Thomas Hurek

Life in grasses: diazotrophic endophytes.

N2-fixing bacteria such as Azoarcus spp., Herbaspirillum spp, and Acetobacter diazotrophicus can infect the interior of gramineous plants without causing symptoms of plant disease but do not survive in soil. Like phytopathogens, they can penetrate into central tissues and spread systemically. There is no evidence for an endosymbiosis in living plant cells; however, the bacteria are physiologically active in the plant apoplast.

Living inside plants: bacterial endophytes

As current research activities have focused on symbiotic or parasitic plant-microbe interactions, other types of associations between plants and microorganisms are often overlooked. Endophytic bacteria colonize inner host tissues, sometimes in high numbers, without damaging the host or eliciting strong defense responses. Unlike endosymbionts they are not residing in living plant cells or surrounded by a membrane compartment. The molecular basis of endophytic interactions is still not well understood. Several traits involved in the establishment of endophytes have been elucidated. Culture-independent methods for community analysis and functional genomic as well as comparative genomic analyses will provide a better understanding of community dynamics, signaling, and functions in endophyte-plant associations.

Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis.

Roots are the primary site of interaction between plants and microorganisms. To meet food demands in changing climates, improved yields and stress resistance are increasingly important, stimulating efforts to identify factors that affect plant productivity. The role of bacterial endophytes that reside inside plants remains largely unexplored, because analysis of their specific functions is impeded by difficulties in cultivating most prokaryotes. Here, we present the first metagenomic approach to analyze an endophytic bacterial community resident inside roots of rice, one of the most important staple foods. Metagenome sequences were obtained from endophyte cells extracted from roots of field-grown plants. Putative functions were deduced from protein domains or similarity analyses of protein-encoding gene fragments, and allowed insights into the capacities of endophyte cells. This allowed us to predict traits and metabolic processes important for the endophytic lifestyle, suggesting that the endorhizosphere is an exclusive microhabitat requiring numerous adaptations. Prominent features included flagella, plant-polymer-degrading enzymes, protein secretion systems, iron acquisition and storage, quorum sensing, and detoxification of reactive oxygen species. Surprisingly, endophytes might be involved in the entire nitrogen cycle, as protein domains involved in N(2)-fixation, denitrification, and nitrification were detected and selected genes expressed. Our data suggest a high potential of the endophyte community for plant-growth promotion, improvement of plant stress resistance, biocontrol against pathogens, and bioremediation, regardless of their culturability.

Azoarcus gen. nov., Nitrogen-Fixing Proteobacteria Associated with Roots of Kallar Grass (Leptochloa fusca (L.) Kunth), and Description of Two Species, Azoarcus indigens sp. nov. and Azoarcus communis sp. nov.

Among the nitrogen-fixing bacteria associated with roots of Leptochloa fusca (L.) Kunth in saline-sodic soils in the Punjab of Pakistan, we repeatedly found yellow-pigmented, straight to curved, gram-negative rods. To group and identify these organisms, we examined morphological, nutritional, and biochemical features and performed polyacrylamide gel electrophoretic analyses of cellular proteins, gas chromatographic analyses of fatty acids, DNA-rRNA hybridizations, and DNA-DNA hybridizations. Our results showed that 11 isolates formed five groups distinct at the species level, with each group containing one to three members. These bacteria constituted a separate rRNA branch in rRNA superfamily III (corresponding to the beta subclass of the Proteobacteria) at a branching Tm(e) level of 67.7°C [Tm(e) is the temperature at which 50% of a hybrid is denatured under standard conditions]. On this branch, the five groups were located in two clusters with Tm(e) values of 79.4 to 80.4°C and around 71.5°C. We propose a new genus, the genus Azoarcus, for these strains. Azoarcus indigens is the type species and has a growth factor requirement; its type strain is strain VB32 (= LMG 9092). A second named species, Azoarcus communis, includes a strain obtained from French refinery oily sludge, strain LMG 5514. Bacteria of this genus have a strictly aerobic type of metabolism, fix nitrogen microaerobically, and grow well on salts of organic acids but not on carbohydrates. Swedish isolates obtained from human sources (E. Falsen group 15 strains LMG 6115 and LMG 6116), as well as “[Pseudomonas] gasotropha” LMG 7583T, were also located on this rRNA branch at a lower Tm(e) level (70.4 to 71.2°C).

Azoarcus Grass Endophytes Contribute Fixed Nitrogen to the Plant in an Unculturable State

The extent to which the N2-fixing bacterial endophyte Azoarcus sp. strain BH72 in the rhizosphere of Kallar grass can provide fixed nitrogen to the plant was assessed by evaluating inoculated plants grown in the greenhouse and uninoculated plants taken from the natural environment. The inoculum consisted of either wild-type bacteria or nifK- mutant strain BHNKD4. In N2-deficient conditions, plants inoculated with strain BH72 (N2-fixing test plants) grew better and accumulated more nitrogen with a lower delta15N signature after 8 months than did plants inoculated with the mutant strain (non-N2-fixing control plants). Polyadenylated or polymerase chain reaction-amplified BH72 nifH transcripts were retrieved from test but not from control plants. BH72 nifH transcripts were abundant. The inocula could not be reisolated. These results indicate that Azoarcus sp. BH72 can contribute combined N2 to the plant in an unculturable state. Abundant BH72 nifH transcripts were detected also in uninoculated plants taken from the natural environment, from which Azoarcus sp. BH72 also could not be isolated. Quantification of nitrogenase gene transcription indicated a high potential of strain BH72 for biological N2 fixation in association with roots. Phylogenetic analysis of nitrogenase sequences predicted that uncultured grass endophytes including Azoarcus spp. are ecologically dominant and play an important role in N2-fixation in natural grass ecosystems.

