The phyllosphere microbiome and its applications in sustainable agriculture

1 The phyllosphere microbiome and its applications in sustainable agriculture Karen Uchida1 1Department of Plant Sciences, University of Cambridge, UK Summary Plants interact with diverse microorganisms that collectively form a microbiome. These microbial communities play an important role in maintaining plant health and offer promising alternatives to synthetic fertilisers and pesticides which harm the environment. The phyllosphere refers to above-ground portion of a plant. This review provides an overview of the current research on the phyllosphere microbiome in major food crops and discusses its potential applications in agriculture. Further studies are needed to expand our knowledge of microbiome functions and link them to plant genetics. This could unlock novel microbiome-based approaches that support sustainable food production for the growing global population. Introduction Current agricultural practices are detrimental to the environment with the extensive use of fertilisers and pesticides disrupting the global biogeochemical cycles and leading to the loss of natural biodiversity (Campbell et al., 2017). With the global population projected to reach 9.7 billion by 2050, food demand is expected to rise by up to 60% between 2010 and 2050 (UN, 2017; van Dijk et al., 2021). Hence it is essential to shift towards sustainable agricultural practices that maximise food production while minimising environmental impacts (Garnett et al., 2013; Richardson et al., 2023). Harnessing plant-associated microbes is proposed as a promising alternative to synthetic chemicals to enable sustainable food production (Hu, Chen and He, 2022). Plants interact with diverse microorganisms including bacteria, fungi, archaea, protists and viruses that form the plant microbiota (Berg et al., 2020; Zilber-Rosenberg & Rosenberg, 2008). The term microbiome, first introduced by Whipps and his team in 1988, describes a collection of genomes, metabolites and functions of the microbiota and their interactions with its host plant and the surrounding environment (Marchesi and Ravel, 2015). Distinct microbiomes can be found across different parts of a plant including the phyllosphere, which encompasses all above- 2 ground parts including leaves, stems (caulosphere), flowers (carposhere) and fruits (anthosphere) (Figure 1A, B) (Lindow and Brandl, 2003). Leaves are among the largest microhabitats on Earth with their total surface estimated to be twice the size of all land area and inhabited by up to 1026 bacteria (Vorholt, 2012). They are colonised by epiphytes on the surface and endophytes within the internal tissues (Figure 1C) (Sohrabi et al., 2023). Microbial species are heterogeneously distributed across the leaf surface (Figure 1D) (Kusstatscher et al., 2020; Remus-Emsermann et al., 2014; Saarenpää et al., 2024). For example, some bacterial species inhabit glandular trichomes on tomato leaves where Figure 1: Plants interact with diverse microorganisms that collectively form the microbiome. (A) Distinct microbial communities are associated with different plant compartments. (Bpd) Microbiomes consist of a complex network of interactions within and between microbial species. (C) Epiphytes colonise the leaf surface while endophytes inhabit the internal leaf tissues. (D) Bacteria colonising leaf trichomes. Microorganisms occupy distinct spatial niches across the plant surface. 3 plant metabolites accumulate (Kusstatscher et al., 2020). Despite the importance of spatial distribution for microbial interactions with plants and other microbes, it is often overlooked in studies that rely solely on bioinformatic approaches. The phyllosphere microbiome is a complex network of plant-microbe and microbe-microbe interactions (Figure 1B) (Chaudhry et al., 2021). Within this network, microorganisms that are consistently found across samples from a specific habitat or a plant are known as the core microbiomes (Shade and Handelsman, 2012). Computational analyses of microbial networks are used to identify the “hub” or “keystone” species, which interact with multiple species and play a central role in shaping the microbiome (Agler et al., 2016). These key species are targets for manipulating the microbiome as changing their abundance can significantly alter the overall microbiome composition and structure (Toju et al., 2018). Most plant microbiome research has focused on below-ground microbiomes which accounted for over 80% of studies in cereal crops (Michl, Berg and Cernava, 2023). Nevertheless, the phyllosphere microbiome is gaining attention for its role in promoting growth and disease resistance (De Mandal and Jeon, 2023). This review will provide an overview of the current state of phyllosphere microbiome