Agronomic and phytochemical aspects of Mikania glomerata Spreng (guaco) inoculated with plant growth-promoting bacteriaAbstract
Mikania glomerata (guaco) is widely used in traditional medicine due to its expectorant, antimicrobial, anti-inflammatory, antitussive, and bronchodilatory properties, which are mainly attributed to coumarins present in its leaves. Despite the growing interest in the use of plant growth-promoting bacteria (PGPB) in medicinal plants, the effects of individual inoculation and co-inoculation with Azospirillum brasilense and Pseudomonas fluorescens on the agronomic and phytochemical responses of M. glomerata remain poorly understood. In this context, we hypothesized that individual inoculation or co-inoculation with A. brasilense and P. fluorescens would differentially influence the agronomic performance and phytochemical profile of M. glomerata. Thus, this study evaluated the agronomic performance and phytochemical profile of guaco following inoculation with A. brasilense and P. fluorescens. A completely randomized 2 × 2 factorial design was employed, including single inoculation and co-inoculation treatments, with ten replicates per treatment. After seven months of cultivation, substrate biological indicators, plant biomass, nutrient content, hydroalcoholic extract yield, total phenolic content, and coumarin concentration were evaluated. P. fluorescens significantly increased plant biomass and shoot nitrogen content, whereas A. brasilense enhanced total phenolic content (7.81 µg gallic acid equivalents per gram of extract) and coumarin accumulation in guaco extracts (88 µg mL-1). Extractive yields ranged from 20.17% to 24.17%, with the highest value observed under co-inoculation. Overall, A. brasilense was the most effective treatment for stimulating the accumulation of pharmacologically relevant metabolites, while co-inoculation improved agronomic performance and extractive yield. These findings demonstrate that microbial inoculation is a sustainable biotechnological strategy for improving guaco cultivation and modulating the production of bioactive metabolites, contributing to the development of medicinal plant production systems with reduced dependence on chemical fertilizers.
Keywords:
co-inoculation; coumarin; plant metabolism; secondary metabolites; sustainability
Resumo
Mikania glomerata (guaco) é amplamente utilizada na medicina tradicional devido às suas propriedades expectorantes, antimicrobianas, anti-inflamatórias, antitussígenas e broncodilatadoras, atribuídas principalmente às cumarinas presentes em suas folhas. Apesar do crescente interesse pelo uso de bactérias promotoras do crescimento vegetal (BPCV) em plantas medicinais, os efeitos da inoculação individual e da coinoculação com Azospirillum brasilense e Pseudomonas fluorescens sobre as respostas agronômicas e fitoquímicas de M. glomerata ainda são pouco compreendidos. Nesse contexto, hipotetizou-se que a inoculação individual ou a coinoculação com A. brasilense e P. fluorescens influenciaria diferencialmente o desempenho agronômico e o perfil fitoquímico de M. glomerata. Assim, este estudo avaliou o desempenho agronômico e o perfil fitoquímico do guaco após a inoculação com A. brasilense e P. fluorescens. Foi empregado um delineamento inteiramente casualizado em esquema fatorial 2 × 2, incluindo tratamentos com inoculação isolada e coinoculação, com dez repetições por tratamento. Após sete meses de cultivo, foram avaliados os indicadores biológicos do substrato, a biomassa vegetal, o teor de nutrientes, o rendimento do extrato hidroalcoólico, o teor de compostos fenólicos totais e a concentração de cumarina. P. fluorescens promoveu aumento significativo da biomassa vegetal e do teor de nitrogênio na parte aérea, enquanto A. brasilense elevou o teor de compostos fenólicos totais (7,81 µg de equivalentes de ácido gálico por grama de extrato) e o acúmulo de cumarina nos extratos de guaco (88 µg mL−1). Os rendimentos extrativos variaram de 20,17% a 24,17%, sendo o maior valor observado na coinoculação. De modo geral, A. brasilense foi o tratamento mais eficaz para estimular o acúmulo de metabólitos de relevância farmacológica, enquanto a coinoculação melhorou o desempenho agronômico e o rendimento extrativo. Esses resultados demonstram que a inoculação microbiana constitui uma estratégia biotecnológica sustentável para otimizar o cultivo de guaco e modular a produção de metabólitos bioativos, contribuindo para o desenvolvimento de sistemas de produção de plantas medicinais com menor dependência de fertilizantes químicos.
