thomas Hurek
Status of the manuscript: In preparation for submission to FEMS Microbial Ecology
Own contribution: - Majority of experiments - Writing of the manuscript
Contribution J.L. Grönemeyer: - Isolates characterization - Southern blot analysis
Study of the microbial community structure and functional diazotrophic diversity in Colophospermum mopane
Claudia Sofía Burbano, Jann Lasse Grönemeyer, Thomas Hurek and Barbara Reinhold-Hurek*
Department of Microbe-Plant Interactions, Center for Biomolecular Interactions Bremen (CBIB), University of Bremen, D-28359 Bremen, Germany
Running title: Microbial and diazotrophic diversity in Mopane
*Corresponding author. Mailing address: Department of Microbe-Plant Interactions, Center for Biomolecular Interactions Bremen, University of Bremen, Postfach 33 04 40, D-28334 Bremen, Germany. Phone: +49 (0)421 218 62868. Fax: +49 (0)421 218 9058. E-mail: [email protected] Abstract
Colophospermum mopane is an indigenous legume tree that grows in Southern Africa and can be the predominant tree of the woodland vegetation. In order to know more about its ecology and how C. mopane has been able to grow in the nitrogen-poor soils of the region, we analyzed the root-associated bacteria to assess the active diazotrophic diversity and total microbial diversity by culture dependent and independent techniques. Root nodules were not detected but in some samples the lateral roots showed an outgrowth-like protuberance. The bacterial isolates recovered were related to Actinobacteria, Firmicutes and Proteobacteria. The total microbial diversity was dominated by Actinobacteria-related phylotypes while the active diazotrophic diversity showed that the majority of the sequences were related to the order Rhizobiales but also to Spirochaetes, Firmicutes, Bacteroidetes and -Proteobacteria, increasing the spectrum of possible phylotypes that can be found in legume trees that are typically nodulated by and -Proteobacteria. Here we report for the first time on the functional diazotrophic community associated with roots of mopane.
1 Introduction
Colophospermum mopane (Kirk ex Benth) (vernacularly known as mopane) is a monotypic genus indigenous to Southern Africa that belongs to the Fabaceae (Legume) family. This legume tree is a secondary colonizer well adapted to dry conditions and covers about 550,000 km2 in South Africa (Mapaure 1994). In the woodlands mopane can be very variable in size ranging from trees as high as 20 meters, passing to 1-2 meters trees, to small trees known as dwarf mopane. Mopane provides many benefits and is of great economic importance for the region. It is used for several domestic purposes such as firewood, construction, ropes and fodder for livestock (Madzibane and Potgieter 1999, Mashabane et al. 2001). Its foliage is also used as a food source by African elephants (Ben- Shahar and Macdonald 2002), eland (Styles and Skinner 1997) and some insects such as the caterpillar of the moth Imbrasia belina, which is used as a food source for the local population in the region. The trees possess an extensive superficial root system (Mlambo et al. 2005). The roots are characterized by coralloid-like lateral roots made of small rootlets of indeterminate growth (Jordaan et al. 2000). It has been suggested that the development of these roots is microbially induced and shows similarities with root nodules (Jordaan et al. 2000), although true nodules have not been found yet. As already mentioned, mopane belongs to the Fabaceae family and is part of the subfamily Caesalpinioideae and the tribe Detarieae. Nodulation in the subfamily is uncommon and many members lack the ability to produce root nodules (Allen & Allen 1981). The occurrence of nodulation in this subfamily is still not clear. In studies that have investigated the production of root nodules in pot cultures, mopane has been unable to produce them (de Faria et al. 1989, Basak and Goyal 1980). Despite the economical importance of mopane in the region, little is known about its ecology and particularly about its roots and their associated bacterial community. Given the fact that mopane is found in mostly nitrogen-poor soils, possible endophytic diazotrophic bacteria might be involved in its growth and development. This group of bacteria is capable to convert the atmospheric nitrogen to ammonia that is then available for the plant. This process –known as biological nitrogen fixation (BNF)– is catalyzed by the nitrogenase enzyme that is a complex composed by two subunits: the dinitrogenase reductase encoded by the nifH gene and the dinitrogenase encoded by nifD and nifK genes. It has been shown that the nifH gene is evolutionary conserved among diverse organisms (Zehr and McReynolds 1989) and its phylogeny largely resembles the 16S rRNA phylogenetic tree (Hennecke et al., 1985; Young, 1992). Therefore, the nifH gene has been used as molecular marker to assess the diversity of nitrogen-fixing bacteria from different environments (Ueda et al. 1995; Zehr et al. 1998, 2003; Hamelin et al. 2002; Tan et al. 2003; Deslippe & Egger
2 2006, Burbano et al. 2011). Knowing that mopane is able to grow in spite of poor soil conditions, we studied the possible role of BNF in the growth of this tree and analyzed the potential diazotrophic bacterial diversity inhabiting the mopane roots (inside or associated with the roots). Rhizobia- like sequences dominated the nifH phylotypes found but sequences related to other phyla were also present. Additionally, we also isolated the bacteria and studied the total microbial community based on the 16S rRNA gene molecular marker (Woese 1987). Rhizobia apparently made up only a small portion of the total bacterial community since the majority of isolates and phylotypes detected by the 16S rRNA approach consisted mainly of Actinobacteria. This is the first in-deep study on mopane roots that analyzed their microbial diversity and shows that there is active diazotrophic bacteria associated to them.
3 Materials and methods
Sample collection Root samples were collected throughout four years (2007-2010) in the Damaraland in Namibia. After collection all root samples were stored in liquid nitrogen and transferred to the laboratory. The samples that were chosen for further culture independent analyses are described in Table S1 and their geographic location is shown in Figure1.
Bacterial isolation and growth conditions Root samples were washed with water, surface-sterilized for 2 min with 70% ethanol, and rinsed twice with sterile water. Roots were homogenized with sterile quartz sand, mortar and pistill. After that, serial dilutions in wash buffer (SM-medium without nitrogen and carbon source (Reinhold et al. 1986)) were used to inoculate mutiple wells of microtiter plates containing isolation medium (modified SM-medium). This media contained per L 1 g of DL- malate, 1 g of glucose, 1 mL of ethanol, 20 mg of yeast extract, 1 mL of vitamins (Hurek et al. 1995), and 8 g of agar. After 7 days of growth at Namibian room temperature (18-30°C), and then incubation of 1-3 weeks at 30°C, single colonies were obtained by streaking cultures on VM-ethanol medium (Hurek et al. 1995). Further cultivation of pure cultures was carried out on VM-ethanol plates at 30°C for 24-48h.
Characterization of isolates Bacterial DNA was isolated following the protocols of Chassy (1976) and Möller et al. (1992) with slight modifications. For the amplification of nifH a nested PCR was performed (Zani et al. 2000) with the internal primer pair ZehrF-LNA and ZehrR-LNA (Burbano et al. 2010) and the external LNA-substituted primers nifH3-LNA (5’-ATRTTRTTNGCNGCRTA-3’, LNA bases are underlined) and nifH4-LNA (5’-TTYTAYGGNAARGGNGG-3’, LNA bases are underlined). PCR amplification of 16S rRNA was carried out using the universal primers Bac8uf (5’-AGAGTTTGATNHTGGYTCAG-3´) and Univ1492r (5´- GGNTCCTTGTTACGACTT-3´). The 50 L reaction mixture consisted of 2,5 U of Taq DNA Polymerase (Molzym), 50 M of each desoxynucleoside triphosphate, 500 nM of each primer (Eurofins MWG Operon) and 0,8 ng of DNA. The thermocycling profile was carried out with an initial denaturation at 95°C (4 min) followed by 35 cycles of denaturation at 95°C (1 min ), annealing at 50°C (30 s), extension at 72°C (1 min) and a final extension at 72°C (10 min). Purification was performed using the Quiaquick PCR purification kit Qiagen kit (Qiagen, Hilden, Germany). Sequencing was carried out at the Sequencing Service Department (LMU, Munich, Germany) with the primers Bac8uf and 530f (5’-GTGCCAGCMGCCGCGG-3’).
4 Microscopy Root samples were embedded with LR White acryl resin soft grade (Agar Scientific Ltd., Essex, United Kingdom), and heat cured at 54°C as described previously (Reinhold and Hurek 1988). Root sections were stained with 4 ,6-diamidino-2-phenylindole (DAPI) as described (Werner et al. 2001). The sections were visualized using a fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).
RNA extraction and purification Total RNA from root samples was extracted following a protocol of Chang and colleagues (1993) with the following modifications: The total amount of buffer used was 735 μl and 25X RNA-secure was added to a final concentration of 1X (Ambion, Austin, TX, USA). An additional step of bead beating at speed 4 for 15 seconds was performed using the Lysing Matrix D (MP Biomecicals, Hessen, Germany) and the FastPrep FP 120 instrument (BIO 101, La Jolla, CA, USA). After precipitation with LiCl, a Proteinase K treatment was performed as described previously (Reinhold-Hurek et al. 2006). RNA was resuspended in 1X RNA-secure (Ambion, Darmstadt, Germany) and further purified removing the DNA by RNeasy plus mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol.
The RNA was finally resuspended in 30 μl of H2O. For each extraction, equal root mass (fresh weight) was used for normalization. RNA concentrations were estimated by Nanodrop (peqlab, Erlangen, Germany) and the RNA quality by agarose gel electrophoresis.
RT- PCR The Titan One Tube RT-PCR system was used for RT-PCR according to the manufacturer’s instructions (Roche, Mannheim, Germany). To synthesize the cDNA, 15 ng of total RNA was used as template. The partial 16S rRNA gene was amplified using the primers 799f and 1492r (Chelius and Triplett 2001, Lane 1991). The 1492r primer was used for the RT step (30 min at 42°C). This primer set amplifies most bacterial sequences to the exclusion of chloroplast DNA and gives a mitochondrial PCR product that is about 1.5 times the size of the bacterial product. The PCR was performed as described previously (Chelius and Triplett 2001) and the bacterial PCR products of approximately 735 bp were visualized on 1% agarose gels with ethidium bromide. For the amplification of nifH and its transcription a nested PCR approach was performed (Zani et al. 2000) where LNA-substituted primers were used. The RT step was done for 30 min at 42°C using the primer nifH3-LNA. The PCR amplification was carried out with an initial inactivation at 95°C (5 min), followed by 30 cycles of denaturation at 95°C (1 min), annealing at 55°C (1 min), and extension at 72°C (1 min), and a final extension at 72°C
5 (10 min) using the nifH3-LNA and nifH4-LNA primers as described by Zani and colleagues (2000). The absence of DNA template was controlled by inactivation of the reverse transcriptase at 95°C (15 min) prior to the reaction. The second round of the nested PCR amplification was performed with 1 μl of the first-round product with the primers Zehr-F-LNA and Zehr-R-LNA (Burbano et al. 2010). The nifH amplification products were about 362 bp long and were visualized on 1.8% agarose gels with ethidium bromide.
