Ethical clearance to conduct this study was obtained from the Cape Peninsula University of Technology Faculty of Applied Sciences Research Ethics Committee on 19 April 2024. The ethical clearance number is 230407862/04/2024.

Plants primarily rely on two N forms, NH₄⁺ and NO₃⁻, which are derived from various soil processes such as mineralisation and nitrification (Zhang, Cai & Müller 2018). Nitrogen is available in the atmosphere, primarily in its gaseous form (N₂), which constitutes about 78% of the Earth’s atmosphere (Glass & Rousk 2024). Nitrogen fixation occurs through a symbiotic relationship between root nodule-dwelling N-fixing bacteria (rhizobia) and plants, where the plant provides the bacteria with carbohydrates while the bacteria fix N₂ into a form that the plant can use (Ahmadi 2023). Another way that some plants may obtain nitrogen for their nutrition is through nitrite (NO₂⁻) from the atmosphere (Bashir et al. 2024). NO₂⁻ is a significant air pollutant produced in the soil when N-containing substances break down under low oxygen conditions (Ye et al. 2022). However, most of it is produced through the combustion of fossil fuels (vehicles, power plants and industrial processes). In soil, NO₂⁻ availability is generally low, and at high concentrations, it becomes toxic to plants (Bashir et al. 2024).

Ammonium N (NH₄⁺) is present in soils through mineralisation of soil organic N and applied as a product of urea hydrolysis. NH₄⁺ uptake is mediated by both high- and low-affinity transport systems, possibly via an NH₄⁺ uniport or K⁺ channel (Jose et al. 2023). NH₄⁺ is the preferred form of N uptake when plants grow under N deficiency; it is rapidly assimilated into amino acids within the roots via the glutamine synthetase and glutamate synthase (GS/GOGAT) pathway (Figure 2), which requires less energy than NO₃⁻ assimilation (Zhang et al. 2018). Because of its positive charge, NH₄⁺ is adsorbed by negatively charged soil colloids (clay and organic matter) and thus is less prone to leaching. Uptake of NH₄⁺ causes rhizosphere acidification because of H⁺ exchange (Imler, Arzola & Nunez 2019). The most used single N (NH₄⁺) is ammonium sulphate, containing 21% N and 24% sulphur (S).

Most agricultural soils allow plant roots to absorb N mainly through NO₃⁻ even though NH₄⁺ might be more accessible in certain soil types. This is mainly because of the higher concentration of NO₃⁻ in soils as compared to NO₂⁻ and NH₄⁺. Additionally, because of its (NO₃⁻) negative charge, it remains in the soil solution rather than binding to negatively charged soil particles, allowing for high mobility and plant uptake (Pinheiro et al. 2020). NO₃⁻ is absorbed via an NO₃⁻/H⁺ symport (Figure 2), involving three transport systems (Muratore, Espen & Prinsi 2021), and the uptake of NO₃⁻ leads to rhizosphere alkalinisation (Imler et al. 2019).

The conversion of NO₃⁻ to NH₄⁺ and amino acid synthesis for protein synthesis depends on nitrate reductase enzyme activity, which is inefficient in blueberries (Kishorekumar et al. 2020). Blueberry plants demonstrate N form and concentration sensitivity in acidic NH₄⁺-dominant soils; however, they thrive best at pH 4.0 to 5.5, which supports acidic soil conditions that favour NH₄⁺ uptake as their preferred N source (Yang et al. 2022). Sensitivity of young blueberry plants to high ammonium sulphate applications may be because of ammonium toxicity, which is linked to increased electrical conductivity (EC) in the soil solution, with growth suppression observed at EC levels above 1.5 dS·m⁻¹ (Machado, Bryla & Vargas 2014).

Table 1 shows that N form and soil acidity are important, with most studies indicating a preference for NH₄⁺ over NO₃⁻ as an N source.

