Application of Azospirillum brasilense and Humic Substances Improves the Nursery Quality of Olive Seedlings in Pots

Abstract

In Brazil due to the establishment of new orchards, olive seedling production is growing strongly, while the use of biostimulants in agriculture has been gaining attention due to their benefits in root formation and nutrient absorption. This study evaluated the use of biostimulants for promoting the growth of 3-month-old rooted olive seedlings in pots and to assess the nursery quality of the seedlings. Rooted cuttings of Arbequina, Maria da Fé, and Ascolano 315 cultivars were treated with Azospirillum brasilense (Az) and humic substances (HS), alone and in combination. Growth parameters, such as height and stem diameter, were measured every month and after 150 days, seedlings per treatment were also analysed for aerial and root fresh and dry biomasses. Arbequina exhibited the highest growth rate with Az and best absolute growth rate with Az + HS treatment. The total dry matter of the olive seedlings, comprising both the aerial and root part, was influenced by both Azospirillum brasilense and humic substances, enhancing nitrogen availability. The three treatments showed their positive effects on aboveground growth and overall plant vigour. Despite increased biomass, treated olive seedlings showed no significant height advantage over controls, suggesting that the effects may appear in later developmental stages.

1. Introduction

Brazil ranks as the world’s second-largest importer of olive oil. In 2023, domestic production reached 700 tons, representing less than 1% of the country’s total olive oil imports. Commercial olive cultivation in Brazil has seen rapid expansion over the past decade with a vast potential for growth, since favourable conditions exist for establishing new olive orchards [1]. Given the increase in domestic demand for olive oil, academic and research institutions are studying the adaptation of various olive cultivars to Brazil’s unique edaphoclimatic conditions [2]. Soil quality, particularly pH, is determinant in ensuring successful olive cultivation. Olive trees thrive in sandy soils with not acidic pH levels, which offer ideal conditions for their growth and productivity [1,3]. Unluckily, in southern Brazil, the leading olive-growing area, soils are typically acidic and require lime applications to improve their suitability [4]. A recent study by [4], carried out on adult orchards, has shown that adjusting soil pH through varying liming levels and applying biostimulants can significantly enhance olive tree growth. Using Azospirillum brasilense, Trichoderma harzianum, and Trichoderma virens resulted in increased plant height, diameter, and dry mass yield, especially under lower acidity soil conditions.

As previously mentioned, the rising demand for olive oil has driven to a crescent interest in olive cultivation in Brazil. The most planted varieties include Arbequina, Ascolano, Barnea, Grappolo, and Koroneiki [5]. Among these, the Arbequina variety, originally from Spain, is one of the most widely cultivated in Brazil, showing excellent adaptation to Brazilian soils and achieving high yields in both productivity and oil content [6].

High-quality seedlings are essential for optimal orchard development and are typically propagated through cuttings. The success of production areas relies on implementing high-quality, well-rooted cuttings to ensure uniformity, rapid growth, and early fruit production [7]. Accelerating the growth rate of young olive trees is a critical goal for nurseries and olive farms [8]. In fact, in nurseries, faster growth would reduce the time required to prepare seedlings for transplanting, while on farms, it would shorten the period needed for trees to reach maturity and begin fruit production [8,9].

In the context of olive oil production, the application of biostimulants promotes plant growth, improves agronomic performance, and supports sustainable agricultural practices [10]. This category encompasses both microbial and non-microbial products, including humic substances [11]. Humic substances are condensed organic compounds produced through microbial degradation, comprising humic acid, fulvic acid, and humin fractions [12]. They represent the most reactive fraction of soil organic matter and are involved in most of the soil chemical reactions [13]. Their application can influence plant hormonal metabolism, promoting growth and development, root elongation, improving nutrient uptake, and increase biomass production [14].

Another widely used biostimulant in Brazil, but not only, is the plant growth-promoting bacterium Azospirillum brasilense [15]. This is a rhizobacterium, that is being increasingly used in agriculture in a commercial scale [16].

Azospirillum brasilense’s key properties, that contribute to its ability to adapt to the rhizosphere habitat and to promote plant growth, are reported by several authors [16]. Although nitrogen fixation is recognized as its primary benefit, increasing evidence highlights its other growth-promoting properties, such as synthesizing auxins, cytokinin, gibberellins, abscisic acid, ethylene, and salicylic acid [17]. These phytohormones stimulate root growth, thereby enhancing water and nutrient uptake [18].

