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Article

Biostimulants Applied in Seedling Stage Can Improve Onion Early Bulb Growth: Cultivar- and Fertilizer-Type-Specific Positive Effects

by
Qianwen Zhang
1,2,
Jun Liu
1,
Sang Jun Jeong
1,
Joseph Masabni
1 and
Genhua Niu
1,*
1
Texas A&M AgriLife Research and Extension Center at Dallas, 17360 Coit Road, Dallas, TX 75252, USA
2
Truck Crops Branch Experiment Station, Mississippi State University, 2024 Experiment Station Road, Crystal Springs, MS 39059, USA
*
Author to whom correspondence should be addressed.
Horticulturae 2025, 11(4), 402; https://doi.org/10.3390/horticulturae11040402
Submission received: 27 February 2025 / Revised: 4 April 2025 / Accepted: 8 April 2025 / Published: 10 April 2025
(This article belongs to the Special Issue Effects of Biostimulants on Horticultural Crop Production)
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Abstract

:
Biostimulants play an active role in sustainable crop production. While biostimulants are thought to have long-term effects on plant growth, little research has been conducted to confirm this hypothesis. In this study, we investigated the long-term residual effects of biostimulants applied exclusively during the onion seedling stage on subsequent plant growth. Three onion cultivars (‘Carta Blanca’, ‘Don Victoro’, and ‘Sofire’) were evaluated with the application of nine microbial biostimulants (LALRISE Mycorrhizae, LALRISE Bacillus velezensis, Mighty Mycorrhizae, MycoApply, Spectrum, Spectrum DS, Spectrum Myco, Tribus Original, and Tribus Continuum), one seaweed extract (Kelpak), and two fertilizer types (conventional and organic fertilizer). Plant morphology and biomass were investigated during the early bulb stage of onion growth. Parameters such as plant height, neck diameter, bulb diameter, and the fresh and dry weights of the shoot, bulb, and root were measured. The results indicated significant cultivar-specific effects of microbial biostimulant and fertilizer type, as well as their interactions, on onion early bulb growth. While seaweed extract exhibited minimal residual impact, specific microbial biostimulants, such as Mighty Mycorrhizae and MycoApply, significantly enhanced bulb growth in the red onion ‘Sofire’. Tribus Continuum was found to increase bulb growth of the yellow onion ‘Don Victoro’. Positive effects of microbial biostimulants on onion growth were also observed with LALRISE Bacillus velezensis, Spectrum Myco, Spectrum, and LALRISE Mycorrizae. Furthermore, microbial biostimulants demonstrated more significant positive effects on onion growth when applied in conjunction with organic fertilizer. In conclusion, microbial biostimulants exhibited long-term positive effects on onion plant growth even when applied solely during the seedling stage prior to transplanting. However, these effects were significantly influenced by onion cultivar and fertilizer type, with the greatest benefits observed when combined with organic fertilizer. We recommend MycoApply and Mighty Mycorrhizae for growers seeking to enhance onion productivity, particularly in organic cultivation, as the two products enhanced bulb and leaf growth in ‘Sofire’ and ‘Don Victoro’.