Nitrogen form plays a critical role in determining blueberry growth and yield responses. As shown in Table 2, numerous studies have investigated the effects of different N sources, including NH₄⁺, NO₃⁻ and combinations thereof, on various blueberry cultivars and developmental parameters. Overall, NH₄⁺-N tends to be more favourable than nitrate-N in most studies (Alt et al. 2017; Anwar et al. 2024; Arias et al. 2024; González et al. 2018; Imler et al. 2019; Messiga et al. 2021; Osorio et al. 2020; Peterson et al. 2022; Rosen, Allan & Luby 2019; Vargas & Bryla 2015; Xu et al. 2021; Yañez-Mansilla et al. 2015; Yuan-Yuan et al. 2021), with consistent improvements in shoot growth, chlorophyll content, leaf dry mass and yield. This trend may be attributed to the limited nitrate reductase activity in Vaccinium species, as well as their preference for acidic soils, which complements the acidifying effect of NH₄⁺ nutrition.

Moreover, a combination of N sources, particularly NH₄⁺: NO₃⁻ ratios of 2:1 or 1:1, has demonstrated synergistic effects on physiological and yield-related traits (Anwar et al. 2024). These ratios often outperform singular forms by enhancing N recovery, leaf area index and net assimilation rate without the adverse effects seen with high NO₃⁻ concentrations (Xu et al. 2021). Table 2 summarises these findings, offering insight into the understanding of blueberry N nutrition. However, recent studies seem to be placing increased emphasis on physiological responses, such as N uptake efficiency and photosynthetic activity, in addition to yield attributes. While cultivar-specific responses and environmental factors (such as soil pH and substrate) can modulate outcomes, the preference for NH₄⁺-dominated nutrition or a combination of forms remains a consistent recommendation for optimising blueberry production.

The antioxidant compounds anthocyanins, phenolic acids and polyphenols, which are present in blueberry plants, provide multiple health advantages (Krishna et al. 2023). Anthocyanin accumulation serves as a protective response for N-deficient plants by making leaves more light-sensitive through chlorophyll reduction. The presence of anthocyanins in plants enhances their ability to withstand N deficiency stress (Liang & He 2018). The accumulation of anthocyanin is triggered by N deficiency but also results from different nutritional imbalances, making it a useful crop nutrient status indicator (Jezek et al. 2023). Low N availability has been shown to enhance secondary metabolite production in plants by redirecting excess carbon (C) energy towards biosynthesis pathways, including flavonoid synthesis (Li et al. 2021).

Contrarily, high N availability can lead to decreased anthocyanin levels and reduced reproductive development. In blueberries, findings vary; while high N may reduce anthocyanin accumulation, Gonzalez et al. (2018) observed increased anthocyanin levels in specific N treatments, such as a balanced 50:50 NH₄⁺:NO₃⁻ ratio, as shown in Table 3. NO₃⁻-based sources generally favour C allocation towards flavonoid production, whereas NH₄⁺ sources tend to enhance N assimilation, potentially at the expense of flavonoid synthesis. Studies on blackberries show that distinct N forms impact the expression of genes involved in flavonoid biosynthesis, specifically dihydroflavonol 4-reductase (DFR) and chalcone synthase (CHS). Ammonium (NH₄⁺) increases gene activity related to phenolic compound production (Duan et al. 2023).

As research specifically investigating the effect of different N sources on phenolic compound accumulation in blueberries is limited, data from studies on related species have been included to provide a broad context. These trends are summarised in Table 3, presenting several studies on how different N sources and conditions influence phenolic compound accumulation across various plants.

The response of blueberry plants to different N forms shows N availability as a critical factor that affects both growth and photosynthesis (González et al. 2018; Osorio et al. 2020; Yuan-Yuan et al. 2021). Adequate N supply remains essential because chlorophyll synthesis depends on N to enable light absorption and photosynthetic efficiency. The use of NH₄⁺ as a nutrient source has been shown to increase stomatal conductance in blueberries, which leads to better gas exchange and supports photosynthesis (Osorio et al. 2020). The application of NH₄⁺ resulted in better gaseous exchange parameters than NO₃⁻, and Yuan-Yuan et al. (2021) demonstrated that a 5:1 NH₄⁺:NO₃⁻ ratio produced the best photosynthetic and stomatal performance.