Notably, Azospirillum brasilense produces indole-3-acetic acid, that is essential for root growth, making it a promising alternative for rooting in fruit-bearing plants [19].

While the use of biostimulant in rooted olive cuttings or young olive orchards remains underexplored, it has shown promising results in plant growth, reducing the time required in the fields to reach maturity and full production [9,20]. Past studies investigated the application methods and effects of biostimulants in different crops like melon [21], bell pepper [22], and tomato [23]. Several research groups in Brazil presented impressive results of increases in root growth, biomass production, grain yield, uptake of nutrients and water, and increased tolerance to abiotic stresses due to the inoculation of Azospirillum brasilense [24]. This rhizobacterium was applied on corn and wheat crops, rice, sugarcane, pasture grasses and soybean [25,26] but none has used it on olive or deciduous tree, even in young plants in the nursery. It was tested in strawberry, in which Azospirillum brasilense was able to promote plant growth [15,27]. In the other hand, Ref. [28] had already tested Azospirillum brasilense to promote the rooting of semi hardwood olive cuttings finding that its use, in combination with IBA, significantly increased rooting success. On the contrary, Ref. [29] found that the inoculation of olive tree cuttings with Rhizophagus clarus, Gigaspora rosea and Acaulospora scrobiculata, combined or not with IBA, did not significantly promote rooting.

To date, data on the effects of biostimulants in promoting the growth of young rooted olive seedling, obtained by cuttings, remain limited, since few studies were focused on 1-year-old potted olive trees [8,9].

Therefore, the objective of this study was to evaluate the use of biostimulants for promoting the growth of 3-month-old rooted olive seedlings in pots and to assess the nursery quality of the seedling. This was carried out using both humic substance and Azospirillum brasilense, either alone or in combination.

2. Materials and Methods

2.1. Location

The experiments were developed and conducted from May to October 2021, at the nursery of the “Professor Mário César Lopes” Experimental Station for Horticulture and Protected Cultivation, at the Western Paraná State University (UNIOESTE), Marechal Cândido Rondon campus (PR), Brazil. The nursery is located at the geographic coordinates of latitude 24°32′22″ S, longitude 54°03′24″ W, and an altitude of 420 m a.s.l.

2.2. Climatic Characteristics

According to the Köppen climate classification, the climate is classified as Cfa, mesothermal, humid subtropical, with well-distributed rainfall throughout the year and hot summers. The average temperatures of the coldest quarter range between 17 and 18 °C, the warmest quarter between 28 and 29 °C, and the annual average between 22 and 23 °C [30]. The relative humidity ranges from 70% to 75%. The average normal total rainfall for the region ranges between 1600 and 1800 mm, with the wettest quarter showing totals ranging from 400 to 500 mm [31].

2.3. Plant Material

Three months old rooted cuttings of the Arbequina, Maria da Fé, and Ascolano 315 cultivars were used (Photo 1). They were obtained from semi-hardwood cuttings rooted with 3000 mg L⁻¹ indolebutyric acid and then planted in polyethylene bags measuring 13 × 19 cm with a total volume of 910 mL, with four perforations at the bottom and six perforations on the sides (Photo 2). The bags were filled with oxisol substrate enriched with soil conditioner based on cattle manure (1:1, v/v) (

Table 1. Chemical and physical analysis of substrate; fertilization applied in pre-transplant.

A	P	OM	pH	H + Al	Al3+	K+	Ca2+	Mg2+	SB	CEC	V	Al
	mg dm−3	g dm−3		----------cmolc dm−3----------							%
A1	443.04	39.63	6.32	2.91	0.09	1.30	9.98	3.96	15.24	15.33	83.96	0.50
A2	298.44	42.38	6.37	2.79	0.00	2.42	7.73	5.72	15.87	18.66	85.05	0.00
A	Sand (%)				Silt (%)				Clay (%)
A1	23.79				13.51				62.70

A = sample, A1 = Red Latosol Eutroferric + soil conditioner (well-composted bovine manure) in a 1:1 ratio, A2 = soil conditioner.

).

