Soil-dwelling Naegleria enhances plant performance by stimulating beneficial bacterial functions in the rhizosphere

Yue, Yang; Xu, Zhihui; Wang, Yijin; Liu, Chen; Sun, Shuo; Ren, Xiangyu; Lv, Qihui; Liu, Jingchi; Dini-Andreote, Francisco; Xiong, Wu; Shen, Qirong

doi:10.1038/s41467-025-64139-x

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Published: 13 October 2025

Soil-dwelling Naegleria enhances plant performance by stimulating beneficial bacterial functions in the rhizosphere

Nature Communications volume 16, Article number: 9079 (2025) Cite this article

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Abstract

Soil protists play vital roles in influencing plant performance, yet their interactions with plant-beneficial bacteria are still poorly understood. Here, we examine how two soil protists (Naegleria sp. and Cercomonas sp.) affect the pathogen Ralstonia solanacearum, both on individual beneficial bacteria and within a synthetic microbial community (SynCom). Combining in vitro and pot experiments, we find that the SynCom together with Naegleria provided significantly greater suppression of the pathogen (enhanced suppressiveness by 74.29% compared to SynCom alone). Additionally, Naegleria increases SynCom biofilm biomass by 2.44 times. Population dynamics tracking revealed that Naegleria enriched Bacillus populations, leading to a positive correlation between Bacillus and Pseudomonas. Metatranscriptomics analysis shows upregulation of genes related to biofilm formation (such as epsA-O and tapA-sipW-tasA operon) and secondary metabolite biosynthesis (e.g., macrolactin H, bacillaene, and difficidin) in the presence of Naegleria. Our study demonstrates that Naegleria enhances plant health by predating on pathogens, promoting beneficial bacteria, and stimulating protective microbial functions in the rhizosphere.

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Introduction

Predatory protists are important members of the soil food web, engaging in trophic interactions with bacteria, archaea, and certain micro-eukaryote taxa¹. These microbial predators exert crucial roles in diverse soil functions, including improving soil fertility and supporting plant growth, health, and performance^2,3. More specifically, predatory protists can enhance soil nutrient availability for plants via the release of nutrients when they consume microbial prey^4,5. In addition, a recent study reported that predatory protists can directly control the Ralstonia solanacearum⁶, a soilborne pathogen that colonizes the xylem of solanaceous plants, leading to wilting symptoms and eventual host death⁷. Due to their predation preference, predatory protists can also increase the abundance of plant-beneficial microbes in the rhizosphere^8,9. For example, this can occur by promoting a greater abundance of taxa involved in biofilm formation, and/or by inducing the synthesis of secondary metabolites in specific microbial taxa with detrimental effects on plant pathogens¹⁰. As such, the study of protist-based biocontrol presents opportunities for sustainable agriculture, yet relatively little is known about the ecological mechanisms governing protist predator-prey interactions with implications for plant-beneficial bacteria in the rhizosphere.

Plant growth-promoting rhizobacteria (PGPR) are beneficial bacteria that colonize the rhizosphere and exhibit multiple traits associated with rhizosphere competence and plant growth. Some of these taxa have been commercialized as sustainable bioinoculants in agriculture^11,12,13. The majority of current commercial products are based on Gram-positive microbes, particularly Bacillus species^14,15, and their field performance has been widely evaluated for phytopathogen control and plant growth promotion¹⁶. For example, Bacillus velezensis SQR9 is a bacterium strain able to exert negative effects on diverse plant pathogens via the synthesis of specific secondary metabolites (e.g., lipopeptides). In addition, this strain has genes involved in plant growth promotion and rhizosphere competence (i.e., spore and biofilm formation)^17,18,19. However, the application of PGPR like B. velezensis as commercial bioinoculants in agriculture is often limited due to unstable field performance, that is, due to the rapid decline in their density and the reduced functional effectiveness in soil over time^20,21.

The effectiveness of plant beneficial bacteria application has been hampered by the biological interactions between beneficial bacteria inoculants and the resident microbial taxa in the rhizosphere^12,22,23. That is, rhizosphere microbes often exert inhibitory effects on the introduced bacteria, thereby reducing their abundance, functionality, and long-term persistence^24,25,26. Bacteria colonization depends on the capacity of inoculated strains to successfully compete for locally available resources in the rhizosphere with multiple other resident microbes, apart from coping with occasional abiotic stresses²⁷.

Biofilm formation by bacteria is a key determinant of successful root colonization^28,29 and plant growth promotion. For instance, the biosynthesis of biofilm has also been reported to be associated with specific plant beneficial functions, such as IAA production, siderophore production, and bio-control activities^17,30. Protists in the rhizosphere can exert significant influence on bacterial biofilm biosynthesis through a variety of mechanisms, including selective grazing on non-biofilm-forming bacteria, indirect signaling via chemical cues, and the induction of anti-predation defense mechanisms in prey bacteria that can promote biofilm formation^31,32. However, the effects of different predatory protist types (i.e., surface-feeding and suspension-feeding modes) on bacterial biofilm formation remain largely unknown^33,34.

Here, we hypothesize that predatory protists can increase the abundance of plant-beneficial bacteria, particularly taxa able to produce biofilms and synthesize secondary metabolites, which positively affect plant performance. To test this hypothesis, we selected two types of predatory protists (one with surface-feeding and one with suspension-feeding mode, based on microscopic observations), i.e., an amoeba Naegleria and a flagellate Cercomonas, both known to directly prey on the phytopathogen Ralstonia solanacearum. We further constructed a synthetic community (SynCom) composed of seven common rhizosphere bacteria, and explored the variations in specific bacterial traits and functions under protist predation. We investigated the effects of trophic interactions between predatory protists and the SynCom on the pathogen growth in vitro and pot experiments using tomato plants as a model system. The impact of predatory protists on bacterial biofilm formation was examined by liquid culture experiments, and the expression of genes associated with biofilm formation and secondary metabolites was examined via metatranscriptomics. Last, we validated some of our findings associated with genes regulating biofilm formation and secondary metabolite biosynthesis using a series of bacterial mutant strains in bacteria-protist co-culture experiments.

