Conservation and Diversity of Seed Associated Endophytes in Zea across Boundaries of Evolution, Ethnography and Ecology

Endophytes are non-pathogenic microbes living inside plants. We asked whether endophytic species were conserved in the agriculturally important plant genus Zea as it became domesticated from its wild ancestors (teosinte) to modern maize (corn) and moved from Mexico to Canada. Kernels from populations of four different teosintes and 10 different maize varieties were screened for endophytic bacteria by culturing, cloning and DNA fingerprinting using terminal restriction fragment length polymorphism (TRFLP) of 16S rDNA. Principle component analysis of TRFLP data showed that seed endophyte community composition varied in relation to plant host phylogeny. However, there was a core microbiota of endophytes that was conserved in Zea seeds across boundaries of evolution, ethnography and ecology. The majority of seed endophytes in the wild ancestor persist today in domesticated maize, though ancient selection against the hard fruitcase surrounding seeds may have altered the abundance of endophytes. Four TRFLP signals including two predicted to represent Clostridium and Paenibacillus species were conserved across all Zea genotypes, while culturing showed that Enterobacter, Methylobacteria, Pantoea and Pseudomonas species were widespread, with γ-proteobacteria being the prevalent class. Twenty-six different genera were cultured, and these were evaluated for their ability to stimulate plant growth, grow on nitrogen-free media, solubilize phosphate, sequester iron, secrete RNAse, antagonize pathogens, catabolize the precursor of ethylene, produce auxin and acetoin/butanediol. Of these traits, phosphate solubilization and production of acetoin/butanediol were the most commonly observed. An isolate from the giant Mexican landrace Mixteco, with 100% identity to Burkholderia phytofirmans, significantly promoted shoot potato biomass. GFP tagging and maize stem injection confirmed that several seed endophytes could spread systemically through the plant. One seed isolate, Enterobacter asburiae, was able to exit the root and colonize the rhizosphere. Conservation and diversity in Zea-microbe relationships are discussed in the context of ecology, crop domestication, selection and migration

Bacterial endophytes in cotton: location and interaction with other plant-associated bacteria

Investigations were conducted to determine if biological control agent Pseudomonas fluorescens 89B-61 could colonize cotton tissues systemically and if internal colonization by a known endophytic bacterium, Enterobacter asburiae JM22, was influenced by the presence of other plant-associated bacteria. Following seed treatment, Pseudomonas fluorescens 89B-61 colonized cotton roots both externally and internally at mean population densities of 8.7 × 105 CFU/g and 1.1 × 103 CFU/g, respectively. However, bacteria were not detected in cotyledons, leaves, or stems. After inoculation onto leaves, Pseudomonas fluorescens 89B-61 established a mean internal population density of 1.6 × 104 CFU/g leaf tissue. Following stem injection, Pseudomonas fluorescens 89B-61 did not colonize roots or leaves. Pseudomonas fluorescens 89B-61 was localized on the root surface concentrated in grooves between epidermal cells, below collapsed epidermal cells, and in intercellular spaces close to the root epidermis, as identified by immunogold labeling of the bacterial membrane. Combined application of E. asburiae JM22 with another endophyte, Paenibacillus macerans Tri2-10, resulted in significantly lower internal populations of E. asburiae JM22 compared with treatment with E. asburiae JM22 alone. However, when coinoculated with a rhizosphere colonist, Micrococcus agilis strain 2RD-11, the colonization density of E. asburiae JM22 was not negatively affected. The results suggest that the internal colonization of cotton by bacteria with biological control activity may be an important aspect in their capacity to protect host plants against plant pathogens. The extent of internal colonization was shown to be influenced by other bacterial colonists.Key words: endophytic bacteria, location, interaction, cotton. </jats:p

Immunological detection and localization of the cotton endophyte <i>Enterobacter asburiae</i> JM22 in different plant species

