Substrate Properties, Vegetative Growth, Chlorophyll Content Index and Leaf Mineral Content of Sweet Cherry Maiden Trees as Affected by Rootstock and Plant Growth-Promoting Rhizobacteria

Abstract

Sweet cherry (Prunus avium L.) is a valuable fruit crop for fresh consumption. Due to its early availability in season, it achieves relatively high prices on the market. Self-fertile cultivar Lapins is one of the world’s leading sweet cherry varieties. Intensive cherry production seeks for new technologies such as using more adaptable rootstocks and microbiological products that could help plants adopt more sustainable growth in different soils/climates. The aim of this work is to determine the substrate properties, vegetative growth, leaf chlorophyll and mineral content of maiden trees grafted on three different rootstocks due to the application of growth-promoting rhizobacteria. A pot experiment was carried out on one-year-old maiden trees of cv. Lapins grafted on SL 64, MaxMa 14 and Gisela 5 and grown in 12 L plant pots filled with commercial substrate. Plant growth-promoting rhizobacteria Azospirillum brasilense was added by watering the plants with 1.12 g L⁻¹ per pot once a month (T₁) or every two months (T₂) from March to September with seven treatments in T₁ and four treatments in T₂. At the same time, control (C) plants were watered with rainwater. Plant height, trunk circumference and leaf chlorophyll content index (CCI) were measured. In addition, shoot growth and internode number were measured in three development stages (BBCH 34, 39 and 91). The substrate and leaf samples were collected and analyzed in the laboratory in accordance with established procedures. Data were processed by ANOVA and the Tukey test. Results have showed that rootstock affected substrate electrical conductivity (EC); nitrate (NO₃⁻), phosphorous (P₂O₅), calcium (Ca) and magnesium (Mg) content, including mineral nitrogen (N) content; tree height, circumference, shoot length and internode number; the leaf chlorophyll content index (CCI); and leaf potassium (K), Ca and Mg content. Furthermore, treatment significantly affected the CCI, average internode length, ammonia (NH₄⁺) and Ca content in the substrate and leaf N, Ca and Mg content. Rhizobacteria A. brasilense can be used as an additional biofertilizer in sustainable agricultural practices for obtaining healthier sweet cherry maiden trees, but microbial biotechnology rules must be respected.

1. Introduction

Sweet cherry (Prunus avium L.) is one of the most prized fruit crops on the market for fresh consumption due to the fruit’s organoleptic properties and nutritional values. It belongs to the group of stone fruits from the genus Prunus and is grown mostly in areas with a moderate climate. According to FAOSTAT data, sweet cherry production in Croatia in 2022 was 1800 tons, half of which was produced in the coastal area [1]. The current production in Croatia is not sufficient for the market needs [2]. A partial reason for the lower production quantities is the widespread use of mahaleb cherry (Prunus mahaleb L.) and wild cherry (Prunus avium L.), two vigorous seedling rootstocks still prevailing as planting material in most Croatian nurseries [3]. Specificities of individual micro-locations, such as soil and climatic conditions, have forced producers to choose optimal rootstocks for sweet cherry production. Rootstock is an essential component in modern fruit cultivation because of its ability to enable the adaptation of a particular cultivar to different environmental conditions and agrotechnical practices. Good rootstock selection provides the grafted tree with important traits such as better root anchoring, root resistance to soil pathogens, improved nutrient absorption and a higher tolerance to limiting ecological and pedological factors [4]. Moreover, it directly affects the vegetative growth, vigor and shape of the tree, as well as the yield, ripening period, size and quality of the fruit [3,5,6,7].

As horticultural production develops and the need for fruits rises, the selection of rootstock is also becoming more important [8]. Clonal rootstocks selected through breeding programs are already replacing outdated seedlings in integrated and organic fruit production programs [9].