Results

Predation preferences and effects of the SynCom on plant performance

We assessed the predation preferences of two protist species—i.e., the amoeba Naegleria sp. and the flagellate Cercomonas sp.—on the bacterial isolates Acinetobacter baumannii XL380, Chryseobacterium rhizoplanae XL97, Comamonas odontotermitis WLL, Enterobacter bugandensis XL95, Pantoea eucrina XL123, Pseudomonas stutzeri XL272, Bacillus velezensis SQR9, Ralstonia solanacearum 1115, and Escherichia coli DH5α. The results showed that the amoeba Naegleria sp. had an optimal growth when predating on R. solanacearum (Supplementary Fig. 1a), while the flagellate Cercomonas sp. had an optimal growth when predating on Acinetobacter baumannii followed by R. solanacearum (Supplementary Fig. 1b). We found both Naegleria sp. and Cercomonas sp. exhibit greater predation on R. solanacearum when compared to the control strain Escherichia coli DH5α in the liquid culture medium (Fig. 1a–d). Besides, the effects of simultaneously inoculating these two protist strains were similar to those of inoculating Naegleria sp. or Cercomonas sp. alone (Fig. 1b–d). We further examined the capacity of Naegleria sp. and Cercomonas sp. to indirectly suppress R. solanacearum pathogen via induced functions in the SynCom taxa using agar plate experiments (Supplementary Fig. 2). Different from the liquid medium experiment, we did not observe predatory activities of Naegleria sp. and Cercomonas sp. on R. solanacearum in the plate experiment. However, we found that the presence of Naegleria sp. significantly enhanced the ability of the SynCom to suppress the pathogen R. solanacearum pathogen (Fig. 1e). Although the population of Cercomonas sp. grew faster than that of Naegleria sp. when feeding on R. solanacearum (Fig. 1b), Naegleria sp. showed a greater effect in predating R. solanacearum than Cercomonas sp. in liquid culture medium (Fig. 1c, d). In line with these findings, the first pot experiment showed that the SynCom with the protist Naegleria sp. resulted in the best tomato performance, including plant height and fresh weight. This resulted in an average plant height and fresh weight increase of 39.23 and 20.61%, respectively (Fig. 1f–h).

**Fig. 1: Effects of the SynCom in the presence of two protists on the pathogen *Ralstonia solanacearum* with implications for plant performance.**

In addition, we tracked the population densities of individual SynCom members in the microcosm experiment. In brief, the abundance of Bacillus velezensis SQR9 decreased 24% after 8 days within the SynCom (Supplementary Fig. 3a). The presence of Naegleria sp. significantly enhanced the population of B. velezensis SQR9 within the SynCom over time. In the presence of Naegleria, the gene copy number of B. velezensis SQR9 increased by 200% compared to the SynCom and by 289% compared to the SynCom with Cercomonas after 8 days. The presence of Cercomonas sp. did not support the population of Bacillus over time (Supplementary Fig. 3b).

Impact of protists on bacterial biofilm formation

The amoeba Naegleria sp. and the flagellate Cercomonas sp. exhibited distinct effects on bacterial biofilm formation (Fig. 2a, b). The crystal violet staining method revealed that the presence of Naegleria sp. significantly increased the biofilm formation of distinct taxa in the SynCom (Fig. 2a, Kruskal-Wallis H test with LSD for multiple comparisons, P-value < 0.001), while the presence of Cercomonas sp. significantly decreased biofilm formation. Compared to the SynCom with Cercomonas, the presence of Naegleria with SynCom increased biofilm production by 244% (Fig. 2a). We also evaluated the impact of these two protists on biofilm formation at the filter interface using a gravimetric measurement. While Naegleria sp. significantly increased the biofilm weight in the SynCom, with the biofilm phenotype revealing pronounced wrinkles (Fig. 2b), Cercomonas sp. significantly decreased biofilm weight in the SynCom (Fig. 2b, one-way ANOVA with LSD for multiple comparisons, P-value < 0.001). Further quantification of individual bacterial strains within the SynCom via the qPCR method revealed a positive correlation between B. velezensis and P. stutzeri in the treatment amended with Naegleria sp. (Fig. 2c, Pearson’s correlation, P-value < 0.01).

**Fig. 2: Effects of two protists on biofilm formation in *Bacillus* and within the SynCom members.**

We further examined the abundance of B. velezensis SQR9 during biofilm formation in 1/2 Msgg+1/2 TSB culture medium. The presence of Naegleria significantly increased the abundance of B. velezensis SQR9 within the SynCom (Fig. 2d, one-way ANOVA with LSD for multiple comparisons, P-value < 0.01). However, the biofilm production by B. velezensis SQR9 alone significantly decreased when co-cultured with Naegleria sp. (Fig. 2e–g). This contrasts with our initial findings that Naegleria sp. promotes biofilm production in the SynCom (Fig. 2a, b). Considering the possible impacts of different nutrient conditions (varying across the used methods) on bacterial growth, we also used the TSB culture medium as an alternative, which revealed consistent results with the 1/2 Msgg+1/2 TSB culture medium (Supplementary Fig. 4).

