The king of bitter plant (Andrographis paniculata Nees.), or sambiloto in Indonesian, is one of the most widely used plants in pharmaceutical formulations (Irianti et al., 2022). Andrographolide is the primary bioactive compound found in A. paniculata and is responsible for the plant’s characteristic bitter taste. This diterpenoid lactone and its derivatives (neo-andrographolide, 14-deoxyandrographolide, and 14-deoxy-11,12-didehydro?>andrographolide) possess a wide range of pharmacological properties (Adiguna et al., 2021; Hao et al., 2020). These include anti-inflammatory, antipyretic, antihyperglycemic, antioxidant, antidiabetic (Islam et al., 2018; Liu et al., 2020), antiviral (Adiguna et al., 2021; Jiang et al., 2021; Shi et al., 2020), and gastrointestinal protective activities (Wang et al., 2021). Due to these properties, andrographolide and its biosynthesis have attracted considerable interest in pharmaceutical and agricultural research.

The concentration of andrographolide in A. paniculata varies among different plant tissues, with the highest levels found in the leaves (ranging from 2.5% to 4.8%), while only small amounts are detected in the roots, stems, and flowers (Fitriansyah et al., 2024). However, several studies have shown that andrographolide content is also influenced by the plant’s age and environmental conditions. For instance, Tajidin et al. (2019) reported that the highest concentration of andrographolide is present when the plants reached 18 WAS (week after sowing).

To improve the biosynthesis of andrographolide and support sustainable cultivation practices, biofertilizers such as endophytic bacteria have emerged as promising alternatives to chemical fertilizers. These bacteria play multiple roles in promoting plant health and productivity. Endophytic bacteria are a type of plant growth-promoting bacteria (PGPB) that can produce phytohormones likeindole acetic acid (IAA), gibberellin (GA3), abscisic acid (ABA), and cytokinin (Egamberdieva et al., 2017). In addition, it fixes nitrogen (N) from the atmosphere (Okamoto et al., 2021), solubilizes phosphate (Li et al., 2017; Alkahtani et al., 2020), and increases the organic matter level released from dead and decomposed bacterial biomass as a biofertilizer (Ali et al., 2022). The endophytic bacteria also generate siderophores (Chen et al., 2016; Maheshwari et al., 2019), which can chelate metals such as Hg, Fe, and Al (Ferreira et al., 2019). These metals, which can hamper plant growth and development, are commonly found in acid soils that dominate the land in Indonesia. Microbes that produce siderophores can also control pathogenic microbes (Kumari et al., 2022), protect plants from environmental stress (Ullah et al., 2019), increase crop productivity, and sustain soil health (Prasad et al., 2020). Endophytic bacteria offer an economical and environmentally friendly approach to increasing plant nutrient supply, reducing chemical fertilizer use, and protecting against abiotic and biotic stresses (Lachu et al., 2022; Prasad et al., 2020).

Recent studies have demonstrated that endophytic microbes can play a significant role in enhancing secondary metabolite production in medicinal plants. For example, Kumari et al. (2023) reported that the bacterial endophyte Micrococcus luteus (ASd6) significantly increased both biomass and andrographolide content in A. paniculata by 68.01% and 44.16%, respectively. Beyond bacterial endophytes, Nair and Sakuntala (2020) highlighted the role of the root-colonizing mutualistic fungus Piriformospora indica as a potent elicitor of andrographolide biosynthesis, achieving a marked increase in andrographolide content up to 58.2 mg/g dry weight compared to 19.5 mg/g in control plants. This improvement was mechanistically associated with the transcriptional upregulation of key genes involved in the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways, including 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxy-D-xylulose 5-phosphate synthase (DXS), 1-Deoxy-D-xylulose 5-Phosphate reductoisomerase (DXR), geranylgeranyl pyrophosphate synthase (GGPS), and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (ISPH), all of which contribute to the biosynthesis of andrographolide precursors. These findings underscore the functional significance of plant and endophyte interactions in modulating secondary metabolism and offer a sustainable strategy to boost the production of pharmacologically important compounds.

One of the essential macronutrients for plant growth is phosphorus (P), which is involved in photosynthesis and primary metabolite processes, serving as an energy source to convert ADP into ATP. Glucose, produced from the primary metabolite process, is then used for secondary metabolite formation, including andrographolide, which is synthesized by A. paniculata. Andrographolide is a secondary metabolite classified under the diterpenoid group and synthesized through the mevalonate and non-mevalonate acid pathways (Bergman et al., 2019). Phosphorus is required in andrographolide biosynthesis for the production of isopentenyl pyrophosphate/diphosphate (IPP/IDP) and dimethylallyl pyrophosphate/diphosphate (DAMPP/DAMPP) (Bergman et al., 2019; Shailaja et al., 2021).