Clone library and sequencing The 16S rRNA and nifH RT-PCR amplicons obtained from the mopane roots were purified with the Quiaquick PCR purification kit (Qiagen, Hilden, Germany) following the manufacture’s instructions. The purified products were cloned into the pJET1.2/blunt plasmid vector included in the CloneJET PCR cloning kit (Fermentas, St- Leon-Rot, Germany) following the instructions provided by the manufacturer and transformed into chemically competent E. coli cells. Clone libraries were screened by colony PCR using primers pJET1.2Forward and pJET1.2Reverse for inserts of the correct size in an agarose gel electrophoresis. Positive nifH clones were additionally screened by spot blot hybridization at medium stringency (2X sodium chloride-sodium citrate (SSC) buffer + 0.1%SDS, 65°C) with a digoxygenin-labelled nifH probe. A clone library was created for every gene and root sample analyzed. The selected clones were sequenced by LGC Genomics (Berlin, Germany).
Phylogenetic and sequence analyses The quality of the obtained 16S rRNA sequences was manually checked using BioEdit 7.0.9.0 (Hall 1999). The presence of chimeric sequences was evaluated using Mallard (Ashelford et al. 2006). The sequences were classified using the RDP classifier (Ribosomal Database Project, Michigan State University, Michigan; Wang et al. 2007) and the taxonomic ascription of the sequences was confirmed by BLASTN. Sequences were imported into the ARB software package (Ludwig et al., 2004) and aligned using the ARB FastAligner. The ARB software package was used to generate phylogenetic trees with the neighbor-joining algorithm. Distance matrices with the Jukes-Cantor correction were generated using DNADIST (http://cmgm.stanford.edu/phylip/dnadist.html). The estimation of operational taxonomic units (OTUs), rarefaction curves of observed OTUs, richness, diversity indices and the differences between libraries were done using mothur (Schloss, 2009). A sequence identity threshold of 98% was used to define OTUs, as this cut-off roughly corresponds to the species level (Stackebrandt and Ebers, 2006). The quality of nifH sequences was manually checked using BioEdit 7.0.9.0 (Hall 1999). Similarity to known nifH sequences was searched using the BLASTN program against the
6 GenBank database (www.ncbi.nlm.nih.gov/blast) (Altschul et al. 1997) and the closest matches found in the GeneBank database were included for phylogenetic analysis. The nucleotide sequences were aligned with MAFFT (http://align.bmr.kyushu- u.ac.jp/mafft/online/server/) using the G-INS-i setting at default (Katoh et al. 2002). The phylogenetic tree was constructed with MEGA 4 (Tamura et al., 2007) using a minimum evolution analysis with a poisson correction, a gamma distribution equal to 0.5285 and the pairwise deletion option. Internal branch test (IBT) confidence values were obtained based on 500 replicates. The aligned sequences were used to generate distance matrices with the Jukes-Cantor correction using DNADIST. Mothur was used for the estimation of OTUs, rarefaction curves of observed OTUs and diversity indices (Schloss et al. 2009). To compare clone libraries of the diazotrophic community, mothur and Unifrac analyses on the web server (Schloss et al. 2009, Lozupone et al. 2006) were carried out.
7 Results and Discussion
Characteristics of mopane tree The mopane tree (Colophospermum mopane) is a legume capable of growing in hot and dry areas of Southern Africa where it is a component of the woodland vegetation, and sometimes the predominant tree. In order to know more about its ecology and particularly its associated bacterial community, root samples were taken which were subsequently analyzed by culture dependent and independent techniques (Figure 1). Samples from trees of approximately 30 cm – 60 cm height were collected (Figure 2A). Although mopane belongs to the Fabaceae family, the roots that were collected did not show the typical nodules found in other legumes. Few samples showed outgrowths like protuberances on lateral roots (Figure 2B). These were found only from samples of the year 2010. Previously, an anatomical study from the roots of mopane stated that the development of the “coralloid-like” structures found in lateral roots of the tree “seems to be microbially induced” similar to nodules in other legumes (Jordaan et al. 2000). These findings could speak for a tight interaction of mopane with root-associated diazotrophic bacteria that might be involved in growth and development of the tree in nitrogen-poor soils of Namibia. Another interesting characteristic was the reddish color found in all roots in the cortex region (Figure 2C, 2D). Active legumes nodules are often characterized by their pink color due to the oxygen-carrier protein leghemoglobin (Sprent 2008). Therefore it seems to be possible that the pink color in mopane roots maybe caused by the presence of this protein but this assumption has to be confirmed. Sections of root samples that were observed under the fluorescence microscope showed that bacteria are present inside the root tissue, which is indicative of a possible endophytic lifestyle (Figure S1).
Spectrum of mopane isolates Root-associated bacteria were isolated from samples in each year of the study. In total eleven bacterial isolates were characterized and were subjected to DNA extraction for 16S rDNA and nifH PCR-mediated assays. According to the 16S rDNA sequence analysis these bacteria were affiliated in almost equal proportions to three different phyla: Proteobacteria ( and ), Firmicutes and Actinobacteria (Table 1). From these eleven isolates a digoxygenin-multilabeled nifH probe detected a highly related nifH gene fragment in DNA preparations of only two of the isolates (data not shown). A comparative sequence analysis of the 16S rDNA sequences of these two isolates showed that these bacteria were related to Agrobacterium tumefaciens (Mop 2-2) and Rhizobium sp. (Mop 3-2) within the order Rhizobiales. The amplification of the nifH gene was only possible for the isolate Mop 3-2, which had 92% DNA sequence identity with the nifH sequence from Mesorhizobium loti
8 confirming the suggestion made by Jordaan et al. (2000) that bacteria resembling rhizobia infect the roots of mopane.
Diversity of active microbial community A 16S rRNA RT-PCR was used to characterize the active microbial community inhabiting mopane roots. The amplified product of approximately 735 bp was obtained in all the samples analyzed (data not shown). A clone library approach was carried out for each of the samples. In total four libraries were obtained from which 618 clones were analyzed. The number of OTUs estimated -with a 98% cut-off value- increased throughout the years. The diversity based on the ChaoI richness estimator varied between the samples and the years and predicted a higher diversity in the samples taken in 2010 and 2009 with respect to the ones in 2008 and 2007. The coverage of the libraries was 62-98% according to the Good’s coverage estimator (Table 2). Rarefaction curves at a 98% similarity cut-off did not reach an asymptote except for the library M1 (Figure S2a). The majority of the sequences in all the libraries except for M1 showed identity values below the species-threshold (98.7-99%) postulated by Stackebrand and Ebers (2006) as follows: 95% for M4, 89% for M31, 77% for M21 and 40% for M1. The phylum Actinobacteria dominated the clone libraries except for the M1 sample where the majority of the phylotypes were related to Pantoea sp. ( -Proteobacteria). The other bacterial groups that were represented in the 16S rRNA clone libraries consisted of -, - and -Proteobacteria (Figure 3). To estimate the similarity between the libraries a clustering analysis was performed (Figure 4a). The M21 and M31 libraries clustered together and were distinctly separated from M4 and M1. The most different community was the one from M1, which was expected from the relative contribution of bacterial groups shown in Figure 3. Nevertheless, when -Libshufft was used to compare the libraries, all of them were found to be significantly different from each other (P<0.0001). The shared OTUs between the pair M21 and M31 libraries were higher than between the M4 and M1 libraries. The same pattern was obtained when we checked for the similarity between the four libraries based on the Jaccard as well as the Sørenson index. Both of them showed that the pair M21-M31 had the highest values. Likewise, the structure of the total bacterial community ( ) was more similar between these two libraries (Table S2).
Active diazotrophic community, species richness and diversity measurements A nifH RT-nested-PCR approach with broad range primers was used to detect the nitrogen-fixing communities associated with the root samples (Zani et al. 2000). The amplification of the nifH from the RNA extracted was very difficult. The only way we were
9 able to amplify the nifH gene was using LNA-substituted primers that have shown to increase the sensitivity of the RT-PCR in rice and sugarcane root samples (Burbano et al. 2010). In this study we also modified the primers used in the first PCR amplification (see materials and methods) to cope successfully with this problem. Amplification products of the appropriate size (364bp) were obtained in all four samples analyzed (one from each year of the sampling). These results were corroborated by a Southern blot hybridization using a digoxygenin-labelled nifH probe (data not shown). All control reactions testing for DNA contamination of the RNA extracts were negative (data not shown). Clone libraries were constructed from each of the samples and a total of 115 clones were analyzed. The identity of their nucleotide sequences was compared to the database entries and revealed that all of the clones represented nifH sequences (Table 3). The majority of clones were related to the order Rhizobiales in all of the samples except from M21 where the majority of the clones were related to Spirochaetes-like phylotypes. The M1 library was the most diverse one, representing sequences related to five different bacterial species. Particularly interesting was the observation that sequences that in BlastN analyses showed similarity to a sequence of Bacillus sp. (Firmicute) grouped together with sequences from Proteobacteria in the NifH protein tree. The Bacillus sp. sequence was deposited in the database as a nifH DNA but no further information was provided. Either this could be a case of lateral gene transfer or a possible PCR contamination, however the answer remains unclear. In the other cases no such discrepancy was found and all the sequences clustered together with the nearest cultivated sequences found in the database (Table 3, Figure 5). Species coverage, richness, and diversity of niH phylotypes were calculated for all the libraries analyzed and the combined data set (Table 4). Using a cut-off of 98% we found a small number of OTUs in all the samples being M1 the one with the more number of OTUs. In general the number of OTUs was in accordance with the phylogenetic distribution of the sequences, which showed the same number of clusters dispersed throughout the Group I and Group II of nitrogenases (Raymond et al., 2004). The highest values from the ChaoI species richness estimator as well as the Shannon diversity index were from the M31 library. It is important to highlight that M31 was the only sample with the protuberances found in the roots. Good s coverage values were greater than 96% in all the libraries suggesting that the majority of the root-associated diazotrophs had been sampled. Nevertheless, rarefaction curves at 98% similarity cut-off did not reach an asymptote (Figure S2b) telling that the sampling effort has to increase. Libraries were compared with different approaches. A cluster analysis showed M1 grouping together with M4 and these two libraries with M31. The most distant library was M21 as was expected from the phylogenetic analysis (Figure 4b). All statistical comparison
10 between the clone libraries with -Libshufft also agreed with the clustering but the pair M1- M4, all the comparisons were significantly different from each other (P<0.0001). Additionally, the PCoA (Principal Coordinate Analysis) revealed patterns of similarity between the samples comparable to the cluster analysis. The first principal coordinate separated the M21 library from the other three samples, putting together M1 with M4 (Figure S3). This pair shared one OTU and its Jaccard and Sørenson index as well as theta values were the highest (Table S2), reflecting the similarity between them, results that were in agreement with the cluster analysis performed.
