This is the accepted version of the article: Chen, Qing-Lin; Cui, Hui-Ling; Su, Jian-Qiang; [et al.]. «Antibiotic Resistomes in Plant Microbiomes». Trends in Plant Science, Vol. 24, Issue 6 (June 2019), p. 530-541. DOI 10.1016/j.tplants.2019.02.010 This version is avaible at https://ddd.uab.cat/record/218327 under the terms of the license Antibiotic Resistomes in Plant Microbiomes Qing-Lin Chen, Hui-Ling Cui, Jian-Qiang Su, Josep Penuelas, and Yong-Guan Zhu Abstract The plant microbiome, the exterior interface between plants and the environment, acts as a biological barrier to prevent invasion by foreign pathogens. Genomic sequencing and “omic” analyses have found that a few bacterial phyla predominate in the aerial components of various plants and have identified the involvement of plant immunological stress responses in the assembly of the belowground habitat. The plant microbiome, the interface between human and environmental microbiomes, represents a major pathway by which humans are exposed to environmental microbes and antibiotic resistomes. In this review, we first highlight the main findings in plant microbiomic research addressing two fundamental questions, who is there and what do they do, and then focus mostly on the dissemination of the resistomes of plant microbiomes by food consumption or direct contact. Microorganisms associated with plants may alter the traits of the human microbiome associated with human health, but this alteration has not received enough attention. 1 Plant microbiomes - who is there? Plants do not grow as axenic organisms in nature but are populated by diverse microorganisms, known as the plant microbiome (Bulgarelli et al., 2013, Muller et al., 2016; Penuelas and Terradas, 2014). Microorganisms can generally colonize all plant tissues (Turner et al., 2013), but we will focus on the phyllosphere, the aerial components of plants, and the rhizosphere, the belowground habitat of plant roots. Our knowledge of phyllospheric microbiology has historically lagged behind our knowledge of the rhizosphere (Vorholt, 2012). This situation has changed in recent years with the advances of next-generation sequencing (NGS) technologies and corresponding bioinformatic tools and with increasing affordability (Bai et al., 2015, Vorholt et al., 2017). A large and rapidly expanding body of phylogenetic information about plant microbiomes is becoming available, which will tell us who is there (Delmotte et al., 2009, Bulgarelli et al., 2012, Horton et al., 2014, Edwards et al., 2015). Recent studies have reported that only a few phyla (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria) are dominant in plant microbiomes across host species such as Arabidopsis thaliana (Lundberg et al., 2012, Bodenhausen et al., 2013), Brassica chinensis (Chen et al., 2017), soybean (Delmotte et al., 2009, Mendes et al., 2014), grapevine (Zarraonaindia et al., 2015) and several tree species (Redford et al., 2010). The presence of some microorganisms may be due to stochastic events (Vorholt et al., 2017), but a core of shared taxa at the phylum and lower levels have been identified within plant microbiotas that consistently and reproducibly establish and are 2 adapted to life in the phyllosphere (Muller et al., 2016). Various studies have also attempted to identify core microbiota across phylogenetically distinct plants or multiple accessions of one plant species or occupying more than one plant compartment (Delmotte et al., 2009, Zarraonaindia et al., 2015, Muller et al., 2016). The evidence suggests a widespread “global” core community across host species, space and time. The phyllosphere is relatively nutrient poor and subject to extremes of temperature, radiation and moisture (Vorholt, 2012). In contrast, the rhizosphere is a region of rich, largely soil-derived, microbial diversity influenced by the deposition of plant mucilage and root exudates (Kent & Triplett, 2002; Preece and Penuelas, 2016). Except for the core microorganisms established in the phyllosphere, plant roots grow within extremely diverse microbial communities but assemble a taxonomically limited microbiome (Lebeis et al., 2015). For example, the root microbiota of A. thaliana, a common model plant for the study of molecular biology and genetics, has substantial compositional consistencies not only across multiple continental soils but also across multiple Arabidopsis lineages (Bulgarelli et al., 2012, Lundberg et al., 2012, Beattie, 2015). We have summarized the bacteria in the phyllospheres and rhizospheres of various plant species based on published data (Table 1). Plant microbiomes - what do they do? The plant microbiome, as the second genome of the plant, can influence host phenotypes such as growth and tolerance to pathogens, pests and environmental stresses (Mendes