What role do biocontrol agents with Mg2+ play in the fate of antibiotic resistome and pathogenic bacteria in the phyllosphere?

Abstract

The contamination of the plant phyllosphere with antibiotics and antibiotic resistance genes (ARGs), caused by application of antibiotics, is a significant environmental issue in agricultural management. Alternatively, biocontrol agents are environmentally friendly and have attracted a lot of interest. However, the influence of biocontrol agents on the phyllosphere resistome remains unknown. In this study, we applied biocontrol agents to control the wildfire disease in the Solanaceae crops and investigated their effects on the resistome and the pathogen in the phyllosphere by using metagenomics. A total of 250 ARGs were detected from 15 samples, which showed a variation in distribution across treatments of biocontrol agents (BA), BA with Mg²⁺ (T1), BA with Mn²⁺ (T2), and kasugamycin (T3) and nontreated (CK). The results showed that the abundance of ARGs under the treatment of BA-Mg²⁺ was lower than that in the CK group. The abundance of cphA3 (carbapenem resistance), PME-1 (carbapenem resistance), tcr3 (tetracycline antibiotic resistance), and AAC (3)-VIIIa (aminoglycoside antibiotic resistance) in BA-Mg²⁺ was significantly higher than that in BA-Mn²⁺ (P < 0.05). The abundance of cphA3, PME_1, and tcr3 was significantly negatively related to the abundance of the phyllosphere pathogen Pseudomonas syringae (P < 0.05). We also found that the upstream and downstream regions of cphA3 were relatively conserved, in which rpl, rpm, and rps gene families were identified in most sequences (92%). The Ka/Ks of cphA3 was 0 in all observed sequences, indicating that under the action of purifying selection, nonsynonymous substitutions are often gradually eliminated in the population. Overall, this study clarifies the effect of biocontrol agents with Mg²⁺ on the distribution of the phyllosphere resistome and provides evolutionary insights into the biocontrol process.

Importance: Our study applied metagenomics analysis to examine the impact of biocontrol agents (BAs) on the phyllosphere resistome and the pathogen. Irregular use of antibiotics has led to the escalating dissemination of antibiotic resistance genes (ARGs) in the environment. The majority of BA research has focused on the effect of monospecies on the plant disease control process, the role of the compound BA with nutrition elements in the phyllosphere disease, and the resistome is still unknown. We believe BAs are eco-friendly alternatives for antibiotics to combat the transfer of ARGs. Our results revealed that BA-Mg²⁺ had a lower relative abundance of ARGs compared to the CK group, and the phyllosphere pathogen Pseudomonas syringae was negatively related to three specific ARGs, cphA3, PME-1, and tcr3. These three genes also present different Ka/Ks. We believe that the identification of the distribution and evolution modes of ARGs further elucidates the ecological role and facilitates the development of BAs, which will attract general interest in this field.

Keywords: antibiotic resistance genes; biocontrol agents; evolution; homologous recombination; metagenome; mutation.

Fig 1

(A). Genes belonging to nine drug classes in different groups (LSD test; P < 0.05). (B). The composition and proportion of top 10 ARGs among four treatment groups. (C). The abundance of ARGs among four treatment groups.

Fig 2

ARGs with a significant difference under different treatments (LSD test; P < 0.05).

Fig 3

(A) The phylogenetic assignment of assembled genome bins; organisms were colored based on the genus level. (B) The co-occurrence network of antibiotic-resistant genes. (C) The heatmap of binnings and ARGs belonging to different drug classes.

Fig 4

(A) The correlation between P. syringae and ARGs (Pearson correlation, P < 0.05). (B) The lm of the abundance of ARGs and P. syringae. The blue shadow indicates the 95% confidence interval.

Fig 5

(A to C) The main structure type of cphA3, tcr3, and PME-1 sequences in the metagenome. The arrows denote genes, and arrow length denotes gene size. (D) The proportion of SNPs N-A, N-T, N-C, and N-G in different sequences. E The Ka/Ks of cphA3, tcr3, and PME-1 (t test; P < 0.05).