Azospirillum bacteria form biofilms with Enterobacter cloacae K7 in the root system of wheat seedlings

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Abstract

Biofilm formation has equal adaptive significance for both epiphytic and endophytic rhizobacteria due to their primary localization on the surface of plant roots. The typical strains Azospirillum brasilense Sp7 and A. baldaniorum Sp245 formed biofilms in the root system of wheat (Triticum aestivum L.) mainly in the zones of the root apex and root hairs, as well as in the places of formation of lateral roots. In the case of the strain Enterobacter cloacae K7 isolated from the roots of Jerusalem artichoke (Helianthus tuberosus L.), biofilm formation in certain root zones was not a characteristic feature. Strain K7 colonized the roots, forming biofilms on the surface of the conduction, absorption, and root tip zones. The strains Sp7/Sp245 and K7 were not antagonists, and in the population of their mixed biofilms (studied on the Sp7 and K7 model), the proportions of subpopulations of each strain were approximately the same. However, in the root system of seedlings inoculated with mixed cultures of K7 and Sp7/Sp245, there were areas in the biofilms in which multicellular clusters of one strain were isolated from the cluster of bacteria of the other. Diffuse distribution of Sp7/Sp245 cells among enterobacteria or K7 cells between azospirillum was not typical.

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About the authors

A. V. Sheludko

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Author for correspondence.
Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

D. I. Mokeev

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

L. P. Petrova

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

E. M. Telesheva

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

I. V. Volokhina

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

Yu. A. Filipcheva

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

I. V. Borisov

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

E. V. Kryuchkova

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

L. Yu. Matora

FRC “Saratov Scientific Center of the Russian Academy of Sciences”

Email: shel71@yandex.ru

Institute of biochemistry and physiology of plants and microorganisms

Russian Federation, Saratov, 410049

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2. Fig. 1. Formation and interactions of macrocolonies of Azospirillum and Enterobacter strains in semi-solid MSM with 1 g/L NH4Cl for 72 h. Panel A: colonies of A. brasilense strains Sp7, Sp7-pJN105TurboGFP, Sp7-pJN105TurboRFP, E. cloacae K7 and K7-pJN105TurboGFP (left); colonies of E. cloacae strains K7 and K7-pJN105TurboGFP (middle); colonies of A. baldaniorum strains Sp245, Sp245-pJN105TurboGFP, Sp245-pJN105TurboRFP and E. cloacae K7 and K7-pJN105TurboGFP (right). Panel B: Effect of nitrogen source on the size of A. baldaniorum Sp245 macrocolonies (1) and their “collision” zones (2). Scale bar corresponds to 10 mm.

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3. Fig. 2. Results of immunochemical studies of biofilms formed by A. brasilense Sp7 (1 and 2), A. baldaniorum Sp245 (6 and 7) and E. cloacae K7 (4 and 5) strains on glass under liquid medium for 7 days of cultivation. Panel A shows a comparison of the relative content (RC%) of Sp7 or K7 antigens in biofilms formed by mixed cultures of these strains. ELISA was used to determine the RC%. One-way analysis of variance (ANOVA) was used to determine the significance of differences in RC%; a indicates no significant differences between the means. Panel B shows the results of the interaction of planktonic culture cell extracts (1, 4 and 6) or biofilm biomass (2, 3, 5, 7 and 8) with strain-specific Abs (Abs to antigens of strains Sp7 (AtSp7), Sp245 (AtSp245) and (AtK7)). Extracts of biomass from biofilms formed by mixed cultures of Sp7 and K7 (3) or Sp245 and K7 (8).

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4. Fig. 3. Confocal microscopy results of the root system of wheat seedlings inoculated with A. brasilense Sp7, A. baldaniorum Sp245, and E. cloacae K7 containing pJN105TurboGFP or pJN105TurboRFP plasmids (7 days after inoculation). Panels A, C, and D show the microscopy results of wheat inoculated with Sp245(pJN105TurboRFP) and K7(pJN105TurboGFP). Panels B and D illustrate the microscopy results of plants inoculated with Sp7(pJN105TurboRFP) and K7(pJN105TurboGFP). Panel E shows the microscopic images of seedlings inoculated with Sp7(pJN105TurboGFP) or Sp245 (pJN105TurboGFP) monocultures. Grayscale images represent visible light. Green or red colors represent the detection of GFP or RFP fluorescence. Panels A, B, D, and E represent the microscopic images of the root absorptive zone. Panels D and B represent the microscopic images of the conduction zone and root tip, respectively.

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