1. Introduction

The application of biostimulants to plant seeds, foliage, or the rhizosphere can benefit plant growth by enhancing nutrient uptake, nutrient use efficiency, resistance to pest and disease problems, and abiotic stress tolerance [1,2]. Biostimulants can be categorized into two groups based on their sources, either microbial or non-microbial substances. Microbial biostimulants include arbuscular mycorrhizal fungi (AMF), yeasts, Trichoderma spp., and plant growth-promoting bacteria (PGPBs) such as genera of Azospirillum, Bacillus, Rhizobium, and Pseudomonas [3,4]. Microbial biostimulants enhance plant growth through various mechanisms, including forming symbiotic relationships with plant roots like AMF that colonize roots and improve nutrient uptake, producing bioactive metabolites similar to plant hormones to stimulate growth and development (PGPBs, Trichoderma spp., and yeasts), and acting as biocontrol agents that repel pests and pathogens around plants (Trichoderma spp. and PGPBs) [5,6,7].
Non-microbial biostimulants include seaweed extracts, humic substances, microalgae extracts, and protein hydrolysates. Seaweed and microalgae extracts are reported to be rich in various growth-promoting compounds, including phytohormones like auxins, gibberellins, and cytokinins, essential macro- and micronutrients for plant growth, and polysaccharides with bacteriostatic properties [8,9,10]. Humic substances can trigger metabolic signaling pathways within plant root cells. At the same time, their rich carbon content promotes the colonization and propagation of beneficial microorganisms [11,12]. Climate change is causing more frequent extreme weather events, posing a significant challenge to crop production [13]. We need efficient methods to mitigate plant stress caused by climate change, but such methods must also be sustainable and eco-friendly. Applying more chemical fertilizers, for example, would only exacerbate climate change [14]. Therefore, the application of biostimulants provides a promising solution to help plants grow better and achieve higher yields while remaining environmentally friendly [15].
Numerous studies have reported the beneficial effects of biostimulant application on plant growth, stress tolerance, and the accumulation of specific health-beneficial compounds across various crops including tomato, Amaranthus, maize, and cucumber [16,17,18,19]. However, the effectiveness of biostimulants can be highly specific, not only to different crops such as onion, carrot, tomato, rapeseed, cauliflower, and lettuce, but even to different phenotypes within the same species, as observed in spinach and onion [20,21,22,23]. In addition, environmental factors like temperature, soil type, and humidity can significantly influence how effectively biostimulants interact with plant growth [24,25]. Therefore, identifying effective biostimulants for specific crops, such as onions, a shallow-rooted crop highly sensitive to various stresses, is a crucial step for successful large-scale implementation. However, current research on biostimulant applications in onions remains limited, with most studies focusing on only one or two types of biostimulants without comparing the effectiveness of different categories, such as microbial versus non-microbial biostimulants. For instance, seaweed extracts have been shown to promote onion germination and seedling growth [26,27], as well as enhancing the yield of onion bulbs [20,28]. Similarly, microbial biostimulants, such as mixtures of rhizobacteria species, have improved onion bulb yield and quality [29]. Other studies have demonstrated that microalgae, humic acid, and their combination at low concentrations can enhance onion growth at both 60 and 150 days after planting [30]. Additionally, inoculating onion plants with mycorrhizal fungi has been shown to increase nutrient uptake, though the efficiency of this process depends significantly on the onion genotype and the specific fungal species employed [31]. These findings show the potential of biostimulants on onion production but also emphasize the need for comprehensive studies comparing the relative effectiveness of different biostimulant categories and their impact across various stages of onion growth.
Organic farming offers another strategy for achieving sustainable agriculture by replacing chemical fertilizers with environmentally friendly alternatives while potentially increasing profitability due to the increasing consumer demand for organic produce [32,33]. However, the slow release of nutrients in organic fertilizers presents a significant challenge for organic farming’s widespread adoption [34]. Given the ability to improve nutrient uptake and utilization in plants, biostimulants have the potential to benefit organic farming, especially since many are certified for organic use [35]. While studies have explored the use of biostimulants in organic farming, few have specifically investigated the synergistic benefits of combining biostimulants with organic fertilizers [35,36]. The release of nutrients from organic fertilizers often relies on microbial activity to stimulate the process [37]. Conversely, organic fertilizers can provide a food source for microbial growth, and these microbes can, in turn, accelerate the mineralization of organic nutrients, making them more readily available for plants [38,39,40]. This mutualistic relationship suggests potential for enhanced nutrient availability and plant growth when organic fertilizers and microbial biostimulants are used in conjunction.
Here, we hypothesized that microbial biostimulants applied during the seedling stage would have lasting growth-promoting effects on onion plants, with effectiveness varying by cultivar and fertilizer type. The objective of this study was to evaluate the residual effects of seedling-stage biostimulant application on subsequent onion growth.

2. Materials and Methods

2.1. Experiment Design

A completely randomized design was employed with eight replicates. The experiment included four factors: three onion cultivars, two seaweed extract treatments, ten microbial biostimulant treatments, and two fertilizer types. Details of each experimental factor are provided in the following section. In total, there were one hundred and twenty treatment combinations with eight replicates each. For data collection on growth parameters, three representative plants were randomly selected and harvested from each treatment combination.

2.2. Plant Materials and Biostimulant Treatments During the Seedling Stage

The research was conducted in a controlled greenhouse environment located at the Texas A&M AgriLife Research and Extension Center in Dallas, TX, USA (32°59′21.4″ N 96°45′56.7″ W). Onion seeds of three cultivars were sown on 17 October 2022 and seedlings were propagated in a greenhouse until 21 December 2022. The three onion cultivars used in this experiment were the white onion ‘Carta Blanca’, yellow onion ‘Don Victoro’, and red onion ‘Sofire’. The onion cultivars were chosen as they represent both the major commercially grown varieties in Texas, USA, and the full range of bulb colors available in the region. Seedlings of the three onion cultivars received ten different microbial biostimulants and two seaweed extract applications during the seedling propagation stage, prior to transplanting, as detailed in our previous study [41]. Microbial biostimulant treatments included no application control, LALRISE Mycorrhizae, LALRISE Bacillus velezensis, Mighty Mycorrhizae, MycoApply, Spectrum, Spectrum DS, Spectrum Myco, Tribus Original, and Tribus Continuum. Additionally, two levels of the seaweed extract Kelpak were applied: with Kelpak and without Kelpak. The treatments of microbial biostimulants and the seaweed extract Kelpak were applied only during the seedling stage, but not after transplanting.

