Africa grapples with the escalating challenge of feeding its rapidly growing population, leading to high rates of hunger and poverty (OECD/FAO, 2016). Addressing this crisis necessitates enhancing agricultural productivity to curb hunger and poverty and ensure food security (Tadele, 2017). One promising approach involves synergizing perennial and feed legumes in agroecological systems, such as push-pull technology (PPT), offering an alternative for elevating agricultural sustainability (Khan et al., 2014; Tadele, 2017). Since its inception in the mid-1990s, PPT has been recognized as a pathway for sustainable cereal crop intensification against lepidopteran pests, parasitic Striga weeds, and biodiversity restoration (Khan et al., 2014; Mutyambai et al., 2023). The strategy provides additional ecosystem services such as soil phytoremediation, moisture content regulation, and shifts in soil microbial and physico-chemical properties (Drinkwater et al., 2021; Jalloh et al., 2024). PPT involves intercropping perennial Desmodium species alongside maize while bordering the plantings with Napier or Brachiaria grass (Khan et al., 2014). The ‘pull' plant (Brachiaria/Napier grass) produces a gummy substance in reaction to the penetration of the stemborer larvae, which causes the death of early instar larvae (Khan and Pickett, 2004). On the other hand, the “push” plant (Desmodium species) emits repellent volatiles against crop pests, limiting oviposition preference of female moths (Cheruiyot et al., 2018; Mutyambai et al., 2019; Peter et al., 2023). Desmodium species also prevent Striga hermonthica parasitism by inducing allelopathic suicidal germination, thereby providing a novel means of in situ reductions of the Striga seed bank in soil (Tsanuo et al., 2003). Despite the existence of several Desmodium species worldwide, only a few species have been utilized in PPT.

Globally, 350 Desmodium species have been delineated, of which only 39 species have been identified in Africa (Ma et al., 2011). Of these Desmodium species, PPT has utilized three, including silverleaf desmodium (SLD) [Desmodium uncinatum (Jacq.) DC] in the conventional PPT (Khan et al., 2000), greenleaf desmodium (GLD) [D. intortum; (Mill.) Urb.] in the climate-smart PPT (Khan et al., 2016a), and African desmodium (AID) (D. incanum DC.) in the third-generation PPT (Cheruiyot et al., 2021). In addition, Desmodium species have specialized interactions with host-specific endophytic microbes such as rhizobia, forming atmospheric N₂-fixing root-nodules that have been reported to yield up to 90 kg N ha⁻¹ seasonally (Gu et al., 2007; Ojiem et al., 2007; Xu et al., 2016). Moreover, Desmodium plants enhance the availability of major nutrients such as phosphorus and carbon in agricultural soils (Drinkwater et al., 2021; Ndayisaba et al., 2022, 2023). AID also effectively remediates petroleum-degraded soils, favoring the multiplication of rhizospheric microbiota (Kitamura and Maranho, 2016). However, the mechanisms through which Desmodium plants affect the below-ground microbial communities have been largely unknown.

Beneath the surface, plant roots engage with a diverse soil microbiome, shaping a localized plant-soil feedback mechanism (Mutyambai et al., 2019; Hannula et al., 2021). This interaction, guided by microbial communities, positively influences soil nutrient cycling, fostering beneficial microbe s survival and plant growth and defense against insects (Muthini et al., 2020; Hannula et al., 2021; Mutyambai et al., 2023; Jalloh et al., 2024). The plant root microbiome is shaped by microbe historical contingency (Carlström et al., 2019), interspecies microbial competition, rhizodeposit signaling cues (flavonoids), and numerous symbiosis (Nod) genes (Curtis et al., 2002; Shi et al., 2011; Oldroyd, 2013). Plants cannot assimilate nitrogen, the primary limiting nutrient in agriculture, due to their inability to break down existing complex bonds (Oldroyd, 2013). However, rhizobia colonizes legume plant roots, forming an endosymbiotic relationship that ensures nitrogen is converted to ammonia via biological nitrogen fixation (BNF) (Jalloh et al., 2020; Wekesa et al., 2022). This happens in specialized organs known as root-nodules, which are formed via an intricate association with rhizobia (Oldroyd, 2013). While nodulation has long been studied as a two-member system (legume plant and rhizobia), recent research highlights complex and distinct microbes residing in separate ecological microniches (Hartman et al., 2017).