Beyond the Rhizobiales Except Gunnera, all flowering plants –including the legumes– that are capable to establish a N2-fixing endosymbioses are part of the single clade Rosid I (Soltis et al. 2000). All microsymbionts of these nodular symbioses are members of Proteobacteria and Actinobacteria phyla. Plants that are nodulated by the filamentous bacteria Frankia are known as actinorhizal plants. In turn, legumes are typically nodulated by - and - Proteobacteria. In the -branch four families (Bradyrhizobiaceae, Methylobacteriaceae, Phyllobacteriaceae, and Rhizobiaceae) include genera capable of this process while in the - branch just one family (Burkholderiaceae) has two genera (Burkholderia and Cupriavidus) able to nodulate them (Sprent 2008). Although we did not find typical nodules in mopane roots, we were able to find diazotrophic bacteria associated with them. The majority of the sequences (57.5%) was related to the order Rhizobiales, an expected result due to the fact that mopane is a legume tree. Apart from that and most interesting was the finding of phylotypes different from - and -Proteobacteria (Table 3, Figure 5). Spirochetes-like sequences were the second most abundant phylotypes found (18%), albeit only in one sample, where Rhizobiales-like phylotypes were not detected. Spirochetes have been found as prominent nitrogen-fixing members (up to 50% of all prokaryotes) of the microbial community in the hindgut of termites (Breznak and Leadbetter 2006). In our study the closest cultured relatives were free-living spirochetes that have been isolated from contaminated water and hydrogen sulfide- containing mud (Canale-Parola et al. 1968) and their ability to fix nitrogen has been evaluated (Lilburn et al. 2001). Sequences related to -Proteobacteria were also found (10.5%) and the closest cultivated bacteria were affiliated with Pelobacter sp. isolated from freshwater sediments (Shinck 1994) and the iron-reducing bacterium Anaeromyxobacter sp. isolated from subsurface sediments (http://genome.jgi-psf.org/ana_f/ana_f.home.html). Both sequences were obtained from their respective genome projects. Firmicutes-related sequences (9%) were also found in two of the samples analyzed increasing the diversity of
11 active diazotrophs in legumes. Heliobacterium-like phylotypes were found in sample M1, this genus is widely distributed in paddy soil and is known as a very active nitrogen-fixing bacterium (Klimble et al. 1992, Enkh-Amgalan et al. 2005). The other sequences from M4 were related to Clostridium beijerinckii that has been found to be a nitrogen-fixing organism in an experiment where the incorporation of 15N was measured (Rosenblum and Wilson 1949). Finally, a small portion of the nifH sequences (5%) from the M21 library had the closest sequence match with the partial nifH sequence from the bacteroidete Paludibacter propionicigenes isolated from rice plant residues (Ueki et al. 2006). In conclusion our study showed that apart from members of the family Rhizobiales also Spirochaetes, Firmicutes and Bacteroidetes contributed to the active diazotrophic bacterial community in mopane roots. Bacteria falling into these groups have probably not been cultivated yet, since the respective expressed nifH genotypes had only nucleotide identities between 74% and 88% to nifH sequences from cultivated representatives. However, the active diazotrophic bacterial community in mopane roots contributed only little to the overall community of root-associated bacteria, which was dominated by Actinobacteria. Again the majority of the 16S rRNA sequences obtained had nucleotide identities below the proposed species threshold value (98.7-99%). Therefore, the root-associated bacterial community of mopane consists mostly of microorganisms, including active diazotrophs that are distinct from cultivated bacteria at the species level. Accordingly, it can be expected that mainly novel bacterial taxa contribute to the growth of mopane in the poor soils of Namibia.
Acknowledgements
This research was supported by grant 01LC0021 from the Bundesministerium für Bildung und Forschung to B.R.-H. and T.H. in the BIOLOG framework. Root samples of mopane were collected in Namibia under the collection permits 1138/2007 and 821/2004, under Material Transfer Agreements with NBRI (National Botanical Research Institute), Winhoek, Namibia.
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14
Figure legends
Fig. 1. Geographical location of the mopane sampling sites in Namibia used for the culture independent analysis.
Fig. 2. (A) Mopane tree growing in the nutrient poor soils in Namibia; (B) lateral roots with the “protuberances” found in samples taken in 2010; (C, D) pigmented mopane roots.
Fig. 3. Relative contribution of major bacterial groups to the 16S rRNA gene clone libraries.
Fig. 4. Clustering analysis of clone libraries based on the 16S rRNA gene (A) and nifH (B).
Fig. 5. Minimum-evolution NifH protein tree. The tree shows the phylogenetic affiliation of 115 nifH cDNA fragments recovered from roots of four mopane root samples. Numbers at branches represent internal branch test (IBT) confidence values >50% from 500 replicates. The scale bars show the number of nucleotide substitutions per site. The parenthesis next to the name of the samples represents the number of identical sequences found.
15 Table 1. Identification of the isolates from Mopane roots by NCBI BlastN
Isolate Closest match (first hit >98%) Accession No. Sequence similarity, % Nucleotides Taxonomic affiliation
Mop1-1 Enterobacter cloacae Z96079 99 1260/1264 -Proteobacteria Mop2-2 Agrobacterium tumefaciens FN433082 99 1328/1332 -Proteobacteria Mop3-2 Rhizobium sp. KD20936 FN645726 99 1375/1376 -Proteobacteria Mop9 Arthrobacter luteolus LNR3 DQ486130 99 1192/1204 Actinobacteria Mop12 Microbacterium sp. R1 AY974047 98 1380/1395 Actinobacteria Mop13 Curtobacterium sp. 3490BRRJ FJ876396 100 1064/1064 Actinobacteria Mop14 Leucobacter sp. JG 31 GU390657 99 1175/1176 Actinobacteria Mop31-8 Bacillus sp. PT401 AB374305 99 1389/1398 Firmicutes Mop31-10 Bacillus sp. PT402 AB374305 99 1367/1371 Firmicutes Mop31-11 Bacillus sp. MB67 AB518979 91 1250/1366 Firmicutes Mop33-5 Microbacterium ginsengisoli GQ181053 99 1351/1358 Actinobacteria
Table 2. 16S rRNA sequence diversity and library coverage estimations
Measurements M1 M4 M21 M31 Combined
No. Clones 151 133 170 164 618 OTUs (cut-off 98%) 8 38 87 95 198 Singletons 3 25 55 62 120 Chao1 estimator of species richness 10 52 174 190 402 Shannon’s index for diversity 1.18 2.16 4.13 4.28 4.25 Simpson’s index for diversity 0.40 0.32 0.01 0.01 0.04 Good’s estimator of coverage, % 98 81 68 62 80
Table 3. Phylotypes found in the nifH transcript clone libraries
nifH transcript sequence Closest cultivated neighbor present in public database
Clone Sequence No. library similarity, % clones Accession no. Name
M1 94.7 – 95.7 32 AB542352 Bradyrhizobium sp. TSA27s 75.7 – 76.4 7 AB126254 Heliobacterium gestii 94.7 – 95.7 6 DQ364803 Bacillus sp. BT97 99.0 3 GQ241353 Rhizobium sp. W3 89.2 1 CP000943 Methylobacterium sp. 4-46 M4 94.3 – 95.2 8 AB542352 Bradyrhizobium sp. TSA27s 81.1 – 82.0 4 CP000142 Pelobacter carbinolicus DSM 2380 87.9 3 AF266462 Clostridium beijerinckii M21 73.9 – 77.7 20 AF325795 Spirochaeta zuelzerae 76.1 – 78.4 6 CP002345 Paludibacter propionicigenes 84.8 1 AF325792 Spirochaeta aurantia M31 79.6 – 81.2 8 CP000769 Anaeromyxobacter sp. Fw109-5 90.3 – 91.0 8 AY428644 Rhizobium daejeonense 98.6 – 99.3 8 DQ364803 Bacillus sp. BT97
Table 4. nifH sequence diversity and library coverage estimations
Measurements M1 M4 M21 M31 Combined
No. Clones 49 15 27 24 115 OTUs (cut-off 98%) 5 3 3 3 12 Singletons 1 0 1 0 2 Chao1 estimator of species richness 5 3 5 9 13 Shannon’s index for diversity 1.06 1.00 0.67 1.09 2.01 Simpson’s index for diversity 0.45 0.35 0.58 0.30 0.17 Good’s estimator of coverage, % 98 100 96 100 98
Figure 2. Figure 3.
Figure 4.
Figure 5.
Table S1. Mopane root samples used in the culture independent approach
Sample name Year Site GPS Description M1 2007 Grootberg pass S 19° 51’ 6.22’’ E 14° 7’ 46.13’’ M4 2008 Grootberg table mountain S 19° 51’ 05’’ E 14° 08’ 03’’ Young tree, 30 cm height, roots dug out 30 cm approximately. M21 2009 Valley S 19° 50’ 42’’ E 14° 06’ 26’’ Small tree, roots from only main root of 3 cm. diameter, upper part red. M31 2010 Road to Sesfontein S 19° 08’ 8.33’’ E 13° 44’ 1.53’’ Young tree, 30 cm height, roots dug out 30 cm. Lateral roots with hypertrophy, deep red.
Table S2. Shared OTUs, abundance-based Jaccard and Sørenson similarity indices and the estimation of community structure similarity (theta) for the Mopane root samples.
Target Comparisons Shared OTUs Jaccard Sørenson theta 16S rRNA M1 – M21 4 0.038 0.074 0.003 M1 – M31 1 0.004 0.009 0.0002 M4 – M21 9 0.096 0.175 0.006 M4 – M31 3 0.014 0.028 0.0001 M21 – M31 17 0.232 0.376 0.229 nifH M1 – M4 1 0.42 0.59 0.68 M1 – M31 1 0.10 0.42 0.05
Figure S1.
Fig. S1. Epiflourescence images of thin sections from embedded Mopane roots stained with DAPI showing bacteria inside root tissue (A, B) and a differential interference contrast image overlaid with the epifluorescence image (C).
A 16S rRNA gene B nifH Figure S2.
Fig. S2. Rarefaction curves at 98% similarity cut-off generated for 16S rRNA gene (A) and nifH (B) sequences from mopane root samples.
Figure S3.
Fig. S3. Principal Coordinate Analysis (PCoA) plot from analysis of nifH transcript diversity in mopane root samples. Chapter 4
CHAPTER 4
Isolation and characterization of root-associated bacteria from agricultural crops in the Kavango region of Namibia
Jann Lasse Grönemeyer, Claudia Sofía Burbano, Barbara Reinhold-Hurek and Thomas Hurek
Status of the manuscript: Submitted to Plant & Soil. In revision.
