Isolation of endophytic bacteria from rice and assessment of their potential for supplying rice with biologically fixed nitrogen

The extension of nitrogen-fixing symbioses to important crop plants such as the cereals has been a long-standing goal in the field of biological nitrogen fixation. One of the approaches that has been used to try to achieve this goal involves the isolation and characterization of stable endophytic bacteria from a variety of wild and cultivated rice species that either have a natural ability to fix nitrogen or can be engineered to do so. Here we present the results of our first screening effort for rice endophytes and their characterization using acetylene reduction assays (ARA), genomic fingerprinting with primers corresponding to naturally occurring repetitive DNA elements (rep-PCR), partial 16S rDNA sequence analysis and PCR mediated detection of nitrogen fixation (nif) genes with universal nif primers developed in our laboratory. We also describe our efforts to inoculate rice plants with the isolates obtained from the screening, in order to examine their invasiveness and persistence (stable endophytic maintenance). Lastly, we review our attempts to tag selected isolates with reporter genes/proteins, such as beta-glucuronidase (gus) or green fluorescent protein (gfp), in order to be able to track putative endophytes during colonization of rice tissues.     

Are common symbiosis genes required for endophytic rice-rhizobial interactions?

Legume plants are able to establish root nodule symbioses with nitrogen-fixing bacteria, called rhizobia. Recent studies revealed that the root nodule symbiosis has co-opted the signaling pathway that mediates the ancestral mycorrhizal symbiosis that occurs in most land plants. Despite being unable to induce nodulation, rhizobia have been shown to be able to infect and colonize the roots of non-legumes such as rice. One fascinating question is whether establishment of such associations requires the common symbiosis (Sym) genes that are essential for infection of plant cells by mycorrhizal fungi and rhizobia in legumes. Here, we demonstrated that the common Sym genes are not required for endophytic colonization of rice roots by nitrogen-fixing rhizobia.     

Low diversity and abundance of root endophytes prevail throughout the life cycle of an annual halophyte

Plants growing in highly saline soils harbor unique communities of fungal root endophytes. We aimed to gain insight into how these communities are established in natural plant populations. We used cultivation-based and molecular approaches to examine root-endophytic colonization in the annual halophyte Salicornia patula at three time points over a 5-month period, from establishment to flowering. At the last sampling, the endophytic community of S. patula was compared to that in the related but perennial halophyte Arthrocnemum macrostachyum. The presence of root endophytes in S. patula was negligible at the first two sampling times, and remained low at the last sampling compared to A. macrostachyum. The latter species showed a well-established endophytic community in its roots that differed from that in S. patula, which was dominated by members of Pleosporales. Although such differences could be partially due to the host lifestyle, the possibility of a strong effect of the substratum could not be excluded. Altogether, our data indicate that the fungal endophytic colonization of roots is a slow process under salt stress. Therefore, we suggest that, in contrast to what is proposed for other systems, endophyte symbioses are unlikely to impact the development of the short-life-cycled S. patula living in these environments.     

Bacterial endophytes and their interactions with hosts

Recent molecular studies on endophytic bacterial diversity have revealed a large richness of species. Endophytes promote plant growth and yield, suppress pathogens, may help to remove contaminants, solubilize phosphate, or contribute assimilable nitrogen to plants. Some endophytes are seedborne, but others have mechanisms to colonize the plants that are being studied. Bacterial mutants unable to produce secreted proteins are impaired in the colonization process. Plant genes expressed in the presence of endophytes provide clues as to the effects of endophytes in plants. Molecular analysis showed that plant defense responses limit bacterial populations inside plants. Some human pathogens, such as Salmonella spp., have been found as endophytes, and these bacteria are not removed by disinfection procedures that eliminate superficially occurring bacteria. Delivery of endophytes to the environment or agricultural fields should be carefully evaluated to avoid introducing pathogens.     

Endophytic bacteria as biocontrol agents against plant pathogens: current state-of-the-art

Most plants co-exist with fungal and bacterial endophytes. Generally, endophytes are beneficial microorganisms that colonize the internal tissues of their host plants. Plants derive several advantages from endophytic colonization, such as the biocontrol of phytopathogens and growth-promoting factors. In this review, we discuss the current knowledge of endophytic bacteria and their potential as natural biocontrol agents for plants.     

