3.1 The occurrence and coexistence of ARGs in the rhizosphere soil
Figure 1
Seventeen categories were identified based on the presence of rhizosphere soil samples under different treatments and at various time points. In total, 96 subtypes of ARGs were considered inherent in the soil. These ARGs were detectable in soil samples from both the planting and harvesting periods, implying their long-term presence and resistance to removal from the experimental soil. Among them, multidrug-resistant genes (36 subtypes) exhibited the highest diversity, followed by tetracycline genes (12 subtypes) and macrolide genes (11 subtypes) (Fig.1A). The application of organic fertilizers increased the abundance of inherent ARGs in the soil during the planting period. Compared to the control group, the use of pig manure organic fertilizer and sheep manure organic fertilizer led to a 154% and 13% increase, respectively, in the total relative abundance of these genes (Fig. 1B). Additionally, both organic fertilizers led to the emergence of 104 new ARG subtypes in rhizosphere soil during the planting period. Specifically, the pig manure organic fertilizer treatment introduced 99 new ARG subtypes, representing 26% of the total relative abundance, while the application of sheep manure organic fertilizer resulted in 29 new ARG subtypes, making up 15% of the total relative abundance (Figs. 1A and S1B). In summary, organic fertilizer application boosted the overall relative abundance and diversity of ARGs in onion root soil during the planting period, with pig manure having a more significant impact. This aligns with the consistent pattern of ARG presence observed in both types of organic fertilizers, where pig manure organic fertilizer exhibited higher ARGs abundance and diversity compared to sheep manure organic fertilizer (Fig. S2). These findings align with earlier research, where organic fertilizers were found to introduce ARBs and ARGs into the soil, resulting in increased diversity and abundance of ARGs in the soil. The magnitude of this effect was notably correlated with the ARGs present in the fertilizer itself(Huang et al 2021).
Figure2
After 8 months of onion cultivation, the total relative abundance of ARGs in the rhizosphere soil of the fertilized groups decreased to levels close to that of the control group. The number of newly introduced ARGs due to fertilization also significantly reduced but remained higher than that in the control group (Fig. 1). During this period, the fertilized groups exhibited similar levels of inherent ARG abundance and the proportion of newly introduced ARGs compared to the control group (Fig. S1). This observation suggests that the ARGs introduced into the soil through fertilization during organic onion cultivation may undergo degradation or transfer. There is a possibility that these “disappearing ARGs” could migrate into the onion. In conclusion, fertilization has been shown to increase the exposure risk of ARGs in onion rhizosphere soil, further elevating the risk of ARGs transmission from the soil to the onion.
3.2 Presence and traceability of ARGs in onion
In the harvested onion roots and bulbs, a total of 53 ARGs from 10 classes were detected. Following the application of pig manure organic fertilizer, onion roots and bulbs exhibited a significant increase in the absolute abundance of ARGs. Specifically, 36 ARGs were detected in onion roots and 17 in bulbs, with abundances 20 - fold and 4 - fold higher than the control treatment, respectively. The use of sheep manure organic fertilizer also led to increased ARG abundance in both onion roots and bulbs, albeit to a lesser extent than pig manure, with increases of 5.3 and 2 times that of the control group, respectively (Figs. S3 and S4). The results showed that application of both organic fertilisers increased the type and abundance of ARGs in onion, with pig manure being greater than sheep manure. In the pig manure organic fertilizer treatment group, the abundance and number of ARGs in the root tissues were 30 times and 2 times higher than in the bulbs, respectively. Additionally, there were only 7 common ARGs in both onion roots and bulbs, which is significantly lower than the total number of ARGs detected in the bulbs. Similar trends were observed in the sheep manure organic fertilizer and control groups (Fig. S3). This observation suggests that the roots of onion exhibit higher ARG abundance and diversity compared to the bulbs, with varying types of enriched ARGs. The root system of plants, being in direct contact with soil microorganisms, possesses a more abundant and readily available nutrient source compared to other plant tissues. Microorganisms in the soil tend to accumulate in the rhizosphere and may colonize plant roots. These microorganisms could potentially carry ARGs, making the root system more susceptible to the influence of ARGs present in the soil (Trivedi et al 2020). The onion's bulb tissue grows in a semi-exposed environment, with half of it buried underground and the other half exposed to the air. In contrast to the roots, ARGs in onion bulbs are influenced not only by the soil but also by biological aerosols in the air. A previous study indicated the presence of a substantial amount of ARGs and ARBs in urban air, and the application of manure fertilizers might potentially impact the abundance of ARBs and ARGs in nearby aerosols (Arfken et al 2015, Li et al 2018).