et al., 2011, Lau & Lennon, 2012, Beattie, 2015, Muller et al., 2016, Bakker 3 et al., 2018). The plant-root system, where nutrients are taken up by the host and plants are exposed to surrounding microbes, has been suggested to be functionally analogous to the animal gastrointestinal system (Berendsen et al., 2012, Haney & Ausubel, 2015). An increasing body of evidence also suggests the importance of this root microbiome, which consists of the entire complex of rhizospheric microbes, their genetic elements and their interactions, in increasing plant growth and decreasing susceptibility to diseases caused by pathogenic fungi, bacteria, viruses and nematodes (Kloepper et al., 2004, Yang et al., 2009, Lebeis et al., 2015, Mendes et al., 2018). Plant microbiomes extend the host phenotypes that affect host reproductive fitness, so plants have likely evolved mechanisms to attract and maintain microbial populations based on their key functional capabilities by innate or induced, stimulatory or inhibitory, activities and may have thus co-evolved with their associated microbes (Panke-Buisse et al., 2015, Theis et al., 2016). In addition to the direct effects on deleterious rhizospheric microbes, many rhizospheric microbiotas elicit beneficial physical or chemical changes to boost the defensive capacity in aboveground plant components, a process referred to as induced systemic resistance (ISR) (van Loon et al., 1998, Bakker et al., 2013). ISR in leaves can be activated by specific rhizospheric microbiotas colonizing roots and is mediated by jasmonic acid and ethylene (Pieterse et al., 2012, Belkhadir et al., 2014, Carvalhais et al., 2015). In contrast, salicylic acid, a key immune regulator in leaves, also drives selection from the available microbial communities to form the root microbiome, indicating that plant immune signaling and root microbiomic assembly are functionally linked and suggesting potential cross-talk between above- 4 and belowground plant components (Turner et al., 2013, Lebeis et al., 2015). Plant microbiomes may also influence ecosystem function and global biogeochemical cycles (Philippot et al., 2013, Turner et al., 2013, Laforest-Lapointe et al., 2017) by their role in extending the host genome and may contribute to the host phenotype (Muller et al., 2016). The diversity of the foliar microbiome has been positively linked to ecosystem productivity, and this link is also maintained after accounting for the contribution of host diversity (Laforest-Lapointe et al., 2017). Phyllospheric microbiome play also a key role in the large amounts of biogenic volatile organic compounds emitted annually by plants to the atmosphere (Penuelas et al 2015, Farre- Armengol et al 2016). Moreover, the soils of agricultural crops can stimulate microbial denitrification and methanogenesis, contributing to emissions of N2O and methane, respectively (Turner et al., 2013). These gases not only represent a loss of carbon and nitrogen from the system but also exacerbate the greenhouse effect. Plant microbiomic resistome – movement and human health The plant microbiome is the interface between the human and environmental microbiomes and represents a major pathway by which humans are exposed to antibiotic-resistant bacteria and resistance genes that are naturally present in the environment. Our knowledge of antibiotic resistomes in nonclinical environments is currently increasing rapidly. Wastewater-treatment plants and operations producing concentrated animal feed are two important reservoirs of antibiotic resistomes associated with humans, and a large body of data is available on the profiles of antibiotic 5 resistomes (Baquero et al., 2008, Rizzo et al., 2013, Zhu et al., 2013, Su et al., 2017). The application of sewage sludge and animal manure to agricultural land are common practices for recycling nutrients and increasing crop productivity and also represent a major method for the disposal of waste. Many studies have investigated the potential spread of resistomes in the environment by agricultural organic fertilization under both laboratory and field conditions using qPCR, high-throughput qPCR (HT-qPCR) and metagenomics. Such data have clearly demonstrated that procedures of organic production facilitate the dissemination of antibiotic resistomes in soil environments (Fahrenfeld et al., 2014, Udikovic-Kolic et al., 2014, Chen et al., 2016), suggesting the need to optimize waste disposal for sustainable agriculture and waste recycling (Burch et al., 2017). These studies, however, have mostly focused on the distribution of antibiotic resistomes in soil or wastewater, with little attention to the subsequent spread of antibiotic resistomes via plant/crop microbiomes. Knowledge of plant microbiomic resistomes has largely lagged behind our knowledge of