However, the advantage depends on concentration because excessive NH₄⁺ leads to metabolic imbalance and oxidative stress and impaired photosynthetic functions (Yañez-Mansilla et al. 2015). Excessive NH₄⁺ stress disrupts electron transport and reduces carboxylation efficiency, thus decreasing CO₂ assimilation (Wang et al. 2019). The assimilation of NO₃⁻ requires more energy than NH₄⁺ but enables sustained photosynthesis through its ability to generate Adenosine Triphosphate (ATP) and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) needed for the Calvin cycle (Kishorekumar et al. 2020). The study by Cárdenas-Navarro et al. (2024) demonstrated that blueberry plants supplied with NO₃⁻ nutrition showed better carbon fixation rates and electron transport activity.

Urea-based fertilisers, which are hydrolysed into NH₄⁺ in the soil, have shown photosynthetic outcomes like NH₄⁺ sources (Nasraoui-Hajaji & Gouia 2014). The controlled N release from urea leads to higher chlorophyll content and better C assimilation (Kozos & Ochmian 2016). The photosynthetic response extends longer because N from urea becomes available more gradually than from NH₄⁺ or NO₃⁻ (Smolander, Martikainen & Henttonen 2022). The most successful approach to maximise photosynthetic efficiency while preventing N-related stress in blueberries involves maintaining balanced NH₄⁺:NO₃⁻ inputs.

Different N sources influence water-use efficiency (WUE), transpiration and osmotic adjustment in blueberry plants; these are key processes for maintaining water status under drought (Ruiz-Romero et al. 2024). NH₄⁺ nutrition enhances blueberry plant drought resistance through multiple physiological processes. The increased root abscisic acid content in drought-stressed NH₄⁺-fed plants leads to better WUE (Ding et al. 2016). Accumulation of osmolytes such as proline and soluble sugars helps sustain root development to reach deeper soil water (Zaher-Ara, Boroomand & Sadat-Hosseini 2016).

Highbush blueberry cultivars showed different levels of drought resistance after drought stress reduced their photochemical efficiency and increased proline content (Balboa, Ballesteros & Molina-Montenegro 2020).

Under water-limited conditions, NH₄⁺ nutrition controls stomatal conductance to minimise excessive water loss through transpiration while allowing sufficient CO₂ uptake for photosynthesis to support plant development (Torralbo et al. 2019). The drought resistance of Malus prunifolia increased with higher NH₄⁺ uptake but lower NO₃⁻ uptake, indicating the importance of NH₄⁺ in drought tolerance (Huang et al. 2018). Similarly, in other crops, high NH₄⁺ concentrations cause ion imbalances, which lead to toxicity and damage the plant’s water stress tolerance (Shilpha et al. 2023). Research conducted by Faralli et al. (2023) demonstrated that NO₃⁻-based fertilisation enhances plant development under sufficient irrigation by improving transpiration efficiency. The positive effects of NO₃⁻-nutrition on transpiration reached their peak when water availability was sufficient, yet NO₃⁻ does not provide drought tolerance at the same level as NH₄⁺. Plants that received NH₄⁺ nutrition demonstrated superior drought tolerance compared to those receiving NO₃⁻ under water-stressed conditions (Ding et al. 2016). However, plants treated with NO₃⁻ still maintained positive hydration status because NO₃⁻ enabled proper stomatal conductance for efficient CO₂ uptake and reduced water loss during photosynthesis (Ding et al. 2016).

Future research should also explore the interactions between N sources and secondary metabolite production, especially phenolic compounds, which are important for blueberry quality and human health benefits. Knowledge of the mechanisms through which N influences phenolic synthesis could provide new ways of improving fruit quality through fertilisation practices. Selection of an N source is the main factor in improving plant growth and physiological performance; hence, it is important to explore this area of research, particularly in blueberry secondary metabolite accumulation, which is relatively scarce in the current available literature.