Plants are the most important organisms on the Earth, whose leaves produce organic carbon (photosynthates) from CO₂ and water, and thus sustain the life on the planet. It is inspiring that the global leaf area has increased by 5.39×10⁶ km² during the years of 2000-2017, reaching 1.71×10⁸ km² (Chen et al., 2019). When plant leaves function as the primary productivity, they meanwhile serve as habitats for diverse microorganisms, which are collectively called phyllosphere microbiome. According Peñuelas and Terradas (2014), up to 10²⁶ bacteria occupy the global plant leaves. Despite of its huge population size, phyllosphere microbiome has been less investigated, compared with rhizosphere microbiome which has received intense attention for decades (Zhu et al., 2022). However, increasingly accumulated evidence indicates that phyllosphere microbiome plays significant roles in benefiting plants with respect to stress tolerance, growth promotion, nutrient uptake, and disease suppression (Stone et al., 2018). For example, a 1-aminocyclopropane -1-carboxylate- (or ACC-) deaminase producing bacterial strain isolated from the leaves of tropical yam significantly promoted the plant growth of tomato after its colonization of the phyllosphere (Herpell et al., 2023). Epiphytic and endophytic N₂-fixers in phyllosphere can contribute greatly to plant N nutrition (Zhu et al., 2023). Considering the necessity of leaf disease control, nutrient supply with foliar spray, and aerial spray of stimulants for stress inhibition in most crops, especially in horticultural crops, it is applausive to apply phyllosphere microbiome-based microbiological technology to achieve sustainable development in agriculture industry.

Since phyllosphere microbime can be helpful in plant growth and development, deep insights into the bacterial or fungal community and their driving force are necessary. It is revealed that phyllosphere bacterial community is highly dynamic, with its composition and structure sensitive to the environments including biotic and abiotic factors (Thapa et al., 2017; Li et al., 2023; Wang et al., 2023; Kong et al., 2024). Among biotic factors, the functional traits of host plants have been intensively studied. Earlier studies focused on the foliar C:N:P stoichiometry, which was demonstrated to affect the phyllosphere nitrogen fixing bacterial community or other functional groups (Martirosyan and Steinberger, 2014; Rico et al., 2014). Thapa et al. (2017) indicated that 83% of the observed variance in phyllosphere microbiome could be assigned to the contents of iron, manganese, and chlorophyll b of leaves. Similarly, Yuan et al. (2023) suggested that the contents of isotope carbon and copper, and the leaf area were the main factors influencing the community structure of phyllosphere microbiome. More recently, by using genome-wide association studies (GWAS), Su et al. (2024) revealed that 4-hydroxycinnamic acid, a compound in the phenylpropanoid biosynthesis pathway and synthesized by a rice gene OsPAL02, was the main driver for the enrichment of Pseudomonadales, which was the key taxa maintaining phyllosphere microbiome homeostasis. These results strongly point out the importance of plant identity in shaping phyllosphere microbiome (Li et al., 2023).

Plant functional traits can be mediated by abiotic factors, which thus can further exert significant influences on phyllosphere microbiome. For example, light intensity modulated the phyllosphere bacterial community of garden lettuce by affecting the functional composition of leaves (Kong et al., 2024). By analyzing 16S rRNA gene sequences from 1453 leaf samples across China, Wang et al. (2023) revealed that phyllosphere microbiome was mostly explained by climate and host plant factors, with abiotic environmental cues more important at low latitudes. Meanwhile, a great deal of literature shows the involvement of soil physicochemical properties in regulating phyllosphere microbiome (Jia et al., 2020; Zhou et al., 2021), probably via their influences on plant growth performance and leaf chemical composition (Zhu et al., 2022). Taken together, it seems recognized that abiotic factors regulate phyllosphere nicrobiome in an indirect way by affecting host plant traits.