Forty ARGs coexist between onion and the soil. Overall, the coexistence of ARGs in onion and rhizospheric soil during the growing season exhibited consistency. Specifically, in the pig manure organic fertilizer treatment group, the abundance of coexisting ARGs was the highest, followed by the sheep manure organic fertilizer treatment group, with both surpassing the control group (Fig. 2). This aligns with previous research, indicating that soil and feces-derived ARGs were significant sources of resistance genes found in vegetables (Chen et al 2017, Zhang et al 2019b, Zhu et al 2017). In order to further predict the transmission of ARGs from fertilized soil to different onion tissues, we attempted to identify potential pathways within the soil-plant system . In the fertilized group, 36 coexisting ARGs, including 17 newly introduced ones, were identified between onion roots and rhizospheric soil (Fig. 2). These newly introduced ARGs likely originated from the soil amendment with poultry manure and appear to have been acquired through endophytic bacteria within the root system. In addition, eight ARGs (aph(6)-Ic, aph(9)-Ib, blaOXY, dfrA14, ermO, erm35, mcr-1, cmlv) were shared among rhizospheric soil, roots, and bulb tissues. These genes likely transferred to onion directly through contact with soil or moved from endophytic bacteria in the root system to the bulb tissues.
3.3 Microbial community composition of rhizosphere soil and onion
Figure 3
The bacterial community of the onion inter-root soil consisted mainly of 12 dominant phyla (relative abundance > 0.1%), of which the Actinobacteria and Ascomycetes phyla accounted for > 80% of the total. The α-diversity indices of the inter-root soil microbial communities of both fertiliser groups at the planting stage were higher than those of the control group, which implies that fertiliser application increased the microbial diversity of the rhizosphere soil (Fig. S6). At harvesting stage, the difference in α index between the inter-root soils of the fertiliser groups and the control group was small (Fig. S6), which was similar to the results of the previous study: the soil microbiome would gradually stabilise after a period of fertiliser application (Ren et al 2020).
A total of 14 phulum comprised the onion endophyte community, with Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Patescibacteria being the dominant phylum (relative abundance > 00.1) (Fig. 3A). PCoA analysis based on weighted single cleavage distances explained 81% of the changes in bacterial community structure (Fig. 3B), and by comparison we found that the application of pig manure organic fertiliser increased the anamorphic phyla by 12% and 28%, decreased the thick-walled phyla by 10% and 23%, and decreased the actinomycetes by 5% and 4% in onion roots and bulbs (Fig.3A). The application of sheep manure reduced the proportion of Ascomycetes and Anaplasma spp. in onion roots by 11 and 2 per cent, increased Ascomycetes by 37 per cent and reduced Thick-walled Ascomycetes by 42 per cent in bulbs (Fig. 3A). The results showed that organic farming practices altered the microbial composition in onion roots and bulbs, which is consistent with previous findings that the effect of fertilisation on the composition of endophytic microbial communities in plants (Huang et al 2021).
Figure 4
Procrustes analysis revealed a significant correlation between the presence and distribution of ARGs in onion roots and bulbs and the bacterial community structure at the phylum level (M2 = 4.372, p < 0.01) (Fig. S4A), suggesting that the composition of bacteria in onion may be a key factor influencing the differences in ARG abundance. To further investigate the relationship between bacterial communities and ARGs, we constructed a network between ARG subtypes and bacterial taxa (Fig. S4B). We identified 20 bacterial genera that could potentially serve as hosts for ARGs. For instance, Lachnoclostridium, typically found in the gut microbiome, was identified as a potential host for aminoglycoside resistance genes (aadA5, aadA16, aac(6')-I, aac(3)-IIIa), as well as MDR (vatA, ceoA), and MLSB (erm35) resistance genes.Additionally, Pantoea, commonly found in the environment, was identified as a potential host for various ARGs, including five MDR (mdtA, mexB, mexE, oprD, ttgB), four aminoglycoside resistance genes (aadA5, aph(6')-Id, aph(3')-Ib, aph(6)-Ic), three β-lactamase genes (blaACT, blaOXY, blMIR), and three tetracycline resistance genes (tetA, tetR, tet44), among others. It's worth noting that among these hosts, including Clostridium_sensu_stricto_1, Pseudomonas, Bacillus, and Stenotrophomonas, there are four potentially pathogenic genera to humans (http://www.globalrph.com/bacterial-strains.htm). Among them, Pseudomonas is a potential host for 19 subtypes of ARGs which could potentially pose a greater threat to the ecological environment and human health.