A rootstock that has been used in sweet cherry orchards throughout the Mediterranean basin is Sainte Lucie^® 64 (SL 64), a medium–vigorous rootstock obtained through the selection of mahaleb at the National Institute for Agronomic Research (INRA) in France. Rootstock SL 64 adapts to well-drained sandy, shallow and skeletal soils with a higher concentration of active lime and is suitable for denser planting [10]. It roots well with good anchorage, rarely produces suckers and produces good-quality fruits. It is drought-resistant, but sensitive to heavy and asphyxiated soils [7].

Another prospective rootstock showing good properties in different growing conditions is MaxMa Delbard^® 14 Brokforest, a cross between mahaleb and wild cherry [11]. Compared to SL 64, MaxMa 14 is less vigorous, while its vigor also depends on grafted variety and soil type. Its branched and deep root network anchors well in soil [7]. Rootstock MaxMa 14 is precocious with a high production capacity for grafted varieties. Unlike SL 64, it tolerates a wider range of soils and is more tolerant to root asphyxia [10].

With the introduction of high-density plantations and intensive cultivation systems, growers are turning to dwarfing rootstocks, among which is Gisela^® 5 (P. cerasus “Schattenmorelle” × P. canescens), selected at the Justus Liebig University in Giessen, Germany [12]. Its root network is dense and branched, but plants need trellis construction for support. It requires fresh and fertile soils with regular irrigation [7]. Due to its dwarfing ability, which has been confirmed by numerous authors [13,14,15,16,17,18], early fruiting and high productivity, Gisela 5 is considered one of the best rootstocks for high-density plantations [3,19,20].

The relationship between the rootstock and the variety is of particular importance. The assortment of cherries is extremely wide. Cv. Lapins is one of the world’s leading sweet cherry varieties: it is selected in Canada by crossing cultivars Van and Stella [21] and is often used in scientific work [22]. Several authors have confirmed the different vegetative growth of cv. Lapins trees grown on SL 64, MaxMa 14 and Gisela 5 in different agroclimatic conditions [18,23,24,25].

Intensive agricultural practices ensure high yields and optimum quality of cultivated crops mainly due to the extensive use of mineral fertilizers and pesticides. However, increasing production costs and negative effects on the environment have awakened interest in environmentally friendly and sustainable agricultural practices [26]. Nowadays, in agricultural production emphasis is placed on the use of various biological, microbiological and biotechnical products that could restore the fertility of depleted soils [27] and preserve rhizosphere values [8]. It is known that the use of microbiological fertilizers containing beneficial microorganisms instead of synthetic chemicals improves plant development by supplying nutrients and contributes to maintaining environmental health and soil productivity [28]. Inoculation with plant growth-promoting rhizobacteria also plays an important role in improving plant growth in unfavorable soil conditions [29,30]. These microorganisms help the plant absorb the necessary nutrients through processes such as N fixation, phosphate solubilization, iron sequestration and the biosynthesis of phytohormones [31]. Their interactions with plants and other rhizosphere life are vital for plant growth and productivity, but also for preserving the health of the environment [32].

In general, the beneficial effects of rhizobacteria on fruit crops are covered to a lesser extent than on other crops. Until now, the positive effects on apples (Malus domestica Borkh.) [33,34,35,36,37], strawberries (Fragaria spp.) [29,38,39,40,41,42,43,44], cherries [45,46,47,48], apricots (P. armeniaca L.) [26,49] and peaches (Prunus persica (L.) Batsch.) [50,51,52,53,54] have been reported. Authors Eşitken et al. [46] applied Pseudomonas and Bacillus strains through foliar and floral treatments on cv. 0–900 Ziraat and obtained significant differences in shoot length, yield and fruit mass. Moreover, bacterial treatments positively influenced N, P and K leaf content.