Palavras-chave:
coinoculação; cumarina; metabolismo vegetal; metabólitos secundários; sustentabilidade
1. Introduction
Mikania glomerata Spreng. (guaco) is a medicinal species native to the Atlantic Forest and used in traditional medicine due to its expectorant, antimicrobial, anti-inflammatory, antitussive, and bronchodilatory properties. These biological activities are mainly associated with coumarins, and other secondary metabolites present in its leaves, compounds of interest in natural product research (Wang et al., 2025). However, secondary metabolite biosynthesis in medicinal plants is non-static and highly sensitive to abiotic cultivation conditions (Marifat et al., 2026), nutritional equilibrium, and rhizosphere microflora dynamics (Diniz et al., 2025).
Despite its pharmacological relevance, few studies have explored cultivation strategies capable of modulating biomass production and the accumulation of bioactive metabolites in guaco. Symbiotic microorganisms, particularly plant growth-promoting bacteria (PGPB), represent a promising approach due to their ability to stimulate plant development and influence secondary metabolism. These microorganisms colonize the rhizosphere and internal tissues, promoting plant growth through biological nitrogen fixation, phytohormone production, and pathogen biocontrol (Rosini et al., 2025).
Specifically, Azospirillum brasilense (AZ) is recognized for its ability to produce indole-3-acetic acid (IAA), which can modulate root system architecture by promoting lateral root formation and root hair elongation, thereby increasing the potential root surface area for water and nutrient acquisition (Spaepen et al., 2014).
In contrast, Pseudomonas fluorescens (PF) can promote plant growth through multiple mechanisms related to nutrient acquisition, including siderophore-mediated iron acquisition and phosphate solubilization through organic acid secretion (Yuliana and Shimizu, 2026), as well as the induction of systemic resistance (Suresh et al., 2022). Root colonization by PF can consequently prime systemic defense responses and activate defense-related genes, including phenylalanine ammonia-lyase (PAL) and other genes associated with the phenylpropanoid pathway (Gómez-Lama et al., 2014). Together, these mechanisms suggest that AZ and PF may differentially influence plant growth and secondary metabolismo potentially reducing the dependence on external nutrient inputs (Fukami et al., 2017). Based on these complementary mechanisms, we hypothesized that inoculation with AZ and PF, either individually or in combination, would differentially influence the agronomic performance and phytochemical profile of M. glomerata.
Despite increasing interest in beneficial microorganisms associated with M. glomerata, the effects of specific plant growth-promoting bacteria on the agronomic and phytochemical responses of cultivated plants remain poorly understood. In particular, the responses of M. glomerata to individual and co-inoculation with AZ and PF have not been investigated in relation to plant growth, nutrient accumulation, and coumarin production.
Therefore, this study evaluated the agronomic and phytochemical responses of M. glomerata following inoculation and co-inoculation with AZ and PF, aiming to assess microbial inoculation as a sustainable strategy to enhance bioactive metabolite accumulation.
2. Materials and Methods
2.1. Experimental design
The experiment was conducted from November 2023 to June 2024 in a private property in Quatro Pontes, Paraná, Brazil (24°34’08.5”S, 53°58’42.0”W; 427 m a.s.l.). Healthy guaco seedlings, previously identified and registered in the Genetic Heritage System (SisGen, code A1DA855), were individually cultivated in 18 × 15 cm pots containing a substrate composed of 50% Rhico, 25% sand, and 25% vermiculite (Urimamã Mining), sieved (4 mm) and fumigated with chloroform (Endlweber and Scheu, 2006). Pots were randomly arranged in a shaded nursery (60% shading) and irrigated daily.
2.2. Treatments
Thirty days after transplanting, the plants were inoculated, co-inoculated, or non-inoculated with A. brasilense (strains Abv5 and Abv6, liquid formulation at 2×108 CFU mL-1, Masterfix®) and P. fluorescens (strain CCT7997, liquid formulation at 1×108 CFU mL-1, Solubio®), inoculating 1 mL plant−1, resulting in four treatments (2 × 2 factorial combination), arranged in a completely randomized design with ten replicates per treatment (total of 40 pots, 4 kg substrate each). The control treatments used 1 mL of deionized water plant−1.