Own contribution: - Bacterial growth and maintenance - DNA isolation - Phylogenetic analysis - Writing of the manuscript
Contribution J.L. Grönemeyer: - SDS-soluble cell proteins - 16S rDNA gene amplification - Plant-growth promotion and biocontrol tests - Writing of the manuscript 1 Isolation and characterization of root-associated bacteria
2 from agricultural crops in the Kavango region of Namibia 3 4 5 6 Jann Lasse Grönemeyer1, Claudia Sofía Burbano1, Thomas Hurek and 7 Barbara Reinhold-Hurek* 8 9 Department of Microbe-Plant Interactions, Center for Biomolecular Interactions 10 Bremen (CBIB), University of Bremen, D-28359 Bremen, Germany 11 12 1Authors contributed equally. 13 14 15 16 Running title: Isolation and characterization of PGPR from Namibia 17 18 Key words: Endophytes, Sorghum, Pennisetum, maize, plant growth-promotion 19 20 21 22 23 24 25 26 27 28 29 30 *Corresponding author. Mailing address: Department of Microbe-Plant Interactions, 31 Center for Biomolecular Interactions Bremen, University of Bremen, Postfach 33 04 32 40, D-28334 Bremen, Germany. Phone: +49 (0)421 218 62868. Fax: +49 (0)421 218 33 9058. E-mail: [email protected] 1
1 Abstract 2 3 Background and aims: Root-associated bacteria may cause beneficial effect on their 4 host plants. Microbial products may promote and stimulate plant growth or lead to 5 bioprotection against pathogens. This study aimed to isolate putatively beneficial 6 bacteria from traditional cereals grown by subsistence farmers in the Kavango of 7 Namibia. 8 Methods: Bacteria were isolated from surface-sterilized roots of Pennisetum 9 glaucum, Sorghum bicolor, and Zea mays, and subjected to phenotypic and 10 phylogenetic analyses. Results: 44 root-associated bacterial strains were isolated. 11 The majority of the distinct isolates belonged to Firmicutes and Actinobacteria, while 12 only nine species were Proteobacteria. Several novel phylotypes were among the 13 isolates. Features known to contribute to plant growth-promotion and biocontrol were 14 tested in vitro and revealed several promising candidates with multiple beneficial 15 characteristics. 16 Conclusions: This is the first report on the characterization of native isolates 17 associated to important agriculture crops in the Kavango region, which have the 18 potential for application as inoculants adapted to poor soils and local crops. 19 Desiccation-tolerant or sporulating gram-positive bacteria are of particular interest for 20 this region characterized by a long dry season. 21 22 23
2
1 Introduction 2 3 Microorganisms in the rhizosphere of plants are important players in the terrestrial 4 ecosystem. They are involved in the cycling of nutrients, the decomposition and 5 mineralization of organic matter, and are essential for long-term sustainability 6 (Germida et al. 1998). More specifically, plant-bacteria associations can help plants 7 to establish in degraded landscapes, protect plants against diseases or even 8 promote plant growth (Glick 1995). Exploring the diversity of plant-bacteria 9 associations is indispensable if these associations can be used further on to increase 10 crop production, conserve biodiversity and sustain agro-ecosystems (Germida et al. 11 1998). 12 Plant growth-promoting rhizobacteria (PGPR) are a group of microorganisms able 13 to colonize the rhizosphere or roots of many plant species, conferring beneficial 14 effects on their host (Kloepper et al. 1980). Currently there are many bacterial genera 15 known to harbor PGPR, some of the most frequently reported being Bacillus, 16 Pseudomonas and Rhizobium (Barriuso et al. 2008). The mechanisms used by 17 PGPR can be either direct or indirect. Direct mechanisms include nitrogen fixation for 18 plant use, provision of bioavailable phosphorus for plant uptake, sequestration of iron 19 for plants by siderophores, and production of plant hormones like auxins, cytokinins 20 and gibberellins. Among the indirect mechanisms are synthesis of enzymes, lysing 21 fungal cell walls, reduction of iron available to phytopathogens in the rhizhospere, 22 antibiotic protection against pathogenic bacteria, and competition with detrimental 23 microorganisms for sites on plant roots (Lucy et al. 2004). 24 Kavango is one of the thirteen regions of Namibia situated in the northeast part 25 that covers approximately 43 000 km2. In this region there are approximately 24 000 26 households that live primarily from agriculture, and their short-term food supply 27 depends on subsistence cultivation. The soils are mainly arenosols with a pH range 28 of 5.5 – 7.5. They are considered nutrient poor with an average organic carbon of 29 0.4%, a total nitrogen range of 0.03 – 0.16% and a very low total phosphorus content 30 that is in most cases below the limit of detection (< 1 mg kg-1). The farmers cultivate 31 several crops of which pearl millet Pennisetum glaucum (locally known as mahangu) 32 is the main one because of its low demands in terms of soil quality and rainfall. Other 33 cultivated crops include maize, cowpea and sorghum (Sorghum bicolor). The fields 34 are not irrigated, herbicides as well as pesticides and fertilizers are not used, and
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1 crop yields are very low (Pröpper et al. 2010). Also in the greater Kavango regions of 2 catchment and Okavango basin in Angola and Botswana, subsistence agriculture 3 with low agrochemical inputs is widespread. Poverty reduction in such small farm 4 households could be obtained by improving yields of their traditional crops. However, 5 there are no studies as yet on crop-associated PGPRs in this region. 6 The application of native, adapted microorganisms might improve the yields by 7 direct plant growth promotion, biocontrol and increasing the plant tolerance to stress. 8 Therefore, we initiated a screening on root-associated bacteria of traditional crops in 9 the Namibian Kavango, mahangu, maize and sorghum. Bacteria were isolated from 10 roots and characterized with respect to their taxonomic affiliation and putative plant- 11 growth-promoting characteristics. A broad range of bacteria was obtained, a large 12 fraction of gram-positive bacteria. These bacteria can be considered promising 13 candidates for application in sustainable agricultural management for the region. 14 15
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1 Materials and methods 2 3 Isolation of bacteria and growth conditions 4 5 The crops selected for the study were pearl millet (Pennisetum glaucum) locally 6 known as mahangu, sorghum (Sorghum bicolor) and maize (Zea mays). They were 7 sampled at the stage of flowering during the rainy season (March 2007) from a field 8 with mixed cultivation of a subsistence farmer near Mutompo in the Kavango region 9 of Namibia. Root systems from 2 -10 cm depth were treated within 1 hour after 10 sampling. Roots were washed with water, surface-sterilized for 2 min with 70% 11 ethanol, and rinsed again twice with sterile water. After homogenization with sterile 12 quartz sand, mortar and pistill, serial dilutions in wash buffer (SM-medium (Reinhold 13 et al. 1986) without nitrogen and carbon source) were used to inoculate mutiple wells 14 of microtiter plates containing isolation medium. The medium consisted of modified 15 SM-medium containing per L 1 g of DL-malate, 1 g of glucose, 1 mL of ethanol, 20 16 mg of yeast extract, 1 mL of vitamins (Hurek et al. 1995), and 8 g of agar. After 1 17 week of growth at Namibian room temperature and further incubation of 1-3 weeks at 18 30°C, cultures were streaked on VM-ethanol medium (Hurek et al. 1995) to obtain 19 single colonies. Further cultivation of pure cultures was carried out on VM-ethanol 20 plates at 30°C. 21 22 Comparison of SDS-soluble cell proteins 23 24 Precultures were grown in VM-ethanol medium at 30°C on a rotary shaker overnight. 25 Cells were washed with saline solution and resuspended to identical cell densities
26 (OD578 of 0.5) in VM-ethanol medium, and 3.5 mL spread onto VM-ethanol agar 27 plates (14 cm in diameter). After incubation at 30°C for 42 hours, biofilms were 28 harvested and the cells were washed twice with PBS buffer. SDS-soluble proteins 29 were extracted according to (Kiredjian et al. 1986) with slight modifications for the 30 gram-positive bacteria that were disrupted by ultrasonication for four times for 30 31 seconds at 50 W before boiling. Protein extracts were separated by SDS- 32 polyacrylamide gel electrophoresis (PAGE) (Laemmli 1970) (12.5% polyacrylamide, 33 30:1) with modifications (Kiredjian et al. 1986). Proteins were stained with 0.25% 34 Coomassie brilliant blue R-250 in 40% ethanol and 10% acetic acid.
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1 2 16S rDNA gene amplification and sequencing 3 4 Bacterial DNA was isolated following the published protocols (Chassy 1976; Möller et 5 al. 1992) with slight modifications for gram-positive or gram-negative bacteria, 6 respectively. Amplification of 16S rDNA by PCR was carried out using the universal 7 primers Bac8uf (5’-AGAGTTTGATNHTGGYTCAG-3´) and Univ1492r (5´- 8 GGNTCCTTGTTACGACTT-3´). The 50 L reaction mixture consisted of 2,5 U of Taq 9 DNA Polymerase (Molzym), 50 M of each desoxynucleoside triphosphate, 500 nM 10 of each primer (Eurofins MWG Operon) and 0,8 ng of DNA. The thermocycling profile 11 was carried out with an initial denaturation at 95°C (4 min) followed by 35 cycles of 12 denaturation at 95°C (1 min), annealing at 50°C (30 s), extension at 72°C (1 min) and 13 a final extension at 72°C (10 min) in a Biometra TGradient thermocycler. Purification 14 was performed using the NucleoSpin Extract II Kit (Machery-Nagel). Sequencing was 15 carried out at the Sequencing Service Department (LMU, Munich) with the primers 16 Bac8uf and 530f (5’-GTGCCAGCMGCCGCGG-3’). 17 18 Phylogenetic assignment 19 20 The quality of the obtained sequences was manually checked using BioEdit 7.0.9.0 21 (Hall 1999). Partial-length sequences were assembled with Sequencher (Gene Code, 22 Ann Arbor, MI, USA). Sequences were imported into ARB software package (Ludwig 23 et al. 2004) and added to the database. Sequences were aligned using the ARB 24 FastAligner, then refined manually. Phylogenetic trees were generated using the 25 maximum parsimony, neighbor joining with Jukes Cantor correction and maximum 26 likelihood algorithm with 1000 bootstrap replicates. Sequences reported in this study 27 were deposited at EMBL under the accession numbers FR750284 to FR750316. 28 29 Screening for biofertilization activities 30 31 Siderophore production was tested on Chrome azurol S (CAS) agar plates (Schwyn 32 and Neilands 1987) with slight modifications: the solutions were not deferrizated and 33 piperazine was not added. A thin layer of approximately 6 mL of modified VM-ethanol 34 medium (Fe-EDTA-free; containing 0.5 g L-1 yeast extract, 1 g L-1 peptone, 18 mM
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1 NH4Cl, 0.1% malate, 0.25% glucose, 2.1 mM K2HPO4 and KH2PO4, 1.7 mM NaCl, -1 2 0.8 mM MgSO4, 0.18 mM CaCl2, 0.06 mM MnSO4, 8 M Na2MoO4 and 17 g L Agar, 3 pH 6.8) was poured over the plates before inoculation. Cultures were grown for up to 4 7 days at 30°C. Orange halos around the colonies were indicative of siderophore 5 activity. 6 Inorganic phosphate solubilization ability was tested on Pikovskaya agar 7 (Pikovskaya 1948) dyed with 20 mg L-1 of bromophenol blue as described (Gupta et 8 al. 1994). Additionally, the medium was supplemented with 5 mL L-1 of ethanol and 9 vitamins (see above). After incubation for up to 7 days at 30°C, formation of yellow 10 halos and/or clearing zones were evaluated.