Naegleria sp. enhances specific gene expression in B. velezensis

Metatranscriptomic analysis revealed that the presence of Naegleria sp. induced significant transcriptional changes in taxa within the SynCom (Fig. 3a). Notably, B. velezensis SQR9 exhibited the most pronounced response to protist predation within the SynCom context (Fig. 3b). Specifically, Naegleria sp. enhanced the expression of key biofilm-related operons in B. velezensis SQR9, including the epsA-O operon responsible for exopolysaccharide production and the tapA-sipW-tasA operon encoding amyloid fibers (Fig. 3c, d). The polyketide synthase (PKS) genes were significantly upregulated within the SynCom with Naegleria sp. (Fig. 3e). Interestingly, all detected PKS gene expression were exclusively detected from B. velezensis SQR9 among SynCom members (Fig. 3b). However, these genes were found to be downregulated when B. velezensis SQR9 was cultured alone with the protist (Fig. 3e). The analysis of secondary metabolite clusters (Supplementary Data 1) showed that Naegleria sp. upregulated led to the upregulation of seven gene clusters in B. velezensis SQR9 within the SynCom (Fig. 3f and Supplementary Fig. 5). However, only macrolactin H was found to have an enhanced expression when B. velezensis SQR9 was cultured alone with the protist, whereas bacillaene and difficidin remained unchanged (Fig. 3f).

**Fig. 3: Effects of the protist *Naegleria* on the gene expression of *Bacillus velezensis* SQR9 within the SynCom.**

The expression of genes associated with non-PKS secondary metabolites revealed distinct patterns when each strain was cultivated individually or within the SynCom context with the protist, with genes associated with bacillibactin, fengycin, and surfactin found to be downregulated, whereas bacilysin was upregulated (Supplementary Fig. 5). Differential expression was observed in the other SynCom members, in P. stutzeri XL272 and E. bugandensis, generally showed downregulation of secondary metabolite genes (amonabactin P750 in P. stutzeri XL272, aryl polyenes and enterobactin in E. bugandensis XL95), except for acinetobactin in A. baumannii which was upregulated (Kruskal-Wallis H test with LSD for multiple comparisons, P-value < 0.01). (Supplementary Fig. 6).

We further employed genetic approaches to validate the functional roles of key genes in Bacillus velezensis SQR9, including mutants deficient in biofilm formation and secondary metabolite production. For that, biofilm-deficient mutants of Bacillus velezensis SQR9 (ΔespD, ΔtasA) and secondary metabolite-deficient mutants of Bacillus velezensis SQR9, including: Δfen (impaired fengycin synthesis), Δsrf (impaired surfactin synthesis), Δdfn (impaired difficidin synthesis), Δbae (impaired bacillaene synthesis), Δmln (impaired macrolactin H synthesis), were used in this study. Co-culture experiments between the SynCom (containing different mutants of Bacillus velezensis SQR9) and Naegleria revealed distinct phenotypic outcomes. The Bacillus abundance was significantly lower in the treatments with Naegleria and Δmln, which was consistent with the control containing the single SynCom without Naegleria, whereas notable changes were observed in all other treatment groups. In general, our findings demonstrate that genes associated with both biofilm formation and secondary metabolite production in Bacillus velezensis SQR9 significantly affected the stability of Bacillus during the co-culture experiment. Most importantly, the results revealed that the Δmln (macrolactin H-deficient) mutant of Bacillus velezensis SQR9 plays an important role in supporting Bacillus relative abundance within SynCom in the presence of protist predation (Fig. 3g).

Naegleria sp. promotes the abundance of B. velezensis in the tomato rhizosphere

To minimize the potential impacts of plant genotypes on the plant growth experiment, we selected the model tomato cultivar ‘Micro-Tom’ (Solanum lycopersicum cv. Micro-Tom) with well-characterized genetic background for the second pot experiment (Supplementary Fig. 7). The data revealed that the SynCom with Naegleria sp. resulted in the best plant performance (including plant height and fresh weight) (Supplementary Fig. 8a). Importantly, this is consistent with the first pot experiment (Fig. 1F). In soil environment, discernible differences in microbial composition were observed between the treatments (Supplementary Fig. 8b and Supplementary Data 2, global ANOSIM analysis P-value < 0.05), except for diversity metrics (Supplementary Fig. 8d and Supplementary Fig. 8e). The treatment of B. velezensis SQR9+Naegleria sp. revealed a higher relative abundance of Bacillota when compared to the treatment with only B. velezensis SQR9 (Supplementary Fig. 8c, one-way ANOVA with LSD for multiple comparisons was performed, where different letters indicate significant differences with P-value = 0.0378). However, no significant difference in the relative abundance of Bacillota was observed when comparing the treatments SynCom and SynCom+Naegleria (Supplementary Fig. 8c). Differential abundance analysis revealed a significant increase of Bacillus in the B. velezensis SQR9+Naegleria treatment, when compared to other treatments (Supplementary Fig. 8f and Supplementary Fig. 8g). This ‘enriched’ Bacillus zOTU had 100% nucleotide similarity with the full-length 16S rRNA gene of B. velezensis SQR9 in the aligned sequence region (Supplementary Fig. 9).

Discussion

In this study, we examined how trophic interactions between protist species and bacterial strains within a SynCom affect bacterial traits associated with rhizosphere competence, control the population of a soil-borne pathogen, and promote plant performance. In brief, our findings revealed that the trophic/cross-kingdom interactions between predatory protists and bacterial taxa result in an increased relative abundance of plant-beneficial bacteria (in this case, Bacillus), both in soil and under liquid medium conditions. Moreover, we also found that the predatory protists promote biofilm formation in specific bacterial strains across different types of culture media and are associated with greater biosynthesis of secondary metabolites (in this case, shown as enhanced expression of specific gene clusters in Bacillus). Our study highlights the potential of the amoeba Naegleria in promoting biofilm formation in specific members within the SynCom, resulting in a greater abundance of plant-beneficial bacteria involved in the biosynthesis of diverse secondary metabolites.