2.3. Determination of microbial biomass carbon, basal respiration, and metabolic quotient
After seven months, microbial biomass carbon (MBC) was determined using a chloroform fumigation-extraction method (Vance et al., 1987; Tate et al., 1988), modified according to Lermen et al. (2019), with 10 g soil subsamples per treatment. Basal substrate respiration (BSR) was measured following Jenkinson and Powlson (1976), as described by Silva et al. (2007a), using 30 g of substrate. The metabolic quotient (qCO2), representing CO2 release per unit of microbial biomass over time, was calculated according to Anderson and Domsch (1993) and Silva et al. (2007b).
2.4. Determination of plant growth parameters
Plant growth parameters were evaluated, including stem diameter, number of tillers, shoots, roots, and total fresh and dry mass (SFM, RFM, TFM, SDM, RDM, and TDM, respectively). Physiological indices measured were the relative chlorophyll index (RCI), anthocyanin index (AI), flavonoid index (FI), and nitrogen balance index (NBI), using the DUALEX SCIENTIFIC+™ device (Quick Start model, FORCE A®, Paris, France) following the manufacturer’s instructions. Ten replicates per treatment were measured on fully expanded leaves in the middle third of each plant.
2.5. Determination of phosphorus and nitrogen contents in shoots and roots
Phosphorus (P) in shoots (PS) and roots (PR) were determined colorimetrically using ammonium molybdate and ascorbic acid at 660 nm, and the nitrogen (N) in shoots (NS) and roots (NR) was estimated via sulfuric acid digestion at 450 °C followed by Kjeldahl distillation (Silva, 2009).
2.6. Determination of extractive yield, total phenolic content, and coumarin content
Dry aerial biomass was oven-dried at 65 °C until a constant weight was achieved, ground, and then used for hydroalcoholic (70% ethanol) extraction by dynamic maceration. Extract yield (EY) was calculated based on the ratio of plant mass to lyophilized extract (w/w%). Hydroalcoholic extraction followed the standard protocol of the Brazilian Pharmacopeia for medicinal plant extracts (Brasil, 2021). Total phenolic content (TPC) was determined using the Singleton and Rossi Junior (1965) method, and coumarin content in leaves and extracts was quantified using high-performance liquid chromatography (Shimadzu UFLC Prominence C, Japan) equipped with a C18 column (250 × 4.6 mm, 3 µm). Samples were prepared at 1000 µg mL−1 in acetonitrile-water (3:7, v/v), filtered through PTFE (0.22 µm × 13 mm, Millex, Millipore), and injected 20 μL (Rocha et al., 2008).
2.7. Statistical analysis
Data collected from the parameters analyzed were first tested for normality using the Shapiro–Wilk test (p ≤ 0.05) and the Levene’s test was applied to data for homogeneity. Following confirmation of normality and homogeneity, data were subjected to analysis of variance (ANOVA). The standard error of the mean was calculated, and treatment means were compared using Duncan’s multiple range test at a significance level of p ≤ 0.05. All statistical analyses were performed using IBM SPSS Statistics® version 22, and the heat maps and Principal Component Analysis (PCA) were generated using SRplot (Tang et al., 2023).
3. Results and Discussion
Inoculation with PF, AZ, and their co-inoculation significantly affected (p ≤ 0.05) eight of the 21 agronomic parameters evaluated (Table 1). Both treatments influenced MBC and qCO2, indicating changes in soil microbial activity. Beneficial microbial inoculation enhances nutrient availability and soil biological functioning, supporting plant development (Pelagio-Flores et al., 2025). PF inoculation also increased BSR (p = 0.02), reflecting higher microbial metabolic activity and improved soil biological status (Andrade et al., 2023).
p-values from two-way ANOVA of soil and plant parameters of M. glomerata inoculated and co-inoculated with A. brasilense (AZ) and P. fluorescens (PF).
MBC increased under AZ and PF inoculation but decreased under co-inoculation (Table 2), suggesting treatment-dependent changes in microbial biomass and C dynamics in the substrate. These results indicate that microbial inoculation can modulate soil C dynamics and contribute to sustainable cultivation systems (Harman et al., 2021).