Biological nitrogen fixation in non-leguminous field crops: Recent advances

There is strong evidence that non-leguminous field crops sometimes benefit from associations with diazotrophs. Significantly, the potential benefit from N₂ fixation is usually gained from spontaneous associations that can rarely be managed as part of agricultural practice. Particularly for dryland systems, these associations appear to be very unreliable as a means of raising the nitrogen status of plants. However, recent technical advances involving the induction of nodular structures on the roots of cereal crops, such as wheat and rice, offer the prospect that dependable symbioses with free-living diazotrophs, such as the azospirilla, or with rhizobia may eventually be achieved.     

Azoarcus sp. CIB,an Anaerobic Biodegrader of Aromatic Compounds Shows an Endophytic Lifestyle

Background

Endophytic bacteria that have plant growth promoting traits are of great interest in green biotechnology. The previous thought that the Azoarcus genus comprises bacteria that fit into one of two major eco-physiological groups, either free-living anaerobic biodegraders of aromatic compounds or obligate endophytes unable to degrade aromatics under anaerobic conditions, is revisited here.

Methodology/Principal Findings

Light, confocal and electron microscopy reveal that Azoarcus sp. CIB, a facultative anaerobe β-proteobacterium able to degrade aromatic hydrocarbons under anoxic conditions, is also able to colonize the intercellular spaces of the rice roots. In addition, the strain CIB displays plant growth promoting traits such nitrogen fixation, uptake of insoluble phosphorus and production of indoleacetic acid. Therefore, this work demonstrates by the first time that a free-living bacterium able to degrade aromatic compounds under aerobic and anoxic conditions can share also an endophytic lifestyle. The phylogenetic analyses based on the 16S rDNA and nifH genes confirmed that obligate endophytes of the Azoarcus genus and facultative endophytes, such as Azoarcus sp. CIB, locate into different evolutionary branches.

Conclusions/Significance

This is the first report of a bacterium, Azoarcus sp. CIB, able to degrade anaerobically a significant number of aromatic compounds, some of them of great environmental concern, and to colonize the rice as a facultative endophyte. Thus, Azoarcus sp. CIB becomes a suitable candidate for a more sustainable agricultural practice and phytoremediation technology.     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium,Gluconacetobacter diazotrophicus that naturally occurs in sugarcane.G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization byG. diazotrophicus, with minimal or zero inputs.     

Functional genomics in the study of seed germination

Several legume genes involved in establishing nitrogen fixation have been discovered using functional genomics; when mutated, the genes affect symbioses, and all encode receptor kinases. This provides long-awaited insights into a complex plant-bacterium interaction and heralds the possibility of extending the range of plants susceptible to nitrogen-fixing nodulation.     

Post-genomic insights into plant nodulation symbioses

Several legume genes involved in establishing nitrogen fixation have been discovered using functional genomics; when mutated, the genes affect symbioses, and all encode receptor kinases. This provides long-awaited insights into a complex plant-bacterium interaction and heralds the possibility of extending the range of plants susceptible to nitrogen-fixing nodulation.     

Endophytic bacteria of the rock-dwelling cactus Mammillaria fraileana affect plant growth and mobilization of elements from rocks

Mammillaria fraileana is a major pioneer, small cactus that harbors endophytic bacteria that have plant growth-promoting traits, including rock-weathering capacity. Our working hypothesis was that this functional group of endophytic bacteria assists in establishing pioneer plants on rocks. When these endophytic bacteria were inoculated on seedlings grown in rock substrate, mobilization of elements from the substrate increased at variable levels across combinations of substrates and inoculants. In plants grown in the rhyodacite substrate, where these cacti naturally grow, increased mobilization occurred in plants inoculated with several strains. Promotion of plant growth, manifested as an increase in dry weight, was greater in cacti inoculated with Enterobacter sakazakii M2PFe. Accumulation of nocturnal acids, indicating photosynthesis by crassulacean acid metabolism, was superior in plants inoculated with the endophytes Azotobacter vinelandii M2Per and Pseudomonas putida M5TSA. Inoculation with endophytes can stimulate plant growth of M. fraileana by mobilizing elements from rock, which can lead to higher photosynthetic activity and accumulation of biomass. Inoculation with P. putida M5TSA also led to accumulation of more total nitrogen than plants inoculated with a control nitrogen-fixing bacteria. Evidence of endophytic colonization is provided after initial inoculation of seedlings and re-isolation and sequencing of 16S DNA of recovered bacteria from developing disinfected plants. The associative interaction between pioneer cacti and their bacterial endophytes enable the host plants to grow in places where plants do not normally grow. Through colonization and establishment of pioneer plants, soil is created, which facilitates colonization by other desert species and contributes to the diversity of dry lands.     