Rhizobia, the nitrogen-fixing bacteria crucial for nodulation, operate in harmony with other representative microbes, forming a balanced association with non-rhizobial endophytes (NREs) like Azospirillum, Devosia, Bacillus, Pseudomonas, and Streptomyces (Mayhood and Mirza, 2021). While information on nodule-colonizing fungi remains limited, various genera, such as Aspergillus, Glomus, Penicillium, Trichoderma, Macrophomina, Fusarium, and Rhizoctonia, have been isolated from legume root-nodules using culture techniques (Muthini et al., 2020; Mayhood and Mirza, 2021). These bacterial and fungal endophytes facilitate nutrient cycling and acquisition, siderophore production, antibiotic resistance, phosphate solubilization, bio-defense against pathogens, regulation of osmotic pressure, hydrolytic enzyme production and secretion of metabolites (Rajendran et al., 2008; Dakora, 2015; Hartman et al., 2017; Drinkwater et al., 2021; Jalloh et al., 2024). These NREs have also been proven to positively impact plant growth when co-inoculated with rhizobia (Mayhood and Mirza, 2021). Consequently, this legume-microbe association holds significant environmental benefits by reducing the overreliance on/and disproportionate use of synthetic chemical fertilizers, which are known contributors to human and environmental hazards (Lupini et al., 2023).

The existing body of research has extensively examined and documented plant-plant and plant-insect interactions within PPT (Khan et al., 2016b; Mutyambai et al., 2016, 2019, 2023). However, the domain of below-ground interactions, particularly focusing on microbial communities inhabiting Desmodium root-nodules and their potential ecological benefits, remains largely unexplored. In this study, we hypothesized that Desmodium species root-nodules serve as habitats for diverse fungal and bacterial communities. Exploring these belowground interactions is anticipated to uncover novel dimensions that could significantly contribute to enhancing the sustainability and productivity of PPT.

The findings of this study revealed diverse NREs within the root-nodules of three Desmodium species, highlighting notable distinctions among them. The observation indicates that Desmodium plants may adapt to soils beyond their native habitat in South America, supported by the recruitment of diverse microbial communities. The diversity of microbes inhabiting the root-nodules of the three Desmodium species showed no significant correlation with the study location. However, variations in bacterial and fungal abundances across sampling locations could result from recurrent differences in climatic factors, including average annual temperatures, humidity and rainfall patterns, as reported by Pang et al. (2021). Remarkably, there was a shift in fungal communities compared to that of bacterial communities. The absence of significant differences in α and β diversity suggest that the overall composition of the Desmodium species root-nodules microbiome among the species was random and not influenced by either the Desmodium species or sampling location. Therefore, these findings contribute to our understanding of the relationships between Desmodium plants and their root-nodule microbiomes, offering insights into potential adaptations and ecological dynamics in diverse soil environments.

Our findings revealed that rhizobia, mainly belonging to the Bradyrhizobium genus, predominated as the primary occupants of Desmodium species root-nodules. This aligns with similar studies on pulse legumes such as Trifolium spp., Pisum sativum, Cicer arietinum, Arachis hypogaea and Glycine max (Hartman et al., 2017; Sharaf et al., 2019; Ilyas et al., 2022). The prevalence of Bradyrhizobium in Desmodium species root-nodules is noteworthy, considering its well-documented robust nitrogen-fixing capability in legumes within tropical and subtropical regions (Hurse and Date, 1992). This genus is a common microsymbiont in major pulses like soybean (Glycine max), peanuts (Arachis hypogaea), common bean (Phaseolus vulgaris), cowpea (Vigna unguiculata), and lima beans (Phaseolus lunatus) (Ormeño-Orrillo and Martínez-Romero, 2019; Muthini et al., 2020), with reported associations in various regions, including sub-Saharan Africa (Wekesa et al., 2022), China (Gu et al., 2007), Argentina (Toniutti et al., 2017) and Mexico (Parker, 2002). Our findings, however, differ from a study by Xu et al. (2016), which highlighted a higher occurrence of fast-growing rhizobia isolates (Rhizobium, Mesorhizobium, and Pararhizobium spp.) over Bradyrhizobium in wild Desmodium sequax, Desmodium elegans, Desmodium gangeticum and Desmodium oldhamii. Importantly, our study did not identify Bradyrhizobium denitrificans, previously reported in AID from Argentina (Toniutti et al., 2017), suggesting potential regional endemism among major rhizobial occupants (Xu et al., 2016). Beyond fixing nitrogen, Bradyrhizobium exhibits other beneficial traits, including acetylene reduction (Parker, 2002), antibiotic resistance (Hurse and Date, 1992), denitrification, and defense against fungal pathogens from the Macrophomina and Sclerotium genera (Chan, 2009). Bacillus spp., the second most abundant NRE known for enhancing heat and freezing tolerance, nodulation and phosphorous solubilization, was exclusively observed in GLD (Xu et al., 2014; Khalifa and Almalki, 2015; Tiwari et al., 2017). This endophyte, when co-inoculated with rhizobia, also serves as a biocontrol agent against Rhizoctonia solani and Sclerotium rolfsii by producing β-1, 3-glucanase enzymes that degrades their cell walls (Compant et al., 2005).