resistomes in their surrounding environments such as soil, water and air (Baquero et al., 2008, Martinez, 2008, Allen et al., 2010, McEachran et al., 2015). Only limited examples are available but nevertheless provide crucial evidence, prompting studies that examine antibiotic resistomes in plant microbiomes. We have summarized the plant microbiomic resistomes based on the current data for food-borne antibiotic-resistant commensal bacteria and resistance genes (Table 2). An important finding among these rare examples was that resistome abundance is approximately eight-fold higher in 6 organically than conventionally produced lettuce (Zhu et al., 2017). The lettuce samples used in this study were collected directly from supermarkets and may be consumed with little processing, so the lettuce poses a substantial risk for the transfer of antibiotic resistance to humans. Antibiotic-resistance genes (ARGs) assessed by PCR-based methods, however, cannot provide information about the functionality and mobility of the ARGs, so more studies using culture-dependent and metagenomic approaches to investigate a broad range of vegetables are still required to reach broader generalizations. Interestingly, some resistomes were detected in plant microbiomes but not their surrounding environments, including soils, suggesting an intrinsic core resistome in the plant microbiome (Chen et al., 2017). This result is very encouraging, because it is important for deriving the baseline of the resistome before evaluating its dissemination via the plant microbiome. Much of the resistome in the plant microbiome clearly overlapped with the surrounding resistome, indicating that these resistomes could be acquired and that soil resistomes might be a major source for plant resistomes. Direct supporting evidence from additional research, however, is needed. Scenario 1 via the food chain Food consumption represents a major route of exposure of the human microbiome to the environmental microbiome; the plant microbiome acts as a bridge connecting these two microbiomes. Recent findings suggested that the daily consumption of food products such as raw leafy vegetables and fruits is likely a major route for the 7 dissemination of antibiotic resistomes from the food chain to human hosts (Zhang et al., 2011, Verraes et al., 2013). For example, HT-qPCR has identified hundreds of ARGs in the plant microbiome, and organic fertilization has markedly increased the occurrence of ARGs, suggesting that plants can acquire exogenous ARGs (Chen et al., 2017). Plants can also take up antibiotics from soil amended with animal manure (Kumar et al., 2005), an organic fertilizer commonly used in agriculture, which may apply selection pressure on the plant microbiome and contribute to the emergence and spread of antibiotic resistance to the human food chain. Most of the plant commensal carriers of ARGs are fortunately nonpathogenic (Zhang et al., 2011), but their potential involvement in the spread of ARGs to other bacterial genera, including human pathogens, by the horizontal gene transfer (HGT) of mobile genetic elements (MGEs) is a concern (Rossi et al., 2014). The class I integron- integrase gene (intI1) and genes encoding transposases are common on harvest vegetables, indicating the possibility of gene transfer in the phyllosphere (Wang et al., 2015). Phyllospheres and rhizospheres are two key areas for horizontal gene transfer in plant and soil habitats due to the high possibility of cells to cluster, forming biofilms in phyllospheres, and bacterial metabolic rates and the mobility of bacteria and MGEs are high in rhizospheres (van Elsas et al., 2003). Scenario 2 via food processing, preservation techniques and transportation Food is often processed and preserved to obtain safe products, extend shelf lives and maximize profits. These processes include drying, heating to reduce water content, 8 cooling, acidification, modified-atmosphere packaging by decreasing oxygen and increasing carbon dioxide, freezing, mild pasteurization and UV irradiation (Gould, 1996, Rico et al., 2007). Their effects on bacterial flora depend on the combination, type and conditions of the technologies used. Most processing methods can reduce the number of food-borne bacteria and thus the potential risks. For example, freezing and UV irradiation can stress or damage bacterial cells and ultimately kill or inactivate the bacteria (Rico et al., 2007). The dead bacterial cells, however, can be lysed due to cell- wall damage. DNA, including possible ARGs, is consequently released into the environment. The transformation of extracellular DNA plays an important role in HGT and the dissemination of antibiotic resistance in the environment (Levy-Booth et al., 2007, Nielsen et al., 2007). The movement of microorganisms has historically mainly been due to natural factors, such as air and water currents or even natural events (Zhu et al., 2017). Globalization has dramatically affected these dynamics, with cheap and highly efficacious long-range transport systems allowing many transnational corporations to manufacture food products in regions with less expensive labor, using ingredients from around the world and then transporting them worldwide (Saker et al., 2004). Bacteria associated with plants cannot be completely eliminated during production, storage, processing and packaging, so they are inevitably transported with their host material by global trading, which has profound impacts on the evolution and dissemination of antibiotic resistomes and may cause unpredictable infections. For example, outbreaks of Salmonella poona infections in the USA associated with eating melons imported from Mexico have been 9 linked to unhygienic irrigation and packaging at source farms (Saker et al., 2004). The situation is exacerbated by the cultivation of non-indigenous crops in some low-income regions to meet market demands, and these crops may be more susceptible to indigenous pathogens. For example, Guatemalan raspberries became contaminated with the protozoan Cyclospora, causing outbreaks of gastroenteritis in the USA and Canada (Saker et al., 2004). The development and implementation of national and international guidelines for assessing the biological risk of microbiomes associated with food is a strategic priority for alleviating these threats. Concluding remarks and outlook Summary 1. The two fundamental questions about plant microbiomes, who is there and what do they do, are becoming increasingly well defined. The phyllosphere has historically been less intensively studied than the rhizosphere, but this situation is beginning to change. The available data suggest that the plant microbiome is dominated by bacteria and bacterial communities on above- and belowground organs, has a defined taxonomic structure and is consistently composed of a few phyla, mainly Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes. The bacterial communities in plant microbiomes have relatively low diversity but have profound consequences on host health and functional traits and even on ecosystem productivity and global climate change. 2. We advocate that more attention should be paid to the antibiotic resistome in plant microbiomes due to the public health threat posed by antibiotic resistance and 10 because the plant microbiomes can act as a bridge connecting the human and environmental microbiomes. Our current understanding of antibiotic resistomes associated with plants is still in its infancy, but experimental evidence suggests that the food chain is the main route by which human microbiomes are exposed to the antibiotic resistomes in plant microbiomes. Globalization may unpredictably disseminate antibiotic resistomes associated with plants, but we believe that different kinds of experiments and surveys and international and interdisciplinary collaborations will be needed to obtain an integrated understanding of plant antibiotic resistomes. Acknowledgements This research was supported by the National Natural Science Foundation of China (21210008, 41571130063) and the Strategic Priority Research Program of Chinese Academy of Sciences (XDB15020402). JP would like to acknowledge the financial support from the European Research Council Synergy grant ERC-SyG-2013-610028 IMBALANCE-P 11 Figures and tables Figure 1 Schematic overview of the plant microbiome. The aboveground components of plants, collectively known as the phyllosphere, represent an inherently open and variable habitat dominated by leaves. Antibiotic resistomes associated with phyllospheres can influence human microbiomes by direct consumption and contact or by the food chain. The belowground components of plant roots are collectively known as the rhizosphere. The good aspects of the rhizospheric microbiome include nutrient acquisition, tolerance to abiotic stress, protection against viruses and other pathogens via induced systemic resistance (ISR) and activation of the plant immune system in response to foliar pathogenic attack (immune response). The bad aspects of the rhizospheric microbiome include possible facilitation of the spread of antibiotic resistomes and risks to human health from contaminated food. 12 Figure 2 Scenario of the plant microbiomic antibiotic resistome. Direct contact and food chains represent major pathways by which human microbiomes are exposed to environmental resistomes. Globalization may exacerbate the dissemination of resistomes worldwide via plant resistomes. 