The rhizobacteria of genus Azospirillum belong to associative microorganisms that are capable of colonizing plant roots, having a beneficial effect on plant growth. This genus mainly colonizes grasses and cereals, but their beneficial effect on perennial plants is known as well [55,56,57]. Azospirillum brasilense associatively inhabits the rhizosphere of the host plant. It is widely used as a microbiological fertilizer with other conventional and organic fertilizers in nutrition programs in fruit crops [58]. The beneficial effects on plants of genus Azospirillum have drawn researchers’ attention because of the need of reducing agricultural chemicals in relation to sustainable agriculture [59]. Regardless of the significant results over the years showing the application potential of Azospirillum, research mostly focuses on indoor conditions [60]. In field in vivo conditions, only a few papers on Azospirillum spp.’s influence on fruit crops have been published [40,44,57,58]. The efficiency of rhizosphere colonization of the host plant in field conditions depends on numerous factors in the agricultural ecosystem, among which are the soil type, pH reaction, salinity and moisture, climatic conditions and soil nutrient content, which play an important role in root colonization and the efficacy of microorganisms [61,62]. Due to unfavorable abiotic and biotic factors, inconsistent results in field research have prevented the widespread use of A. brasilense for significant commercial purposes.

Still, previous papers on fruit growing have shown the influence of A. brasilense in in vitro and post vitro conditions on cuttings’ growth, transplant adaptation, plant vegetative growth and fruit characteristics. For instance, inoculated cuttings of GF 677 (P. persica × P. amygdalus) and Mr.S 2/5 (P. cerasifera × P. spinosa) rootstocks under controlled conditions showed significant differences in the node number, stem length, fresh stem mass and fresh root mass [57,63].

Furthermore, A. brasilense improved strawberry fruit’s nutritional value in hydroponics [44] and invigorated plant metabolism [42]. In field cultivation, Salazar et al. [40] reported the effects of A. brasilense in the early stage of strawberry production. Rhizobacteria also promoted the growth and yield of pomegranate (Punica granatum L.), white mulberry and custard apple (Annona squamosa L.) [58,64,65].

For similar reasons, A. brasilense is considered an important rhizobacteria that promotes the growth of the investigated fruit crops. The above-mentioned studies proved the existence of a positive influence of certain rhizobacteria on fruits including sweet cherry, but also a positive influence of some A. brasilense strains on Prunus spp. rootstocks and others. However, an analysis of A. brasilense’s influence on vegetative growth and leaf mineral content of cv. Lapins maidens grown in pots until now has not been reported.

Therefore, the aim of this study was to determine the content of N, P, K, Ca and Mg in the substrate where maidens were growing and also in the leaves of cv. Lapins maidens grafted on SL 64, MaxMa 14 and Gisela 5 rootstocks as a result of different amounts of A. brasilense applied in total. Secondly, the aim was to test maidens’ vegetative growth and chlorophyl content index due to different amounts of A. brasilense applied.

2. Materials and Methods

2.1. Experiment Location

A pot experiment was conducted in 2022 at the location Poljana on the island of Ugljan (44°5′23.103″ N, 15°11′1.806″ E), Zadar County, Croatia. The experiment was set up in pots on an area of 120 m². Location climatological data for the experiment period were obtained from the nearest meteorological station of Croatian Meteorological and Hydrological Service [66].

2.2. Climatic Conditions

In 2022 (Figure 1), the mean annual temperature was 16.9 °C. The absolute maximum temperature was 39.0 °C (August), while the absolute minimum temperature was −0.7 °C (January). The total amount of precipitation in 2022 was 671 mm. According to the Koppen classification, the climate of this area is characterized as the sub-Mediterranean type, with mild, rainy and moderately windy winters and hot and dry summers (Csa) [67]. Furthermore, according to Miljković [68] and Gonçalves et al. [69] the experiment location is suitable for sweet cherries.

2.3. Planting Material

One-year-old plants of three different rootstocks (SL 64, MaxMa 14 and Gisela 5) were obtained from commercial nurseries as certified virus-free planting material. Plants were 70 cm tall on average and cut down to 20 cm prior to budding. Rootstock plants were chip-budded at the beginning of March with buds from cv. Lapins by a grafting expert. The budding height was 10 cm above the root collar. A total of 45 plants per rootstock were budded.