Although the use of predatory protists as microbial bioinoculants in agriculture is still in the early stages of development, some studies have provided supporting evidence that these organisms can promote plant growth and health^3,35,36,37. For instance, this can occur by the direct consumption of soil-borne pathogens, thereby reducing plant disease. As shown in our study, the protist Naegleria sp. efficiently predated the phytopathogen Ralstonia solanacearum, resulting in a pathogen population decrease of 74.29% (Fig. 1e). Corroborating this finding, a previous study showed that predatory protists can control tomato bacteria wilt by consuming plant pathogens⁶. In addition, our results highlighted the protist Naegleria sp. as being able to promote higher densities of the biocontrol agent Bacillus within bacterial communities. This bacterial genus is often associated with the control of plant pathogens³⁸, which supports the notion that predatory protists in soils can indirectly suppress soil-borne pathogens via enhancing the survival³⁹ and activity of pathogen-suppressive microbes^6,40.

Our study also provides evidence that predatory protists can induce the expression of genes involved in secondary metabolite biosynthesis in bacteria, thereby positively affecting plant performance. Several studies have reported multiple specific bacterial secondary metabolites associated with plant health promotion, e.g., iturin (in Bacillus), and 2,4-Diacetyl phloroglucinol (DAPG) (in Pseudomonas)^41,42. Members within the genus Bacillus can secrete diverse antibacterial secondary metabolites that contribute to soil-borne disease suppression (such as Fusarium oxysporum and Rhizoctonia solani)³⁸. In our study, despite the cell number of Bacillus was found to decrease in the presence of the amoeba Naegleria sp., the gene expression of macrolactin H, and the expression of the PKS biosynthetic gene clusters (BGCs), were found to be upregulated (Fig. 3f). These findings indicate that the predation pressure exerted by protists can promote the biosynthesis of antibiotics in specific microorganisms⁴³, therefore contributing to plant pathogen suppression.

Although several genes associated with the biosynthesis of secondary metabolites (e.g., bacillibactin, fengycin, and surfactin) were found to be downregulated in B. velezensis+Naegleria when compared with B. velezensis alone, the expression of all seven studied secondary metabolite genes (see methods for details) was found to be upregulated in the SynCom+Naegleria, which better aligns with the expected biological conditions in the plant rhizosphere (Fig. 3f). Collectively, these results demonstrate that the predatory activity of the amoeba Naegleria sp. contributes to an increase in secondary metabolite gene expression in Bacillus within bacterial communities, especially in a complex ecological community context. Given the fact that the predatory protists can likely result in an increase in the potential biocontrol functions of specific plant-beneficial bacteria in this system, these results suggest a crucial role for predatory protists in enhancing the effectiveness of microbial biocontrol agents within complex soil ecosystems⁴⁴.

Ensuring the survival and persistence of PGPR bioinoculants is crucial for obtaining desirable beneficial functions supporting plant growth and health⁴⁵. In line with this, our results revealed a rapid decline in Bacillus within the SynCom over time (Supplementary Fig. 3a), which is consistent with previous findings^46,47,48. However, we found that the presence of Naegleria sp. effectively sustains the population of Bacillus in soils. This could be partly explained by the selective predation of predatory protists, as Naegleria sp. feeds preferentially on Ralstonia, Acinetobacter, and Escherichia species (Supplementary Fig. 1), thus reducing their competition with Bacillus taxa. In addition, this may also be associated with the protist-mediated effect on the enhanced biosynthesis of antibiotics by bacterial taxa in the rhizosphere. Although biofilm formation and secondary metabolite biosynthesis can influence the population dynamics of Bacillus in the rhizosphere, the biosynthesis of macrolactin H seems to exert the most significant effects (Fig. 3g). As a macrocyclic antimicrobial compound, some studies have shown the effects of this antimicrobial molecule on soil bacterial communities^49,50. In addition to inducing bacterial antibiotic production, our results demonstrated that the presence of Naegleria sp. resulted in a higher relative abundance of B. velezensis, apart from inducing biofilm formation within the SynCom. Several studies have shown that protist grazing can stimulate biofilm formation, which serves as an important protective prey mechanism against predation^51,52,53,54. Biofilm formation in PGPR is also linked with rhizosphere competence and diverse other traits associated with plant growth-promotion, such as induction of systemic resistance, and biosynthesis of secondary metabolites^29,55.

Previous studies have shown the association between bacterial biofilm formation and beneficial PGPRs^56,57. Here, we found the expression of specific biofilm-related genes in Bacillus—including the epsA-O operon⁵⁸ and the tapA-sipW-tasA operon⁵⁹—to be upregulated (Fig. 3d, e) at both experimental conditions in the presence of protists (i.e., Bacillus single strain or in the SynCom context). The presence of protists enhanced the biofilm formation of bacterial species in the SynCom and altered the interactions between taxa, for instance, leading to positive associations between Bacillus and Pseudomonas (Fig. 2e). In line with that, it has been shown that metabolic interactions between Bacillus and Pseudomonas can lead to biofilm formation⁶⁰ and further promote plant health. This metabolic activity might play a critical role in enabling Bacillus to evade protist predation through cooperative behavior, thus sustaining their fitness in a complex community^61,62. Overall, the enhanced biofilm formation observed under protist predation might largely depend on specific Bacillus strains⁴⁶.

Despite the protist Naegleria sp. being found to enhance biofilm formation in the SynCom, the flagellate Cercomonas sp. had the opposite effect (Fig. 2a, b). These contrasting effects of the two types of protists on bacterial biofilm formation may be partly explained by the differences in feeding preferences and motility of the two selected protists in this study (e.g., a surface-feeding protist and a suspension-feeding protist)^34,63. Previous studies have reported that the free-swimming filter feeder Tetrahymena sp. had a larger impact than the surface-associated predator Chilodonella sp. on reducing biofilm formation³⁴. However, the opposite pattern also exists; for example, surface-feeding grazer protists were found to cause a strong reduction in biofilm biomass, while suspension-feeding ciliates have been shown to promote biofilm biomass in single-species assays, such as those performed with Pseudomonas⁶³. Additionally, our contrasting results on the effects of Naegleria and Cercomonas on bacterial biofilm formation might be explained by the complex interspecies interactions within SynCom members and with the different protists. In line with that, we have also found evidence supporting that Naegleria promotes positive interactions between Bacillus and Pseudomonas (Fig. 2e). Of key importance, it is worth mentioning that biofilm formation in the rhizosphere does not necessarily imply beneficial microbial metabolisms associated with plant growth performance (Fig. 1g, h). This should be seen as an important microbial metabolism associated with rhizosphere competence. Besides, biofilms can support bacterial population growth and stability and enhance protection against predatory protists⁶⁴.