13 Dominant Host species Phyllosphere Rhizosphere Methodology Reference phyla Proteobacteria  Illumina sequencing Actinobacteria Ruiz-Perez et Espeletia of 16S rRNA gene Proteobacteria al. (2016)  amplicons Acidobacteria Acidobacteria Bulgarelli et  Planctomycetes al. (2012) Proteobacteria Pyrosequencing of Arabidopsis Acidobacteria 16S rRNA gene thaliana Actinobacteria amplicons Lundberg et  Bacteroidetes al. (2012) Proteobacteria Proteobacteria Illumina shotgun Hordeum vulgare Actinobacteria sequencing and Bulgarelli et (barley)  Bacteroidetes pyrosequencing of al. (2015) 16S rRNA gene amplicons Proteobacteria Bodenhausen Pyrosequencing of Bacteroidetes et al. (2013),  16S rRNA gene Actinobacteria Horton et al. amplicons Arabidopsis (2014) thaliana Proteobacteria Actinobacteria Pyrosequencing of Bodenhausen  Bacteroidetes 16S rRNA gene et al. (2013) amplicons Proteobacteria 16S rRNA gene Delmotte et  Actinobacteria clone libraries al. (2009) Glycine max Bacteroidetes (soybean) Proteobacteria 454 metagenomic Mendes et al.  Actinobacteria shotgun sequencing (2014) Firmicutes Pinus flexilis Proteobacteria Pyrosequencing of  Pinus strobiformis Bacteroidetes 16S rRNA gene Catalpa speciosa Bacteroidetes amplicons Fraxinus Firmicutes Redford et al. pennsylvanica Actinobacteria (2010)  Fraxinus americana Magnolia Proteobacteria soulangeana 14 Table1 Dominant bacterial phyla in rhizospheres and phyllospheres. Proteobacteria Pyrosequencing of Peiffer et al. Maize  Bacteroidetes 16S rRNA gene (2013) Actinobacteria amplicons  Proteobacteria Illumina sequencing Proteobacteria Zarraonaindia Grapevine of 16S rRNA gene  Acidobacteria et al. (2015) amplicons Bacteroidetes Proteobacteria Rastogi et al.  Firmicutes Pyrosequencing of (2012) Lettuce Proteobacteria 16S rRNA gene Cardinale et  Bacteroidetes amplicons al. (2015) Chloroflexi Proteobacteria Illumina sequencing Edwards et al.  Acidobacteria of 16S rRNA gene (2015) Chloroflexi amplicons Proteobacteria 454 metagenomic Rice  Actinobacteria shotgun Knief et al. Proteobacteria sequencing, 16S (2012)  rRNA gene clone Chloroflexi libraries Proteobacteria  Firmicutes Illumina sequencing Chen et al. Edible rape Proteobacteria of 16S rRNA gene (2017)  Chloroflexi amplicons Actinobacteria Proteobacteria Pyrosequencing of Yeoh et al. Sugarcane  Bacteroidetes 16S rRNA gene (2016) Actinobacteria amplicons Actinobacteria Pyrosequencing of Inceoglu et al. Potato  Proteobacteria 16S rRNA gene (2011) amplicons 15 Table 2 Recent findings for antibiotic resistomes in plant microbiomes. Host Major findings Methodology Reference 1) All targeted genes were detectable on vegetables in the presence of sewage but not detectable on Marti et al. Lettuce vegetables in Culture- (2014), Rahube Tomato unamended soil. dependent method and et al. (2014), Radish 2) A target was never quantitative PCR Rahube et al. Carrot more abundant on a (2016) vegetable grown in the presence than the absence of sludge a year after the application of sewage sludge. 1) Over a hundred unique ARGs were detected in the phyllosphere and foliar endophyte of market High-throughput Lettuce Zhu et al. (2017) lettuce. quantitative PCR 2) Organic lettuce harbored more ARGs than conventionally produced lettuce. 1) Soil receiving manure was enriched in ARBs and ARGs, but the abundance of ARBs for Tomato vegetable grown in Radish manure-fertilized soil did Carrot not correspondingly Culture- Marti et al. Cucumber increase. dependent method and (2013) Pepper 2) Numerous ARGs were quantitative PCR Lettuce detected on vegetables grown in control soil. Only a smaller number of ARGs were additionally detected on vegetables grown only in manured Edible rape 1) Detected ARGs High-throughput Chen et al. (2017) 16 enriched after quantitative PCR and application of struvite. Illumina sequencing 2) Twenty-five ARGs were innate in phyllosphere but not detectable in soil 3) Bacterial taxa shared between rhizosphere and phyllosphere were significantly correlated with ARGs. Biochar amendment can be a practical strategy to protect crops from ARGs, Quantitative PCR and Ye et al. (2016), Lettuce and human pathogenic Illumina sequencing Duan et al. (2017) bacteria (HPB) contamination 1) Five subtypes of ARGs (sulI, tetG, tetC, tetA and tetM) and a class I integron-integrase gene Lettuce (intI 1) were detected in Wang et al. Quantitative PCR Endive both endophytes and (2015) phyllospheres. 