Our previous study on rhizosphere microbiome found that soil amino acids could profoundly regulate rhizosphere bacterial community (Feng et al., 2021), because amino acids can serve as both carbon source and nitrogen source for these soil organisms. Particularly, application of exogenous phenylalanine enriched functional groups promoting nitrogen cycling and plant growth (Feng et al., 2023a). In contrast, however, the regulation of phyllosphere microbiome by leaf-derived amino acids has not been fully elucidated yet. Moreover, perennial woody plants undergo seasonal changes in the plant traits as affected by the dynamics in weather parameters. Thus, we investigated the annual dynamics of phyllosphere microbiome and plant functional traits of pomelo (Citrus maxima (Burm.) Merr.) in this study, which will provide novel insights into the respective contribution of biotic factors and abiotic factors and the regulation of phyllosphere microbiome by amino acids in leaves. We aimed i) to explore the importance of leaf derived amino acids in shaping phyllosphere microbiome, ii) to compare the effects of abiotic environmental cues (weather parameters) and biotic factors on phyllosphere microbiome, and iii) to reveal the annual dynamics of phyllosphere microbiome in pomelo, which will facilitate the rational management and utilization of phyllosphere microbiome for plant growth and health in pomelo.

Bacterial and fungal communities colonizing phyllosphere are critical components of plant microbiome, which play a essential role in maintaining plant health, nutrient acquisition and stress resistance (e.g. N) (Chen et al., 2020; Li et al., 2022b; Zhu et al., 2023; Li et al., 2024). However, phylosphere microbiome has been less explored so far compared to rhizosphere microbiome. Phyllosphere microbiome is highly dynamic in response to environmental cues, which include both biotic and abiotic factors (Thapa et al., 2017; Li et al., 2023; Wang et al., 2023; Kong et al., 2024). In this study, we demonstrate that both weather parameters (precipitation and air temperature) and biological factors (leaf traits) shaped the bacterial and fungal communities of pomelo leaves, with plant factors exerting a greater influence. This is similar to the results by Zhou et al. (2023), who indicated that environmental factors (geographic location and climatic conditions) and host genotype affected the epiphytic bacterial and fungal communities of wild soybeans across China. However, plant traits contributed 12%-19% to the variation of microbial community, much higher than weather parameters (4%-15%) in our study, while environmental factors contributed 19.9%-25.8% to the variation, much higher than host genotype (0.4%-3.6%) in the study by Zhou et al. (2023). This suggests that the relative importance of environmental factors and plant factors might depend on context, such as plant species, sampling area. It is well established that plant microbiome can be regulated both directly by plant traits (internal factors) and indirectly by environments (external factors) (Li et al., 2023; Kong et al., 2024), and external factors normally work via their influences on internal factors. The seasonal fluctuations of amino acids and sugars in citrus leaves have been reported, normally with low contents in the actively growing seasons (e.g. summer) (Yildiz et al., 2013; Xiong et al., 2024). Additionally, plant leaves can release volatile organic compounds (VOCs), which might possess antimicrobial activity and act as carbon sources, thereby regulating phyllosphere microbiome (Farré-Armengol et al., 2016). Specifically, Citrus plants are well recognized for their fragrance (namely VOCs), which was demonstrated to strongly structure their phyllosphere bacterial community (Wang et al., 2022). In this study, it is possible that the weather parameters greatly affected the plant traits (such as amino acids and sugars in leaves), especially in summer when the vegetative growth of pomelo plants was vigorous with both high temperature and high precipitation. Moreover, the VOC profile of Citrus plants varies much depending on seasonality (Lin et al., 2022), thereby probably contributing to the seasonal pattern of phyllosphere microbiome in this study. Considering the coupled effects of appropriate climate conditions and N fertilizers in promoting plant vegetative growth, N fertilizer application is necessary to regulate the phyllosphere microbiome even with appropriate temperature and precipitation in citrus production systems.