11 The ability to fix N2 was tested on N-free semisolid SM medium (Reinhold et al. 12 1986) supplemented with the carbon sources used in the isolation medium. 13 Additionally, presence of the gene encoding the iron protein of nitrogenase (nifH) in 14 the genome was tested by nifH-targeted PCR amplification using the degenerate 15 primers ZehrF and ZehrR (Zehr and McReynolds 1989). The reaction mixture 16 consisted of 2,5 U of Taq DNA Polymerase (Molzym), 50 M of each 17 desoxynucleoside triphosphate, 500 nM of each primer (Eurofins MWG Operon) and 18 0,8 ng of DNA in a 50 L reaction, amplified as described (Hurek et al. 2002) in a 19 Biometra TGradient thermocycler. 20 21 Production of enzymes for phytostimulation 22 23 The qualitative detection of indole-3-acetic acid (IAA) production was carried out 24 based on a colorimetric method (Gordon and Weber 1950). Precultures were grown 25 in VM-ethanol medium supplemented with 6 mM L-tryptophane at 30°C on a roller at
26 100 rpm until the optical density OD578 was about 1. Subsequently, cell free
27 supernatants were mixed 2:1 with Salkowski reagent (0.01 M FeCl3 in 35% perchloric 28 acid) and incubated in the dark for 45 min at RT. UV-Vis absorption spectra were 29 determined using a Nanodrop 2000 photospectrometer (Thermo Scientific). IAA 30 containing solutions were indicated by a reddish color with an absorption peak at 530 31 nm. 32 Aminocyclopropane-1-carboxylate (ACC) deaminase activity was tested with ACC 33 as the sole nitrogen source. The isolates that grew on SM agar containing 1 mL L-1 of 34 vitamins (Hurek et al. 1995) and 4 mM ACC were considered to express ACC-
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1 deaminase. As control the bacteria were grown on media where ACC was omitted or -1 -1 2 replaced by 0.1 g L of yeast extract and 0.5 g L NH4Cl. Plates were incubated for 3 up to 3 days at 30°C. 4 5 Screening for hydrolytic activity and antibiotic resistance 6 7 Endo-1,3--D-glucanase activity was carried out using chromogenic azurine- 8 crosslinked substrate AZCL-Pachyman (Megazym). VM-ethanol medium was 9 supplemented with 0.1% of AZCL-substrate. Agar plates were incubated for up to 7 10 days at 30°C, and the formation of a blue halo around the colonies was recorded. 11 Extracellular protease activity was determined according to the methodology of 12 Berg et al. (2002) with slight modifications. The tryptic soy agar was replaced by VM- 13 ethanol medium. The formation of clearing zones on the skim milk agar was 14 registered. 15 The isolates were tested for resistance to antibiotics by growing them on VM- 16 ethanol medium supplemented individually with the following antimicrobial agents: 17 kanamycin or streptomycin (final concentration of 10 g mL-1); ampicillin or 18 chloramphenicol (final concentration of 50 g mL-1). Cultures without antibiotics 19 served as references. Plates were incubated for up to 3 days at 30°C until the 20 antibiotic-free cultures showed visible growth. 21 22 Production of N-acylhomoserine lactones 23 24 Production of long and short chain N-acylhomoserine lactones was tested using 25 strains with reporter plasmids pKR-C12 (Riedel et al. 2001) and pJBA89 (Andersen 26 et al. 2001). Both plasmids mediate AHL-dependent expression of the green 27 fluorescent protein. Pseudomonas putida F117 (pKR-C12) and E. coli JM105 28 (pJBA89) were plated next to an isolate on VM-ethanol medium. Following an 29 incubation for 4 days at 30°C, the plates were illuminated with blue light and the 30 appearance of green fluorescent sensor strains was documented with help of a 31 Hamamatsu Color Chilled 3CCD camera mounted on an Olympus SZX12 binocular. 32 AHL-production was also investigated by use of the reporter strain Agrobacterium 33 tumefaciens NTL4 (pCF372) (pCF218) (Fuqua and Winans 1996) which possesses 34 AHL-dependent expression of the -galactosidase. The test was performed
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1 according to Szenthe and Page (2003). In short, cells from a 50 mL overnight culture 2 of the reporter strain were included in 100 mL soft agar (0.7%) medium containing 3 the -galactosidase substrate X-gal (40 g mL-1), and overlayed on top of solid 4 medium. Small amounts (10 L) of the test strains grown overnight in VM-ethanol 5 were injected, and the formation of blue halos within 2 days of incubation at 30°C 6 was documented. 7 8
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1 Results 2 3 Isolation and grouping of root-associated bacteria 4 5 A total of 44 root-associated bacteria were isolated from agricultural crops in the 6 Kavango region in Namibia. Ten of them were obtained from sorghum (designated as 7 SO), 14 from maize (MA) and 20 from mahangu (MH). The first number in the strain 8 designation represented the decimal root tissue dilution from which the strain was 9 obtained, giving a rough estimate on the abundance. Isolates were grouped on the 10 basis of morphology data at first (Table S1). In order to further discriminate the 11 morphologically identical isolates at the putative species level, patterns of SDS- 12 soluble cellular protein extracts in a SDS-PAGE were compared (Fig.1). 13 The isolates that showed indistinguishable protein patterns were considered to 14 have a high degree of overall genomic similarity, suggesting that the respective 15 strains within a group belonged to the same species (Kersters 1985). Based on this 16 grouping we chose as representative strains for further tests: isolate MA5-1 for MA5- 17 1 and MA6-2; isolate MH5-7 for MH5-6 and MH5-7; isolate MH5-5 for the group of 18 MH5-4, MH5-5, MH6-4. Unexpected was the similarity of protein patterns from a 19 group of four isolates from sorghum (SO3-3, SO5-1, SO6-2, SO3-1) together with 20 five isolates from mahangu (MH4-3, MH5-3, MH5-8, MH5-9, MH6-2). To represent 21 strains from both different crops, both, SO6-2 and MH5-8, were included in the 22 following analyses. Subsequent tests were carried out on 33 isolates which were 23 either differentiable by morphology or by protein profiles. 24 25 Phylogenic assignment based on partial 16S rDNA analysis 26 27 Phylogenetic assignment was based on sequence analysis of partial 16S rDNA 28 fragments which were in most cases around 1000 - 1400 bp in length. The overall 29 isolation spectrum consisted of 19 gram-negative strains (four Alphaproteobacteria 30 and 15 Betaproteobacteria) and 25 gram-positive strains (12 Actinobacteria and 13 31 bacilli) (Figure 2). The number of unique strains included 11 gram-negative and 22 32 gram-positive isolates out of which the following isolates were identified to be closely 33 related due to high partial 16S rDNA sequence similarities: MA5-2 and MA5-3 34 (99.9%); MA3-4 and MA3-6 (100%); MA5-1 and MA5-4 (99.8%); MH5-2 and MH5-5
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1 (99.9%); SO3-5 and MA3-1 (99.5%); SO6-2, MH5-8, and MH4-4 (99.9-100%); SO3-4 2 and MH4-1 (100%); SO3-2 and MH5-11 (99.9%). 3 Remarkably, 16S rDNA sequence comparisons revealed that several isolates from 4 sorghum were closely related to isolates observed from mahangu. As expected, 5 identical partial 16S rDNA sequences were obtained for the Ralstonia - related 6 isolates SO6-2 and MH5-8 which already showed matching protein patterns. With the 7 isolates SO3-4 and MH4-1 belonging to Arthrobacter and isolates SO3-2 and MH5- 8 11 belonging to Paenibacillus, two more pairs of very similar strains could be 9 detected in both millets (Table 1). 10 11 Affiliation of isolates obtained from maize 12 13 Out of 14 isolates from maize, six were identified as bacilli belonging to the genus 14 Bacillus and Brevibacillus. Additionally, three Actinobacteria (Curtobacterium, 15 Microbacterium), two Alphaproteobacteria (Sphingomonas) and three 16 Betaproteobacteria (Massilia) were isolated. The isolates assigned to Massilia, 17 Brevibacillus and Microbacterium were observed to be closely related sharing either 18 (almost) identical partial 16S rDNA sequences or non-differentiable protein patterns. 19 In contrast, phylogenetic analysis revealed higher diversity of Bacillus-related strains, 20 with all four isolates identified as separate species by showing less than 95.9% 21 partial 16S rDNA sequence identity to each other. While in most instances partial 22 16S rDNA sequence identities to the closest related species type strain of around 99 23 – 99.8% were observed, three isolates belonging to Microbacterium and Massilia 24 showed values within or below the species-threshold of 98.7 - 99% recently 25 postulated (Stackebrandt and Ebers 2006) (Table 1). 26 27 Affiliation of isolates obtained from mahangu 28 29 The isolation spectrum from mahangu consisted of six bacilli (Bacillus, Paenibacillus, 30 Lactococcus), five Actinobacteria (Leifsonia, Arthrobacter, Humihabitans, 31 Nocardioides), 2 Alphaproteobacteria (Bradyrhizobium, Balneimonas) and seven 32 Betaproteobacteria (Ralstonia, Ramlibacter), where most strains were related to 33 Ralstonia with five out of six related isolates according to protein patterns.
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1 Additionally, the two Leifsonia isolates together with three out of four Lactococcus 2 isolates were found to be indistinguishable in protein pattern analysis. 3 This first cultivation of root-associated bacteria from mahangu revealed four 4 candidates for unknown species: partial 16S rDNA sequence identity to the closest 5 related type strain was 97.78% for MH4-1 and Arthrobacter ramosusT / Arthrobacter 6 pascensT, 98.69% for MH5-1 and Humihabitans oryzaeT, 98.28% for MH5-7 and 7 Leifsonia shinshuensisT, and 97.98% for MH5-10 and Balneimonas flocculansT. 8 Furthermore, isolate MH6-5 and the closest related Nocardioides exalbidusT shared 9 only 96.5% of the available 16S rDNA sequence. However, comparison was based 10 on only 790 (sometimes ambiguous) positions. For the residual isolates from 11 mahangu, high relatedness to known species with partial sequence identity values 12 ranging from 99.3 - 99.8% were observed. 13 14 Affiliation of isolates obtained from maize 15 16 From sorghum, one Paenibacillus, four Actinobacteria and five Betaproteobacteria 17 (Ralstonia, Burkholderia) were isolated. The fraction of Betaproteobacteria again 18 largely consisted of four very similar Ralstonia isolates which were closely related to 19 the ones cultivated from mahangu according to protein patterns (Fig. 1). As a result, 20 most diverse isolates belonged to the class Actinobacteria. Two Arthrobacter - 21 related isolates sharing 97.7% partial 16S rDNA identity and one Burkholderia - 22 related isolate were found to exhibit sequence identities to the closest species type 23 strain below the proposed threshold for species (Stackebrandt and Ebers 2006) 24 (Table 1). 25 26 Characteristics putatively related to plant-growth promotion in the root-associated 27 isolates 28 29 A range of features which are known to contribute to plant-growth promotion or 30 biocontrol were tested for the isolates associated with roots of Namibian crops. The 31 results are summarized in Table 2. 32 Production of indole-3-acetic acid (IAA). Out of 34 individual isolates tested, a total 33 of 11 strains were detected to produce IAA in vitro (Table 2). Their distribution among 34 plant species was found to be uniform. While appearing to be widespread in isolated
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1 Actinobacteria (eight out of 12), IAA-production could be shown only for two out of 11 2 bacilli and two out of 11 gram-negative isolates. All the isolates classified as 3 Arthrobacter together with members of the genera Bacillus, Balneimonas, 4 Leucobacter, Microbacterium, Nocarioides and Sphingomonas were observed to be 5 IAA-positive. 6 ACC-deaminase activity. The ability to grow on medium containing ACC as the 7 sole source of nitrogen could only be observed for the isolate SO1-1, which showed 8 closest 16S rDNA sequence to the species type strains Burkholderia phenoliruptrixT 9 and Burkholderia graminisT. Both strains are known to express ACC-deaminase 10 (Onofre-Lemus et al. 2009) and were used as positive controls in this test.