2) Planting can affect the distribution of ARGs in manure-amended soil 17 References Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J & Handelsman J (2010) Call of the wild: antibiotic resistance genes in natural environments. Nature reviews Microbiology 8: 251-259. Bai Y, Muller DB, Srinivas G, et al. (2015) Functional overlap of the Arabidopsis leaf and root microbiota. Nature 528: 364-369. Bakker P, Pieterse CMJ, de Jonge R & Berendsen RL (2018) The Soil-Borne Legacy. Cell 172: 1178-1180. Bakker PA, Doornbos RF, Zamioudis C, Berendsen RL & Pieterse CM (2013) Induced systemic resistance and the rhizosphere microbiome. Plant Pathol J 29: 136-143. Baquero F, Martinez JL & Canton R (2008) Antibiotics and antibiotic resistance in water environments. Curr Opin Biotechnol 19: 260-265. Beattie GA (2015) Microbiomes: Curating communities from plants. Nature 528: 340-341. Belkhadir Y, Yang L, Hetzel J, Dangl JL & Chory J (2014) The growth-defense pivot: crisis management in plants mediated by LRR-RK surface receptors. Trends Biochem Sci 39: 447-456. Berendsen RL, Pieterse CM & Bakker PA (2012) The rhizosphere microbiome and plant health. Trends Plant Sci 17: 478-486. Bodenhausen N, Horton MW & Bergelson J (2013) Bacterial communities associated with the leaves and the roots of Arabidopsis thaliana. PloS one 8: e56329. Bulgarelli D, Schlaeppi K, Spaepen S, Ver Loren van Themaat E & Schulze-Lefert P (2013) Structure and functions of the bacterial microbiota of plants. Annual review of plant biology 64: 807-838. Bulgarelli D, Garrido-Oter R, Munch PC, Weiman A, Droge J, Pan Y, McHardy AC & Schulze-Lefert P (2015) Structure and function of the bacterial root microbiota in wild and domesticated barley. Cell Host Microbe 17: 392-403. Bulgarelli D, Rott M, Schlaeppi K, et al. (2012) Revealing structure and assembly cues for Arabidopsis root-inhabiting bacterial microbiota. Nature 488: 91-95. Burch TR, Sadowsky MJ & LaPara TM (2017) Effect of Different Treatment Technologies on the Fate of Antibiotic Resistance Genes and Class 1 Integrons when Residual Municipal Wastewater Solids are Applied to Soil. Environmental science & technology. Cardinale M, Grube M, Erlacher A, Quehenberger J & Berg G (2015) Bacterial networks and co- occurrence relationships in the lettuce root microbiota. Environmental microbiology 17: 239-252. Carvalhais LC, Dennis PG, Badri DV, Kidd BN, Vivanco JM & Schenk PM (2015) Linking Jasmonic Acid Signaling, Root Exudates, and Rhizosphere Microbiomes. Mol Plant Microbe Interact 28: 1049-1058. Chen Q, An X, Li H, Su J, Ma Y & Zhu YG (2016) Long-term field application of sewage sludge increases the abundance of antibiotic resistance genes in soil. Environment international 92-93: 1-10. Chen QL, An XL, Zhu YG, Su JQ, Gillings MR, Ye ZL & Cui L (2017) Application of Struvite Alters the Antibiotic Resistome in Soil, Rhizosphere, and Phyllosphere. Environmental science & technology 51: 8149-8157. Delmotte N, Knief C, Chaffron S, Innerebner G, Roschitzki B, Schlapbach R, von Mering C & Vorholt JA (2009) Community proteogenomics reveals insights into the physiology of phyllosphere bacteria. Proceedings of the National Academy of Sciences of the United States of America 106: 16428-16433. Duan M, Li H, Gu J, Tuo X, Sun W, Qian X & Wang X (2017) Effects of biochar on reducing the abundance of oxytetracycline, antibiotic resistance genes, and human pathogenic bacteria in soil and lettuce. Environmental pollution 224: 787-795. 18 Edwards J, Johnson C, Santos-Medellin C, Lurie E, Podishetty NK, Bhatnagar S, Eisen JA & Sundaresan V (2015) Structure, variation, and assembly of the root-associated microbiomes of rice. Proceedings of the National Academy of Sciences of the United States of America 112: E911-920. Fahrenfeld N, Knowlton K, Krometis LA, Hession WC, Xia K, Lipscomb E, Libuit K, Green BL & Pruden A (2014) Effect of manure application on abundance of antibiotic resistance genes and their attenuation rates in soil: field-scale mass balance approach. Environmental science & technology 48: 2643-2650. Gould GW (1996) Methods for preservation and extension of shelf life. International journal of food microbiology 33: 51-64. Haney CH & Ausubel FM (2015) MICROBIOME. Plant microbiome blueprints. Science 349: 788-789. Horton MW, Bodenhausen N, Beilsmith K, et al. (2014) Genome-wide association study of Arabidopsis thaliana leaf microbial community. Nature communications 5: 5320. Inceoglu O, Al-Soud WA, Salles JF, Semenov AV & van Elsas JD (2011) Comparative analysis of bacterial communities in a potato field as determined by pyrosequencing. PloS one 6: e23321. Kent AD & Triplett EW (2002) Microbial communities and their interactions in soil and rhizosphere ecosystems. Annu Rev Microbiol 56: 211-236. Kloepper JW, Ryu CM & Zhang S (2004) Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp. Phytopathology 94: 1259-1266. Knief C, Delmotte N, Chaffron S, Stark M, Innerebner G, Wassmann R, von Mering C & Vorholt JA (2012) Metaproteogenomic analysis of microbial communities in the phyllosphere and rhizosphere of rice. The ISME journal 6: 1378-1390. Kumar K, Gupta SC, Baidoo SK, Chander Y & Rosen CJ (2005) Antibiotic uptake by plants from soil fertilized with animal manure. Journal of environmental quality 34: 2082-2085. Laforest-Lapointe I, Paquette A, Messier C & Kembel SW (2017) Leaf bacterial