2.4. Treatments and Growing Conditions

The applied treatments were: untreated seedling only nursery irrigation water (later named—Control); application of 0.5 mL per plant of inoculant containing Azospirillum brasilense (later Azospirillum brasilense or Az); application of 0.4 mL of humic substance (later humic substance or HS) per seedling; application of Azospirillum brasilense + humic acids (at the same concentrations as the previous treatments) (Azospirillum brasilense + humic acids or later Az + HS).

The commercial product used for humic substances was provided by SoloHumics^®, containing 25% humic substances (253 g L⁻¹) in its composition, and the Azospirillum brasilense inoculant was provided by Nitro 1000^® (containing strains AbV5, AbV6, and 200 million cells mL⁻¹).

For each treatment, 30 mL of inoculant and 24 mL of humic substance were used, diluted in water to form a final solution volume of 3000 mL, in such way that each plant received 50 mL of the solution. All treatments consisted of 45 plants, 15 of each cultivar. The seedlings received monthly treatments. The seedlings were trained as a single stem (Photo 3). Other cultivation management practices, such as insecticide application for whitefly (Bemisia tabaci) control and lateral shoot thinning to maintain the plant as a single stem, were carried out as needed.

Every 30 days, on average, measurements were carried out evaluating seedling height (cm), stem diameter (mm), number of nodes on the main axis. Height was measured using a graduated ruler, and stem diameter was measured with the aid of a digital caliper five centimeters above the soil. The number of nodes was counted manually, from the first sprouting of the cutting to the apex of the seedling.

2.5. Parameters

To evaluate vegetative growth, the Growth Rate (GR) and the Absolute Growth Rate (AGR), referred to seedling height, stem diameter and nodes of the main axis, were used.

Growth rate (GR), expressed in percentage, was calculated as the difference of the seedling height (SH), stem diameter (SD) or main axis node values at time T₁ and T₀, relative to the growing season, with respect to values at the time T₀ [32]:

GR = (SH_t1) − (SH_t0)/(SH_t0) × 100.

Absolute Growth Rate (AGR), expressed as cm day⁻¹, mm day⁻¹, node number day⁻¹ following the equation by [33]:

AGR = P₂ − P_1ΔT

P₂ = height, diameter, and number of main axis nodes at the end of the trial, at 150 days after transplanting (DAT).

P₁ = height, diameter, and number of main axis nodes at the beginning of the trial, and Δt = time interval between collections.

2.6. Aerial and Root Parts Weight and Distribution

When the seedlings were about 8 months old, approximately 150 days after the first treatment application, the final monthly evaluation was performed, and 15 plants per treatment were removed for both experiments as samples for fresh biomass (g) of both aerial and root parts. The destroy time was chosen considering that olive trees in Brazil are typically sold at 8 months of age.

The plants were removed from the substrate, the roots were washed in running water and separated from the aerial part of the plant. Each part was placed in properly labelled paper bags, weighed on a precision scale, and taken to a drying oven at 65 °C until they maintained a constant weight (four days). Subsequently, they were weighed for quantification of dry biomass (g).

Then the water content and the dry matter of the aerial and root part was calculated as follows:

Water Content (%) = (fresh weight − dry weight)/fresh weight × 100

Dry matter (%) = fresh weight (g) − water content (g) × 100

In addition, the fresh weight and dry weight of the aerial part of the seedling was divided by the total length of the seed to obtain the weight of 1 cm of seedling.

The distribution between the aerial part and root part of the seedling, in term of fresh and dry weight, was expressed as percentage ratio between part weight (aerial part or root part) and seedling weight as follows:

Aerial part distribution in fresh weight (%): (aerial part fresh weight/seedling fresh weight) × 100.

Root part distribution in fresh weight (%): (root part fresh weight/seedling fresh weight) × 100.

2.7. Experimental Design and Data Analysis

The experimental design used was randomized blocks in a 3 × 4 factorial system (3 cultivars × 4 treatments), with a split-plot arrangement over time, containing four replications of 5 plants and three replications of 5 plants.

Data were subjected to analysis of variance, and the means were compared using the Duncan test at a 5% probability of error using the Infostat software version 2020 [34].

Principal component analysis (PCA) was performed using the most significant seedling parameters as input variables to explore the variability among samples and to detect the most discriminating variables. PCA summarizes the information contained in the data matrix in fewer independent PCs, obtained as linear combinations of the original variables, lying in the direction of maximum variance.