Immunological methods were used to study the colonization of internal tissues of different plant species by the endophytic bacterium Enterobacter asburiae JM22. Polyclonal and monoclonal antibodies applied in enzyme-linked immunosorbent assay (ELISA), dot blot assay, tissue printing, or immunogold labeling were sensitive and specific enough to detect JM22 in plant tissues. Detection limits were 1.0 × 103 colony-forming units (CFUs)/mL for tissue printing, 1.0 × 104 CFUs/mL for ELISA and 1.0 × 105 CFUs/mL for dot blot assay. Polyclonal and monoclonal antibodies showed a positive immunological reaction with nearly all tested Enterobacter spp. In contrast with polyclonal antibodies, the monoclonal antibodies differentiated Enterobacter spp. and closely related genera like Pantoea or Serratia. Other bacterial genera, plant sap from nontreated field-grown crops, and soil solutions did not react with the antisera. When applied as a seed treatment, JM22 colonized roots, stems, and cotyledons of bean, cucumber, and cotton plants. Fourteen days after inoculation of cotton cotyledons or leaves, JM22 was detected inside the inoculated plant tissue and the bacteria moved to the roots. JM22 reached concentrations up to 1.0 × 105 CFUs/g in roots, 1.0 × 104 CFUs/g in stems, and 1.0 × 103 CFUs/g in cotyledons or leaves. Population densities of JM22 varied between the different plant species, being highest in bean and lowest in cotton. JM22 was detected with ELISA in different plant growth media. While sand, ground clay, and loamy sand showed high and comparable ELISA readings, the extinctions of sandy loam and Promix were significantly lower than the ones of the other three growth media, indicating a strong influence of soil mixes on immunological reactions. JM22 showed an intensive gold label in drop preparations of bacterial suspensions in phosphate buffer, plant sap, and ultrathin sections of plant tissue. After seed treatment, the bacteria were located on the root surface, concentrated in grooves between epidermal cells, below collapsed epidermal cells, within epidermal cells, and inside intercellular spaces in the root cortex close to conducting elements. Inoculation of leaves or cotyledons resulted in the occurrence of many gold labeled cells of JM22 on the petiole surfaces. Enterobacter asburiae colonizes different plant species and establishes endophytic populations in various tissues.Key words: immunology, endophytic bacteria, colonization, localization, plant species. </jats:p

Bacterial endophytes in agricultural crops

Endophytic bacteria are ubiquitous in most plant species, residing latently or actively colonizing plant tissues locally as well as systemically. Several definitions have been proposed for endophytic bacteria; in this review endophytes will be defined as those bacteria that can be isolated from surface-disinfested plant tissue or extracted from within the plant, and that do not visibly harm the plant. While this definition does not include nonextractable endophytic bacteria, it is a practical definition based on experimental limitations and is inclusive of bacterial symbionts, as well as internal plant-colonizing nonpathogenic bacteria with no known beneficial or detrimental effects on colonized plants. Historically, endophytic bacteria have been thought to be weakly virulent plant pathogens but have recently been discovered to have several beneficial effects on host plants, such as plant growth promotion and increased resistance against plant pathogens and parasites. In general, endophytic bacteria originate from the epiphytic bacterial communities of the rhizosphere and phylloplane, as well as from endophyte-infested seeds or planting materials. Besides gaining entrance to plants through natural openings or wounds, endophytic bacteria appear to actively penetrate plant tissues using hydrolytic enzymes like cellulase and pectinase. Since these enzymes are also produced by pathogens, more knowledge on their regulation and expression is needed to distinguish endophytic bacteria from plant pathogens. In general, endophytic bacteria occur at lower population densities than pathogens, and at least some of them do not induce a hypersensitive response in the plant, indicating that they are not recognized by the plant as pathogens. Evolutionarily, endophytes appear to be intermediate between saprophytic bacteria and plant pathogens, but it can only be speculated as to whether they are saprophytes evolving toward pathogens, or are more highly evolved than plant pathogens and conserve protective shelter and nutrient supplies by not killing their host. Overall, the endophytic microfloral community is of dynamic structure and is influenced by biotic and abiotic factors, with the plant itself constituting one of the major influencing factors. Since endophytic bacteria rely on the nutritional supply offered by the plant, any parameter affecting the nutritional status of the plant could consequently affect the endophytic community. This review summarizes part of the work being done on endophytic bacteria, including their methodology, colonization, and establishment in the host plant, as well as their role in plant–microbe interactions. In addition, speculative conclusions are raised on some points to stimulate thought and research on endophytic bacteria.Key words: endophytic bacteria, methods, localization, diversity, biological control.</jats:p