diversity mediates plant diversity and ecosystem function relationships. Nature 546: 145-147. Lau JA & Lennon JT (2012) Rapid responses of soil microorganisms improve plant fitness in novel environments. Proceedings of the National Academy of Sciences of the United States of America 109: 14058-14062. Lebeis SL, Paredes SH, Lundberg DS, et al. (2015) PLANT MICROBIOME. Salicylic acid modulates colonization of the root microbiome by specific bacterial taxa. Science 349: 860-864. Levy-Booth DJ, Campbell RG, Gulden RH, Hart MM, Powell JR, Klironomos JN, Pauls KP, Swanton CJ, Trevors JT & Dunfield KE (2007) Cycling of extracellular DNA in the soil environment. Soil Biol Biochem 39: 2977-2991. Lundberg DS, Lebeis SL, Paredes SH, et al. (2012) Defining the core Arabidopsis thaliana root microbiome. Nature 488: 86-90. Marti R, Tien YC, Murray R, Scott A, Sabourin L & Topp E (2014) Safely coupling livestock and crop production systems: how rapidly do antibiotic resistance genes dissipate in soil following a commercial application of swine or dairy manure? Applied and environmental microbiology 80: 3258-3265. Marti R, Scott A, Tien YC, Murray R, Sabourin L, Zhang Y & Topp E (2013) Impact of manure fertilization on the abundance of antibiotic-resistant bacteria and frequency of detection of antibiotic resistance genes in soil and on vegetables at harvest. Applied and environmental microbiology 79: 5701-5709. Martinez JL (2008) Antibiotics and antibiotic resistance genes in natural environments. Science 321: 365-367. McEachran AD, Blackwell BR, Hanson JD, Wooten KJ, Mayer GD, Cox SB & Smith PN (2015) Antibiotics, bacteria, and antibiotic resistance genes: aerial transport from cattle feed yards via particulate matter. 19 Environmental health perspectives 123: 337-343. Mendes LW, Kuramae EE, Navarrete AA, van Veen JA & Tsai SM (2014) Taxonomical and functional microbial community selection in soybean rhizosphere. The ISME journal 8: 1577-1587. Mendes LW, Raaijmakers JM, de Hollander M, Mendes R & Tsai SM (2018) Influence of resistance breeding in common bean on rhizosphere microbiome composition and function. The ISME journal 12: 212-224. Mendes R, Kruijt M, de Bruijn I, et al. (2011) Deciphering the rhizosphere microbiome for disease- suppressive bacteria. Science 332: 1097-1100. Muller DB, Vogel C, Bai Y & Vorholt JA (2016) The Plant Microbiota: Systems-Level Insights and Perspectives. Annu Rev Genet 50: 211-234. Nielsen KM, Johnsen PJ, Bensasson D & Daffonchio D (2007) Release and persistence of extracellular DNA in the environment. Environ Biosafety Res 6: 37-53. Panke-Buisse K, Poole AC, Goodrich JK, Ley RE & Kao-Kniffin J (2015) Selection on soil microbiomes reveals reproducible impacts on plant function. The ISME journal 9: 980-989. Peiffer JA, Spor A, Koren O, Jin Z, Tringe SG, Dangl JL, Buckler ES & Ley RE (2013) Diversity and heritability of the maize rhizosphere microbiome under field conditions. Proceedings of the National Academy of Sciences of the United States of America 110: 6548-6553. Philippot L, Raaijmakers JM, Lemanceau P & van der Putten WH (2013) Going back to the roots: the microbial ecology of the rhizosphere. Nature reviews Microbiology 11: 789-799. Pieterse CM, Van der Does D, Zamioudis C, Leon-Reyes A & Van Wees SC (2012) Hormonal modulation of plant immunity. Annu Rev Cell Dev Biol 28: 489-521. Rahube TO, Marti R, Scott A, Tien YC, Murray R, Sabourin L, Duenk P, Lapen DR & Topp E (2016) Persistence of antibiotic resistance and plasmid-associated genes in soil following application of sewage sludge and abundance on vegetables at harvest. Can J Microbiol 62: 600-607. Rahube TO, Marti R, Scott A, Tien YC, Murray R, Sabourin L, Zhang Y, Duenk P, Lapen DR & Topp E (2014) Impact of fertilizing with raw or anaerobically digested sewage sludge on the abundance of antibiotic-resistant coliforms, antibiotic resistance genes, and pathogenic bacteria in soil and on vegetables at harvest. Applied and environmental microbiology 80: 6898-6907. Rastogi G, Sbodio A, Tech JJ, Suslow TV, Coaker GL & Leveau JH (2012) Leaf microbiota in an agroecosystem: spatiotemporal variation in bacterial community composition on field-grown lettuce. The ISME journal 6: 1812-1822. Redford AJ, Bowers RM, Knight R, Linhart Y & Fierer N (2010) The ecology of the phyllosphere: geographic and phylogenetic variability in the distribution of bacteria on tree leaves. Environmental microbiology 12: 2885-2893. Rico D, Martin-Diana AB, Barat JM & Barry-Ryan C (2007) Extending and measuring the quality of fresh-cut fruit and vegetables: a review. Trends Food Sci Tech 18: 373-386. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, Michael I & Fatta-Kassinos D (2013) Urban wastewater treatment plants as hotspots for antibiotic resistant bacteria and genes spread into the environment: a review. The Science of the total environment 447: 345-360. Rossi F, Rizzotti L, Felis GE & Torriani S (2014) Horizontal gene transfer among microorganisms in food: Current knowledge and future perspectives. Food microbiology 42: 232-243. Ruiz-Perez CA, Restrepo S & Zambrano MM (2016) Microbial and Functional Diversity within the Phyllosphere of Espeletia Species in an Andean High-Mountain Ecosystem. Applied and environmental microbiology 82: 1807-1817. 20 Saker L, Lee K, Cannito B, Gilmore A & Campbell-Lendrum D (2004) Globalization and infectious diseases: A review of the linkages. Geneva: World Health Organization; (TDR/STR/SEB/ST/04.2). Su JQ, An XL, Li B, Chen QL, Gillings MR, Chen H, Zhang T & Zhu YG (2017) Metagenomics of urban sewage identifies an extensively shared antibiotic resistome in China. Microbiome 5: 84. Theis KR, Dheilly NM, Klassen JL, et al. (2016) Getting the Hologenome Concept Right: an Eco- Evolutionary Framework for Hosts and Their Microbiomes. mSystems 1. Turner TR, James EK & Poole PS (2013) The plant microbiome. Genome biology 14: 209. Udikovic-Kolic N, Wichmann F, Broderick NA & Handelsman J (2014) Bloom of resident antibiotic- resistant bacteria in soil following manure fertilization. Proceedings of the National Academy of Sciences of the United States of America 111: 15202-15207. van Elsas JD, Turner S & Bailey MJ (2003) Horizontal gene transfer in the phytosphere. New Phytol 157: 525-537. van Loon LC, Bakker PA & Pieterse CM (1998) Systemic resistance induced by rhizosphere bacteria. Annual review of phytopathology 36: 453-483. Verraes C, Van Boxstael S, Van Meervenne E, et al. (2013) Antimicrobial resistance in the food chain: a review. International journal of environmental research and public health 10: 2643-2669. Vorholt JA (2012) Microbial life in the phyllosphere. Nature Reviews Microbiology 10: 828-840. Vorholt JA, Vogel C, Carlstrom CI & Muller DB (2017) Establishing Causality: Opportunities of Synthetic Communities for Plant Microbiome Research. Cell Host Microbe 22: 142-155. Wang FH, Qiao M, Chen Z, Su JQ & Zhu YG (2015) Antibiotic resistance genes in manure-amended soil and vegetables at harvest. Journal of hazardous materials 299: 215-221. Yang J, Kloepper JW & Ryu C-M (2009) Rhizosphere bacteria help plants tolerate abiotic stress. Trends in Plant Science 14: 1-4. Ye M, Sun M, Feng Y, et al. (2016) Effect of biochar amendment on the control of soil sulfonamides, antibiotic-resistant bacteria, and gene enrichment in lettuce tissues. Journal of hazardous materials 309: 219-227. Yeoh YK, Paungfoo-Lonhienne C, Dennis PG, Robinson N, Ragan MA, Schmidt S & Hugenholtz P (2016) The core root microbiome of sugarcanes cultivated under varying nitrogen fertilizer application. Environmental microbiology 18: 1338-1351. Zarraonaindia I, Owens SM, Weisenhorn P, et al. (2015) The soil microbiome influences grapevine- associated microbiota. mBio 6. Zhang L, Kinkelaar D, Huang Y, Li YL, Li XJ & Wang HH (2011) Acquired Antibiotic Resistance: Are We Born with It? Applied and environmental microbiology 77: 7134-7141. Zhu B, Chen Q, Chen S & Zhu YG (2017) Does organically produced lettuce harbor higher abundance of antibiotic resistance genes than conventionally produced? Environment international 98: 152-159. Zhu YG, Gillings M, Simonet P, Stekel D, Banwart S & Penuelas J (2017) Microbial mass movements. Science 357: 1099-1100. Zhu YG, Johnson TA, Su JQ, Qiao M, Guo GX, Stedtfeld RD, Hashsham SA & Tiedje JM (2013) Diverse and abundant antibiotic resistance genes in Chinese swine farms. Proceedings of the National Academy of Sciences of the United States of America 110: 3435-3440. 21 Figure legend Figure 1 Schematic overview of plant microbiome. The aboveground parts of plants, collectively called the phyllosphere, represent an inherently open and variable habitat that is dominated by leaves. The phyllosphere-associated antibiotic resistome can influence human microbiome through direct consumption and intact or through food chain. The belowground parts of plants, collectively called the rhizosphere. The good aspects of rhizosphere microbiome including nutrient acquisition, tolerance to abiotic stress, protection against pathogen and viruses via induced systemic resistance (ISR), and activation of the plant immune system in response to foliar pathogen attack (immune response). The bad aspects of rhizosphere microbiome including it may facilities the spread of antibiotic resistome and pose risks to human health through contaminated food. Figure 2 The scenario of plant microbiome antibiotic resistome. Direct intact and food chain represents a major pathway by which expose human microbiome to nature resistome. While globalization may exacerbate the dissemination of resistome worldwide via plant resistome 22