Screening, Identification and Efficacy Evaluation of Antagonistic Bacteria for Biocontrol of Soft Rot Disease Caused by Dickeya zeae

Abstract

Dickeya zeae is the causal agent of bacterial soft rot disease, with a wide range of hosts all over the world. At present, chemical agents, especially agricultural antibiotics, are commonly used in the prevention and control of bacterial soft rot, causing the emergence of resistant pathogens and therefore increasing the difficulty of disease prevention and control. This study aims to provide a safer and more effective biocontrol method for soft rot disease caused by D. zeae. The spot-on-lawn assay was used to screen antagonistic bacteria, and three strains including SC3, SC11 and 3-10 revealed strong antagonistic effects and were identified as Pseudomonas fluorescens, P. parafulva and Bacillus velezensis, respectively, using multi-locus sequence analysis (MLSA) based on the sequences of 16S rRNA and other housekeeping genes. In vitro antimicrobial activity showed that two Pseudomonas strains SC3 and SC11 were only antagonistic to some pathogenic bacteria, while strain 3-10 had broad-spectrum antimicrobial activity on both pathogenic bacteria and fungi. Evaluation of control efficacy in greenhouse trials showed that they all restrained the occurrence and development of soft rot disease caused by D. zeae MS2 or EC1. Among them, strain SC3 had the most impressive biocontrol efficacy on alleviating the soft rot symptoms on both monocotyledonous and dicotyledonous hosts, and strain 3-10 additionally reduced the occurrence of banana wilt disease caused by Fusarium oxysporum f. sp. cubensis. This is the first report of P. fluorescens, P. parafulva and B. velezensis as potential bio-reagents on controlling soft rot disease caused by D. zeae.

Keywords: antagonistic bacterial screening; determination of biocontrol effect; species identification.

Figure 1

Inhibitory activities of antagonistic strains and Dickeya zeae pathogens in spot-on-lawn assay. The plates were incubated at 28 °C for 24 h. (A) the antagonistic activities of strains SC3, SC11 and 3-10 against pathogenic D. zeae strains EC1, MS2 and MS3; (B) inhibitory activities of D. zeae pathogens against antagonistic strains SC3, SC11 and 3-10.

Figure 2

Joint phylogenetic trees based on the concatenated nucleotide sequences of the 16S rRNA, gyrB, rpoB and rpoD genes of strains SC3 (A), SC11 (B), and the 16S rRNA, gyrA and gyrB genes of strain 3-10 (C). Consensus sequences of every gene from related strains were aligned with ClustalW and trimmed in the same sizes. All the sequences from the same strain were assembled to construct a joint neighbor-joining tree. Bootstrap values after 1000 replicates are expressed as percentages. Scale bar denotes nucleotide substitutions per site.

Figure 3

Biocontrol efficacy of antagonistic bacterial strains against soft rot disease on dicotyledonous carrot (A), potato (B) and Chinese cabbage (C). Both MS2 and antagonistic bacteria were grown in lysogeny broth (LB) medium till OD₆₀₀ ≈ 1.8. One microliter of LB, LB+antagonistic bacteria, LB+MS2 and MS2+antagonistic bacterium was respectively spotted onto the center of tissue slices. The rotting area was measured by Image J 1.52a, and the data were subjected to unpaired two-tailed t-test analysis by Graphpad Prism 8.4.1 (GraphPad Software, San Diego, CA, USA) (ns: no statistical significance, *** p < 0.001). The marks in the plot area represent the diseased area on 10 inoculated plant slices, and the red lines represent the average values of diseased area.

Figure 4

Biocontrol efficacy of antagonistic bacterial strains against rice foot rot and soft rot diseases on monocotyledonous rice (A) and banana seedlings (B). EC1, MS2 and antagonistic bacteria were grown in LB medium till OD₆₀₀ ≈ 1.8. Ten milliliters of LB, EC1+LB and EC1+antagonistic bacterium was respectively irrigated into the pots with 20 rice seedlings after needle punctures on stem bases. Two hundred microliter of LB, MS2+LB and MS2+antagonistic bacteria was respectively injected into the pseudostems of banana seedlings. Plants were incubated at 28°C with 12 h alternating light-dark cycles for 7 days, and disease was assessed by using virulence scoring method described previously. Data were subjected to unpaired two-tailed t-test analysis by Graphpad Prism 8.4.1 (*** p < 0.001). The marks in the plot area represent the virulence scores of the inoculated plant seedlings, and the red lines represent the average virulence scores.

Figure 5

Biocontrol efficacy of strain 3-10 against banana wilt caused by Fusarium oxysporum f. sp. cubensis FOC4. Strain 3-10 was cultured in LB medium overnight and resuspended with ddH₂O to a final concentration of OD₆₀₀ = 0.1. FOC4 strain was cultured on PDA plates for about 5 days and washed with ddH₂O to collect hypha and conidia. Half roots of each banana seedling (30 seedlings) were randomly cut off, 20 seedlings were soaked in pure FOC4 suspension and 10 were soaked in ddH₂O for 20 minutes, and re-planted in sterilized pots. Then 100 mL of 3-10 cell suspension and ddH₂O was respectively drenched into soils of each 10 FOC4-soaked seedlings, while 100 mL of ddH₂O was also drenched into the soils of the ddH₂O-soaked seedlings. Symptoms were observed every day and pictures were taken in the 16^th day after inoculation. Data were subjected to unpaired two-tailed t-test analysis by Graphpad Prism 8.4.1 (*** p < 0.001). The marks in the plot area represent the virulence scores of the inoculated seedlings, and the red lines represent the average virulence scores.

Figure 6

Inhibitory effect of strains SC3, SC11 and 3-10 and their metabolites on the growth of D. zeae MS2. (A) Inhibitory activities of the SC3, SC11 and 3-10 supernatants (up panel) and 1 µL of overnight cultures (bottom panel) against MS2 growth; (B) Inhibitory activity of SC3 supernatant (SSC3) and its Trypsin enzymatic hydrolysate (SSC3 + Trypsin) against MS2 growth; (C) MS2 (OD₆₀₀ = 1.5) dilutions respectively grown on the LB agarose, SC3, SC11 and 3-10 metabolite strips; (D) MS2 (OD₆₀₀ = 1.5) dilutions respectively grown on the LB agarose, and microwave melted SC3, SC11 and 3-10 metabolite strips.