Antagonistic activity of endophytic actinobacteria from native potatoes (Solanum tuberosum subsp. tuberosum L.) against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum

Abstract

Background: The native potatoes (Solanum tuberosum subsp. tuberosum L.) grown in Chile (Chiloé) represent a new, unexplored source of endophytes to find potential biological control agents for the prevention of bacterial diseases, like blackleg and soft rot, in potato crops.

Result: The objective of this study was the selection of endophytic actinobacteria from native potatoes for antagonistic activity against Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum, and their potential to suppress tissue maceration symptoms in potato tubers. This potential was determined through the quorum quenching activity using a Chromobacterium violaceaum ATCC 12472 Wild type (WT) bioassay and its colonization behavior of the potato plant root system (S. tuberosum) by means of the Double labeling of oligonucleotide probes for fluorescence in situ hybridization (DOPE-FISH) targeting technique. The results showed that although Streptomyces sp. TP199 and Streptomyces sp. A2R31 were able to inhibit the growth of the pathogens, only the Streptomyces sp. TP199 isolate inhibited Pectobacterium sp. growth and diminished tissue maceration in tubers (p ≤ 0.05). Streptomyces sp. TP199 had metal-dependent acyl homoserine lactones (AHL) quorum quenching activity in vitro and was able to colonize the root endosphere 10 days after inoculation.

Conclusions: We concluded that native potatoes from southern Chile possess endophyte actinobacteria that are potential agents for the disease management of soft rot and blackleg.

Keywords: Blackleg; Confocal laser microscopy; Endophytic actinobacteria; Potato; Quorum quenching; Soft rot; Streptomyces sp..

Fig. 1

Scanning electron microscopy. A Nocardia sp. CR34 growth in liquid ISP1 culture after 120 h of incubation at 120 rpm and 28 °C. B Nocardia sp. CR34 growth in ISP2-agar culture after 14 days of incubation at 28 °C. C Streptomyces sp. TP199 substrate mycelium growth in liquid ISP1 culture after 120 h of incubation at 120 rpm and 28 °C. D Streptomyces sp. TP199 aerial mycelium growth in ISP2-agar culture after 14 days of incubation at 28 °C. E Streptomyces sp. A2R31 substrate mycelium growth in liquid ISP1 culture after 120 h of incubation at 120 rpm and 28 °C. F Streptomyces sp. A2R31 aerial mycelium growth in ISP2-agar culture after 14 days of incubation at 28 °C. Scale bar: 5 μm

Fig. 2

Assessment of the antagonistic activity of Streptomyces sp. TP199 and Streptomyces sp. A2R31. Isolates against P. carotovorum subsp. carotovorum and P. atrosepticum in vitro. A Inhibition growth of pathogens using the cross-streak method, negative control: pathogens without antagonistic; B agar plug method, positive control: ampicillin disc (10 μg). Plot mean with SD. Different letters on the top of error bars indicate significative statistical differences (Tukey-test, p ≤ 0,05), n = 4

Fig. 3

Phylogenetic tree based on an analysis of partial sequences of the 16S ribosomal gene. The maximum likelihood method shows relations between isolates H2T199, HP171, DP143, NP199, TP199, A2R31, KR31, MP136, CR34, and selected members of the phylum Actinobacteria from the NCBI database; Corynebacterium sp. (AY211127) was used as the tree root. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The bar indicates 0.02 substitutions per nucleotide

Fig. 4

Maceration assay in tuber slices. Maceration tissue halos measured (mm) on tuber slices inoculated with Pectobacterium carotovorum subsp. carotovorum and Pectobacterium atrosepticum. These at a concentration of 10⁸ cells/mL in combination with Streptomyces sp. A2R31 and Streptomyces sp. TP199 at a concentration of 10⁸ spores/mL, incubated at 28 °C for 72 h; the treatments were: (i) P. carotovorum subsp. carotovorum, (ii) P. atrosepticum, (iii) Streptomyces sp. A2R31, (iv) Streptomyces sp. (v) P. carotovorum subsp. carotovorum + Streptomyces sp. A2R31 and (vi) P. carotovorum subsp. carotovorum + Streptomyces sp. TP199. Error bars indicate standard deviation of the media. CN: Sterile Nutrient Broth. Pba: P. atrosepticum. Pcc: P. carotovorum subsp. carotovorum. Plot mean with SD. Different letters on the top of error bars indicates statistical significative differences (Tukey-test, p ≤ 0,05), n = 3

Fig. 5

Tuber slice assay. a. Wells inoculated with P. carotovorum subsp. carotovorum. b. Wells inoculated with Streptomyces sp. TP199. c. Wells inoculated with P. carotovorum subsp. carotovorum + Streptomyces sp. TP199. d. Wells inoculated with sterile nutrient broth. e. Uninoculated slice of the tuber

Fig. 6

Inhibition of the violacein production of Chromobacterium violaceum ATCC 12472 (WT) by culture of Streptomyces sp. TP199. This according to the metal used. The bars represent the diameter sizes generated (inhibition halo in mm) of the colorless areas. Plot mean with SD. Different letters on the top of error bars indicate statistical significative differences (Tukey-test, p ≤ 0,05), n = 3

Fig. 7

Streptomyces sp. TPP199 root colonization patterns targeted by 23S rRNA with Cy5 dye double-labeled HGC69a. Probe visualized by two-photon laser scanning microscopy where autofluorescence of the plant tissue is shown in blue (B, F, L, N, R) and green (C, G, K, O, S) separately, while the probe is in red separately (D, H, J, P, T) and in a merged image (A, E, I, M, Q). A-D Apical root from a not-inoculated plant, scale bar of 50 μm (day 3 of the assay). E-H Longitudinal histological section root from a not-inoculated plant, scale bar of 90 μm (day 10 of the assay). I-L Rhizodermis from a 3 day post-inoculated plant, scale bar of 20 μm. M-P Root hair growth area from a 3 day post-inoculated plant, scale bar of 60 μm. Q-T Longitudinal histological section root from a 10 day post-inoculated plant, scale bar of 60 μm. A, E, I, M, and Q merged image. B, F, J, N, and R blue (excitation-emission: 405/410–480 nm). C, G, K, O, and S green (excitation-emission: 488/490–560 nm) separately, while the 23S rRNA with Cy5 dye double-labeled HGC69a probe is in D, H, L, P, and T red separately (excitation-emission: 633/638–747 nm). Rz: rhizodermis, Pr: root hair. Mx: metaxylem. Yellow arrows indicate Streptomyces sp. TP199 targeted with Cy5 dye double-labeled HGC69a probe fluorescence, and white arrows indicate the plant morphology