It is interesting that the contribution of weather parameters to bacterial community (4.0%) was much lower than that to fungal community (15%). It is possible that fungal community is more sensitive to weather parameters, especially to environmental moisture (monitored as precipitation in this study) than bacterial community (Kaisermann et al., 2015; He et al., 2023). Moreover, it is notable that the overlap of weather parameters and biological factors for bacterial community (30%) was much higher than that for fungal community (11%). This suggests that the influence of weather parameters on the bacterial community was more likely dependant on their effects on plant traits, while weather parameters influenced the fungal community in a relatively independant manner.

The core taxa of microbiome are defined as the members shared by all or most microbial communities with similar backgrounds, and play essential roles in the community functioning (Shade and Handelsman, 2012; Ren and Wu, 2016). For example, Shen et al. (2022) demonstrated that the core taxa (mainly belonging to Myxococcales, Pseudomonadales, Xanthomonadales) of suppressive soils from six banana plantation sites showed protective effects against banana Fusarium wilt disease, compared to the core taxa of conducive soils. In this study, we explored the core taxa of phyllosphere microbiome according to occupancy, and identified Methylobacterium-Methylorubrum, Pseudomonas, Hymenobacter, Sphingomonas, and Massilia as the top 5 core genera of bacterial community for pomelo, among which Methylobacterium-Methylorubrum, Pseudomonas, and Sphingomonas were also the core genera of wild soybean (Zhou et al., 2023). Methylobacterium-Methylorubrum is one of the most commonly reported phyllosphere bacteria promoting growth performance of many plant species, such as cucumber (Zhang et al., 2025) and rice (Oeum et al., 2024), which has been intensively investigated regarding its colonization capacity and functionality (Abanda-Nkpwatt et al., 2006; Yurimoto et al., 2021). The mechanisms underlying the plant growth promotion by Methylobacterium-Methylorubrum in phyllosphere mainly include nitrogen fixation, secretion of auxin, cytokinin, and 1-aminocyclopropane-1-carboxylate deaminase, and etc (Zhang et al., 2021). Recently, Zhang et al. (2024) demonstrated that the phosphoribosylpyrophosphate synthetase of Methylorubrum extorquens AM1 facilitated its superior colonization capability and functionality. It is possible that Methylobacterium-Methylorubrum bacteria assimilate methanol emitted from phyllosphere and then provide carbon sources to other members in the community. Pseudomonas is frequently recognized as beneficial member of phyllosphere community. Li et al. (2025) inoculated P. fluorescens to Salix matsudana and showed a increase of 90.51% in plant biomass. In detail, inoculation increased the asymbiotic nitrogen-fixation, improved photosynthetic traits (e.g. net photosynthetic rate, intercellular CO₂ concentration, stomatal conductance, transpiration rate) and the root traits (e.g. root length, root branching) and modified the phyllosphere microbiome beneficial for plant health, thereby promoting the plant nutrient uptake and biomass. Su et al. (2024) indicated that the compound 4-hydroxycinnamic acid synthesized by OsPAL02 in rice plants enriched Pseudomonadales in phyllosphere, while the reduced Pseudomonadales abundance in the knockout mutant of OsPAL02 resulted in the dysbiosis of phyllosphere microbiome and higher susceptibility to disease. These studies suggest that Pseudomonas, either native or inoculated, might function via maintaining homeostasis of phyllosphere microbiome in most cases.

Our study reveals that Acrodontium, Hyphozyma, Inocybe were the top 3 core fungal genera of pomelo leaves. Acrodontium is the frequent colonizer of citrus leaves, which was enriched in healthy trees compared to HLB-infected trees and thus was regarded as the keystone taxa of phyllosphere fungal microbiome (Ginnan et al., 2020). It is interesting that a Hyphozyma species (H. roseoniger) can convert sclareol to ambradiol (Ncube et al., 2022), which might contribute to the production of fragrance compounds of pomelo leaves. Surprisingly, however, Inocybe has been frequently reported as dominant and ectomycorrhizal fungus (Nara, 2009; Bohorquez et al., 2021; Khan and Reshi, 2022), occasionally occurring in leaf litter (Liber et al., 2022).