2.8. Seedling Characteristics at the Beginning of Trial

At the time of the first treatment application the rooted cuttings of Maria da Fé cultivar were significantly higher than those of Arbequina cultivar, while those of Ascolano 315 cultivar showed the thicker stem and lowest node number per seedling (

Table 2. Characteristics of rooted cuttings at the time of treatments (means ± s.e.). Mean values followed by different letters are significantly different per α < 0.05.

Cultivar	Seedling Height (cm)	Stem Diameter (mm)	Seedling Node (n.)
Arbequina	22.33 ± 0.35 ab	2.35 ± 0.05 b	14 ± 0.32 a
Ascolano 315	21.68 ± 0.43 b	2.64 ± 0.10 a	12 ± 0.34 b
Maria da Fé	23.78 ± 0.30 a	2.53 ± 0.09 ab	15 ± 0.28 a

).

3. Results

3.1. Size of the Seedlings over Time According to Biostimulant Treatments and to Cultivars

In general, the plants treated with biostimulants showed higher values for assessed variables, such as seedling height, compared to the control treatment. However, for certain variables, the differences were not statistically significant (Figure 1 and Figure 2).

No significant differences were observed for the seedling height variable across the various treatments. At the end of the experiment, plants treated with Azospirillum brasilense and Azospirillum brasilense plus humic substances reached a height of 77.1 cm. Plants treated only with humic substances reached 71.8 cm in height, while control plants reached 76.4 cm (Figure 1a). After 120 days of planting, the Arbequina and Maria da Fé seedlings were significantly taller (with an average height of 65.9 cm) compared to the Ascolano 315 variety, which had an average height of 61.2 cm (Figure 1b). 150 days after transplanting, there was no significant difference between the seedling heights of the three cultivars, treated and untreated, with an average height of 75.6 cm (Figure 1b). The Arbequina plants treated with Az+HS showed significant results, with an average height 80.2 cm (Figure 1c), higher length compared to results seen in the different treatments. On the contrary, the Arbequina treated with only HS were significantly different for showing lowest plants height average of 69.4 cm. The Ascolano 315 plants treated with Az +HS did not show a significant difference among the different treatments (Figure 1d). The cv. Maria da Fé plants did not present a significant difference among treatments for seedling height, throughout all the periods of days after transplanting (Figure 1e).

For the stem diameter variable (Figure 2a), no differences were observed among the seedlings across different treatments. At the end of 150 days after transplanting (DAT), the Azospirillum brasilense treatment and the HS treatment have led the highest values; however, they did not differ significantly from the other treatments (Figure 2a).

At 120 DAT, the stem diameters were significantly different, mainly between the Ascolano 315 and Arbequina cultivars. Although, at 150 days, there was no significant difference in stem diameter among the varieties (Figure 2b). At 120 DAT the Arbequina cultivar showed a greater response to the HS treatment, significantly different from the others. At 150 DAT, there was a significant difference between control Arbequina cultivars and those treated with HS (Figure 2c), while no significant difference was detected between those treated with Azospirillum brasilense and Az + HS. The Ascolano 315 cultivar showed no significant differences in any treatment throughout over the whole trial period (Figure 2d). At 120 DAT, the Maria da Fé cultivar showed a higher value of stem diameter when the seedlings were treated with Azospirillum brasilense compared to the other treatments; however, at 150 DAT, the different treatments did not significantly affect stem diameter of the seedlings (Figure 2e).

Concerning the number of main axis nodes from 30 to 150 DAT, considering the treatment effect (Figure 3a) on olive seedlings, no significant differences are observed, except after 120 DAT, where a significant difference is observed between the control and both of HS and Az + HS, and a less significant difference between the control and Az treatment. Considering the cultivar effect (Figure 3b), Ascolano 315 cv. showed a significant difference throughout all the DAT compared to Arbequina cv. and Maria da Fé cv. This could be explained by the mean value of the three cultivars for the count of main axis nodes character, which are 30, 34 and 35 for Ascolano 315 cv., Arberquina cv. and Maria da Fé cv. respectively.

The number of main axis nodes on Arberquina cv., when different treatments were applied (Figure 3c), showed not significant difference during the 120 and 150 DAT. Same results were observed in both Ascolano 315 cv. and Maria da Fé cv. as no significant differences were observed in all treatments applied for this parameter (Figure 3d,e).