2.3. Fertilizer Treatments After Transplanting

Sixty-four days after sowing, sixteen representative healthy and uniform seedlings were randomly selected from each of the above treatment combinations (seedling stage) and were transplanted into square plastic pots with a volume of 250 mL filled with BM6 All-Purpose Mix substrate containing peat moss, perlite, limestone, and wetting agent (Berger, Saint-Modeste, QC, Canada). Among the sixteen plants of each treatment combination during the seedling stage, eight seedlings were subjected to organic slow-release fertilizer Sustane 4-6-4 (4%N-1.31%P-3.31%K, Sustane Corporate, Cannon Falls, MN, USA) and the other eight seedlings were subjected to conventional slow-release fertilizer Osmocote Plus 15-9-12 (15%N-3.9%P-9.9%K, ICL Specialty Fertilizers, Summerville, SC, USA). The fertilizer application rate was determined by our previous experience using Sustane 4-6-4 in watermelon seedling production and the manufacturers’ recommendation [32]. The conventional slow-release fertilizer Osmocote Plus 15-9-12 was applied only once through incorporating with the substrate at 2.13 g/L on 0 day after transplanting (DAT), while the organic slow-release fertilizer Sustane 4-6-4 was applied four times based on our previous experience using this organic fertilizer, at 2 g/L on 0 DAT through incorporating with substrate and 2 g/L on 22, 50, and 65 DAT through top-dressing application [32]. The total nitrogen (based on label) provided to each plant from both fertilizers through the experiment was equivalent. To encourage upright growth after transplanting, seedlings were pruned to 3 inches (7.62 cm) at 0 DAT. Throughout the experiment, irrigation frequency was determined by daily assessment of visual substrate dryness (top 2 cm layer) and weight-based moisture estimates from representative pots. Tap water was applied uniformly when pots reached approximately 60–70% of field capacity. To minimize microenvironmental variation, pot position was randomized weekly within the greenhouse. The greenhouse temperature was controlled between 17 °C and 22 °C using heating and ventilation systems. The average temperature was measured at 20.59 ± 0.11 °C. Additionally, the average daily light integral (DLI) was 12.60 ± 0.64 mol m−2 d−1. The average DLI was calculated from the daily average photosynthetically active radiation (PAR) by the following equation [42]:
A v e r a g e   D L I = D a i l y   a v e r a g e   P A R × 0.0864
where the PAR was measured using two quantum sensors strategically positioned inside the greenhouses. These sensors measured the PAR every 10 s and the Campbell datalogger (Campbell Scientific CR 1000, Logan, UT, USA) recorded the hourly and daily average PAR values.

2.4. Growth Measurements

Three representative onion plants were randomly selected and harvested from each treatment combination at 92 DAT. The standard for selecting representative plants involves randomly selecting individuals from the replicates, while avoiding visually extreme plants (biggest and smallest). Selected representative plants were immediately transported to the laboratory for processing, where all measurements were conducted under controlled conditions. Plant height was measured from the substrate surface to the tip of the farthest leaf tip using a tape measure. Each plant was removed from the pot and the roots were gently washed with tap water and patted dry with a paper towel. The neck diameter (the thinnest part of the white-to-pale green subterranean stem) and bulb diameter (the thickest part of the swollen bulb) were measured using a digital caliper. The plant was dissected into three parts using a razor blade: shoot, bulb, and root. The top of the bulb was separated from the unswollen subterranean stem, while the bottom of the bulb was carefully separated from the root, ensuring minimal bulb tissue remained on the root to prevent the roots from separating. Fresh weight (FW) of the shoot, bulb, and root were measured and then collected into separate paper bags and put into 70 °C oven until each sample reached constant weight. Then, the dry weight (DW) of the shoot, bulb, and root were recorded. Total FW was determined by summing the fresh weights of the shoot, bulb, and root.

2.5. Statistical Analysis

To investigate the main effects of the onion cultivar, seaweed extract treatments, microbial biostimulant treatments, and fertilizer type, an analysis of variance (ANOVA) was conducted. A four-way ANOVA was initially performed (Table S1). Subsequently, a dimension reduction was carefully considered, and a three-way ANOVA was conducted to effectively present the main effects and interactions of three key factors: onion cultivar, microbial biostimulant treatments, and fertilizer type. Post hoc analysis was then performed using Duncan’s multiple range test at a significance level of p < 0.05. Both ANOVA and mean separation analyses were conducted using SAS software (version 9.4, SAS Institute, Cary, NC, USA). The results are presented graphically in bar plots generated using Microsoft Excel (Microsoft 365, Seattle, WA, USA). Furthermore, the variation in collected parameters was analyzed using principal component analysis (PCA) to reduce dimensionality. The results were visualized by plotting the data along the first two principal components (PC1 and PC2), with data points separated based on different levels of onion cultivar, microbial biostimulant treatments, fertilizer type, and seaweed extract treatments. PCA and biplot analyses were performed and visualized using R (version 4.3.3, R Foundation for Statistical Computing, Vienna, Austria).