research with a focus on agriculturally important food crops and discuss their potential applications in sustainable agriculture. Main Current phyllosphere microbiome research To gain an overview of the current state of the phyllosphere microbiome research, a literature search was conducted on the Web of Science by using the keywords “Plant,” “Phyllosphere” and “Microbiome.” This resulted in 584 primary research articles and 89 reviews as of 1st March 2025. Within the research articles, entries that were miscategorised, such as reviews, book chapters or studies focused exclusively on the rhizosphere were excluded, resulting in a list of 503 publications. While phyllosphere microbiome research began in the early 2000s as evident by key reviews including Lindow and Brandl (2003) and Vorholt (2012), the earliest articles from this search was from 2013 (Figure 2A). This could be due to changes in terminology over time, highlighting the limitation of this search method. Nevertheless, the list was used for further analysis to gain an insight into the trends in phyllosphere microbiome research over the past decade. 4 The growing interest in phyllosphere microbiome research was evident in the increasing number of publications (Figure 2A). Overall, 57.5% of the articles focused on food crops such as cereals, vegetables and fruits or cash crops including rapeseed, cacao and cotton, while 39.1% covered non-agricultural plants including wild trees, grasses and shrubs. The emphasis on agriculturally relevant crops is promising as this could drive progress towards harnessing microbiomes in agriculture. Figure 2: Overview of current phyllosphere microbiome research. (A) Number of research articles published per year. Articles were categorised as agriculture (food crops or cash crops) or ecology (non-agricultural plant species) based on the plant species studied. (B) Diversity of food crops studied with the number of articles indicated for each crop. (C) Proportion of studies using amplicon or metagenomic sequencing. (D) Types of microorganisms studied using amplicon sequencing. 5 Although the phyllosphere microbiome has been studied in over 70 different food crops, the research primarily focused on globally important staple crops such as rice, wheat and maize (Figure 2B). Tomatoes and lettuce were among the most extensively studied vegetables. Grapevines were widely researched in Europe due to their economic value for fruit and wine production (Awad et al., 2023). Highly studied crops such as rice had extensive resources, including high-quality sequencing datasets that can be used for further studies (Masuda et al., 2024; Su et al., 2022). Research focused on specific crops can help build robust model systems to advance our understanding of microbiomes and apply them to other plants. A key step in microbiome research is using sequencing to characterise the composition, abundance, diversity, and structure of a given microbiome. Two commonly used sequencing techniques are amplicon or metagenomic sequencing (Liu et al., 2021). Amplicon sequencing involves the amplification of a variable genetic region (Regalado et al., 2020). For prokaryotes and archaea, this locus is in the 16S ribosomal RNA (rRNA), while in eukaryotes, the internal transcribed spacer (ITS) region of the 18S rRNA is used (Schoch et al., 2012). In contrast, metagenomic approaches use shotgun or long-read sequencing to characterise the entire genetic content of a sample, including plasmids and viruses (Handelsman et al., 1998; Masuda et al., 2024; Portik, Brown & Pierce-Ward, 2022). While some studies categorise amplicon sequencing as ‘metagenomics,’ it is important to distinguish these terminologies as amplification-based methods do not characterise the entire genome (Aguiar-Pulido et al., 2016; Quince et al., 2017). In this review, the term 'metagenomics' specifically refers to whole-genome sequencing. Over 80% of studies relied on amplicon sequencing (Figure 2C). Although it is high-throughput and cost-effective, major drawbacks include low taxonomic-level resolution and primer bias which can hinder the detection of certain taxa (Liu et al., 2021). Nevertheless, amplicon data are often used in correlation-based network analyses to predict microbial interactions and identify key taxa (Faust and Raes, 2012; Sapkota, Jørgensen and Nicolaisen, 2017). Metagenomics can overcome these limitations by providing species-level classification, enabling advanced functional characterisation (Su et al., 2022). Integrating sequencing with multi-omics approaches, such as transcriptomics and metabolomics can expand our understanding of microbiome functions (Wu et al., 2024). However, it is important to note that these bulk analysis methods fail to capture the spatial distribution of microbe-microbe interactions