3.2. Growth Rate of the Seedlings over Time According to Biostimulant Treatments and to Cultivars

The seedling height Growth Rate, expressed in percentage, resulted affected by Azospirillum brasiliensis throughout the entire of 5 months growth period (Figure 4a). Moreover, the seedlings of the cv. Ascolano 315 showed a greater increase in height than those of the other two cultivars (Figure 4b). The cultivars have responded differently to treatments. The significant effects were observed only in Arbequina’s seedlings, where Azospirillum brasiliensis determined the highest height Growth Rate (Figure 4c). Effects were also found in Ascolano 315’s seedlings, although with not significant differences (Figure 4d), while in Maria da Fé the treatments did not affect the Growth Rate (Figure 4e).

The stem diameter Growth Rate resulted not significantly different both among the treatment and cultivars (Figure S1). Only in Maria da Fè’s seedlings, the humic substances determined the highest Growth Rate (Figure S1e).

The increase in the number of nodes when considering the effect of treatment showed a significant difference between Azospirillum brasiliensis and humic substances after 150 DAT (Figure 5a).

No differences were observed among the cultivars in terms of node number growth rate (Figure 5b). Azospirillum brasiliensis treatment showed the highest values of node growth rate for both Arbequina cv. (Figure 5c) and Ascolano 315 cv. (Figure 5d), however there was no significant difference among the treatments (Figure 5e).

The Absolute Growth Rate results showed a significant difference in seedling height for plants treated with humic substances where the lowest values were observed. Treatments with Azospirillum brasiliensis and Azospirillum brasiliensis and humic substances showed greater efficiency compared to the humic substances treatment (Figure 6a).

No significant differences were observed in stem diameter and node number based on results of Absolute Growth Rate. No significant differences were noted in seedling height and stem diameter

Absolute Growth Rate values across all cultivars, although a slightly significant difference was observed in node number Absolute Growth Rate values for Ascolano 315 which showed lower Absolute Growth Rate values than the other two cultivars (Figure 6b).

Regarding the interaction between treatments and cultivars, Arbequina showed greater height growth with Azospirillum brasiliensis and humic substances treatments; however, no significant differences in stem diameter and node number were found across all treatments after 150 DAT (Figure 6c).

No significant differences were observed in Absolute Growth Rate values for Ascolano 315 and Maria da Fé cultivars throughout all treatments (Figure 6d,e).

3.3. Aerial and Root Biomasses of the Seedlings at the End of Trial According to Biostimulant Treatments and to Cultivars

No differences were observed among the four treatments and the three cultivars about fresh and dry biomass of aerial part, which consists of stem and leaves (Figure S2).

Significant differences were observed between control plants and treated plants (Figure 7a), as well as among the Arbequina cultivar and the Ascolano 315 and Maria da Fé cultivars (Figure 7b), with respect to fresh and dry root biomass.

Specifically, control Arbequina seedlings exhibited significantly higher fresh and dry root biomass compared to the treated samples (Figure 7c).

Similarly, notable differences were observed in both the Ascolano 315 and Maria da Fé cultivars under different treatments considering fresh and dry root biomass (Figure 7d,e).

Significant differences were observed in the fresh biomass seedling weight, which combines the root seedling weight and the aerial seedling part weight (Figure 8a). Control seedlings exhibited a lower aerial part fresh biomass weight, but a higher root seedling fresh biomass weight compared to treated seedlings.

The total fresh biomass weight of Arbequina cultivars significantly differed from that of the other two cultivars (Figure 8b).

Additionally, control Arbequina seedlings displayed significantly different total fresh biomass weights compared to the treated counterparts (Figure 8c).

No significant differences were observed in the fresh biomass weight between control and treated Ascolano 315 seedlings (Figure 8d).

Significant differences were observed in the root seedling and aerial part fresh biomass weight in Maria da Fé seedlings in the different treatments (Figure 8e).

Significant differences were observed in the dry biomass seedling weight, which combines the seedling root and the aerial part weight (Figure 9a), between control seedlings compared to treated seedlings.

Untreated seedlings exhibited a lower aerial part dry biomass weight, but a higher root seedling dry biomass weight compared to treated seedlings. The total dry biomass weight of Arbequina cultivars differed significantly from that of the other two cultivars (Figure 9b). No significant differences were observed among the different treatments applied to the three cultivars (Figure 9c–e), except for the aerial seedling part dry biomass weight in control Arbequina seedlings.