Our work highlights the amoeba Naegleria sp. to exert direct predation on the pathogen Ralstonia solanacearum, and to enhance plant-beneficial bacteria in the rhizosphere. The presence of this protist also enhances biofilm formation in specific rhizosphere-competent bacterial taxa and induces the expression of gene clusters associated with secondary metabolite biosynthesis (Fig. 4). In addition, the presence of the amoeba Naegleria sp. results in positive interactions between beneficial bacteria (Bacillus and Pseudomonas) within the rhizosphere microbiota. The amoeba Naegleria sp. may serve as an important organism maintaining the population of plant-beneficial bacteria with the genus Bacillus in soil/rhizosphere systems. We foresee the utilization of specific protists in agricultural systems as potential mediators of beneficial soil and rhizosphere-competent organisms that can enhance plant health and performance.

**Fig. 4: Conceptual model illustrating the ecological mechanisms of interaction between protists and bacterial taxa in the rhizosphere.**

Methods

Selection and cultivation of protists

Two predatory protist lineages obtained from soil (the amoeba Naegleria sp. and the flagellate Cercomonas sp.), following comparison with the NCBI 18S rRNA gene database. The sequence from Naegleria sp. (GenBank accession PX373292 [https://www.ncbi.nlm.nih.gov/nuccore/PX373292]) showed the closest affinity to that of Naegleria gruberi, a non-pathogenic amoeboflagellate; and the sequence from Cercomonas sp. (GenBank accession PX372085 [https://www.ncbi.nlm.nih.gov/nuccore/PX372085]) was most similar to that of Cercomonas longicauda. We hereafter refer to them as amoeba Naegleria sp. and flagellate Cercomonas sp. throughout this study. Each protist was cultivated in PAS buffer (0.136 g/L KH₂PO₄, 0.142 g/L Na₂HPO₄, 0.120 g/L NaCl, 4 mg/L MgSO₄·7H₂O, 4 mg/L CaCl₂·2H₂O) at 20 °C, using standard protocols⁶⁵, with heat-inactivated Escherichia coli (E. coli) DH5α (10⁵ Colony Forming Unit (CFU) mL^-1) used as a food source. To avoid bacterial contamination, the PAS buffer was supplemented with penicillin/streptomycin at both 500 μg/mL for short-term treatment (20 minutes), as both protist species are insensitive to these antibiotics. Protist cells were collected by filtration through 5 μm membranes to remove bacterial cells. The filtered protist suspensions were plated to check for potential contamination and bacterial-protist co-culture. Samples containing <5 bacterial CFU were considered as successful protist preparations (Supplementary Fig. 10). Protist counting was performed under an inverted microscope (Mshot, MF52-N).

Construction of the SynCom

We focused on designing a rhizosphere SynCom capable of forming stable microbial biofilms – as an important mechanism associated with rhizosphere competence. For that, we constructed a SynCom composed of seven common rhizosphere bacteria based on the following principles: The synthetic microbial community (SynCom) was constructed from bacterial isolates originating from the cucumber rhizosphere. First, co-existence with Bacillus velezensis SQR9 in biofilm assays was adopted as the primary selection criterion. Amplicon sequencing revealed 14 genera and two families co-existing under these conditions, from which 11 representative isolates across three phyla (Firmicutes, Proteobacteria, Bacteroidetes) were obtained. These isolates were assembled into an initial 11-member biofilm community and monitored through biofilm development stages. Species unable to establish or persisting at low abundance were subsequently excluded. To rationally reduce the complexity, microbial co-occurrence network analysis identified taxa with high abundance or strong centrality, resulting in a six-member SynCom composed of Chryseobacterium rhizoplanae, Pantoea eucrina, Comamonas odontotermitis, Enterobacter bugandensis, Acinetobacter baumannii, and Pseudomonas stutzeri. Functional importance of each member was experimentally validated using a “removal strategy” and strain-specific qPCR quantification. Keystone species were defined based on their positive (metabolic donor) or negative (resource competitor) contributions to community biomass. This stepwise approach—natural co-occurrence, tractability, and network-guided reduction—ensured the construction of a stable and experimentally manageable SynCom for investigating metabolic interactions in multi-species biofilms^46,66.

Based on co-culture compatibility testing, we established a simplified stable synthetic community (SynCom) consisting of seven bacterial strains. Each of these strains had previously undergone complete genome sequencing^46,66. These strains are Acinetobacter baumannii XL380, Chryseobacterium rhizoplanae XL97, Comamonas odontotermitis WLL, Enterobacter bugandensis XL95, Pantoea eucrina XL123, Pseudomonas stutzeri XL272, and Bacillus velezensis SQR9. The strain B. velezensis SQR9 has been extensively studied as a PGPR^67,68. All these strains are capable of co-existing within the SynCom⁴⁶. For the establishment of the SynCom, each bacterial strain was cultured in TSB (tryptic soy broth) medium at 30 °C and 170 rpm for 24 h to obtain bacterial suspensions. These suspensions were diluted to OD₆₀₀ = 1 and mixed at equal volumes. Note: distinct strains may vary in cell density based on OD measurement. As such, we standardize our SynCom based on Od values rather than absolute cell count numbers.