Another notable finding was the association of Labrys neptuniae with Desmodium species root-nodule, a bacterium belonging to the family Xanthobacteraceae. While this is the first report of Labrys neptuniae in Desmodium species, it has been previously isolated from root-nodules of wild Acaciella spp. in Mexico and Entada phaseoloides in Japan (Chou et al., 2007; Bai et al., 2011; Chávez-Ramírez et al., 2022). Variovarax, a genus capable of producing a broad spectrum of hydrolytic enzymes (like lipase, cellulase, and protease) and demonstrating various beneficial traits such as phosphorous solubilization, indoleacetic acid (IAA) synthesis, and heavy metal phytoremediation, was detected in SLD root-nodules (Aserse et al., 2013; De Almeida Lopes et al., 2016; Bessadok et al., 2020; Khan et al., 2023). However, the low occurrence of Variovarax in the studied Desmodium species suggests a lack of specific mechanisms to select these endophytes, limiting their potential role in enhancing plant growth (Mayhood and Mirza, 2021).

The study identified phytopathogenic bacteria, including Enterobacter kobei, Macrophomina neoaurum, Pseudomonas spp. and Dyadobacter fermentans, exclusively associated with SLD root-nodules. Enterobacter kobei, previously isolated from the root-nodules of wild Hedysarum genera, was reported to inhibit anthracnose causing Colletotrichum musae in bananas and improve zinc and phosphate solubilization in Barleria lupulina and lentils (Muresu et al., 2010; Damasceno et al., 2019). Macrophomina neoaurum, known for enhancing in vitro iron acquisition under iron-deficiency conditions via exochelin secretion, was also identified by Chan (2009). Pseudomonas spp. and Chryseobacterium indologenes reported as phosphate-solubilizing plant growth-promoting rhizobacteria (PGPR), IAA synthesis and biocontrol agents against Phytophthora, were associated with SLD root-nodules (Chelius and Triplett, 2001; Sang et al., 2018).

Fungal abundance within Desmodium species root-nodules uncovered a diverse assembly of filamentous fungi coexisting with bacterial occupants. These fungi, identified across all Desmodium species, colonize root-nodules by developing abruptly ending, thin-walled hyphae within cortical cells (Russell et al., 2002). Notably, pathogenic and non-pathogenic fungi have been identified in asymptomatic legumes, with potential mitigating effects by cohabiting bacteria (Da Silva et al., 2021). Talaromyces spp. was found across all three Desmodium species with higher abundance in GLD. This fungus is an antagonist against Sclerotinia sclerotiorum solubilizes phosphorous and produces IAA (Sahu et al., 2019). Fusarium was more abundant in the root-nodules of AID compared to SLD and GLD, a contrast to previous reports identifying it as a major fungal genus in the roots of forage legume Sainfoin (Onobrychis viciifolia) (Slabbert et al., 2023). While Fusarium has been isolated from various plant roots, including Medicago species and chickpeas (Cicer arietinum L.) (Lamprecht et al., 1988; Moparthi et al., 2021), its presence as Desmodium species root-nodule occupants is novel. SLD has also been reported to mediate the inhibition of Fusarium oxysporum growth (Were et al., 2022). Interestingly, the pathogen was not observed in all the Desmodium species, indicating that the mediation effect could be present in all three Desmodium species. Fusarium solani and F. chlamydosporum were previously isolated from the roots and nodules of Medicago species (Lamprecht et al., 1988), while F. solani and F. sporotrichioides were also isolated and identified from chickpeas (Cicer arietinum), dry pea (Pisum sativum), lentil (Lens culinaris) and Axonopus compressus roots (Moparthi et al., 2021). SLD and GLD exhibited enrichment of phytopathogenic microbes, including F. sporotrichioides and F. sacharri, known to cause root rot, lag-leaf sheath spot, wilt, and blights in legume plants, rice and potatoes compared to AID (Moparthi et al., 2021).