Azospirillum halopraeferens sp. nov., a nitrogen-fixing organism associated with roots of Kallar grass (Leptochloa fusca (L.) Kunth)

Among the nitrogen-fixing bacteria associated with the roots of Leptochloa fusca (L.) Kunth in salt-affected soils in the Punjab region of Pakistan, we found a homogeneous group of eight diazotrophs. Cells are vibrioid to S shaped, are motile by one polar flagellum, and produce granules of poly-β-hydroxybutyrate. They have a respiratory type of metabolism, show microaerophilic growth when fixing nitrogen, grow well on salts of organic acids, and can also use fructose and mannitol. On nitrogen-free semisolid media, they require biotin, utilize mannitol, but not glucose or sucrose, and cannot acidify glucose aerobically or anaerobically. Optimal growth occurs at 0.25% NaCl and 41°C. Deoxyribonucleic acid (DNA)-ribosomal ribonucleic acid (rRNA) hybridizations show that the organisms belong to the Azospirillum rRNA branch, where they cluster together with Azospirillum amazonense. They form a phenotypically and protein electrophoretically homogeneous group of bacteria, clearly distinct from Azospirillum amazonense, Azospirillum lipoferum, and Azospirillum brasilense. As no DNA-DNA binding was found with any of the three Azospirillum species, we propose a fourth Azospirillum species for this group of isolates. Because of better growth at increased NaCl concentrations, we named the new species Azospirillum halopraeferens. Strain Au 4 (= LMG 7108) is the type strain, which has been deposited at the Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany, as DSM 3675.

Type IV pili are involved in plant–microbe and fungus–microbe interactions

Adherence of bacteria to eukaryotic cells is essential for the initiation of infection in many animal and human pathogens, e.g. Neisseria gonorrhoeae and Pseudomonas aeruginosa. Adhesion‐mediating type IV pili, filamentous surface appendages formed by pilin subunits, are crucial virulence factors. Here, we report that type IV pilus‐dependent adhesion is also involved in plant–bacteria and fungus–bacteria interactions. Nitrogen‐fixing, endophytic bacteria, Azoarcus sp., can infect the roots of rice and spread systemically into the shoot without causing symptoms of plant disease. Formation of pili on solid media was dependent on the pilAB locus. PilA encodes an unusually short (6.4 kDa) putative pilin precursor showing 100% homology to the conserved N‐terminus of the Pseudomonas aeruginosa type IV pilin. PilB encodes for a 14.2 kDa polypeptide showing similarity to FimF, a component of type I fimbriae of Escherichia coli. It was found to be extruded beyond the cell surface by immunofluorescence studies, and it may, therefore, be part of a pilus assembly complex or the pilus itself. Both genes are involved in the establishment of bacteria on the root surface of rice seedlings, as detected by fluorescence microscopy. Moreover, both genes are necessary for bacterial adhesion to the mycelium of an ascomycete, which was isolated from the same rhizosphere as the bacteria. In co‐culture with the fungus, Azoarcus sp. forms complex intracytoplasmic membranes, diazosomes, which are related to efficient nitrogen fixation. Adhesion to the mycelium appears to be crucial for this process, as diazosomes were absent and nitrogen fixation rates were decreased in pilAB mutants in co‐culture.

Endophytic expression of nif genes of Azoarcus sp. strain BH72 in rice roots

Transcriptional fusions of gusA and gfp to the nifH gene as well as immunogold labeling with antibodies against the iron protein of nitrogenase revealed high nitrogenase gene expression levels of the endophyte Azoarcus sp. strain BH72 inside infected rice roots (Oryza sativa) in the laboratory. Thus, environmental conditions inside rice roots are permissive for endophytic nitrogen fixation in bacterial microcolonies in the aerenchyma.

Specific Detection of Bradyrhizobium and Rhizobium Strains Colonizing Rice (Oryza sativa) Roots by 16S-23S Ribosomal DNA Intergenic Spacer-Targeted PCR

ABSTRACT In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata(IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in theRhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens;emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genusBradyrhizobium, as illustrated here for strains ofBradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Use of green fluorescent protein to detect expression of nif genes of Azoarcus sp. BH72, a grass-associated diazotroph, on rice roots

A gfp (green fluorescent protein) cassette for transcriptional fusions has been developed to study gene expression in Azoarcus sp. BH72 in association with plant roots. The bacteria expressed nitrogenase genes (nifHDK) in the rhizosphere, on root tips, and in epidermal cells of rice seedlings. Green fluorescent protein fusions also visualized promoter activity of single cells in soil.