Given these facts, there is a strong need to explore the combined use of biofertilizers and phosphate fertilizer to maximize A. paniculata growth and andrographolide yield. Therefore, this study aimed to determine the most effective combination of endophytic bacteria and phosphate fertilizer dosage by evaluating physiological traits, agronomic performance, and secondary metabolite content. This work supports the application of sustainable cultivation technologies for medicinal plants, as promoted by the Indonesian Ministry of Agriculture.

The results indicate that the application of endophytic bacteria Bacillus sp. and phosphate fertilizer significantly improved plant growth, biomass production, and andrographolide accumulation, supporting the hypothesis that biofertilizers can reduce dependency on chemical inputs while sustaining or even improving yield and compound content. The improvement in agronomic traits such as plant height, branch number, and herbage yield is attributed to the role of Bacillus sp. in improving nutrient availability, particularly through phosphorus solubilization, as well as the production of plant growth-promoting phytohormones such as indole acetic acid (IAA) and gibberellic acid (GA3) (Adeleke et al., 2021). Similar findings have been reported in previous studies. For instance, Bacillus strains have been shown to positively influence maize growth and increase phosphorus (P) and nitrogen (N) content in the shoots (Alkahtani et al., 2020). Additionally, endophytic bacteria isolated from the medicinal plant Paris polyphylla var. yunnanensis have been found to enhance its growth (Tao et al., 2022). Likewise, the use of endophytic bacteria improved plant height in tomatoes (Abdallah et al., 2018) and peanuts (Lucero et al., 2021).

Endophytic bacteria not only release phytohormones but also play a crucial role in stimulating plant growth and improving overall plant performance. Previous studies have reported that bacterial endophytes can enhance tryptophan availability and produce substantial amounts of IAA, up to 60 µg mL^-1 (Alkahtani et al., 2020), and 68 mg L^-1 (Herlina et al., 2017). These phytohormones stimulated an increase in plant height and the number of primary branches in A. paniculata. IAA plays essential roles in regulating various plant cellular mechanisms, including cell orientation, division, organ development, differentiation, and elongation (Asgher et al., 2015; Labeeuw et al., 2016). Meanwhile, GA3 contributes significantly to the formation of lateral shoots (Cai et al., 2022), regulates stomatal behavior, supports metabolic activity under both normal and stress conditions (Zhou et al., 2024) and enhances root number and length (Choudhary et al., 2022).

Beyond promoting plant growth, endophytic bacteria also improve soil fertility by increasing nutrient availability. Previous studies have demonstrated that endophytic bacteria enhance pepper growth and soil fertility, leading to more efficient nutrient absorption (Gusmaini et al., 2020). In the present study, nutrient uptake patterns varied across treatments, reflecting the differential influence of Bacillus sp. and phosphate fertilizer on nutrient availability and transport. For example, the combination treatment (B1+P1) resulted in higher P and K uptake due to microbial phosphate solubilization and improved root architecture, which may enhance potassium mobility (Timofeeva et al., 2022). In contrast, 1.35 g P₂O₅ treatment (P2) led to the highest N and Mg uptake and the 0.675 g P₂O₅ treatment (P1) showed increased calcium uptake, likely because of the higher external availability of nutrients rather than microbial mediation.

These nutrient dynamics translated into higher biomass accumulation. At 14 weeks after planting (WAP), the application of endophytic Bacillus sp. and/or phosphate fertilizer led to increases in fresh herb production by 12.17–41.74% and in dry herb production by 20.92–51.13% at 14 WAP. The combined treatment of Bacillus sp. and 0.675 g P₂O₅ plant^-1 (B1+P1) yielded the highest fresh (41.74%) and dry (51.13%) herb production compared to the control and showed no significant difference from the 1.35 g P₂O₅ treatment ( Tables 3 , 4 ). This indicates that microbial support may allow for reduced chemical input without compromising productivity. These results are consistent with previous findings where endophytic bacteria increased biomass production by up to 68.01% (Kumari et al., 2023).