Clonostachys spp., observed in GLD and SLD, is recognized as a mycoparasitic fungus and biocontrol agent against pathogens, including Rosellinia root rot of cocoa by Clonostachys byssicola and banana crown rot by C. byssicola (Krauss et al., 2013). It also acts as an antagonist to Phytophthora palmivora (Krauss et al., 2013), Moniliophthora roreri (Harry et al., 2003), and F. graminearum (Nygren et al., 2018). Clonostachys rosea, secretes secondary metabolites with biotic activity against Sclerotinia sclerotiorum (Cabrera et al., 2011). Monosporascus spp., reported in higher relative abundance in GLD, has been previously isolated from the roots of xerophytic shrubs and as an endophyte of Astragalus adsurgens, contributing to soil organic carbon accumulation (Zuo et al., 2022). Sistotrema spp. also found in GLD and SLD, has conferred resilience to nutrient deficiency and drought stress in blueberries (Vaccinium corymbosum) (Ye et al., 2023). Metacordyceps chlamydosporia, a nematophagous fungal endophyte isolated from soybean root tips, (Strom et al., 2020) was only detected in SLD and GLD. Acrocalymma, Atractiella rhizophila, Serendipita, Cadophora and Melanconiella were selectively identified in the three Desmodium species root-nodules. Acrocalymma and Melanconiella were only observed in GLD and SLD, Atractiella rhizophila only observed in SLD and AID, and Cadophora only observed in AID. These microbes are classified as dark septate root endophytes (DSEs), which, through strain-dependent symbiotic associations with plants, enhance host plant tolerance to various environmental stresses, such as water deficit and salt stress (Farias et al., 2020; He et al., 2021). Melanconiella spp. and A. rhizophila have been reported to improve nitrogen and phosphorous nutrition in cowpeas under elevated salinity (Bonito et al., 2017; Farias et al., 2020; Gama et al., 2020; Muazzam and Darah, 2020). To the best of our knowledge, this is the first report of DSEs in Desmodium species root-nodules, although their agroecological services still need further study.

Penicillium rubidurum, Cladosporium subuliforme, Ceratobasidium ramicola and Sarocladium kiliense had varied relative abundance across the root-nodules of the three Desmodium species. Ceratobasidium ramicola, which exhibits both phytopathogenic and endophytic characteristics and confers antibiotic resistance to plants (Muazzam and Darah, 2020), was only reported in SLD. Sarocladium kiliense is only observed in GLD and has been reported as a Brachiaria endophyte conferring resistance against pathogenic Sclerotinia sclerotiorum (Gama et al., 2020). Cladosporium subuliforme, which was more relatively abundant in GLD than SLD and AID, has been identified as a Diaphorina citri entomopathogen (Wang et al., 2023). Penicillium rubidurum, observed only in AID, has been isolated from soil in Korea (Adhikari et al., 2017) and Colorado cropping soils, respectively, with biocontrol activity against phytopathogenic Penicillium expansum and in the roots of Artemisia annua (Yuan et al., 2011).

The root-nodules in our study displayed a notable potential for robust nitrogen fixation, attributable to the higher abundance of rhizobia. However, diverse NREs, suggested potential metabolic functional variances that warrant further exploration. In the context of legume symbiosis, substantial energy inputs are essential, with a critical reliance on carbon sources supplied by legume host cells for effective nodulation and nitrogen fixation (Makoudi et al., 2018). Here, we identified upregulated metabolic functions for rhizosphere fitness, including carbon metabolism, carbohydrate metabolism, and amino acid biosynthesis. Carbon metabolism is vital as it facilitates the transfer of photosynthates, primarily carbohydrates, from plant leaves to root-nodules for cellular respiration (Liu et al., 2018). Our findings indicate the involvement of sucrose invertase in sucrose degradation, as well as the aromatic degradation of protocatechuate and a meta-cleavage pathway of catechol, vanillin and vanillate (C1 Compounds). These processes are identified as characteristic features of Bradyrhizobium japonicum USDA110, and Rhizobium leguminosarum in pea (Pisum sativum) (Sudtachat et al., 2009; Garcia-Fraile et al., 2015; Jalloh et al., 2024). Moreover, the super pathway of D-glucarate and D-galactarate catabolism was reportedly enhanced in Burkholderia-Caballeronia-Paraburkholderi spp. Following this, carbon sources are transported across the bacteroid and peribacteroid membranes, entering the tricarboxylic acid cycle (TCA), glycolysis, and Entner-Doudoroff pathways for glucose catabolism (Liu et al., 2018). The Entner-Doudoroff pathway also aids Rhizobium spp. in enduring sulfur-abundant soils by breaking down abundant phototroph-derived carbohydrates (Li et al., 2020).