which influence 6 their functions (Remus-Emsermann and Schlechter, 2018). Therefore, additional experiments are needed to validate the inferred observations. Despite being termed ‘microbiome’, most studies have primarily focused on bacteria and fungi, leaving a significant knowledge gap regarding the diversity and functions of phyllosphere archaea, protists and viruses (Figure 2D). This is due to the widespread use of bacterial and fungal-specific amplicon sequencing and the lower abundance of archaea and protists in the phyllosphere compared to bacteria and fungi, which makes them difficult to detect (Taffner et al., 2019). Nevertheless, archaea and protists have key roles in plant nutrient cycling and pathogen control (Sun et al., 2021; Taffner et al., 2019). This underscores the need to diversify research methods to gain a more complete understanding of the microbiome. 7 Box 1: Hotspots of phyllosphere microbiome research To investigate the global distribution of phyllosphere microbiome research, the study locations provided in the methods section were mapped (Figure 3). This revealed that research on food crops was concentrated in China, the United States and India. The distribution mirrored trends observed in a meta-analysis of over 300,000 plant science publications, which highlighted the geographic disparities in current research (Marks et al., 2023). The study also pointed out the bias towards studying a limited number of plant species, primarily model systems and major crops, which was also observed in phyllosphere microbiome research. Microbial products are often proposed as promising tools for sustainable agriculture as part of achieving global goals such as the United Nations Sustainable Development Goal Zero Hunger (Hu, Chen and He, 2022). However, harnessing microbes would require expanding research to diverse geographic locations and plant species, including orphan crops and wild varieties of commercial crops (Cernava, 2024; Yin et al., 2023). This would facilitate the development of microbiome-based strategies tailored to different regions (Ayeni et al., 2024). Figure 3: Geographic distribution of phyllosphere microbiome research. Green circles representing include studies on major food and cash crops. 8 Numerous studies have investigated the effect of biotic, abiotic and anthropogenic factors on the composition of the phyllosphere microbiome as reviewed by Bashir et al. (2022). Building on these findings, the remainder of this review will focus on their potential applications in sustainable agriculture. Three main strategies for harnessing the phyllosphere microbiome include: (1) engineering microbial communities to enhance beneficial traits, (2) manipulating host plant genetics to naturally recruit favourable microbiomes and (3) adapting agricultural practices to promote the formation of beneficial phyllosphere microbiome. Engineering the phyllosphere microbiome The knowledge of phyllosphere microbiome diversity and composition can be applied to design microbial communities with desired functions using microbiome engineering (Figure 4). (Lawson et al., 2019). This include bottom-up approaches where the individual microorganisms are isolated and characterised (Ke, Wang and Yoshikuni, 2021). Microbial strains with beneficial traits can be used to build synthetic communities (SynComs) or their metabolites can be applied as probiotics to recruit beneficial microbiomes (Figure 4A, B). In contrast, top-down approaches utilise existing microbiomes, either by direct transplantation or modifying them through selective passaging or virus-mediated engineering to enrich beneficial traits (Figure 4C-E). This section will discuss key findings in phyllosphere microbiome research on food crops under the framework of microbiome engineering. 9 Designer microbiomes as probiotics A synthetic community (SynCom) is an assembly of functionally characterised microorganisms (Vorholt et al., 2017). It is commonly used as a simplified model of a complex microbial community to investigate factors that influence the microbiome assembly (Niu et al., 2017). Beyond fundamental studies, SynComs can be assembled from beneficial microorganisms to produce probiotics that enhance plant health by promoting growth and providing protection against diseases (Vassileva et al., 2020). Combining several strains in a SynCom produces an additive effect that enhances the beneficial traits of individual strains. For example, different combinations of Pseudomonas strains, isolated from potatoes infected with the late blight oomycetes Phytophthora infestans, were tested using a leaf disk assay (De Vrieze et al., 2018). This identified combinations of two strains that exhibited higher protection against P. infestans than when applied individually (De Vrieze et al., 2018). Likewise, in Arabidopsis thaliana, bacterial strains that suppress Pseudomonas syringae Figure 4: Microbiome engineering consists of bottom-up and top-down strategies. Bottom-up approaches include (A) synthetic communities (SynComs) and (B) the use of metabolites as prebiotics. Top-down approaches include (C) microbiome transplant, (D) successive passaging and (E) virus-mediated engineering. 16 that reduced use of pesticides and herbicides could help preserve beneficial microbial communities on crop surfaces. Additionally, no-till farming systems demonstrated a higher recovery of fungal networks following fungicide application, with 61% of core microbiome recovered compared to 34% in conventional systems (Noel et al., 2022). This was likely due to aerial spore dispersal by yeasts persisting in residual crops from the previous season in no-till fields (Noel et al., 2022). Although the underlying molecular mechanisms remain unclear, these findings suggest that principles of regenerative agriculture, such as minimal chemical input and no-till management may support the resilience and health of the phyllosphere microbiome (Khangura et al., 2023). While the use of livestock manure as fertilisers enhances microbiome richness in organic farming, it is also associated with increased abundance of phyllosphere bacteria carrying antibiotic resistance genes (ARGs), raising concerns about the spread of antibiotic resistance (Chen et al., 2018, 2020). This was seen in lettuce and bok choy and the proposed mechanisms of ARG transmission to the leaf included soil-to-root and root-to-leaf transfer or aerial dispersal (Li et al., 2025; Zhang et al., 2019). Biochar, or charcoal, could serve as an alternative to manure for enhancing microbiome richness without contributing to the spread of ARGs (Li et al., 2025). Various strategies for manipulating the microbiome can be integrated into existing farming practices. This was demonstrated in a study that tested a combination of synthetic fertilisers and a SynCom, consisting of three yeast strains with growth-promoting traits isolated from rice (Muthukrishanan et al., 2024). Greenhouse and field trials showed that combining the SynCom with 75% of the recommended fertiliser dosage most effectively increased the growth and yield of rice compared to using fertilisers or SynComs alone. Such integrated research methods help identify ways to rapidly incorporate new findings into current farming practices. These can help reduce the use of chemicals while complementing current limitations of microbiome-based methods, promoting the transition towards sustainable agricultural practices. 17 Conclusion Phyllosphere microbiome research is still its early stages with most studies focusing on characterising the composition and diversity of microbial communities with a bias towards bacteria and fungi. Combining sequencing with multi-omics approaches is key to address the current knowledge gaps in the genetic and molecular basis of microbiome traits, including enhanced plant health and resilience against disease. Recent advances in identifying plant genes linked to beneficial microbiomes present a promising strategy for incorporation into breeding programs compared to microbiome engineering approaches, which pose significant challenges in scalability. Further studies are required to validate these findings under field conditions. Moving forward, most widely studied crops, such as rice and tomatoes, could serve Figure 5: Strategies for harnessing phyllosphere microbiome in sustainable agriculture. Based on previous findings that demonstrated effects of biotic, abiotic and anthropogenic factors in shaping the phyllosphere microbiome, three major strategies for harnessing the microbiome in sustainable agriculture were discussed in this review. 18 as model systems for demonstrating proof-of-concept for microbiome-based strategies. Ultimately, integrated research methods that incorporate current findings into existing farming practices will be key to promote transition towards sustainable agriculture to meet the growing global food demand. Acknowledgements I would like to thank Professor Sebastian Eves-van den Akker for his guidance during the initial discussion. I would also like to express my gratitude to Dr Albin Teulet, Dr Darius Kosmützky and Dr Alex Guyon from the Schornack group for their valuable advice on the draft of this essay. 19 References Agler, M.T., Ruhe, J., Kroll, S., Morhenn, C., Kim, S.-T., Weigel, D. & Kemen, E.M. (2016) Microbial Hub Taxa Link Host and Abiotic Factors to Plant Microbiome Variation. PLOS Biology. 14 (1), e1002352. doi:10.1371/journal.pbio.1002352. Aguiar-Pulido, V., Huang, W., Suarez-Ulloa, V., Cickovski, T., Mathee, K. & Narasimhan, G. 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