3. Results

The four-way ANOVA results (Table S1) showed no statistically significant main effect of seaweed extract on any morphological or biomass parameters. Most interactions between seaweed extract and the other experimental factors were also not significant. Based on these findings, the effect of seaweed extracts to be minimal for onion early bulb growth. The biplot of PCA, separated by seaweed extract application, also revealed overlapping patterns in morphology and biomass between treated and untreated onion plants (Figure S1). Consequently, the data from treatments with and without seaweed extract were pooled together.
The results of the subsequent three-way ANOVA, analyzing the main effects and interactions among onion cultivar, microbial biostimulants, and fertilizer type, are presented in Table 1. It can be seen that the main effects of onion cultivar, microbial biostimulants, and fertilizer type were statistically significant for all morphological and biomass parameters measured, except for microbial biostimulants on root FW and fertilizer type on root DW. The interaction between onion cultivar and microbial biostimulants, as well as the interaction between onion cultivar and fertilizer type, were not statistically significant for neck diameter or root FW. Significant interactions were observed for the remaining parameters, except for the interaction between cultivar and microbial biostimulants on leaf DW and the interaction between cultivar and fertilizer type on root DW. A significant interaction between microbial biostimulants and fertilizer type was observed only for bulb diameter, bulb FW, bulb DW, and leaf DW. The three-way interaction among onion cultivar, microbial biostimulants, and fertilizer was found to be significant for plant height, leaf FW, bulb FW, total FW, and leaf DW. Significant interactive effects were observed among onion cultivars, microbial biostimulants, and fertilizers on most measured parameters. Therefore, the results are presented separately for two combinations: onion cultivar in conjunction with microbial biostimulants, and fertilizer type in conjunction with microbial biostimulants.

3.1. Plant Height and Mini Bulb Parameters

The impact of microbial biostimulants on plant height varies across onion cultivars (Figure 1A). For red and white onion cultivars, microbial biostimulants did not affect plant height significantly. However, for the yellow onion cultivar, MycoApply increased plant height by 10%. Furthermore, cultivar significantly influenced plant height when treated with LALRISE Mycorrhizae, Mighty Mycorrhizae, and Tribus Continuum, with the white cultivar showing the highest plant height. Biostimulant application increased plant height when combined with organic fertilizer but had no effect when applied with conventional fertilizer (Figure 1B). Specifically, MycoApply resulted in a 7% increase in plant height when used with organic fertilizer. Microbial biostimulants did not significantly affect neck diameter across all onion cultivars and fertilizer types (Figure 1C,D). However, bulb diameter, an important parameter for assessing onion yield and quality, were affected by cultivar, microbial biostimulants, and fertilizer type interactively (Figure 1E,F). In the red cultivar, Mighty Mycorrhizae and MycoApply increased bulb diameter by 24% and 20%, respectively (Figure 1E). For the yellow cultivar, Tribus Continuum and LALRISE Bacillus velezensis led to a 34% and 22% increase in bulb diameter, respectively. Cultivar affected bulb diameters when applied with certain microbial biostimulants including LALRISE Bacillus velezensis, Spectrum Myco, and Tribus Continuum, with the yellow cultivar resulting in the highest bulb diameter compared to the red and the white cultivars. When applied with conventional fertilizer, microbial biostimulants had no significant effect on bulb diameter (Figure 1F). However, when applied with organic fertilizer, MycoApply, Tribus Continuum, Spectrum Myco, and Mighty Mycorrhizae increased bulb diameter by 25%, 22%, 20%, and 19% compared to the control, respectively.

3.2. Fresh Weight Parameters

Leaf FW was significantly affected by onion cultivar, microbial biostimulants, fertilizer type, and their interactions (Table 1). While microbial biostimulants did not significantly affect leaf FW in red and white cultivars compared to the control, MycoApply led to a 32% increase in leaf FW in the yellow cultivar (Figure 2A). Among the cultivars, white onions exhibited the highest leaf FW when treated with most of the microbial biostimulants, except for LALARISE Bacillus velezensis and MycoApply. When applied with conventional fertilizer, Mighty Mycorrhizae resulted in the highest leaf FW, although this difference was not significant (Figure 2B). However, when applied with organic fertilizer, MycoApply resulted in a 24% increase in leaf FW. Bulb fresh weight is another important parameter directly associated with the yield of onion plants, which was interactively affected by onion cultivar, microbial biostimulants, and fertilizer type (Table 1).
The positive effects of microbial biostimulants on bulb FW showed different trends among various onion cultivars (Figure 2C). In the red cultivar, Mighty Mycorrhizae and MycoApply increased bulb FW by 55% and 50%, respectively. In contrast, microbial biostimulants did not significantly impact bulb FW in the white cultivar. In the yellow cultivar, Tribus Continuum and LALRISE Bacillus velezensis increased bulb FW by 97% and 67%, respectively. Among the cultivars, the yellow cultivar resulted in the highest bulb FW when treated with LALRISE Bacillus velezensis, Spectrum Myco, and Tribus Continuum. The positive influence of microbial biostimulants on bulb FW was significant, regardless of fertilizer type (Figure 2D). When applied with conventional fertilizer, Mighty Mycorrhizae increased bulb FW by 52% when compared to the control. When applied with organic fertilizer, MycoApply, Tribus Continuum, and Spectrum Myco increased bulb FW by 70%, 49%, and 41%, respectively. Microbial biostimulants did not affect root FW when compared to the control regardless of the onion cultivars (Table 1 and Figure 2E). However, when applied with organic fertilizer, Spectrum was found to increase root FW by 41% (Figure 2F). Total FW served as an indicator of overall plant growth, encompassing the contributions of the shoot, bulb, and root. In the red cultivar, MycoApply increased total FW by 33% (Figure 2G), while in the yellow cultivar, Tribus Continuum led to a 45% increase in total FW. When applied with conventional fertilizer, Mighty Mycorrhizae increased total FW by 29%. When applied with organic fertilizer, MycoApply and Spectrum increased total FW by 40% and 20%, respectively.