In terms of siderophore biosynthesis, we observed higher upregulation in SLD compared to AID and GLD, potentially indicating the presence of more microorganisms in SLD producing high-affinity Fe (III)-chelating compounds. Fatty acid and lipid biosynthesis pathways play a role in generating fatty acids and other lipids while converting nutrient-derived carbons into fatty acids (Chandel, 2021). Amino acid biosynthesis regulates ammonium assimilation and asparagine synthesis (Okumoto and Pilot, 2011). Vitamin biosynthesis is essential for various metabolic processes inside root-nodules and transporting selected decarboxylases, carboxylases and transcarboxylases. Examples of vitamins include thiamine, vitamin B12, and biotin production by R. leguminosarum and R. etli (Guillén-Navarro et al., 2005). Additionally, protocatechuate catabolism has been shown to increase the fitness of R. leguminosarum in the rhizosphere by providing a constant supply of dicarboxylates to the bacteroids (Strodtman et al., 2018). PICRUSt predicts functions of multiple 16S genes within genera using defined marker-gene metagenomic reference data. However, the lack of direct metagenome data on root-nodule microbiomes limits comprehensive prediction of functional gene categories within the root nodule microbiomes. Therefore, further research is necessary to delineate the 16S root-nodule metagenome to enable comprehensive prediction of functional gene categories within the root-nodule microbiomes.

This study unveiled diverse microbial communities co-inhabiting root-nodules of three Desmodium species used in push-pull cropping systems with Bradyrhizobium spp. bacterial symbionts being predominant within the plant organ. Additionally, it reveals the coexistence of diazotrophic bacteria with non-nodulating fungi within Desmodium root-nodules, although their precise functions remain unclear warranting further investigation. Our findings further reveal no apparent differences in the microbiomes of AID, SLD, and GLD reflecting similarities in symbiotic partner selection and adaptability among the three Desmodium species to the local environment, leading to convergence in their root-nodule microbiomes. Microbiome diversity was generally higher in root-nodules collected from Siaya county compared to those collected from Homabay, Vihiga, and Kisumu counties. Further assessment of soil geochemical properties and temporal climatic fluctuations from all sampling locations is necessary to determine if and how these abiotic factors contribute to the observed variation in microbial diversity. The findings of our study also provide insights into other potential functions of Desmodium root-nodule-associated microbiome such as carbon metabolism and amino acid biosynthesis. These functions are potentially involved in the endosymbiotic relationship while also conferring rhizosphere fitness. Thus, we recommend that future research investigates and harnesses the microbes responsible for these novel functions for improved soil health, plant growth and agroecosystem functioning.

The raw metagenome amplicon sequences data from the root-nodules of the three Desmodium species were submitted to NCBI Sequence Read Archives (SRA) under BioProject accession number PRJNA1102675 for the 16S dataset. Specifically, the 16S (V3-V4) metagenome data were assigned Biosample accession numbers SAMN41026867-SAMN41026890, while the ITS (ITS1-ITS2) metagenome data were registered under BioProject PRJNA1102689 with accession numbers SAMN41027037-SAMN41027060. GPS coordinates was provided in the Additional file 1. Additionally, we also provided the R scripts for data analysis along with all the necessary input files, Additional files 2A, B.

IA: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. GA: Data curation, Formal analysis, Methodology, Writing – review & editing. SN: Conceptualization, Investigation, Writing – review & editing. AJ: Data curation, Formal analysis, Methodology, Writing – review & editing. JM: Data curation, Formal analysis, Investigation, Methodology, Writing – review & editing. FC: Investigation, Writing – review & editing. FK: Writing – review & editing. ZK: Supervision, Writing – review & editing. SS: Funding acquisition, Supervision, Writing – review & editing. TD: Supervision, Writing – review & editing. DM: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing.