Bacteriophage LDT325 enhances Pseudomonas syringae tolerance by improving antioxidant defense in tea plant [ Camellia sinensis (L.) O. Kuntze]

Abstract

Bud blight caused by Pseudomonas syringae is a serious disease affecting tea plants and causing severe damage to production output and quality. Phages play an important role in controlling the development of bacterial diseases in plants. Previous studies have shown that the tolerance of phage-treated tea plants to bud blight was notably greater compared with that of the control group. In the present study, we determined the effect of bacteriophage therapy on physiological and biochemical parameters of tea leaves. Transmission electron microscopy (TEM) was used to analyze the cellular structure of tea leaves, and bioinformatics was used to analyze the phage. Results revealed that bacteriophage treatment can enhance the expression of antioxidant enzyme genes (CsSOD, CsCAT, and CsPOD). The levels of osmotic adjustment compounds, including proline and soluble sugars, were also elevated, suggesting that bacteriophage enhances the osmotic adjustment capacity in tea plants. TEM analysis revealed that the integrity of the cell structure of the tea leaves treated with phage was notably better compared with that of the control group. Interestingly, we also observed that the phage lysed the animal pathogen Salmonella as well as the plant pathogen P. syringae. Using NCBI BLASTn to compare the entire genome with other nucleotide sequences, we found that the phage LDT325 exhibited cross-species characteristics that had not been previously reported. In summary, our findings demonstrate that bacteriophages can protect tea plants from damage caused by bacterial diseases by regulating antioxidant systems.

Keywords: Camellia sinensis; Pseudomonas syringae; antioxidant enzyme; bacteriophage; physiological characters.

Figure 1

Primers used for gene expression analysis.

Figure 2

Effects of phage on antioxidant enzyme activity and gene expression in tea at designated time. (A) Superoxide dismutase (SOD), (B) catalase (CAT), (C) peroxidase (POD), (D) CsSOD, (E) CsCAT, (F) CsPOD. Vertical bars indicate the standard error of the mean values. Asterisks imply statistically significant differences (p < 0.05) between the control and phage treated group in the same day.

Figure 3

Phage affecting on physiological parameters of tea leaves. (A) Proline content, (B) soluble sugar content, (C) chlorophyll content. Vertical bars indicate the standard error of the mean values. Asterisks imply statistically significant differences (p < 0.05) between the control and phage treated group in the same day.

Figure 4

Transmission electron microscopy images of ultrathin sections of tea leaves. (A, B) Transmission electron microscopy images of ultrathin sections of tea leaves in sterile water group. (A) Observed at 1,000×. (B) Observed at 5,000×. (C, D)Transmission electron microscopy images of ultrathin sections of tea leaves in the control group. (C) Observed at 1,000×. (D) Observed at 5,000×. (E, F) Transmission electron microscopy images of ultrathin sections of tea leaves in phage treatment group. (E) Observed at 1,000×. (F) Observed at 5,000×. cw, cell wall; cm, cell membrane; m, mitochondria; t, thylakoid.

Figure 5

Genomic analysis. (A) Genome map of phage vB_PsS_LDT325, the genome of phage vB_PsS_LDT325 depicted in the circular. These arrows represent 61 CDS. In addition, the map shows GC skew and content about the genome. (B) An expanded view of the region of the tree containing the most closely related phages. The location of phage LDT325 is indicated in the red triangle.

Figure 6

Genome structure. Arrows indicate the direction of transcription for predicted ORFs. ORFs with different functions are shown in different colors.

Figure 7

Several animal pathogenic Salmonella strains lysed by phage LDT325. The number below the picture is the number of Salmonella. Image S73-1 showed that after 12 h of culture, the phage suspension showed a clear cleavage area on the double-layer plate covered with Salmonella suspension numbered S73, showing the lysing bacteria. Picture S73-2 was further purified from the clear cleavage region of phage in picture S73-1. The same below.