Immunological detection and localization of the cotton endophyte Enterobacter asburiae JM22 in different plant species

Abstract

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.