Soil microcosm experiments

We added 100 g of sterilized soil (after autoclave sterilization) into 250 ml tissue culture flasks (sealed with breathable screw caps) for the soil microcosm experiment. Sterile water was added to maintain the water-holding capacity at 30%. The soil microcosms were pre-incubated in the dark at 24 °C for 1 week before a test for sterility by the method of soil suspension plate spreading to prevent contaminants. After the pre-incubation, each flask was inoculated with 1 ml of the SynCom (total bacterial cells of ~1.0 × 10⁷ CFU g^-1 sterilized soil). Sampling of the soil was performed at 2, 4, and 8 d post-inoculation using 6 replicates at each time point.

Total soil DNA was extracted using the DNeasy PowerMax^® Soil Kit (Qiagen, Germany) following the manufacturer’s instructions. The qRT-PCR was used to determine the absolute copy number of the single-copy housekeeping gene for each bacterium using the ChamQ SYBR qPCR Master Mix (Q711, Vazyme, China). qPCR was performed on a QuantStudio 5 (Thermo Fisher, USA) using the standard curve method, following the standard protocol recommended by the manufacturer. Standard plasmids constructed using the pMD19-T vector (Takara, Japan) and primers for each bacterium were obtained from a previous study⁴⁶.

Protist predation experiment

The predation activities of the protists Naegleria sp. and Cercomonas sp. were assessed using the plant pathogen Ralstonia solanacearum (including both wild Ralstonia solanacearum and RFP-labeled R. solanacearum 1115-RFP-1) and E. coli DH5α as prey. Both protists were added at 10⁴ cells mL^-1 in a 96-well microplate containing 100 μL PAS buffer per well. Ralstonia solanacearum and E. coli DH5α were added at a final concentration of 10⁶ CFU mL⁻¹, and each treatment consisted of six biological replicates. Plates were incubated in a dark chamber at 20°C. Protist populations were measured using an inverted microscope (Mshot, MF52-N), and total bacterial quantification was performed using spectrophotometric measurements at an OD₆₀₀ at 24 h. In addition, fluorescence intensity of RFP-labeled R. solanacearum was measured to quantify the abundance with excitation at 561 nm and emission at 600 nm in a microplate reader (BioTek, Synergy H1) at 24 h. The unit of fluorescence intensity is RFU (Relative Fluorescence Unit).

Impact of the SynCom on R. solanacearum

A volume of 2 μL of the SynCom culture, combined with 100 individuals of the protists (Cercomonas sp. or Naegleria sp.), was inoculated on a piece of sterile filter paper at the center of the NA plates. For control samples not containing the SynCom culture or protists, an equal volume of PAS buffer was used as a substitute. After the filter paper was completely dry, the plates were incubated at 30 °C for 12 h. After that, a suspension of R. solanacearum (OD₆₀₀ = 1) was sprayed onto the NA medium. These plates were incubated at 30 °C for 24 h. The impact on R. solanacearum was based on the visual observation of an inhibition zone, which was determined as [Inhibition zone diameter (D) - Inoculated colony diameter (d)]/2.

Biofilm assays and metatranscriptome analysis

After thoroughly mixing individually each strain culture of the SynCom (each bacterium at an OD₆₀₀ = 1) in equal amounts in PAS buffer, 100 μL of the mixed SynCom culture was taken and co-inoculated with 10,000 protists (Cercomonas sp. or Naegleria sp.) into 6-well cell culture plates (Thermo Fisher, USA). Each well contained 10 mL of TSB medium (or 1/2 Msgg+1/2 TSB medium, a culture medium to form biofilm), and was incubated at 30 °C for 48 hours. The experiment included treatments with SynCom+Naegleria, Bacillus+Naegleria, and the respective controls without protists. For the control treatment without the SynCom, an equal volume of B. velezensis SQR9 culture was used as a substitute, while for the treatment without protists, an equal volume of PAS buffer was used as a substitute. After cultivation, a sterile 100 μm nylon cell filter (Biologix, USA) was immediately added to each well to provide a physical structure for biofilm formation. After 48 h of incubation, we collected the cell filters, eliminated visible droplets with paper, and measured the biofilm weight using the gravimetric method. The fresh weight of the collected biofilm (biofilm production) was determined as the total weight minus the weight of the nylon mesh. Phenotype images of the biofilm were captured under an inverted microscope (Mshot, MF52-N) using a CCD camera (Mshot, China).

After removing the biofilm, the entire liquid fraction of the culture system was collected and transferred to a new sterile EP tube. These collected samples were subjected to centrifugation at 10,000 x g, the supernatant was removed, and the cell pellet was frozen in liquid nitrogen and stored at -80 °C. Total RNA was extracted using the Qiagen RNA PowerSoil^® Total RNA Isolation Kit (Hilden, NRW, Germany), following the manufacturer’s instructions. Each treatment contained four biological replicates. The obtained RNA of the samples was sent to Personalbio Genomic Technology Limited (Nanjing, China) for metatranscriptome library construction and sequencing.

In addition, biofilm formation was measured using the crystal violet staining method⁶⁹. In brief, each of the seven bacteria from the SynCom was cultured overnight in a culture medium, and then mixed (each bacterium at an OD₆₀₀ = 1). A volume of 160 μL of the mixture was amended with 2000 protists in 96-well culture plates, and incubated at 30 °C for 48 h. The cultures were washed with 200 μL PBS solution to remove free cells. Then, the biofilm in each well was transferred to a new plate containing 180 μL of crystal violet solution for 20 min and transferred to another plate containing 200 μL of 96% ethanol for 30 min. Finally, the biofilm formation was measured in a microplate reader (BioTek, Synergy H1) at an OD₅₉₀.