3.3. Dry Weight Parameters

Leaf DW was significantly influenced by onion cultivar, microbial biostimulants, and fertilizer type (Table 1). Among the cultivars, the white cultivar exhibited the highest leaf DW (Figure 3A). While there were differences in leaf DW among the various microbial biostimulants, their application did not significantly increase leaf DW (Figure 3A,B). However, Tribus Continuum increased bulb DW by 66% in the yellow cultivar (Figure 3C). When applied with conventional fertilizer, microbial biostimulants did not significantly increase bulb DW (Figure 3D). However, when applied with organic fertilizer, MycoApply and Tribus Continuum increased bulb DW by 77% and 55%, respectively. In the red cultivar, it was found that LALRISE Mycorrhizae increased root DW by 83% (Figure 3E). When applied with conventional and organic fertilizers, LALRISE Myco increased root DW by 43% and 39%, respectively (Figure 3F).

3.4. Principal Component Analysis

The relationships among onion cultivars, microbial biostimulants, fertilizer types, and measured plant morphological and biomass parameters were visualized using principal component analysis (PCA) biplots in Figure 4, Figure 5, Figure 6 and Figure S1. The first two principal components (PC1 and PC2) explained 76.2% of the total variance, indicating that they captured a substantial portion of the data variability (Figure 4, Figure 5, Figure 6 and Figure S1). In the second quadrant, parameters including neck diameter, leaf DW, root DW, root FW, leaf FW, and plant height showed positive associations with PC2. Conversely, in the third quadrant, bulb FW, bulb DW, and bulb diameter exhibited negative associations with PC2. All measured parameters were negatively associated with PC1. This indicates an inverse correlation between bulb development and the growth of the shoot and root systems in onion plants. Figure 4 revealed a clear separation of the three onion cultivars. The white cultivar tended to cluster towards the positive end of PC2, while red and yellow cultivars were more closely grouped towards the negative direction of PC2. This suggests that the white onion ‘Carta Blanca’ may exhibit better shoot and root growth, whereas the yellow onion ‘Don Victoro’ may exhibit better bulb growth. In Figure 5, while some overlap existed among microbial biostimulants, certain treatments, such as Mighty Mycorrhizae and MycoApply, clustered towards the negative directions of PC1 and PC2 in the third quadrant. In contrast, the control treatment clustered towards the positive directions of PC1 and PC2 in the first quadrant. This suggests that Mighty Mycorrhizae and MycoApply may have promoted bulb growth, as well as shoot and root growth. Figure 6 showed that onion plants grown with conventional fertilizer (red dots) tended to cluster towards the negative end of PC1, while those grown with organic fertilizer (green triangles) were more scattered towards the positive end of PC1. This suggests that fertilizer type had a significant influence on onion plant growth, with conventional fertilizer generally resulting in better overall plant growth compared to organic fertilizer. However, in Figure S1, the clusters of plants treated with and without seaweed extract overlapped extensively, indicating that seaweed extract application during the seedling stage had minimal residual effects on later early bulb development in onions.

4. Discussion

Biostimulants have emerged as a promising solution for sustainable crop production, particularly as climate change intensifies and plants face increasing stress [15]. As most biostimulants are derived from microbial sources or natural products like plants and minerals, they offer an environmentally friendly alternative to conventional fertilizers and pesticides [43]. Not only can biostimulants enhance plant growth and stress tolerance, but they also have a minimal environmental impact. While it has been hypothesized that biostimulants exert long-term effects on plant growth, limited research has explored this aspect [41,44]. This study demonstrates that microbial biostimulants, when applied solely during the onion seedling stage, can have enduring positive effects on plant growth, persisting into the early bulb stage. In addition, the combination of organic fertilizer and microbial biostimulants can increase onion plant growth during the early bulb stage.

4.1. Multiple Applications of Seaweed Extract May Be Necessary

Biostimulants contain a diverse range of substances or mixtures derived from various sources. In this study, microbial biostimulants and the non-microbial biostimulant seaweed extract were classified as two independent experimental factors. Seaweed extracts like the Kelpak product used in this study are known to enhance plant growth through multiple mechanisms: they contain natural growth regulators such as auxins and cytokinins that stimulate root development, increase lateral root formation, and improve nutrient uptake efficiency [8,45,46]. These compounds can significantly enhance seedling vigor and survival rates after transplantation by promoting stronger root systems and mitigating transplant shock [47]. In our previous study, during the seedling stage, a synergistic interaction was observed between microbial biostimulants and seaweed extract, resulting in enhanced onion plant growth [41]. However, the residual impact of seaweed extract was not significant (Table S1), suggesting its effects may be transient. This observation does not negate the potential of seaweed extract to positively influence onion growth. The transient nature of these effects may be attributed to the rapid metabolism of bioactive compounds in seaweed extracts. For example, in Phaseolus, auxin elongated leaf strips within 10 h of application, after which the strips became insensitive to it [48].
Alternatively, additional applications of seaweed extract after transplanting to field, rather than a single application during the seedling stage, might be necessary to sustain its beneficial effects. For example, a previous study that investigated the same seaweed extract used in this study, Kelpak, found that it improved onion plant growth when applying three times at 2, 4, and 6 weeks after transplanting [49]. Multiple application likely maintained adequate concentrations of bioactive compounds to support continued root development and nutrient assimilation throughout critical growth stages. An even more frequent application of seaweed extract on tomato plants, twice daily through the irrigation system, was found to significantly enhance plant growth, fruit yield, microbial colony count, and nutrient availability in the substrate [50]. These findings suggest that application frequency and method are critical factors in realizing the full potential of seaweed extracts, which may explain the limited residual effects observed in our single-application experimental design. The effects of application frequency of seaweed extract on plant growth warrant further investigations.