Bacterial endophytes in cotton: mechanisms of entering the plant

Investigations were conducted to determine how a systemic plant-colonizing bacterium Enterobacter asburiae JM22 enters cotton plant tissues. Passive uptake was excluded for JM22 by experimentation with glutaraldehyde-fixed (killed) bacterial cells applied to seeds and leaves; no bacteria were found internally or externally on roots or leaves. In contrast, application of live JM22 cells led to colonization of external and internal root and leaf tissues. Active penetration of JM22 in the absence of external wounding was demonstrated for cotton seedlings germinated on water agar and inoculated with the bacterial suspension. The mean internal bacterial population density for seedlings was 3.8 × 103 CFU/g surface-disinfected radicle tissue. Studies of in planta enzymatic activity demonstrated hydrolysis of wall-bound cellulose in the vicinity of JM22 bacterial cells. The same phenomenon was observed for a cortical root colonizing bacterium, Pseudomonas fluorescens 89B-61, a plant growth-promoting strain with biocontrol potential against various pathogens.Key words: endophytic bacteria, cotton, cell wall hydrolysis. </jats:p

Bioprospecting endophytic bacteria for biological control of coffee leaf rust Bioprospecção de bactérias endofíticas como agentes de biocontrole da ferrugem do cafeeiro

Suppression of plant diseases due to the action of endophytic microorganisms has been demonstrated in several pathosystems. Experiments under controlled conditions involving endophytic bacteria isolated from leaves and branches of Coffea arabica L and Coffea robusta L were conducted with the objective of evaluating the inhibition of germination of Hemileia vastatrix Berk. & Br., race II, urediniospores and the control of coffee leaf rust development in tests with leaf discs, detached leaves, and on potted seedling of cv. Mundo Novo. The endophytic bacterial isolates tested proved to be effective in inhibiting urediniospore germination and/or rust development, with values above 50%, although the results obtained in urediniospore germination tests were inferior to the treatment with fungicide propiconazole. Endophytic isolates TG4-Ia, TF2-IIc, TF9-Ia, TG11-IIa, and TF7-IIa, demonstrated better coffee leaf rust control in leaf discs, detached leaves, and coffee plant tests. The endophytic isolates TG4-Ia and TF9-Ia were identified as Bacillus lentimorbus Dutky and Bacillus cereus Frank. & Frank., respectively. Some endophytic bacterial isolates were effective in controlling the coffee leaf rust, although some increased the severity of the disease. Even though a relatively small number of endophytic bacteria were tested, promising results were obtained regarding the efficiency of coffee leaf rust biocontrol. These selected agents appears to be an alternative for future replacement of chemical fungicide.<br>Supressão de doenças de plantas por microrganismos endofíticos tem sido demonstrada em diversos patossistemas. Neste trabalho foram selecionados isolados de bactérias endofíticas de folhas e ramos de cafeeiro com potencial para o controle biológico da ferrugem do cafeeiro, pois é conhecido que esses microrganismos podem possuir essa característica. Bactérias endofíticas isoladas previamente de folhas e ramos de Coffea arabica L e Coffea robusta L foram avaliadas quanto ao seu potencial de biocontrole da ferrugem do café causada pelo fungo Hemileia vastatrix Berk. & Br., raça 2. As bactérias foram testadas para a inibição da germinação de urediniosporos do fungo e em bioensaios para o controle do desenvolvimento da ferrugem alaranjada do cafeeiro em discos de folhas, folhas destacadas e mudas da cv. Mundo Novo. Os isolados de bactérias endofíticas testados demonstraram eficácia na inibição da germinação de urediniosporos e/ou no desenvolvimento da ferrugem, com valores acima de 50%, embora os resultados obtidos nos testes de germinação de urediniosporos tenham sido inferiores ao tratamento com propiconazole (testemunha padrão). Nos testes em discos de folhas, folhas destacadas e em plantas de cafeeiro, os isolados endofíticos TG4-Ia, TF2-IIc, TF9-Ia, TG11-IIa e TF7-IIa demonstraram melhor controle da ferrugem do cafeeiro. Os isolados endofíticos TG4-Ia e TF9-Ia foram identificados como Bacillus lentimorbus Dutky e Bacillus cereus Frank. & Frank., respectivamente. De acordo com os resultados verifica-se que alguns isolados foram eficientes em controlar a ferrugem do cafeeiro, embora alguns tenham aumentado a severidade da doença. Apesar do número relativamente baixo de bactérias endofíticas testadas, resultados promissores foram obtidos em relação ao controle biológico da ferrugem, sendo que esses poderão no futuro apresentar uma alternativa aos fungicidas