11 Nitrogen fixation. None of the isolates was positive for growth on N2 or acetylene 12 reduction in semisolid medium. Concordantly, attempts to amplify dinitrogenase 13 reductase (nifH) gene fragments did not yield PCR-products of the appropriate size 14 (not shown). 15 Solubilization of inorganic phosphates. The ability to solubilize tricalcium 16 phosphates was detected by growing the isolates on bromophenol blue containing 17 Pikovskaya Agar. The formation of a halo around the colonies was observed for nine 18 of the isolates, originating from mahangu in majority (four), followed by maize (three) 19 and sorghum (two). They were related to Burkholderia, Ralstonia, Lactococcus, 20 Bacillus, Curtobacterium and Microbacterium. As the Microbacterium-related isolate 21 MA5-3 was positive in this test, it can be discriminated from the isolate MA5-2 for 22 which an identical partial 16S rDNA sequence was determined. 23 Production of siderophores. As HDTMA in test agar plates appeared to be toxic for 24 many isolates, a thin layer of HDTMA-free medium was poured on top of the 25 HDTMA-containing CAS medium before inoculation. While this prevented direct 26 contact to toxic HDTMA, siderophores were able to traverse the layer by diffusion 27 and induced a color shift in the CAS medium. Ten of the isolates were able to 28 produce this color shift and therefore they were considered as siderophores 29 producers. The isolates were originating from all three plants (four from sorghum, 30 three from maize or mahangu, respectively). They were related to Burkholderia, 31 Ralstonia, Arthrobacter, Bacillus and Brevibacillus. 32 Endoglucanase-activity. Five isolates were able to degrade azurine-crosslinked 33 1,3--D-Glucane, generating a release of diffusible blue dye particles into the culture
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1 medium. They were originating from maize (three) and from mahangu and sorghum 2 (one each). The isolates were related to Curtobacteria, Massilia and Paenibacillus. 3 Extracellular proteases. Fifteen isolates induced a clear zone on skim milk agar 4 within 5 days of incubation. Protease secretion was found out to be especially 5 widespread in isolated Actinobacteria (eight out of 11), with all positively tested 6 isolates observed from sorghum and mahangu belonging to this class. However, only 7 a minority of isolates from sorghum (three out of seven) and from mahangu (three out 8 of 12) secreted proteases, while this was the case for a remarkably high quantity of 9 nine out of 12 tested isolates observed from maize. The accumulation among maize 10 isolates resulted from the comparatively large fraction of bacilli, which 11 unexceptionally showed extracellular proteolytic activities in contrast to bacilli 12 observed from the millets. Apart from two Massilia timonae - related isolates, none of 13 the Proteobacteria showed extracellular protease activity. 14 Detection of AHL-like autoinducer molecules. To detect whether the isolates were 15 likely to communicate via common quorum sensing autoinducers, they were 16 screened for the production of a variety of short and long chain AHLs with the help of 17 GFP-based reporter plasmids. On pKR-C12, the AHL-sensor cassette containing a
18 PlasB-gfp(ASV) translational fusion is based on the las-system of P. aeruginosa and 19 therefore shows sensitivity to several long chain AHLs. The sensor cassette on
20 pJBA89 carrying PluxI-RBSII-gfp(ASV) is based on the lux-system of Vibrio fischeri 21 and is sensitive to several short chain AHLs. After growth in a coculture, three out of 22 11 gram-negative isolates induced expression of GFP by both reporter strains. These 23 Betaproteobacteria belonged to Burkholderia and Massilia. Furthermore, a 24 -galactosidase-based reporter strain that carries the plasmid pCF372 with a PtraI- 25 lacZ fusion and the TraR overexpressing plasmid pCF218 was used, allowing the 26 detection of at least 29 autoinducers (Zhu et al. 1998). The application of the - 27 galactosidase-based reporter strain revealed that two Alphaproteobacteria isolates 28 identified as Balneimonas sp. and Sphingomonas sp. produced AHL-like molecules 29 that were not recognized by the GFP-based biosensors. 30 31 Other characteristics of the root-associated isolates 32 33 Resistance to antibiotics. Isolates showing visible growth on medium containing 34 antibiotics were regarded to be resistant to the particular antibiotic at the given
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1 concentration. Thirteen isolates did not grow in the presence of any antibiotic, while 2 12 isolates displayed multidrug resistance (Table 2). Four gram-negative isolates 3 were detected to be resistant to all antibiotics tested. 4 Endospore formation. The ability to produce endospores was observed by 5 microscopic inspection for all isolated members of Bacillus and Brevibacillus, leading 6 to a high proportion of endospore- producing isolates from maize. Furthermore, the 7 isolate MH5-11 belonging to Paenibacillus formed endospores and was thus 8 differentiated from the closely related isolate SO3-2, which was not sporulating. None 9 of the isolated Actinobacteria produced endospores. 10 11
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1 Discussion 2 3 Our study delivers the first insights into the root-associated bacterial community from 4 agricultural crops in the Kavango region of Namibia. We characterized 44 isolates by 5 analyzing their phenotypic properties, their taxonomic position, and furthermore their 6 features likely to contribute to promote plant growth. 7 Initially, the isolates were compared with respect to morphology data and 8 comparison of whole-cell protein patterns in SDS-PAGE, which has proven to be a 9 powerful method for discrimination and grouping of closely related strains (Kersters 10 1985). These comparisons and phylogenetic 16S rDNA sequence analysis of 33 11 representative strains revealed that sorghum (Sorgum bicolor) and mahangu 12 (Pennisetum glaucum) shared several closely related strains belonging to Ralstonia, 13 Arthrobacter and Paenibacillus. The isolates assigned to Ralstonia showed 14 indistinguishable protein patterns and were isolated frequently. Additionally, two 15 Curtobacterium sp. that had almost identical 16S rDNA sequences occurred in both, 16 sorghum and maize. As several members of the mentioned genera have already 17 been isolated from various plants (Lacava et al. 2007), colonization by the same 18 bacterial species seems plausible especially for both millets. These findings might be 19 related to the traditional agricultural practice in the Kavango region, where crops are 20 grown in mixed cultivation on the same fields as the plants used in the current study. 21 The differences in the root-associated bacterial populations which were nevertheless 22 observed might therefore depend on plant specificity rather than on abiotic, soil- 23 dependent factors. 24 The phylogenetic analysis revealed the association of the isolates to three highly 25 supported main phyla: Proteobacteria, Firmicutes and Actinobacteria (Figure 2). In 26 total 25 gram-positive and 19 gram-negative strains were isolated from the three 27 agronomic crops. These results are in agreement with results on endophytes from 28 maize and sorghum in Nebraska (Zinniel et al. 2002), where an almost equal 29 distribution of gram-positive and gram-negative isolates was found. In our case the 30 gram-negative bacteria were mainly composed of nine very similar Ralstonia isolates 31 and thus not very diverse. In contrast, a higher diversity was observed for the gram- 32 positives strains with 22 unique isolates belonging to Actinobacteria and bacilli. 33 Bacillus and Pseudomonas are the most commonly reported genera that represent 34 the dominant isolates in many plants studied (Hallmann and Berg 2006). Studies on
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1 the diversity of root-associated bacteria in maize that were carried out at different 2 geographical regions, revealed extensive colonization by Bacillus sp. during the 3 plants active growth stage (Lalande et al. 1989; Lambert et al. 1987). Both studies 4 also reported a substantial or rather predominant fraction of Pseudomonas that in our 5 sampling was not found. 6 The higher diversity of gram-positive bacteria isolated might reflect their ability to 7 persist the seasonal drought periods in the Kavango region. Accordingly, 8 approximately 75% of the Firmicutes were able to form endospores indicating the 9 capacity to deal with environmental stress. Additionally, a similar quantity of isolates 10 was assigned to the phylum Actinobacteria, which includes many common soil 11 bacteria that are well adapted to environmental fluctuations such as desiccation. For 12 instance, four or two isolates were identified to belong to the genus Arthrobacter or 13 Microbacterium, respectively, that are known to persist drought periods by formation 14 of resting cocci (Goodfellow and Williams 1983) or highest level of desiccation 15 tolerance (Narváez-Reinaldo et al. 2010), or may even promote drought-stress 16 tolerance to plants (Narváez-Reinaldo et al. 2010). 17 Among our isolates, there are several candidates for new species, as may be 18 expected when a new habitat is analyzed. Within the high diversity of gram-positive 19 isolates, most candidates belonged to the phylum Actinobacteria and were especially 20 isolated from mahangu. Seven isolates showed 16S rDNA similarities below the 21 recently recommended (Stackebrandt and Ebers 2006) species-threshold value. 22 There were three isolates related to Arthrobacter pascensT, two others related to 23 Microbacterium testaceumT, one to Leifsonia shinshuensisT, and one related to 24 Humihabitans oryzaeT (Table 1). The discrimination of the Arthrobacter and 25 Microbacterium isolates from the closest related type strain was supported by the 26 phylogenetic tree topology, the isolates forming separate clusters (Fig. 2). Other 27 isolates being possibly new species were gram-negatives related to Burkholderia 28 phenoliruptrixT, Balneimonas flocculansT, and Massilia timonaeT. 29 Some of the isolates add considerable diversity to genera with so far few 30 representatives. According to 16S rDNA identity, the isolate MH5-1 might represent a 31 defined species harboring plant-associated strains within the genus Humihabitans or 32 Intrasporangium. While both genera consist of only one species, Humihabitans 33 oryzaeT was isolated from a paddy field soil (Kageyama et al. 2007), whereas 34 Intrasporangium calvumT was isolated from air in a school dining room (Kalakoutskii
17
1 et al. 1967). A similar situation occurs for the isolate MH5-10, where the closest 2 related species is Balneimonas flocculansT. This strain is until now the only validly 3 published representative of this genus and was isolated from a hot spring (Takeda et 4 al. 2004). In addition, two isolates from maize belonged to the genus Massilia that 5 has been typically isolated from air, soil, drinking water or human blood (Weon et al. 6 2010; Zul et al. 2008). The first evidence from a plant-associated Massilia sp. was in 7 stems of sweet pepper (Rasche et al. 2006) or in soybeans stems (Ikeda et al. 2009). 8 Thus our study of an as yet underexplored habitat in the Kavango region revealed 9 several novel phylotypes with putatively plant-growth-promoting characteristics (see 10 below). 11 However, also several common plant-associated genera were detected, with some 12 isolates showing close relation to known PGPR and the ability to exert PGPR- 13 activities in vitro. One example is the isolation of a Burkholderia species from 14 sorghum. Even though the genus comprises many plant-, animal- and human- 15 pathogens, a growing number of Burkholderia species that exert beneficial effects to 16 several plants -including sorghum- has been identified in recent years (Compant et 17 al. 2008). Isolate SO1-1 was found to be related to the commercial nitrogen fixers B. 18 cepacia SEMIA 6417 and 6422, while known pathogens (e.g. the B. cepacia complex 19 and the cluster of B. gladioli, B. glumae, B. plantarii) were more distantly related. 20 Although no nifH gene was detected, our isolate showed beneficial effects with 21 respect to the bioavailability of iron and phosphates in vitro. Additionally, ACC- 22 deaminase activity and the production of a wide variety of AHLs were observed. 23 Thus, the isolate combines promising biofertilization, phytostimulation as well as 24 biocontrol properties (sequestration of siderophores). These bacteria might be 25 promising candidates for application as a specific pest control via competition and 26 niche exclusion with their related pathogenic Burkholderia spp. 27 In terms of biocontrol the isolation of two Curtobacterium and two Microbacterium 28 strains is of particular interest. Isolates from both genera have been shown to 29 persistently colonize various agronomic crops and prairie plants in high numbers 30 without causing symptoms of disease (Zinniel et al. 2002). Apart from the well known 31 phytopathogen Curtobacterium flaccumfaciens, members of this genus have been 32 described to exert beneficial effects on plant growth. Among strains capable of 33 producing IAA and solubilizing phosphates (Merzaeva and Shirokikh 2010; 34 Rodriguez-Diaz et al. 2008), several Curtobacterium sp. have been successfully