Microbial biomass carbon (MBC, µg C g−1), basal substrate respiration (BSR, µg C-CO2 g−1 h−1), and metabolic quotient (qCO2, µg CO2 µg−1 microbial C h−1) of the substrate cultivated with M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
Stem diameter and number of tillers were not significantly affected (p > 0.05) (Table 1). However, PF inoculation significantly increased RFM, TFM and RDM (Table 3 and Table 4), consistent with previous studies linking PGPB to improved nutrient and water uptake (Andrade et al., 2023; Hashim and Khan, 2024; Pelagio-Flores et al., 2025). This response may be associated with the multiple plant-growth-promoting traits reported for PF, including nutrient solubilization and acquisition, siderophore and phytohormone production, which can contribute to root development (Mehmood et al., 2023).
Shoot fresh mass (SFM – g plant−1), root fresh mass (RFM – g plant−1), and total fresh mass (TFM – g plant−1) of M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
Dry shoot mass (SDM – g plant−1), dry root mass (RDM – g plant−1), and total dry mass (TDM – g plant−1) of M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
Nutritional parameters were also influenced by microbial treatments. PF significantly increased SN content, while co-inoculation reduced P levels in shoots (Table 5), possibly reflecting changes in nutrient allocation. The higher SN content observed under PF inoculation was accompanied by increased RBS and greater root biomass, suggesting an association between enhanced microbial activity in the substrate, root development, and N accumulation in the shoot.
Nitrogen in roots (NR) and shoots (SN) (mg g−1), and phosphorus in roots (RP) and shoots (SP) (mg g−1) of M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
PF is known to enhance nutrient uptake through increased rhizosphere activity and root biomass (Andrade et al., 2023; Hashim and Khan, 2024). Similarly, Oliveira et al. (2025) reported that PF inoculation increased root and shoot biomass and N accumulation in hydroponically grown lettuce, together with increased ammonium accumulation, reduced nitrate accumulation, and enhanced nitrate reductase activity. Co-inoculation reduced shoot P concentration, indicating that the combined bacterial treatment altered plant P status. The effects of bacterial inoculation on P nutrition are dependent on the plant species, inoculant combination, and P availability (Rosa et al., 2022).
Physiological indices related to photosynthetic performance were also affected. The RCI and NBI were influenced by AZ inoculation and co-inoculation (Table 1). Increased chlorophyll content may result from improved nutrient uptake and enhanced photosynthetic capacity (Maxwell and Johnson, 2000). Lower NBI values observed in AZ-treated plants (Table 6) may reflect altered N assimilation dynamics associated with microbial interactions.
Relative chlorophyll index (RCI), nitrogen balance index (NBI), anthocyanin index (AI), leaf flavonoid index (FI), and total phenolic content of the extract (TPC – µg GAE g ext−1) of M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
Cluster analysis based on soil parameters, plant biomass, and nutrient content revealed distinct treatment groupings (Figure 1). The control formed an independent cluster, followed by AZ, whereas co-inoculated plants grouped separately, indicating distinct functional roles of microbial treatments. These patterns were associated with the distinct responses observed for AZ and PF including differences in N-related and microbial parameters.
The heatmap, combined with hierarchical clustering, revealed distinct profiles among treatments based on soil and plant parameters in M. glomerata inoculated with A. brasilense (AZ), P. fluorescens (PF), co-inoculated with both, and a non-inoculated control. Abbreviations in Table 1.
No significant differences were observed for AI or FI. As these pigments are associated with plant stress responses (Kumar et al., 2023), the results suggest that microbial inoculation may not have significantly altered flavonoid-related stress metabolism under the conditions tested (Kumar et al., 2022).
PGPB enhance plant metabolism through mechanisms including N fixation, P solubilization, and phytohormone production (Santos et al., 2021). AZ stimulates root development through auxin-mediated pathways, improving N uptake (Cassán et al., 2020), whereas PF promotes rhizosphere activity and P mobilization (Andrade et al., 2023), which may contribute to the nutrient responses observed in the present study.
From a phytochemical perspective, microbial inoculation also influenced bioactive metabolite accumulation in M. glomerata. Coumarins are pharmacologically relevant phenolic compounds involved in plant defense and environmental adaptation (Wang et al., 2025).
Hydroalcoholic extracts of guaco leaves showed that AZ inoculation markedly increased TPC, reaching values nearly 3.8-fold higher - the control and nine-fold higher than co-inoculation (Table 7). This increase was accompanied by higher coumarin accumulation.
Extract yield (EY) (%), coumarin content in the extract (µg mL−1), and estimated coumarin concentration in the hydroalcoholic extract (mg g−1 fresh leaves) of M. glomerata inoculated and co-inoculated with A. brasilense and P. fluorescens.