4.2. Different Microbial Biostimulants Have Cultivar-Specific Effects on Onion Plant Growth

In contrast to seaweed extract, microbial biostimulants demonstrated significant and sustained positive effects on onion growth. Based on the results from Figure 1, Figure 2 and Figure 3, we found that arbuscular mycorrhizal fungi (AMF) products, including MycoApply, Mighty Mycorrhizae, LALRISE Mycorrhizae, and Spectrum Myco led to increases in six parameters in the red onion ‘Sofire’, two in the yellow onion ‘Don Victoro’, three with conventional fertilizer application, and ten with organic fertilizer application across the measured parameters. The superior performance of AMF products, particularly with organic fertilizer application, can be attributed to their symbiotic relationship with plant roots. AMF hyphae extend the root absorption zone, enhancing phosphorus uptake and water-use efficiency, while also producing phytohormones that stimulate lateral root development [40]. These mechanisms likely contributed to the improved bulb growth observed in ‘Sofire’ and ‘Don Victoro’ onions. Among the bacteria-based biostimulant products, Tribus Continuum, LALRISE Bacillus velezensis, and Spectrum also showed significant positive effects on onion vegetative and bulb growth, with increases in six parameters in yellow cultivar and five with organic fertilizer application across the measured parameters. This aligns with studies showing that PGPBs flourish in organic systems, such as those found in the carbon-rich soils of a temporary pond, where these conditions promote biofilm formation and metabolite production, ultimately increasing maize tolerance to drought stress [51].
Overall, as also evidenced by the clustering tendencies of Mighty Mycorrhizae and MycoApply in Figure 5, AMF products had broader positive effects on onion growth across both red and yellow cultivars, as well as under both conventional and organic fertilization. Our findings are consistent with a study on the yellow onion ‘Stalagmit’ F1, where root colonization of AMF increased shoot growth [52]. While microbial biostimulants showed significant positive effects on red and yellow onion cultivars, no such benefits were observed in the white onion cultivar in this study, highlighting strong cultivar-specific responses to these biostimulants. This aligns with previous research demonstrating that AMF effects on plant nutrient uptake depend on both onion genotype and AMF species [31]. Therefore, small-scale testing of microbial biostimulant products for specific onion cultivars is essential before large-scale application, as demonstrated by this study’s screening of nine microbial biostimulants across three different onion cultivars.

4.3. Organic Fertilizer Enhanced Microbial Biostimulant Benefits

Onion plants grown with organic fertilizer exhibited less growth than those grown with conventional fertilizer (Figure 6). This is likely due to the slower nutrient release of organic fertilizers, where nutrients are not as readily available to plant roots as in conventional fertilizers [37,52]. Consequently, despite equal nitrogen application across both groups based on label on the product, the available nitrate-nitrogen in organic fertilizer may be lower than that in the conventional fertilizer, resulted in reduced plant growth [37]. However, in previous studies we have observed a critical compensatory mechanism: microbial biostimulants significantly improved nutrient mineralization in organic systems [37]. Specifically, AMF hyphae extended beyond the root depletion zone to access immobilized nutrients [40].
Our data demonstrate that microbial biostimulants led to increases in many measured parameters compared to the control when plants received organic fertilizer. In contrast, the same microbial biostimulants did not enhance plant growth when plants received conventional fertilizer (Figure 1B,F, Figure 2B,F and Figure 3D). This suggest that organic fertilizers may create a favorable microenvironment for microbial biostimulant activity by enriching the substrate with carbon for microbial colonization, improving habitat through larger pore space and water retention, and reducing chemical stress from conventional fertilizers [53,54]. Similar findings were reported in previous studies, which showed that substrates rich in carbon resulted in better AMF root colonization, leading to improved onion plant growth [52]. Another soil ecology study also found that the application of organic substances can significantly enhance Mycorrhizae growth [55]. This effect is not only observed in mycorrhizal fungi (MycoApply, Mighty Mycorrhizae, and Spectrum Myco) but also in bacteria-based biostimulants containing Bacillus (Tribus Continuum and Spectrum). A more organic-rich substrate attracts Bacillus bacteria, promoting colonization and propagation, which in turn improves nutrient availability in the substrate and enhances plant stress resistance [56,57]. Microorganisms utilize organic substances for growth, colonization, and propagation [3]. Through their metabolic processes, organic nutrients are converted into inorganic forms, thereby enhancing nutrient availability for plant growth [58]. This explains the observed synergy where organic fertilizers provide the habitat and resources for microbial biostimulants to express their full growth-promoting potential, while microbial activity compensates for organic fertilizer’s slower nutrient release. The results of this study provide evidence supporting the combined application of organic fertilizers and microbial biostimulants to optimize plant growth. Further research is needed to investigate the specific mechanisms underlying this synergistic effect and to determine the optimal application rates and ratios for different organic fertilizers and microbial biostimulants.