Seedling treated with humic substances showed significant differences compared to other treatments, displaying higher dry and fresh aerial part biomass weight per cm (Figure 10a).

Maria da Fé showed significantly higher fresh aerial biomass weight per cm compared to other cultivars, while Ascolano 315 showed significantly lower dry aerial biomass weight per cm compared to other cultivars (Figure 10b).

Arbequina seedlings showed significantly higher dry and fresh aerial biomass weight per cm when treated with humic substances compared to other treatments (Figure 10c). Significant differences were observed in Maria da Fé seedlings with all treatment regarding fresh aerial part biomass weight per cm (Figure 10e).

Significant differences were observed in aerial part dry matter (%) when comparing the results between the different treatments, while only the control differed in the root part dry matter (%) (Figure 11a).

No significant differences were detected in root and aerial part dry matter between the different cultivars (Figure 11b). Significant differences were noted in aerial part dry matter (%) when comparing the results between the different treatments applied to Arbequina, while only the control differed in the root part dry matter (%) (Figure 11c).

No significant differences were detected in aerial part dry matter between the different treatments applied to Ascolano 315 cv., while significant differences were observed in the roots dry matter (Figure 11d).

No significant differences were detected in root and aerial part dry matter between the different treatments applied to Maria da Fé seedlings (Figure 11e).

3.4. Principal Component Analysis

The PCA results revealed clear differences in seedling growth traits, with distinct variations visible across the different treatments. Notably, the untreated seedlings were clearly distinguished from those of the three treatment groups, highlighting the influence of Azospirillum brasilense and humic substances and their combination on seedling growth promotion (Figure 12).

PC1, which accounts for 28.8% of the total variance, was primarily influenced by aerial part fresh and dry weight, by biomass aerial part fresh and dry weight per 1 cm of seedling height, absolute growth rate. These compounds were positioned on the right side of the plot, indicating that biostimulants application may affect biomass increasing as well as seedling growth. PC2, explaining 24.0% of the variance, showed a clear effect of humic substances on increasing biomass. In addition, the PCA biplot reveals correlations between growth parameters. As can be seen from the Figure 12, the variables related to root part distribution resulted negatively correlated with parameters tied to aerial biomass, as they point in opposite directions on the PCA plot. Growth rates and seedling height showed a positive correlation along PC2. The distribution of treatments on the PCA plot further demonstrates how these correlations are influenced by the treatments. HS, AH, and Az-treated plants align with growth rates and aerial biomass parameters, indicating their positive effects on aboveground growth and overall plant vigour. In contrast, the control plants are more associated with root biomass growth, suggesting a trade-off between aerial and root development in untreated conditions.

4. Discussion

The effect of Azospirillum brasilense and humic substances, alone or together, on the growth promotion of 3 months old seedlings was assessed on three olive cultivars (Arbequina, Ascolano 315 and Maria da Fé).

In general, seedling height of olive was not significantly affected by the treatments; however, in Arbequina seedlings, the combination of humic substances with A. brasilense, or Azospirillum brasilense alone, determined the increase of the overall plant vigour. This result may be due to two main factors. Firstly, this study involved very young seedlings—only three months old at the start, growing up to eight months old—reflecting typical nursery practices in the area. Previous studies used seedlings that were 8 months old or even 1 year old [1,8]. Secondly, the differences observed among the three cultivars could be due to both genetic factors [28] and variations in root system size. In fact, the species Olea europaea L. exhibits different growth and development behaviours across genotypes, due to many years of empirical selection and the collection of specimens from different regions during the domestication of the plant [35,36]. Among the studied cultivars, differences arise from the intrinsic characteristics of each. In particular, Ascolano 315 is characterized by longer internodes and has fewer nodes compared to the other cultivars.

Refs. [28,37] showed that Arbequina, at the end of the cutting period, forms a greater number of roots, almost twice as many, than those of the Maria da Fè and of the Ascolano 315. This greater rooting ability enhances the Arbequina cultivar’s nutrient uptake capacity. In fact, at the end of the trial, Arbequina seedlings exhibited higher root biomass than in the other cultivars. In addition, the biomass distribution in Arbequina seedlings was predominantly allocated to the roots, which accounted for 41.83% of the fresh biomass and 46.48% of dry biomass. In contrast, Maria da Fé and Ascolano 315 averaged 35.65% and 40.93% for fresh and dry root biomass, respectively. The increasing of biomass may be due to the production of phyto-hormones, such auxin, which is a promoter of cell expansion and can influence a greater accumulation of biomass, in agreement to that observed in strawberry by [27].