Greenhouse experiments

Two greenhouse experiments were performed to examine the direct and indirect effects of protist applications in promoting plant growth, both using tomato as a model plant. The first experiment with the dwarf red cultivar ‘Jiangshu’ (Solanum. lycopersicum cv. Jiangshu)⁷⁰ consisted of the following treatments: (1) Cercomonas sp.: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Cercomonas sp. per gram soil; (2) Naegleria sp.: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Naegleria sp. per gram soil; (3) Cercomonas+SynCom: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Cercomonas sp. and 1.0 × 10⁶ cells of each bacterium of the SynCom per gram soil; (4) Naegleria+SynCom: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Naegleria sp. and 1.0 × 10⁶ cells of each bacterium of the SynCom per gram soil; (5) SynCom: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10⁶ cells of each bacterium of the SynCom per gram soil; and (6) Control (CK): soil was supplemented with 50 mL of PAS buffer. The second experiment with the dwarf red cultivar ‘Micro-Tom’ (S. lycopersicum cv. Micro-Tom)^71,72 consisted of the following treatments: (1) Naegleria sp.: soil was supplemented with 50 mL of PAS buffer containing 1.0×10³ cells of Naegleria sp. per gram soil; (2) B. velezensis SQR9: soil was supplemented with 50 mL of PAS buffer containing 7.0 × 10⁶ cells of B. velezensis SQR9 per gram soil; (3) SQR9+Naegleria: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Naegleria sp. and 7.0 × 10⁶ cells of B. velezensis SQR9 per gram soil; (4) SynCom: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10⁶ cells of each bacterium of the SynCom per gram soil; (5) SynCom+Naegleria: soil was supplemented with 50 mL of PAS buffer containing 1.0 × 10³ cells of Naegleria sp. and 1.0 × 10⁶ cells of each bacterium of the SynCom per gram soil; and (6) Control (CK): soil was supplemented with 50 mL of PAS buffer. The two pot experiments were conducted in a greenhouse at the Weigang Campus of Nanjing Agricultural University, Nanjing, China. Each treatment consisted of six replicates in a random block design, with each pot containing 500 g of natural field soil (the soil used is classified as yellow-brown soil, collected at the Nanjing Institute of Vegetable Sciences, Nanjing, China, 32.03 N, 118.78 E). These experiments were conducted in the greenhouse, with a daytime temperature of 30 °C for 12 h and a nighttime temperature of 28 °C for 12 h, with approximately 50% relative humidity. Each pot received regular watering during the duration of the experiment.

Rhizosphere soil sampling and bacterial 16S rRNA gene sequencing

Plant rhizosphere soil samples were collected in each pot following the procedures outlined in a previous study³⁶. Total DNA extraction was performed using the DNeasy PowerMax^® Soil Kit (Qiagen, Germany), following the manufacturer’s instructions. The V4 region of the bacterial 16S rRNA gene was PCR-amplified with the primer set 515 F (5’-GTGCCAGCMGCCGCGG-3’) and 907 R (5’-CCGTCAATTCMTTTRAGTTT-3’). PCRs were performed in a final volume of 20 μL, including 4 μL of 5 × reaction buffer, 2 μL of dNTP (2.5 mM), 0.8 μL of each primer (10 μM), 0.4 μl of 2 × Phanta Max Master Mix (Vazyme, China), 10 ng of DNA template, and the remaining volume completed with ddH₂O. Amplifications were conducted using an initial denaturation at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, and a final extension step at 72 °C for 10 min. PCR products were purified and eluted to a final concentration of 10 ng/μl. Paired-end sequencing was performed on an Illumina Novaseq 6000 platform at Personalbio Genomic Technology Limited (Nanjing, China).

Bioinformatics and statistical analysis

For metatranscriptome analysis, the bbduk tool available at the bbtools suite⁷³ was used to remove residual rRNA sequences from the raw fastq data using an rRNA reference library (Silva138+RFAM)^74,75. De novo assembly of metatranscriptome reads was performed using Trinity (version 2.14.0)⁷⁶. The assembled unigenes sequences were aligned against the NR database (version 20230513) using Diamond⁷⁷ for both functional and taxonomic annotation. Gene functions were annotated using the eggNOG-mapper (version 2.1.7, eggNOG database version 5.0.2)⁷⁸. Secondary metabolite gene clusters of each strain within the SynCom were predicted using antiSMASH (Supplementary D 1, version 7.0.0)⁷⁹. Secondary metabolites with similarity greater than 80% were selected as subjects for quantification. We collected all gene clusters that met the criteria and mapped the raw reads to these clusters to determine expression levels – performed using Kallisto⁸⁰. Genomes of the SynCom members were referenced from a previous study⁴⁶.

The analysis of the bacterial 16S rRNA gene amplicon sequences was performed using the USEARCH pipeline (version 11). In brief, paired sequences with expected errors > 1.0 or a length <200 bp were removed. Zero-radius Operational Taxonomic Unit (zOTU) was obtained using UNOISE3⁸¹ with the simultaneous removal of chimeras. Taxonomic annotation was performed using the NCBI 16S ribosomal RNA database, with sequence alignment performed using Blastn (version 2.12). The taxID of the sequence with the highest annotation score (bitscore) was used for taxonomic annotation, and if mSynComultiple taxIDs were found. The LCA (lowest common ancestor) algorithm was used for taxonomic classification using the LCA option⁸² in the taxonkit software⁸³. In addition, pairwise sequence alignments were conducted using the SnapGene software (v6.0.2), using the local alignment method.