Extractive yields ranged from 20.17% to 24.17%, with the highest value observed under co-inoculation. These values are consistent with previously reported yields for hydroalcoholic extracts of guaco leaves (Celeghini et al., 2007), indicating that microbial management did not compromise extraction efficiency while improving metabolite accumulation.
The stimulation of phenolic metabolism under AZ inoculation may be related to increased N availability and phytohormone-mediated regulation of secondary metabolic pathways (Pelagio-Flores et al., 2025). Estimated coumarin concentrations ranged from 0.35 to 1.21 mg g−1 of fresh leaves (Table 7), consistent with previous reports (Vieira et al., 2020). AZ inoculation increased coumarin levels to 1.21 mg g−1 (88 µg mL−1), exceeding concentrations reported under organic cultivation (0.126 mg g−1; Santos et al., 2019). This increase may be associated with the reported capacity of AZ to contribute to biological N fixation and to influence phytohormone production, including indole-3-acetic and salicylic acids, which stimulate secondary metabolism (Santos et al., 2021).
Because coumarin biosynthesis is connected to the phenylpropanoid pathway, changes in the regulation and metabolic flux of this pathway may contribute to the accumulation of phenolic compounds and coumarins in response to physiological and environmental conditions (Wang et al., 2025). In the present study, this broader modulation of phenylpropanoid metabolism may have contributed to the simultaneous increase in TPC and coumarin accumulation observed under AZ inoculation, although phenylpropanoid pathway activity was not directly assessed.
Multivariate analysis supported these findings. Heatmap and hierarchical clustering based on extract yield, AI, FI, TPC, and coumarin content revealed two clusters: AZ with control, and PF with co-inoculation. TPC and coumarin variables formed a closely related subgroup, indicating coordinated metabolic responses. PCA explained 96.6% of the total variance (PC1 = 55.5%, PC2 = 41.1%), clearly separating AZ from the other treatments (Figure 2).
The heatmap combined with hierarchical clustering (A) and Principal Component Analysis (PCA) (B) shows anthocyanin index (AI), flavonoid index (FI) in leaves, total phenolic content (TPC), extract yield (EY) (%), extract coumarin content (Cum) (mg mL−1), and coumarin concentration (CC) (mg kg−1) in hydroalcoholic extracts from fresh leaves. Principal Component Analysis (PCA) (B) of the main parameters in M. glomerata inoculated with A. brasilense (AZ), P. fluorescens (PF), co-inoculated with both, and a non-inoculated control.
Although co-inoculation resulted in the highest extractive yield, it did not produce a corresponding increase in coumarin accumulation. This lack of a consistent additive response suggests that the combined inoculants did not necessarily reproduce the effects observed with their individual applications. The absence of a consistent additive response may reflect interactions between the bacterial inoculants; however, these interactions were not directly assessed in the present study and therefore remain a possible explanation for the observed responses.
The increase observed under AZ inoculation highlights the potential of microbial management strategies to enhance the phytochemical value of medicinal plants. Microbial inoculation therefore represents a sustainable approach to improve agronomic performance and the production of pharmacologically relevant natural products, with possible implications for reducing dependence on chemical fertilizers (Pelagio-Flores et al., 2025).
4. Conclusion
Inoculation with AZ and PF modulated microbial activity, plant growth, nutrient accumulation, and secondary metabolism in M. glomerata. AZ inoculation was the most effective treatment, significantly enhancing phenolic and coumarin accumulation, while co-inoculation increased extractive yield without improving coumarin content.
These results identify AZ as a key driver of pharmacologically relevant metabolite biosynthesis in guaco. Microbial inoculation thus emerges as a sustainable strategy to optimize phytochemical production and agronomic performance in guaco. Further studies under different nutritional conditions and fertilization regimes are needed to determine whether microbial inoculation can contribute to reducing chemical inputs.
Acknowledgements
The authors acknowledge UNIPAR for financial support. D. J. N. Boufleuer and M. Dalmagro thank PROSUP/CAPES for the scholarship. M. M. Pinc thanks “Programa de Desenvolvimento da Pós-Graduação” (PDPG)/CAPES for the scholarship. O. Alberton acknowledges a research fellowship from the National Council for Scientific and Technological Development (CNPq) (grant number 310981/2023-9).
Data Availability Statement
All the data supporting the results of this study were published in the article itself.
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