Humic substances had lower effects on seedlings growth, in terms of height, stem diameter and node number, in comparison to the control. This agrees with the result of a study carried out in pot—grown olive cuttings of the Cobrancosa cultivar, where leonhardite-based treatments did not significantly affect any variables [38]. On the other hand, Azospirillum brasilense inoculation significantly affected the height growth rate of Arbequina seedlings. This result agrees with that obtained by [1] using seedlings six months old and 40 cm tall.

The total dry matter of the olive seedlings, comprising both the aerial and root part, was influenced by both Azospirillum brasilense and humic substances. This result supports what observed by [8], indicating improved nursery quality. In specific, the dry matter of the aerial part was more strongly affected by Azospirillum brasilense, whereas the dry matter of root was influenced by all the treatments, in agreement to what reported in strawberry [15]. These effects are likely related to the role of Azospirillum brasilense and humic substances, alone or in combination, in enhancing nitrogen availability for seedlings [1,38]. These results agree with other studies, showing that Azospirillum brasilense can supply more than 50% of the N needed by plant, increasing in water and nutrient absorption [39].

On the other hand, studies carried out on older seedlings, 1 year old at the beginning of the trial, showed that all the components of the plant were positively affected by the application of biostimulant, based on protein hydrolysates [8]. The authors attributed this to the hormone—like action of biostimulants on plant physiology, which improved the overall growth of olive trees. Other studies [9,38,40], carried out on seedlings in pot, had used older seedling and/or for more time. Therefore, the fact that the olive seedlings treated with Azospirillum brasilense and humic substances did not become higher than the untreated plants may also be attributed to the use of very young plants, potted for only 3 months, as well as the limited duration of the experiment, which lasted only 6 months.

Considering that olive trees in Brazil are typically sold at 8 months of age, whereas European nurseries sell 10–12-month-old plants, a longer nursery period might better reveal the effects of Azospirillum brasilense and humic substances. Further trials are required to determine the precise timing where biostimulant effects can be observed. In fact, longer time to see the effects explains why in the field, Azospirillum brasilense has been used many times on several annual crops such as soybean [25], wheat [41], purple maize [26] and other more, but none tested it on perennial crops, such as olive or other fruit tree except on strawberries [15,27] and forest tree seedling such as Cordia alliodora [39]. This may also be due to the faster growth of herbaceous roots than tree roots, that takes longer time to expand and to form many roots that can be colonized by rhizobacteria [27,42,43].

Regarding humic substances, it has been reported that most biostimulant effects of humic substances refer to the improvement of root nutrition, through different mechanisms: increased uptake of macro- and micronutrients, hormonal effects and stress protection [9]. Therefore, the response of olive seedlings to the application of Azospirillum brasilense and humic substances may be due and to the higher efficiency of absorption of the mineral nitrogen (N) available in the substrate, as observed in Cordia alliodora [39], and to substances based on phytohormones, such as auxins and cytokinins, that can be released by Azospirillum brasilense and later improve the efficiency of N use by plants, consequently leading to greater gain in biomass production. This was observed in the present study with the variables aerial dry mass and root dry mass. Nevertheless, although bacterial inoculation did not always result in measurable positive results, we can assume that such biotized olive seedlings may exhibit an improved resistance against environmental stress after out—planting and subsequent growth in the orchard, as suggested even for strawberry [15].

5. Conclusions

The application of Azospirillum brasilense and humic substances, either alone or in combination, on very young olive seedlings showed that, while these treatments may enhance the seedlings’ ability to grow in the field, by promoting higher biomass, in the short term, they did not produce bigger plants than control. However, Azospirillum brasilense has led to higher growth rates, which may indicate a potential reduction in the time seedlings spend in the nursery. By improving the dry matter content of the olive seedlings, Azospirillum brasilense and humic substances, led to obtain plants with better quality of nursery, which in turn can enhance rooting success after transplantation into the field.

To better understand the effect of Azospirillum brasilense and humic substances on the growth of young olive seedlings in pot, both in quantitative and qualitative terms, further research will be conducted, analysing also other parameters such as chlorophyll content and sugar contents of aerial and root parts, after longer time.