Endophytic Xylaria (Xylariaceae) among liverworts and angiosperms: phylogenetics, distribution, and symbiosis

Nuclear ribosomal 18S and internal transcribed spacer (ITS) sequence data were used to identify endophytic fungi cultured from six species of liverworts collected in Jamaica and North Carolina. Comparisons with other published fungal sequences and phylogenetic analyses yielded the following conclusions: (1) the endophytes belong to the ascomycete families Xylariaceae, Hypocreaceae, and Ophiostomataceae, and (2) liverwort endophytes in the genus Xylaria are closely related to each other and to endophytes isolated from angiosperms in China, Puerto Rico, and Europe. Liverwort endophytes are expected to be foragers or endophytic specialists, although little is known about the role of these fungi in symbioses. Features that may indicate a mutualistic role for these endophytes are discussed.     

Microbial influence on gene-for-gene interactions in legume-Rhizobium symbioses

Recent advances in our understanding of the molecular genetics of legume-Rhizobium symbioses have indicated that relatively few bacterial genes are required for nodulation. While some of these genes are functionally similar and shared among microsymbionts nodulating genetically diverse legumes, others appear to encode host-specific nodulation (hsn) functions which allow for nodulation of plants within a given legume genus. More recently, genotype-specific nodulation (GSN) determinants have been identified in R. leguminosarum bv. viceae strain TOM and in B. japonicum strain USDA 110. GSN determinants refer to those bacterial sequences that allow for nodulation of specific plant genotypes within a given legume species. In contrast to the avr loci of several plant pathogens, rhizobia host-range determinants (hsn and GSN) have been shown to positively affect nodulation. That is, the introduction of exogenous hsn and GSN loci extends host-range. Since GSN loci have been reported to interact with single host plant alleles, it suggests that gene-for-gene interactions occur in rhizobial-legume symbioses and contribute to nodulation specificity at the host genotype level.     

Interactions of Gramineous Plants with Azoarcus spp. and Other Diazotrophs: Identification,Localization, and Perspectives to Study their Function

Nitrogen-fixing bacteria colonize the roots of many gramineous plants from different geographic regions. The discovery that diazotrophs can be isolated from surface-sterilized roots or other plant material led to studies of their potential to inhabit plant tissue. For some diazotrophs, their endophytic character has been documented. This review summarizes current methods to identify endophytes and to characterize the colonization of plants by endophytic bacteria. Taxonomy, occurrence, diversity, and mechanisms of plant infection of Azoarcus spp. is discussed in relation to Herbaspirillum spp. and Acetobacter diazotrophicus. Perspectives how to study their functions and metabolism in association with plants are discussed.     

OBPC Symposium: Maize 2004 &; beyond—Intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

Summary The problem of environmental nitrogen enrichment is most likely to be solved by reducing the inputs of synthetic nitrogen fertilizers through the creation of cereals that, like legumes, are able to fix nitrogen. In legumes, rhizobia present intracellularly in vesicles in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Rhizobia within these membrane-bounded compartments are supplied with energy from plant photosynthates and, in return, the bacteria provide the plant with biologically fixed nitrogen. Recently, we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of the root meristems of maize, rice, wheat, and other major non-legume crops can be colonized intracellularly by the non-rhizobial, non-nodulating, nitrogen-fixing bacterium, Gluconacetobacter diazotrophicus, that occurs naturally in sugarcane. G. diazotrophicus expressing nitrogen-fixing genes is present in membrane-bounded compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume species, similar to the intracellular colonization of legume root nodule cells by rhizobia. In order to obtain an indication of the likelihood of adequate growth and yield of maize, for example, with reduced inputs of synthetic nitrogen fertilizers, we are determining the extent to which nitrogen fixation is correlated with systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs of synthetic nitrogen fertilizer.     

Wheat lectin as a factor in plant-microbial communication and a stress response protein

Wheat lectin (wheat germ agglutinin, WGA), a representative of a broad group of cereal lectins, is excreted by plant roots into the surrounding medium and interacts with both pathogenic microflora and growth-stimulating rhizobacteria. WGA was found to serve as a molecular signal for the rhizobacterium Azospirillum brasilense, which forms endophytic and associative symbioses with wheat plants. The bacterial response to the lectin was pleiotropic: WGA at concentrations from 10(-10) to 10(-6) M exerted a dose-dependent effect on a range of processes in the bacterium that are important for the establishment and functioning of symbiosis. Plants with different WGA content differed in their responses to severe nitrogen starvation and to seed treatment with Azospirillum.     

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.