5. Conclusions

Studies conducted under controlled conditions of a greenhouse showed that the duration and intensity of growth promotion in onion plants following seedling-stage biostimulant applications depended on the type of biostimulant and background fertilizer (organic or mineral nature) and their combinations. This study showed that seaweed extract did not have significant residual effects on onion growth. In contrast, microbial biostimulants including MycoApply, Mighty Mycorrhizae, Tribus Continuum, LALRISE Mycorrhizae, LALRISE Bacillus velezensis, Spectrum Mycorrhizae, and Spectrum significantly enhanced onion growth after transplanting, particularly combined with organic fertilizer. These beneficial effects were cultivar-specific, with the red ‘Sofire’ and yellow ‘Don Victoro’ onions responding more positively than the white onion ‘Carta Blanca’. Applying biostimulants represents a promising strategy for sustainable crop production, but their high cost can be a limiting factor for growers. Our study found that microbial biostimulants exhibit long-term residual effects on plant growth, applying them only during the seedling stage could reduce production costs. Their effectiveness varied by cultivar and fertilizer type, showing the greatest improvement with organic fertilizer. MycoApply and Mighty Mycorrhizae proved particularly effective, increasing bulb and leaf growth in ‘Sofire’ and ‘Don Victoro’ onions, making them recommended choices for organic onion production.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11040402/s1, Figure S1: PCA biplot of onion morphological and biomass parameters grouped by seaweed extract treatments; Table S1: Four-way ANOVA test results showing levels of significance for morphology and biomass parameters.

Author Contributions

Conceptualization, G.N., Q.Z. and J.M.; methodology, G.N. and Q.Z.; formal analysis, Q.Z., J.L. and S.J.J.; investigation, Q.Z., J.L. and S.J.J.; resources, G.N. and J.M.; writing—original draft preparation, Q.Z.; writing—review and editing, G.N., S.J.J., J.M. and J.L.; supervision, G.N.; funding acquisition, G.N. and Q.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Specialty Crop Multi-State Program, USDA Agricultural Marketing Service (grant number TX-SCM-21-05), and the USDA National Institute of Food and Agriculture (NIFA) Hatch Project MIS-300011. The Mississippi Agricultural and Forestry Experiment Station also contributed to this research.

Data Availability Statement

The original contributions presented in this study are included in the article and Supplementary Materials. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