18
1 applied as bioprotectants e.g. by triggering induced systemic resistance or by 2 antibiosis (Raupach and Kloepper 2000; Sturz and Matheson 1996). The 3 Curtobacterium species isolated in this study were distinguishable in their ability to 4 solubilize phosphate and they also showed extracellular endoglucanase and 5 protease activity. Therefore they have the potential to act antagonistic against fungi 6 and insects as well as against bacterial pathogens. 7 In the genus Microbacterium, strains with fungicidal and nematicidal activity have 8 been reported (Dikin et al. 2007; Sturz and Kimpinski 2004). The high colonization 9 levels observed (Zinniel et al. 2002) suggest its potential for competitive exclusion of 10 bacterial pathogens. Another Microbacterium strain isolated from soil has been 11 recently described to exhibit ACC deaminase activity, to produce siderophores and 12 IAA and to solubilize phosphates (Sheng et al. 2009). The last two capacities were 13 also observed for one of the two isolates from this study. 14 A high amount of Ralstonia isolates originated from both millets. Most of the 15 described Ralstonia spp. are either plant pathogens or of clinical importance. R. 16 pickettii and R. mannitolilytica were mainly isolated from clinical sources and human 17 patients, respectively. The isolates observed in our study were assigned to R. 18 mannitolilytica exhibiting at least 99.8% 16S rDNA identity to the corresponding type 19 strain. Besides, they can be discriminated from pathogenic R. solanacearum strains 20 by their inability to produce hexanoyl- and octanoyl- AHL, which have been identified 21 as determinants for its pathogenicity (Flavier et al. 1997). While for R. pickettii a plant 22 pathogenic (Polizzi et al. 2008) and an endophytic lifestyle were recently discovered 23 (Kuklinsky-Sobral et al. 2005), plant-associated R. mannitolilytica strains have not yet 24 been described. The isolates were able to sequester siderophores, solubilize 25 tricalcium phosphates and showed resistance to all antibiotics tested, and have thus 26 PGPR potential. 27 For three related Arthrobacter sp. isolates, phylogenetic analysis revealed highest 28 16S rDNA similarities to A. pascensT (97.7% - 98.2%), indicating they belong to a yet 29 unknown species which occurs in both, sorghum and mahangu. Even though the 30 genus Arthrobacter is generally known to consist of ubiquitous soil bacteria, a range 31 of endophytes including A. pascens strains and PGPR have been reported 32 (Hallmann and Berg 2006). Descriptions of PGPR-relevant capacities include free 33 nitrogen fixation, phosphate solubilization, IAA synthesis and sequestration of 34 siderophores. The latter two activities were also observed for the isolates from our
19
1 study. Moreover, A. pascens strains have been intensively investigated concerning 2 their ability to encounter water deficit via accumulation of betaine, which is a highly 3 efficient osmoprotectant (Le Rudulier et al. 1984). The availability of desiccation- and 4 salt- tolerant PGPR appears to be advantageous especially in seasonally dry regions 5 or regions with salinity problems, which is 40% of the world land surface (Cordovilla 6 et al. 1994). 7 8 In conclusion, several novel putative PGPRs were detected in our screening in 9 addition to known phylotypes. Especially gram-positive, putatively desiccation- 10 resistant isolates may be of high potential as plant-beneficial inoculants for areas with 11 seasonal drought. Strains that are adapted to these conditions and to low-fertility 12 soils in Kavango sands are of high interest for agricultural applications in these 13 regions. These microorganisms should be tested in planta in order to confirm their 14 capacities for improvement of yields of traditional African crops. 15 16 17 Acknowledgements 18 19 This research was supported by grants 01LC0021 and 01LL0912G from the 20 Bundesministerium für Bildung und Forschung to B.R.-H. and T.H. in the BIOLOG 21 and TFO (The Future Okavango) framework. We thank Wolfgang Streit and Leo 22 Eberl for the supply of AHL reporter strains, and Richard Hahnke for helping in the 23 phylogenetic tree reconstruction. Materials were collected in Namibia with Research 24 and Collection permit 1138/2007 and export permit 61911 from the Ministry of 25 Environment and Tourism, Windhuk, Namibia. 26 27 28
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1 Figure legends 2 3 Fig. 1 Profiles of SDS-soluble cellular proteins extracted from bacterial isolates and 4 separated by SDS-PAGE in a 12.5% polyacrylamid separation gel. Patterns that 5 were recognized to be identical are marked with brackets. 6 7 Fig. 2 Maximum likelihood 16S rDNA tree of representative isolates. The tree shows 8 the phylogenetic affiliation of 31 partial 16S rDNA sequences of strains isolated from 9 roots of Zea mays (maize), Sorghum bicolor (sorghum), and Pennisetum glaucum 10 (mahangu). For isolates, accession numbers of 16S rDNA sequences are given next 11 to strain designations (in bold). Numbers at branches represent bootstrap values 12 >50% from 1000 replicates. The scale bar shows the number of nucleotide 13 substitutions per site. 14 15 16 17
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Table 1. Maximal 16S rDNA sequence identity between bacterial isolates and type strains of most closely related species
Isolate Na Type strain Genbankb Hits %c Matching isolatesd
SO1-1 0 Burkholderia phenoliruptrix (T); AC1100 AY435213 1350/1365 98.90% SO6-2 0 Ralstonia mannitolilytica (T); LMG 6866 AJ270258 1364/1367 99.78% SO3-1, SO3-3, SO5-1, MH5-8 group SO3-2 0 Paenibacillus lactis (T); DSM 15596 AY257868 1038/1045 99.33% SO3-5 0 Curtobacterium citreum (T); DSM 20528 X77436 1051/1057 99.43% SO3-4 0 Arthrobacter pascens (T); DSM 20546e X80742 996/1014 98.22% e SO6-3 1 Arthrobacter pascens (T); DSM 20546 X80742 1047/1072 97.67%
SO6-4 0 Arthrobacter tumbae (T); DSM 16406 AJ315069 1298/1308 99.24% MH4-2 0 Bradyrhizobium betae (T); LMG 21987 AY372184 1268/1274 99.53% MH5-10 0 Balneimonas flocculans (T); TFB AB098515 1166/1190 97.98% MH4-4 0 Ralstonia mannitolilytica (T); LMG 6866 AJ270258 1319/1321 99.85%
MH5-8 0 Ralstonia mannitolilytica (T); LMG 6866 AJ270258 1212/1214 99.84% MH4-3, MH5-3, MH5-9 MH6-2, SO6-2 group
MH6-3 0 Ramlibacter henchirensis (T); DSM 14656 AF439400 1154/1162 99.31%
MH6-1 1 Bacillus niacini (T); DSM 2923 AB021194 617/619 99.68% MH5-11 0 Paenibacillus lactis (T); DSM 15596 AY257868 1035/1041 99.42% MH5-2 0 Lactococcus lactis ssp. lactis (T); DSM 20481 AB100803 1233/1236 99.76% MH5-5 0 Lactococcus lactis ssp. lactis (T); DSM 20481 AB100803 1078/1082 99.63% MH5-4, MH6-4 MH5-7 0 Leifsonia shinshuensis (T); DSM 15165 DQ232614 965/973 99.18% MH5-6 MH4-1 0 Arthrobacter pascens (T); DSM 20546d X80742 1236/1261 98.02%
MH5-1 0 Humihabitans oryzae (T)*; KV-657 AB282887 1358/1378 98.55%
MH6-5 3 Nocardioides exalbidus (T); RC825 AB273624 762/790 96.46% MA3-2 0 Sphingomonas kaistensis (T) DSM 16846 AY769083 1340/1345 99.63% MA4-1 0 Sphingomonas dokdonensis (T); DS-4 DQ178975 966/974 99.18% MA5-1 4 Massilia timonae (T); CCUG 45783 U54470 1298/1306 99.39% MA6-2 MA5-4 4 Massilia timonae (T); CCUG 45783 U54470 1278/1296 98.61%
MA-AZ 1 Bacillus cereus (T); DSM 31 AE016877 1347/1350 99.78%
MA3-3 0 Bacillus megaterium (T); DSM 32 D16273 1323/1327 99.70% MA3-5 0 Bacillus circulans (T); DSM 11 AY043084 1156/1168 98.97% MA3-7 1 Bacillus acidiceler (T); DSM 119 DQ374637 1423/1434 99.23% MA3-4 0 Brevibacillus brevis (T); DSM 30 AB271756 1219/1223 99.67% MA3-6 0 Brevibacillus brevis (T); DSM 30 AB271756 1218/1222 99.67% MA3-1 0 Curtobacterium citreum (T); DSM 20528 X77436 1270/1273 99.76%
MA5-2 0 Microbacterium testaceum (T); DSM 20166 X77445 1248/1264 98.73% MA5-3 0 Microbacterium testaceum (T); DSM 20166 X77445 1258/1274 98.74%
a, number of indistinct nucleotides in sequence b, GenBank accession number c, putative new species in bold d, isolates sharing identical colony morphology and patterns of SDS-soluble cell proteins e, Arthrobacter pascensT shares an identical 16S rDNA sequence with Arthrobacter ramosusT
Table 2. PGPR-related properties of bacterial isolates.a
d e f g h c phytostimulation biofertilization lytic enzymes AHL production Antibiotic resistance Isolate Nb Identification spores IAA ACC nifH Ca3(PO4)2 SID GLUC PROT short long NTL4 AMP CHL STR KM SO1-1 1 Burkholderia sp. ND - + - + + - - + + + + + - - SO6-2 4 Ralstonia mannitolilytica ND - - - + + - - - - - + + + + SO3-2 1 Paenibacillus lactis ------ND + - - - SO3-5 1 Curtobacterium citreum ------+ + - - ND - - - - SO3-4 1 Arthrobacter sp. - + - - - + - + - - ND - - - - SO6-3 1 Arthrobacter sp. - + - - - + - - - - ND - - - - SO6-4 1 Arthrobacter tumbae - + - - - - - + - - ND - - - - MH4-2 1 Bradyrhizobium betae ND ------++++ MH5-10 1 Balneimonas sp. ND + ------+ - - - - MH4-4 1 Ralstonia mannitolilytica ND - - - + + - - - - - + + + + MH5-8 5 Ralstonia mannitolilytica ND - - - + + - - - - - + + + + MH6-3 1 Ramlibacter henchirensis ND ------MH6-1 1 Bacillus niacini + + ------ND - - - -
MH5-11 1 Paenibacillus lactis + - - - - - + - - ND + - + -
MH5-2 1 Lactococcus lactis - - - - + - - - - - ND - - - -
MH5-5 3 Lactococcus lactis - - - - + - - - - - ND - - - -
MH5-7 2 Leifsonia sp. ------ND + - - +
MH4-1 1 Arthrobacter sp. - + - - - + - + - - ND - - - -
MH5-1 1 Humihabitans sp. ------+ - - ND - - - +
MH6-5 1 Nocardioides sp. - + - - - - + - - ND - - - - MA3-2 1 Sphingomonas kaistensis ND +------+- MA4-1 1 Sphingomonas ND ------+ + - + - MA5-1 2 Massiliadkd tim ionae ND - - - - - + + + + + + - + + MA5-4 1 Massilia sp. ND - - - - - + + + + + + - + +
MA-AZ 1 Bacillus cereus + - - - - + - + - - ND + - - -
MA3-3 1 Bacillus megaterium + + - - + - - + - - ND + - - - MA3-5 1 Bacillus sp. + ------ND - - - -
MA3-7 1 Bacillus acidiceler + - - - - + - + - - ND - - - -
MA3-4 1 Brevibacillus brevis + - - - - + - + - - ND + - + +
MA3-6 1 Brevibacillus brevis + ------+ - - ND + - + +
MA3-1 1 Curtobacterium citreum - - - - + - + + - - ND - - - +
MA5-2 1 Microbacterium sp. - + - - - - + - - ND - - - +
MA5-3 1 Microbacterium sp. - + - - + - - - - - ND - - - +
a, All tests were carried out with 1 or 2 repetitions b, Number of isolates showing identical morphology and patterns of SDS-soluble cell protein c, Taxonomic identification based on 16S rDNA gene similarity to the closest related species type strain and phylogenetic tree analysis d, IAA, Indole-3-acetic acid production; ACC, Aminocyclopropane-1-carboxylate deaminase activity e , nifH, nifH gene detected by PCR approach and acetylene reduction; Ca3PO4; solubilization of anorganic calciumphosphate; SID, siderophore production f, CHIT, Chitinase activity; GLUC, Endo-1,3--D-Glucanase activity; PROT, protease activity g, Production of N-Acylhomoserine lactones; short, short chain AHL; long, long chain AHL; NTL4; AHL reporter strain A. tumefaciens NTL4 h, AMP, ampicillin (50 g/ml); CHL, chloramphenicol (50 g/ml); STR, streptomycin (10 g/ml); KM, kanamycin (10 g/ml) +, positive; -, negative; ND, not determined.
