Structural Interaction Between GFP-Labeled Diazotrophic Endophytic Bacterium Herbaspirillum seropedicae RAM10 and Pineapple Plantlets 'VitóRia'

Abstract

The events involved in the structural interaction between the diazotrophic endophytic bacterium Herbaspirillum seropedicae, strain RAM10, labeled with green fluorescent protein, and pineapple plantlets 'Vitória' were evaluated by means of bright-field and fluorescence microscopy, combined with scanning electron microscopy for 28 days after inoculation. After 6 hours of inoculation, H. seropedicae was already adhered to the roots, colonizing mainly root hair surface and bases, followed by epidermal cell wall junctions. Bacteria adherence in the initial periods occurred mainly in the form of solitary cells and small aggregates with pleomorphic cells. Bacteria infection of root tissue occurred through the cavities caused by the disruption of epidermal cells during the emergence of lateral roots and the endophytic establishment by the colonization of intercellular spaces of the cortical parenchyma. Moreover, within 1 day after inoculation the bacteria were colonizing the shoots. In this region, the preferred sites of epiphytic colonization were epidermal cell wall junctions, peltate scutiform trichomes and non-glandular trichomes. Subsequently, the bacteria occupied the outer periclinal walls of epidermal cells and stomata. The penetration into the shoot occurred passively through stoma aperture followed by the endophytic establishment on the substomatal chambers and spread to the intercellular spaces of spongy chlorenchyma. After 21 days of inoculation, bacterial biofilm were seen at the root hair base and on epidermal cell wall surface of root and leaf, also confirming the epiphytic nature of H. seropedicae.

Keywords: Ananas comosus; microscopy; plant-growth promoting bacteria.

Figure 1

Fluorescence microscopy (A, B, C, D, E) and bright-field microscopy (F) of the initial stages of interaction between H. seropedicae RAM10 and pineapple plantlet roots. (A) 6 h after inoculation, predominance of bacteria in filamentous shape (arrow) colonizing preferentially the root hair surface, solitary or in small aggregates. (B) 1 day after inoculation, the bacteria also colonized the root hair base. (C, D) 2 days after inoculation, the bacteria were arranged on the root hair surface in the form of aggregates of different sizes and rod-shaped bacteria were predominant (arrow). (E, F) 7 days after inoculation, bacteria colonized epidermal cell walls junctions.

Figure 2

Fluorescence microscopy (A, B), scanning electron microscopy (C) and bright-field microscopy (D) showing the epiphytic colonization, infection and endophytic colonization of H. seropedicae RAM10 on pineapple plantlets roots 14 days after inoculation. (A) Intense epiphytic bacterial colonization on the periclinal wall of epidermal cells. (B) Colonization mainly in the regions of emergence of lateral roots (asterisk). (C) Bacterial infection through the cavity (arrow) resulting from the rupture of epidermal cells during the emergence of lateral roots (asterisk). (D) Endophytic colonization (arrow) in the intercellular spaces of cortical parenchyma.

Figure 3

Scanning electron microscopy of biofilms of H. seropedicae RAM10 located epiphytically on the root and shoot surface of pineapple plantlets 21 days after inoculation. (A) Biofilms on the root hairs and (B) on the periclinal wall of root epidermal cells. (C) Biofilms on the outer periclinal wall of epidermal leaf cells and (D) detail of the bacterial cells forming the biofilm.

Figure 4

Fluorescence microscopy (A, C), scanning electron microscopy (B, E, F) and bright-field microscopy (D) of leaf epiphytic colonization of H. seropedicae RAM 10 preferably on the trichomes of pineapple. (A, B) Bacterial colonization on and at the base of non-glandular trichomes. (C, D, E, F) Bacterial colonization on and at the basis of peltate scutiform trichomes.

Figure 5

Fluorescence microscopy (A), bright-field microscopy (B) and scanning electron microscopy (C, D) of leaf epiphytic colonization of H. seropedicae RAM 10. (A, B, C) Bacterial colonization in epidermal cell walls junctions and (D) on the outer periclinal wall of epidermal cells.

Figure 6

Scanning electron microscopy (A, B) and bright-field microscopy (C, D) of epiphytic colonization, infection and endophytic colonization of H. seropedicae RAM 10 in shoots of pineapple plantlets. (A) Epiphytic colonization of bacteria on the surface of the outer periclinal walls of epidermal cells. (B) Bacterial colonization on the stomatal complex and infection via stoma (asterisk). (C) Bacterial infection through stoma (asterisk) and endophytic colonization in the substomatal chamber (arrow). (D) Endophytic bacterial colonization in the intercellular spaces of the chlorophyll parenchyma (arrow).

Figure 7

Log of the most probable number (MPN) of H. seropedicae RAM10 on pineapple plantlets ‘Vitória’ in response to time.