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Figure 1. Effects of microbial biostimulants and fertilizer type on plant morphological parameters of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Plant height of different onion cultivars treated with microbial biostimulants. (B) Plant height of onion under conventional and organic fertilizer with microbial biostimulants. (C) Neck diameter of different cultivars treated with microbial biostimulants. (D) Neck diameter of onion under conventional and organic fertilizer with microbial biostimulants. (E) Bulb diameter of different onion cultivars treated with microbial biostimulants. (F) Bulb diameter of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
Figure 1. Effects of microbial biostimulants and fertilizer type on plant morphological parameters of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Plant height of different onion cultivars treated with microbial biostimulants. (B) Plant height of onion under conventional and organic fertilizer with microbial biostimulants. (C) Neck diameter of different cultivars treated with microbial biostimulants. (D) Neck diameter of onion under conventional and organic fertilizer with microbial biostimulants. (E) Bulb diameter of different onion cultivars treated with microbial biostimulants. (F) Bulb diameter of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
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Figure 2. Effects of microbial biostimulants and fertilizer type on fresh weights of leaf, bulb, root, and total plant of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Leaf fresh weight of different onion cultivars treated with microbial biostimulants. (B) Leaf fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. (C) Bulb fresh weight of different onion cultivars treated with microbial biostimulants. (D) Bulb fresh weight under conventional and organic fertilizer with microbial biostimulants. (E) Root fresh weight of different onion cultivars treated with microbial biostimulants. (F) Root fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. (G) Total fresh weight of different onion cultivars treated with microbial biostimulants. (H) Total fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
Figure 2. Effects of microbial biostimulants and fertilizer type on fresh weights of leaf, bulb, root, and total plant of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Leaf fresh weight of different onion cultivars treated with microbial biostimulants. (B) Leaf fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. (C) Bulb fresh weight of different onion cultivars treated with microbial biostimulants. (D) Bulb fresh weight under conventional and organic fertilizer with microbial biostimulants. (E) Root fresh weight of different onion cultivars treated with microbial biostimulants. (F) Root fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. (G) Total fresh weight of different onion cultivars treated with microbial biostimulants. (H) Total fresh weight of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
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Figure 3. Effects of microbial biostimulants and fertilizer type on dry weights of leaf, bulb, and root of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Leaf dry weight of different onion cultivars treated with microbial biostimulants. (B) Leaf dry weight of onion under conventional and organic fertilizer with microbial biostimulants. (C) Bulb dry weight of different onion cultivars treated with microbial biostimulants. (D) Bulb dry weight under conventional and organic fertilizer with microbial biostimulants. (E) Root dry weight of different onion cultivars treated with microbial biostimulants. (F) Root dry weight of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
Figure 3. Effects of microbial biostimulants and fertilizer type on dry weights of leaf, bulb, and root of three onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). (A) Leaf dry weight of different onion cultivars treated with microbial biostimulants. (B) Leaf dry weight of onion under conventional and organic fertilizer with microbial biostimulants. (C) Bulb dry weight of different onion cultivars treated with microbial biostimulants. (D) Bulb dry weight under conventional and organic fertilizer with microbial biostimulants. (E) Root dry weight of different onion cultivars treated with microbial biostimulants. (F) Root dry weight of onion under conventional and organic fertilizer with microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum. Error bars represent standard error of the mean. Different letters within the same color of bars indicate significant differences at p ≤ 0.05 according to Duncan’s multiple range test. Asterisks (*) indicate significant differences between cultivars within a microbial biostimulant treatment at p ≤ 0.05 according to Duncan’s multiple range test.
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Figure 4. PCA biplot of onion morphological and biomass parameters grouped by onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). FW: fresh weight; DW: dry weight.
Figure 4. PCA biplot of onion morphological and biomass parameters grouped by onion cultivars: red (Sofire), white (Carta Blanca), and yellow (Don Victoro). FW: fresh weight; DW: dry weight.
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Figure 5. PCA biplot of onion morphological and biomass parameters grouped by microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum; FW, fresh weight; DW, dry weight.
Figure 5. PCA biplot of onion morphological and biomass parameters grouped by microbial biostimulants. Abbreviations: LALb, LALRISE Bacillus velezensis; LALMyco, LALRISE Mycorrhizae; MightyMyco, Mighty Mycorrhizae; SpecDS, Spectrum DS; SpecMyco, Spectrum Myco; Tribus, Tribus Original; TribusC, Tribus Continuum; FW, fresh weight; DW, dry weight.
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Figure 6. PCA biplot of onion morphological and biomass parameters grouped by fertilizer types. FW: fresh weight; DW: dry weight. Conventional: conventional fertilizer Osmocote Plus 15-9-12; Organic: organic fertilizer Sustane 4-6-4.
Figure 6. PCA biplot of onion morphological and biomass parameters grouped by fertilizer types. FW: fresh weight; DW: dry weight. Conventional: conventional fertilizer Osmocote Plus 15-9-12; Organic: organic fertilizer Sustane 4-6-4.
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Table 1. Three-way ANOVA test results showing levels of significance for morphology and biomass parameters.
Table 1. Three-way ANOVA test results showing levels of significance for morphology and biomass parameters.
Plant HeightNeck DiameterBulb DiameterLeaf FWBulb FWRoot FWTotal FWLeaf DWBulb DWRoot DW
Cultivar****************************
Micro************NS*******
Fertilizer*************************NS
Cultivar × Micro*NS******NS***NS******
Cultivar × Fert**NS*****NS***NS
Micro × FertNSNS*NS*NSNS***NS
Cultivar × Micro × Fert***NSNS***NS***NSNS
Abbreviations: Micro, microbial biostimulants; Fert, fertilizer; FW, fresh weight; DW, dry weight. Level of significance: NS, not significant; * significant at p < 0.05; ** significant at p < 0.01; *** significant at p < 0.001.
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Zhang, Q.; Liu, J.; Jeong, S.J.; Masabni, J.; Niu, G. Biostimulants Applied in Seedling Stage Can Improve Onion Early Bulb Growth: Cultivar- and Fertilizer-Type-Specific Positive Effects. Horticulturae 2025, 11, 402. https://doi.org/10.3390/horticulturae11040402

AMA Style

Zhang Q, Liu J, Jeong SJ, Masabni J, Niu G. Biostimulants Applied in Seedling Stage Can Improve Onion Early Bulb Growth: Cultivar- and Fertilizer-Type-Specific Positive Effects. Horticulturae. 2025; 11(4):402. https://doi.org/10.3390/horticulturae11040402

Chicago/Turabian Style

Zhang, Qianwen, Jun Liu, Sang Jun Jeong, Joseph Masabni, and Genhua Niu. 2025. "Biostimulants Applied in Seedling Stage Can Improve Onion Early Bulb Growth: Cultivar- and Fertilizer-Type-Specific Positive Effects" Horticulturae 11, no. 4: 402. https://doi.org/10.3390/horticulturae11040402

APA Style

Zhang, Q., Liu, J., Jeong, S. J., Masabni, J., & Niu, G. (2025). Biostimulants Applied in Seedling Stage Can Improve Onion Early Bulb Growth: Cultivar- and Fertilizer-Type-Specific Positive Effects. Horticulturae, 11(4), 402. https://doi.org/10.3390/horticulturae11040402

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