2.4. Characteristics of the Substrate

The medium basic substrate TS3 (Klasmann-Deilmann GmbH, Geeste, Germany) has a fine structure and according to the manufacturer’s specifications contains moderately decomposed white peat and clay particles. It retains water well and has a good porosity. Fertilizer NPK 16:10:18 was added to the substrate at the factory in the amount of 1.0 g L⁻¹. The substrate sample was analyzed before planting maidens in the laboratory of the Institute for Plant Nutrition of the Faculty of Agriculture, University of Zagreb. Analysis was carried out according to the following methods: pH [70], EC [71] and N content [72]. From the water extract [73], the amounts of NO₃⁻, NH₄⁺ and P₂O₅ ions were obtained spectrophotometrically, the amount of K₂O was obtained by flame photometry and the amounts of Ca and Mg were obtained by an atomic absorption spectrometer [74]. According to the obtained data from

Table 1. Analysis of the commercial substrate by an authorized laboratory.

Parameter	Measuring Unit	Value
pH	H2O	7.29
EC	mS cm−1	0.19
NH4+	mg L−1	23.85
NO3−	mg L−1	15.10
N-min.	mg L−1	21.93
P2O5	mg L−1	84.75
K2O	mg L−1	36.23
Ca	mg L−1	9.09
Mg	mg L−1	1.28

, it is evident that the substrate is very well supplied with P₂O₅, but very poorly supplied with N and K₂O.

2.5. Experimental Design

Maiden trees budded on three different rootstocks were planted in 12-liter pots with Klassman TS3 substrate. All pots had plant saucers. The experiment consisted of 135 plants in total: 45 plants per rootstock and 15 plants per treatment, where one replication was represented by 5 plants. Pots were arranged in a randomized block design. Microbiological fertilizer AZOS^® was purchased from the Reforestation Technologies International (Gilroy, CA, USA). The product contains a pure culture of rhizobacteria A. brasilense in a concentration of 10⁶ CFU g⁻¹. Treatment included the following:

C—watering with rainwater (0 kg of AZOS per ha);

T₁—watering with a suspension of AZOS once a month (1.12 kg of AZOS per ha);

T₂—watering with a suspension of AZOS every two months (1.12 kg of AZOS per ha).

According to the manufacturer’s instructions, each pot in T₁ and T₂ was watered with 1.12 g L⁻¹ of AZOS suspended in rainwater. For the watering treatments, each pot was watered manually with the same volume of suspension (T₁ and T₂) or rainwater (C). Treatments started in March and ended in September. Maidens in T₁ were treated seven times in total while maidens in T₂ were treated four times in total. During growth, all maiden plants were protected from pests and diseases according to integrated pest management practice equally. Pots were irrigated by drip irrigation with well water. The amount of irrigated water per pot was estimated at the beginning of an experiment by testing the substrate’s field capacity.

2.6. Substrate Analysis

In autumn, substrate samples were collected and averaged out for each replication before analysis. Substrate samples were packed, labeled and delivered to the Laboratory of the Plant Nutrition Institute of the Faculty of Agriculture, University of Zagreb. Water extraction (1:2 vol.) was performed in the laboratory, and the extracts were used for further analysis.

The pH reaction was determined according to HRN EN 13037:2012 [70], and the electrical conductivity (EC) was determined according to HRN EN 13038:2012 [71]. The amount of NO₃⁻, NH₄⁺ and P₂O₅ ions was determined from the water extract according to the official method [73] using a spectrophotometer [74]. Furthermore, the amount of K₂O ions was determined using a flame photometer, and the amounts of calcium and Mg ions were determined using an atomic absorption spectrometer [74]. Also, the amount of total mineral N was determined according to the modified Kjeldahl method [72].

2.7. Vegetative Growth Characteristics

Maiden growth was determined by measuring the trunk diameter at 5 cm above the root collar and 5 cm above the budding union and by measuring plant height at the end of the trial. Shoot growth and its internode number were measured in three different developmental stages according to the BBCH scale [75].