3.4 The impact of root exudates on ARGs in onion
Figure 5
Onion, rich in phenolic compounds, release phenolic metabolites from their roots, affecting their growth and influencing the assembly and maintenance of the rhizosphere microbial community (Baetz and Martinoia 2014). We selected 53 phenolic compounds closely related to plants and analyzed a total of 29 of these compounds. Principal component analysis of root exudates among treatments revealed that PC1 and PC2 components accounted for 27% and 16.7% of the total variance, respectively (Fig. 5A). The volcano plot based on metabolites indicated that pig manure organic fertilizer application increased the levels of Quercetin, Protocatechuic acid, Apigenin, and P-coumaric acid while decreasing the levels of Rutin in onion roots during harvesting compared to the control group (Fig. 5B). In the sheep manure organic fertilizer treatment group, Catechin, Quercetin, Syringic acid, Prunin, Kaempferol, and Epicatechin and Rutin were upregulated, while Luteolin was downregulated relative to the control group (Fig. 5C). The results indicate that fertilization alters the composition and content of phenolic compounds in onion roots. Quercetin and Kaempferol may recruit beneficial microorganisms in the rhizosphere, epicatechin can act as an inhibitor for cell wall-invading enzymes and plant lipoxygenases, Rutin can reduce the damage caused by pathogenic rhizosphere microbial communities to plants, and Syringic acid plays a crucial role in local and systemic resistance against plant pathogens. These phenolic compounds that significantly changed in the root system of the fertilized group further influence the presence of ARGs in onion by affecting the composition of rhizospheric soil microorganisms and their potential colonization in plants (Hassan and Mathesius 2012, Jain et al 2015, Nautiyal et al 2002, Rivas-San Vicente and Plasencia 2011). To further demonstrate the relationship between onion root phenolic secretion and ARGs in onion roots, we conducted a network analysis of the 29 phenolic secretions in onion roots, rhizospheric soil, and potential hosts of ARGs in onion (Fig. S7). We found that the rhizospheric soil and onion root endophytic microbial communities, which act as carriers and transmitters of ARGs, are significantly influenced by root secretions. Upregulated ions are positively correlated with various potential hosts. For example, Quercetin is positively correlated with Bradyrhizobium and Mesorhizobium in rhizospheric soil, and with Bacillus and Pantoea in onion roots. Additionally, the relative abundance of these four potential hosts of ARGs in onion from the fertilized group is higher than that in the control group. In conclusion, we infer that the differences in phenolic compounds in onion root systems after fertilization are one of the reasons for the differential enrichment of ARGs in onion roots in different treatment groups. This is likely mediated indirectly through the regulation of phenolic compounds on the host microorganisms in onion rhizospheric soil and onion roots.
3.5 The influence of MGEs, trace elements and soil physicochemical properties on ARGs in onion
Figure 6
The selected variables explained 97.95% and 97.45% of the total variation in ARGs in onion root and bulb tissues, respectively. Among the factors we've taken into account, the shifts in ARGs were predominantly driven by MGEs, accounting for a substantial 43.36% in root tissues and an even more significant 49.59% in bulb tissues (Figs. 6A and 6B). This underscores the tight relationship between ARG abundance and integrase genes and plasmids, aligning with prior research findings(Chen et al., 2013). We observed a significant positive correlation between intl1 (23%) and intl2 (10%) and ARGs. Similarly, previous research found that intl1 can carry various ARGs and HGT among microorganisms(Zhang et al., 2018). IS1247 and IncI1_repI1 detected in onion roots and scales were also significantly positively correlated with ARGs. Integratrons are typically found on widely distributed transposons and plasmids. Transposons can capture integrons to form new species with greater potential for ARG mobility(Wolters et al., 2014). Integrons were typically found on widely distributed transposons and plasmids, with transposons capable of capturing integrons to create new species with enhanced ARG mobility. Therefore, the detected ARGs in onions might co-occur with transposases, allowing for their simultaneous spread through HGT. Fertilization altered the abundance of MGEs in both onion roots and scales. Application of pig manure organic fertilizer increased the abundance of intl1 by 7.7 times in onion root tissues and by 22 times in scales, while in the sheep manure organic fertilizer treatment, the abundance of intl1 in scales was 1.98 times that of the control group. This pattern mirrors the presence of ARGs in onions. In conclusion, MGEs are likely a factor influencing the dynamics of ARGs in soil.