In addition to producing phytohormones, Bacillus sp. also enhanced nutrient availability through nitrogen fixation and phosphorus solubilization (Lucero et al., 2021). These mechanisms allow nutrients to be utilized more efficiently by plants, thereby supporting their growth. Previous studies have also reported that endophytic bacteria from Bacillus strains improved dry biomass and phosphorus use efficiency in wheat cultivars (Wang et al., 2022), as well as enhanced phosphorus uptake in pearl millet under low-phosphorus conditions (Ribeiro et al., 2018).

Enhanced nutrient absorption positively influences plant growth and development, as demonstrated by the increased dry herb yield. The application of phosphate fertilizer, either alone or in combination with endophytic bacteria, significantly improved the uptake of N, P, K, Ca, and Mg, thereby boosting both fresh and dry herb yields ( Tables 3 , 4 ). These findings align with previous studies highlighting the role of phosphate-solubilizing endophytic bacteria in enhancing phosphorus uptake.

The net assimilation rate (NAR) describes the relationship between leaf area and dry matter accumulation over time, serving as an indicator of the plant’s efficiency in producing dry biomass per unit of leaf area per unit time. A reduction in leaf area adversely impacts dry biomass production. Similarly, the relative growth rate (RGR), which quantifies the rate of dry weight increase over time, is closely influenced by NAR (Lamont et al., 2023).

Endophytic bacteria significantly enhanced the accumulation of andrographolide and its derivatives in A. paniculata, with andrographolide content increasing by up to 44.16% following bacterial application (P. Kumari et al., 2023). This enhancement is attributed to the ability of Bacillus sp. to produce IAA and GA3, as well as to improve nitrogen and phosphorus acquisition. These factors collectively act as plant growth regulators, thereby promoting biomass production and stimulating the biosynthesis of secondary metabolite (Sales and Rigobelo, 2024).

In this study, the highest total andrographolide yield was observed at 14 weeks after planting (WAP). It is primarily due to two factors, i.e increased biomass and developmental stage. Plant age strongly influences secondary metabolite biosynthesis, with older plants often exhibiting higher metabolite accumulation due to metabolic shifts from growth to defence and storage (Nair and Sakuntala, 2020). Furthermore, enhanced nutrient availability at 14 WAP may have supported the mevalonate (MVA) and methylerythritol phosphate (MEP) pathways involved in andrographolide biosynthesis, especially under Bacillus sp. treatment (Kumari et al., 2023; Nair and Sakuntala, 2020).

Additionally, Bacillus strains are known to produce a wide range of bioactive compounds, including aromatic compounds, lipopeptides, phytohormones, polysaccharides, and enzymes involved in phenylpropanoid metabolism (Ek-Ramos et al., 2019). These metabolites play critical roles in promoting plant growth and supporting effective crop management strategies. Importantly, the inoculation of endophytic bacteria can influence the biosynthetic pathways of host plants, modulating the production of pharmacologically active compounds while simultaneously supporting vegetative growth (Singh et al., 2017).

Reducing the use of chemical fertilizers is a key priority for sustainable agriculture, particularly in the cultivation of medicinal plants, where minimizing chemical residues is crucial. In tropical soils, chemical phosphorus fertilizers are often inefficient due to their strong fixation by soil particles, which limits their availability to plants. In contrast, microbial biofertilizers such as Bacillus sp. offer a more sustainable solution by mobilizing fixed soil nutrients, enhancing root development, and lowering the risk of environmental pollution (Ribeiro et al., 2018; Wang et al., 2022). In this study, Bacillus sp. alone or in combination with half-dose phosphate (0.675 g P₂O₅) achieved results comparable to full chemical application (1.35 g P₂O₅), indicating the potential to reduce chemical inputs by up to 50% without compromising plant growth or andrographolide production. These results reinforce the value of microbial biofertilizers as eco-friendly tools to enhance biomass and secondary metabolite production in A. paniculata, aligning with broader efforts to promote low input and sustainable medicinal plant cultivation.

The agronomic and physiological characteristics, as well as andrographolide yield of Andrographis paniculata were significantly improved by applying the endophytic bacteria Bacillus sp. and phosphorus fertilizer. The combination of Bacillus sp. and 0.675 g P₂O₅ plant^-1 produced the best agronomic and physiological performance and andrographolide yield, with the best results observed at the flowering stage (14 weeks after planting). This suggests that using Bacillus sp. can reduce the use of chemical phosphorus fertilizer by up to 50%, benefiting farmers and contributing to more environmentally friendly practices in support of sustainable agriculture. These results also align with the Ministry of Agriculture’s regulation that encourages the use of organic fertilizers as part of Good Agricultural Practice (GAP) for medicinal plants.