The chemical properties of plant leaves are primary factors shaping phyllosphere microbiome (Yadav et al., 2005; Li et al., 2022a; Luo et al., 2023). When nutrient contents such as N, P, K and their stoichiometry have been explored for a long time, in this study, we focused on the amino acids in leaves and found that amino acids contributed much to the variation in microbial community, which has been less reported before. Our previous work on rhizosphere microbiome revealed that amino acids greatly regulated bacterial community in rhizosphere, and phenylalanine was the most effective in promoting soil N cycling (Feng et al., 2021; Feng et al., 2023a). This study reveals that amino acids played much more important roles in shaping bacterial community than non-amino acid constituents, such as several kinds of mineral nutrients, with Lys, Arg, and Ser more effective than others. Our results put the special importance on the amino acids in leaves for the first time although other chemical properties of leaves have been investigated. This importance can be attributed to the fact that amino acids can provide both N and C sources to the phyllosphere microbiome (Feng et al., 2021, 2023a). Similarly, several studies also shed lights on amino acids. Proline, tyrosine, serine and phenylalanine showed important influence on the phyllosphere microbiome of nettle (Urtica cannabina), with the affected taxa including both bacteria (Enterococcus, Hymenobacter, Sphingomonas, Sphingobacterium, Massilia, Ochrobactrum, Oxalobacteraceae) and fungi (Pezizella, Udeniomyces, Filobasidium, Didymellaceae, Glomerellales, Helotiales) (Jia et al., 2023). Total fee AAs were one of the most outstandingly determined factors interacting with phyllosphere microbiome of garden lettuce, with the functional taxa (Kinetoplastibacterium, Natronococcus, Bacillus, Bradyrhizobium, Methanococcus) harboring mdh or glyA genes significantly affected (Kong et al., 2024). In contrast, fungal community (e.g. Taphrina, Cylindrocladiella, Aspergillus, Boletus, Malassezia, Cladosporium, Xenocylindrocladium, Cordyceps, Pyrenochaeta) in the phyllosphere of tea plants was more sensitive to sugars than bacterial community (Chen et al., 2024), which might be due to their differential trophism. Since the phyllosphere microbime is significantly regulated by the amino acids and sugars in leaves, the future research can focus on the fine regulatory patterns of these compounds on some specific taxa, such as Methylobacterium-Methylorubrum, Pseudomonas, Sphingomonas, which represent beneficial taxa for plant performance (Li et al., 2022b, 2024; Zhang et al., 2024). As such, it is possible to develop amino acid-based biostimulants enriching the beneficial phyllosphere microbial taxa, which supports the sustainability of agricultural production.

Phyllosphere microbime can promote plant disease resistance and growth performance. Therefore, the understanding of the annual dynamics and drivers of phyllosphere microbime is pivotal to the utilization of it. Therefore, we characterized the phyllosphere bacterial and fungal communities across annual cycle, and identified Methylobacterium-Methylorubrum, Pseudomonas, Hymenobacter and Acrodontium, Hyphozyma, Inocybe as the top core taxa of pomelo phyllosphere microbiome. The bacterial community in summer and the fungal community in autumn were much different from those in other seasons, respectively. Both biological factors (including 24 leaf traits) and weather parameters (temperature and precipitation) affected microbiome assembly, with the former (12%-29%) contributing more to the assemblage than the latter (4%-15%). Furthermore, we demonstrated for the first time that amino acids and sugars in leaves were the main drivers of bacterial and fungal communities, respectively, highlighting the importance of amino acids in manipulating phyllosphere microbiome. In general, our data suggest that biological factors (e.g. amino acids and sugars in leaves) and weather parameters regulate the phyllosphere microbiome in direct and indirect ways, respectively.