Statistical analyses of data were performed in R (version 4.3.0). By default, error bars in all data analyses throughout the text are presented as the 95% confidence interval (95% CI). In brief, alpha diversity (Shannon index and Simpson index) was computed using the “vegan” package, and linear discriminant analysis effect size (LEfSe) analysis was performed using the “microeco” package⁸⁴. In the LDA analysis, the LDA score values were log₁₀-transformed, with the significance threshold set at LDA > 2.0 for feature selection. Principal coordinate analysis (PCoA) based on Bray-Curtis distances was performed to explore differences in bacterial communities across treatments using the “vegan” package. One-way analyses of variance (ANOVA) with LSD multiple range test were used for multiple comparisons. Normality of the data was assessed using the Shapiro-Wilk test. When the assumption of normality was violated, the Kruskal-Wallis H test was applied instead, and exact P-values for all comparisons are reported in the Source Data. Student’s t-test was used to test for differences in different parameters of data obtained between two treatments. Data visualization was conducted using the “ggplot2” package.

Isolation and identification of bacterial taxa co-cultured with protists

We isolated and characterized bacterial taxa retrieved in the medium used for protist growth. For that, the preserved protist stock (Naegleria sp.) was initially reactivated in PAS buffer at 20 °C. After that, the protist culture was streaked onto LB agar plates and incubated at 30 °C for 48 hours to facilitate potential bacterial growth. The isolated single colonies of bacteria were purified by streaking onto fresh LB agar plates. Genomic DNA was extracted from these purified bacterial isolates using the Universal Genomic DNA Kit (CWBIO, China). The nearly full-length 16S rRNA genes were amplified via PCR using the universal primers 27 F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492 R (5′-TACGGCTACCTTGTTACGACTT-3′). Sanger sequencing of the PCR products was performed at Tsingke Biotechnology (Nanjing, China). The identification was performed using Blastn (version 2.12) against the NCBI 16S ribosomal RNA database. A total of 100 obtained bacterial isolates resulted in the identification of only two bacterial species: Pseudomonas protegens (Supplementary Data 3) and Variovorax sp. (Supplementary Data 4). Furthermore, we trimmed the respective V4-V5 hypervariable region of these isolates’ 16S rRNA sequences, corresponding to amplicon positions 515F-907R (Supplementary Data 3 and Supplementary Data 4), using SnapGene (v6.0.2). The amplicon sequencing data obtained from the protist culture media were processed using the same bioinformatics pipeline (Supplementary Data 5). These isolated bacteria were identified in our sequencing dataset based on 100% sequence identity. The results showed the relative abundances of the two co-cultured bacterial species to be <0.03% within the total rhizosphere bacterial community (Supplementary Fig. 11).

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw metatranscriptome data and the derived gene catalogs and the raw 16S rRNA gene sequences (i.e., amplicon sequencing data) are publicly available at the NCBI Sequence Read Archive (SRA) under the BioProject accession number PRJNA1165671 [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1165671]. The BioProject accession numbers for the seven strains in SynCom are as follows: Acinetobacter baumannii XL380 (PRJNA593376) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA593376], Chryseobacterium rhizoplanae XL97 (PRJNA721858) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA721858], Comamonas odontotermitis WLL (PRJNA762695) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA762695], Enterobacter bugandensis XL95 (PRJNA761942) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA761942], Pantoea eucrina XL123 (PRJNA762676) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA762676], Pseudomonas stutzeri XL272 (PRJNA593625) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA593625], and Bacillus velezensis SQR9 (PRJNA227504) [https://www.ncbi.nlm.nih.gov/bioproject/PRJNA227504]. Source data are provided with this paper.

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Acknowledgements

We thank Prof. George A. Kowalchuk from Utrecht University, and Prof. Rong Li and Prof. Zhong Wei from Nanjing Agricultural University for the helpful suggestions on this manuscript. This project received funding from the National Natural Science Foundation of China (42377296, 42090064 and 42107141), the National Key Research and Development Program of China (2023YFD1901402 and 2023YFD1901105), and the Fundamental Research Funds for the Central Universities (YDZX2025046 and RENCAI2025022). Francisco Dini-Andreote is financially supported by the USDA National Institute of Food and Agriculture (grant 2024-67019-42476) and Hatch Appropriations under Project PEN04908 (7006279).

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These authors contributed equally: Yang Yue, Zhihui Xu, Yijin Wang.

Authors and Affiliations

Jiangsu Provincial Key Lab for Solid Organic Waste Utilization, Key Lab of Organic-Based Fertilizers of China, Jiangsu Collaborative Innovation Center for Solid Organic Wastes, Educational Ministry Engineering Center of Resource-Saving Fertilizers, Nanjing Agricultural University, Nanjing, People’s Republic of China
Yang Yue, Zhihui Xu, Yijin Wang, Chen Liu, Shuo Sun, Xiangyu Ren, Qihui Lv, Jingchi Liu, Wu Xiong & Qirong Shen
Department of Plant Science & Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA
Francisco Dini-Andreote
The One Health Microbiome Center, Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA
Francisco Dini-Andreote

Authors

Yang Yue
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Zhihui Xu
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Yijin Wang
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Chen Liu
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Shuo Sun
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Xiangyu Ren
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Qihui Lv
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Jingchi Liu
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Francisco Dini-Andreote
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Wu Xiong
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Qirong Shen
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Contributions

Y.Y., Z.X. and Y.W. contributed equally to this work. Y.Y., Y.W., C.L., S.S., X.R., Q.L. and J.L. performed the experiments and data analysis. W.X., Z.X. and F.D.-A. provided guidance on microbial ecology and data interpretation. W.X. and Q.S. conceived and supervised the project. Y.Y., Y.W. and W.X. wrote the manuscript with input from all authors. All authors read and approved the final manuscript.

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Correspondence to Wu Xiong.

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Nature Communications thanks Rasit Asiloglu, Linfang Huang, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

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Yue, Y., Xu, Z., Wang, Y. et al. Soil-dwelling Naegleria enhances plant performance by stimulating beneficial bacterial functions in the rhizosphere. Nat Commun 16, 9079 (2025). https://doi.org/10.1038/s41467-025-64139-x

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Received: 24 November 2024
Accepted: 05 September 2025
Published: 13 October 2025
DOI: https://doi.org/10.1038/s41467-025-64139-x