The explanation percentages of trace elements on the total variation of ARGs in onion roots and scales were 39.5% and 37.2%, respectively. B and Cu in onion root tissues explained a significant portion of the variation in ARGs, accounting for 12.1% and 9.0%, respectively (p < 0.05) (Fig. 6A). In bulb, Cu and B explained 10% and 5% of the ARG variation (p < 0.05) (Fig. 6B). This suggests a certain relationship between these two trace elements and the presence of ARGs in onions. Previous studies have indicated that boron plays a crucial role in cell wall formation, while copper is involved in some reactions related to cell wall synthesis(Han et al., 2008; Printz et al., 2016). The cell wall plays a vital role in various aspects of plant growth and development, as well as in responding to external environmental factors and interactions with symbiotic organisms and pathogens(Ishida & Noutoshi, 2022). Excess levels of boron and copper may impact the structure and stability of the cell wall, potentially affecting the colonization of bacterial hosts in onions. This could alter interactions between bacteria and plants, indirectly influencing the presence of ARGs in onions. Elevated concentrations of boron and copper can potentially induce oxidative stress, leading to an excessive production of reactive oxygen species within cells, which can damage cell structures and functions(Liu et al., 2014; Riaz et al., 2018). This could potentially impact the plant's own immune system, as well as its growth, development, and metabolism, which may further influence the expression and activity of ARGs within plant cells. Furthermore, studies have indicated that copper can induce metal resistance genes in plant-associated microorganisms. When ARGs are found on the same genetic elements as MGEs or within the same bacterial strains, they can exhibit co-resistance, thereby increasing the frequency of horizontal gene transfer. This makes ARGs more likely to be transferred among microorganisms(Baker-Austin et al., 2006). Fertilization has altered the trace element levels in onions. Pig manure organic fertilizer increased boron (B) and copper (Cu) content in onions by 28% and 18%, respectively. Both pig and sheep manure fertilizers had similar effects on B and Cu levels. This could influence ARGs in onions, but further research is needed to confirm the complex mechanisms.
3.6 Risk assessment of ARG in onion bulbs
Onion bulbs are the main part of people's daily consumption, and we isolated a total of five bacterial strains exhibiting three antibiotic resistances, namely Bacillus, Paenibacillus, Fictibacillus, Streptomyces and Weizmannia, of which Bacillus and Streptomyces accounted for greater than 1% of the endophytic bacterial community of onion bulbs.The five strains carried a total of 12 ARGs and all carried at least one ARG, with Fictibacillus and Streptomyces carrying seven and five ARGs, respectively (Fig. S8). The ARGs analysed on these organisms were located on the chromosome, but there was still a risk of transferring them to human pathogens and the risk was less than if the ARGs appeared on the plasmid.
The onion scales, a primary edible part, showed an average gene copy number of approximately 1.1×106, 4.2×106, and 2.4×106 per gram of tissue in the control, pig manure organic fertilizer-treated group, and sheep manure organic fertilizer-treated group, respectively. To better evaluate the effect of fertilization on the edible risk of ARGs in onion, we calculated the potential edible risk of ARGs in onion scales for each treatment. The assessment values for pig manure organic fertilizer, sheep manure organic fertilizer, and the control group were approximately 214.4, 292.7, and 258.6, respectively. This indicated that fertilization significantly increased the edible risk of ARGs in onion, with pig manure organic fertilizer having a greater impact compared to sheep manure organic fertilizer. Currently, research on raw vegetables has shown that the average gene copy number of ARGs in carrot root tissues in the control group and manure-treated group is approximately 4.8×104 and 1.1×106, respectively(Guo et al 2021). For Coriandrum sativum, Cichorium endivia, Lactuca sativa, and Brassica chinensis treated with chicken manure fertilization, the average gene copy numbers of ARGs in their leaves are approximately 1.0×108, 3.0×108, 2.6×109, and 3.0×109 (Guo et al 2021). The study shows that ARGs' average gene copy numbers differ between vegetables with edible tissues above ground (Chinese cabbage and lettuce) and those with edible tissues partially or entirely underground (onion and carrot). However, these comparisons are influenced by various factors like planting conditions, fertilizers, and detection methods. Nonetheless, research on ARGs in raw vegetables is vital for assessing food safety and monitoring environmental health, warranting attention (Lin et al 2023). Furthermore, it's undeniable that fertilization increases the consumption risk of ARGs in the edible parts of vegetables.