Grönemeyer et al. Figure 1 100 AY078053 Microvirga subterranea 63 FR750292 MH5-10 AB098515 Balneimonas flocculans 100 EU727176 Microvirga guangxiensis 63 AJ558025 Bradyrhizobium canariense 95 U69638 Bradyrhizobium japonicum 100 X87273 Rhizobium lupini AF363132 Bradyrhizobium liaoningense 100 59 FR750291 MH4-2 100 DQ4520 Sphingomonas roseiflava Alpha-Proteobacteria 83 AJ429240 Sphingomonas aerolata 94 AB264131 Sphingomonas jaspi AB277583 Sphingomonas astaxanthinifacie 95 FR750304 MA3-2 98 AY769083 Sphingomonas kaistensis FR750305 MA4-1 99 AM229669 Sphingomonas mucosissima 100 AM231588 Massilia aurea FR750306 MA5-1 68 EF546777 Massilia brevitalea EU808006 Massilia niabensis 52U54470 Massilia timonae 100 95 FR750307 MA5-4 AY677088 Burkholderia cepacia 98 90 FR7500284 SO1-1 AY741356 Burkholderia phenoliruptrix 66 98 AJ30231Burkholderia phymatum Beta-Proteobacteria 53 AY741342 Ralstonia pickettii FR7500285 SO6-2 68 FR750294 MH5-8 57 FR750293 MH4-4 100 AJ270258 Ralstonia mannitolilytica 68 EF016361 Ralstonia solanacearum 83 AF144383 Ramlibacter tataouinensis FR750295 MH6-3 65 94 AF439400 Ramlibacter henchirensis 100 AB112718 Brevibacillus invocatus AB271756 Brevibacillus brevis 73 FR750312 MA3-4 FR750313 MA3-6 96 90 AB112717 Brevibacillus limnophilus 100 AF021924 Paenibacillus campinasensis 100 AY257868 Paenibacillus lactis 73 FR750286 SO3-2 FR750297 MH5-11 100 D85609 Paenibacillus amylolyticus GQ337866 Lactococcus lactis FR750299 MH5-5 AB100803 Lactococcus lactis subsp. lactis 56 AB100804 Lactococcus lactis subsp. hordniae 91 GQ337878 Lactococcus lactis 79 FR750298 MH5-2 Firmicutes 51 AB021185 Bacillus flexus 97 FR750309 MA3-3 73 GU120639 Bacillus megaterium 96 AB271747 Bacillus circulans FR750310 MA3-5 93 EU656111 Bacillus nealsonii 69 99X60611 Bacillus benzoevorans AB245380 Bacillus panaciterrae FR750311 MA3-7 AB271745 Bacillus cereus 99 FR750308 MAA-Z EF528290 Bacillus cereus 61 AM747227 Bacillus pseudomycoides AY998119 Bacillus niabensis 99 FR750315 MA5-2 67 FR750316 MA5-3 X77445 Microbacterium testaceum 99 68 AJ491806 Microbacterium paraoxydans AJ853908 Microbacterium xylanilyticum Y11928 Terracoccus luteus 97 AJ566282 Intrasporangium calvum 61 53 FR750302 MH5-1 AJ278870 Arthrobacter roseus 97 X83409 Arthrobacter sulfureus AB279889 Arthrobacter oryzae 77 AB279890 Arthrobacter humicola X80740 Arthrobacter pascens 98 X80742 Arthrobacter ramosus FR750288 SO3-4 Actinobacteria 78 FR750301 MH4-1 52 FR750289 SO6-3 100 AJ315069 Arthrobacter tumbae DQ097525 Arthrobacter subterraneus 93 FR750290 SO6-4 X80736 Arthrobacter globiformans 67 EF587758 Curtobacterium gingensoli 72 AJ312209 Curtobacterium flaccumfaciens 16S rDNA sequences FR750314 MA3-1 FR750287 SO3-5 obtained from roots of: AM410682 Leifsonia poae DQ232614 Leifsonia shinshuensis Maize 100 DQ232612 Leifsonia naganoensis FR750300 MH5-7 Mahangu 0.10 Sorghum
Grönemeyer et al. Figure 2
Table S1. Morphology description of bacterial isolates. a Isolate Colony morphology
SO 1-1 whitish-translucent, shiny, convex 0.5 mm SO 3-2 shiny, creamy, white, tiny <0.1 mm SO 3-4 yellowish, shiny, convex 1 mm SO 3-5 translucent, creamy, rough, tiny <0.1 mm
SO 6-2 translucent, smooth ring, 0.5 mm
SO 3-1 as SO 6-2 SO 3-3 as SO 6-2 SO 5-1 as SO 6-2 SO 6-3 white, translucent, convex, 1 mm SO 6-4 yellowish, shiny, tiny <0.1 mm MA -AZ white, pink colored stripes, rough MA 3-1 whitish, translucent, tiny <0.2 mm
MA 5-2 as MA 3-1, but yellowish
MA 3-2 small, red-orange, rough MA 3-3 white, silky, convex 1-2 mm MA 3-4 white, silky, flat, 1-2 mm MA 3-7 as MA 3-4 MA 3-5 white, rough, fringy spreading, 0.5 mm MA 3-6 creamy, silky, round, 0.5 mm MA 4-1 shiny, yellow, rough, tiny, 0.1 mm
MA 5-1 yellowish-translucent, flat, concave
MA 5-4 as MA 5-1 MA 6-2 as MA 5-1 MA 5-3 slightly orange, rough, small 0.2 mm MH 4-2 white, small, grows better inside agar, 0.2 mm MH 4-4 translucent, rough, small 0.2 mm MH 5-1 yellowish-whitish, smooth, tiny <0.1 mm MH 5-2 translucent ring, flat, concave, sticky, 0.5-1 mm MH 5-7 whitish-translucent, grows better inside agar, <0.1
MH 5-6 as MH 5-7 MH 5-8 translucent whitish-creamy, smooth, convex 0.5-1 MH 5-9 as MH5-8 MH 4-3 as MH5-8 MH 5-3 as MH5-8 MH 6-2 as MH5-8 MH 6-3 as MH5-8
MH 5- brownish, diffusible pigments, round, rough
MH 5-4 as MH 5-10
MH 5-5 as MH 5-10 MH 6-4 as MH 5-10 MH 5- translucent, smooth, small, 0.1 mm MH 6-1 white, rough, slightly convex, 1-2 mm MH 4-1 creamy, slightly convex, 1-2 mm, similar to MH 6-1
MH 6-5 creamy, rough, slightly convex, 1-2 mm aGrown on VM-Ethanol agar for 4 days at 30°C Acknowledgments
Acknowledgments
I would like to start thanking Prof. Dr. Barbara Reinhold-Hurek for the opportunity to join her lab in Bremen and work on the interesting topics studied in this project. I want to specially thank her for all the time she invested discussing ideas, always aiming to improve the project and successfully achieving promising results. Along with Prof. Dr. Reinhold-Hurek, a fundamental person for this project was Dr. Thomas Hurek; to him my thankfulness for all the knowledge he has shared with me, and for all the fruitful talks and discussions we had during both easy and hard times.
I want to express my acknowledgments to Prof. Dr. Michael Friedrich for kindly taking the time to evaluate my dissertation. Also, my gratitude goes to Prof. Dr. Friederike Koenig, who took the time to start to study together with me the compound possibly causing the particular coloration to the mopane roots, and who also kindly accepted to participate in the commission for my thesis defense.
I also want to thank all the people who worked or helped me at some point in the development of this project throughout the years: Yuan Liu, Asim Aute, Ariana Neumann, Kim Rösner, Elvira Prugger, Thea Fründ, Janina Ötjen, Jessica Döring and specially to Jann Grönemeyer, who worked together with me in many parts of the project. I express my gratitude to all the members of the AG-Reinhold-Hurek lab, specially the people who worked with me at the corner lab; thank you for the nice atmosphere and work environment. Andrea Krause, Abhijit Sarkar and Teja Shidore, thank you for always having the time to listen to me. Anne Rose and Stefanie Haack, thanks to you for your help with the administration papers.
I also would like to mention my friends in Bremen who had always time to encourage me: Lena, Sabrina, Jessi, Janina, Frauke, Anne, Birte and Mela; and also Jean, James, Guillem, Julien, Tobias, Cyril and Hendrick. I also want to thank Christiane Schreiweis and Jann Grönemeyer for helping me with the German translation of the summary of this thesis, as well to Jessica Döring for the revision. I thank James Collins and Theresa Dinse for the language editing of the summary. I want to mention my good friend Andrés Felipe Santacruz; I thank him for listening me since my first science project back in 2002. Acknowledgments
I am fortunate to have a marvelous family that has always been there for me. I thank my parents Elssy Sofía and Hernán! It is because of their education, support and most importantly, because of their love that I have come to achieve this PhD. I am grateful to my brother Hernán Andrés, he had the patience to listen to me everyday during these four years and he has always encouraged me. Herny thank you for your advice, support and love. I want to finally thank Domenico, he arrived at the perfect moment, when the publications were just a dream and decisively helped me to make them come true. For all his support, care and love. Grazie amore mio!
Declaration
Declaration
I hereby assure that I prepared my dissertation entitled “Molecular analysis of root- associated diazotrophs in important plants from Southern Africa and South America” independently, without any prohibited aid and that I used no other but only the sources and aid, specifically designated for this work.
I also admit that this dissertation, in the present form or in a similar form was never submitted to another University and has never served for other examination purposes.
Bremen, date 04.04.2011
Erklärung
Ich versichere, dass ich meine Dissertation „ Molecular analysis of root-associated diazotrophs in important plants from Southern Africa and South America “ selbständig ohne unerlaubte Hilfe angefertigt und mich dabei keiner anderen als der von mir ausdrücklich bezeichneten Quellen und Hilfen bedient habe.
Die Dissertation wurde in der jetzigen oder einer ähnlichen. Form bei keiner anderen Hochschule eingereicht und hat noch keinen sonstigen Prüfungszwecken gedient.
Bremen, den 04.04.2011