2.8. Chlorophyll Content Index (CCI)

Evaluation of chlorophyll content in the leaves was determined by measuring the CCI at the beginning of summer. Values were measured with a Chlorophyll Content Meter Model CCM-200 (Opti-Sciences, Inc., Hudson, NH, USA) [76]. This field device measures the CCI, which is a non-destructive and rapid method for measuring chlorophyll content changes and stress effects on different plants. A noninvasive measurement was carried out on five fully developed and healthy leaves selected from the central part of the shoot. The CCI values were measured at the same time, always in the morning at 8:00. The mean value was calculated for each plant and determined as the CCI.

2.9. Analysis of Macronutrients in Leaves

Leaves were sampled during summer and averaged out for each replication before analysis. Fully developed leaves without visible damage were selected and picked from the middle part of the developed shoot and placed in pre-weighed and marked paper bags. The leaf mass for each replication was averaged to 6.50 g of fresh matter per sample. The leaves were transported to the Laboratory of the Plant Nutrition Institute of the Faculty of Agriculture, University of Zagreb. In the laboratory, samples were dried at 105 °C. After drying, samples were weighed on a precision scale, and the percentage of dry matter for each sample was calculated as the ratio of the fresh and dry mass. Prepared leaf samples were decomposed in a 1200 Mega microwave digester (Milestone Srl, Sorisole, Italy) with concentrated HNO₃ and HClO₄. After decomposition, P₂O₅ concentration was determined on a spectrophotometer, K₂O concentration was determined by a flame photometer, while Ca and Mg concentrations were determined using an atomic absorption spectrometer [74]. The total N content was determined by the modified Kjeldahl method [72].

2.10. Statistical Data Analysis

The data were analyzed using the software Statistica 14th edition (TIBCO, Santa Clara, CA, USA). Data were statistically processed by a two-way ANOVA while significant differences used for comparing treatment means in pairs were investigated by the Tukey HSD test (p ≤ 0.05).

3. Results

3.1. Physicochemical Properties of the Substrate

The results in

Table 2. Mean values of pH, EC and macronutrient content of the substrate in which cv. Lapins maidens were grown on different rootstocks (SL 64, MaxMa 14 and Gisela 5) and watered with AZOS suspension at different doses and intervals (C, T₁ and T₂) during the 2022 growing season.

Parameter	Rootstock			Treatment			R	T	R × T
	SL 64	MaxMa 14	Gisela 5	C	T1	T2	p	p	p
pH (H2O)	6.46 ± 0.15	6.22 ± 0.09	6.40 ± 0.09	6.23 ± 0.09	6.40 ± 0.14	6.45 ± 0.10	n.s.	n.s.	n.s.
EC (mS cm−1)	0.52 ± 0.10 ab	0.69 ± 0.07 a	0.34 ± 0.09 b	0.62 ± 0.09	0.50 ± 0.10	0.42 ± 0.06	**	n.s.	*
NH4+ (mg L−1)	4.68 ± 0.67	4.48 ± 0.94	3.29 ± 0.41	5.32 ± 0.66 a	4.43 ± 0.72 ab	2.69 ± 0.41 b	n.s.	*	n.s.
NO3− (mg L−1)	18.56 ± 2.31 a	19.15 ± 1.85 a	10.99 ± 1.43 b	18.70 ± 3.18	15.57 ± 1.35	14.43 ± 1.68	**	n.s.	n.s.
Mineral N (mg L−1)	7.82 ± 0.95 a	7.81 ± 0.86 a	5.04 ± 0.34 b	8.36 ± 1.12 a	6.96 ± 0.65 ab	5.35 ± 0.40 b	**	**	n.s.
P2O5 (mg L−1)	29.19 ± 2.86 a	22.18 ± 1.91 ab	19.19 ± 3.03 b	28.74 ± 3.36	22.02 ± 2.27	19.80 ± 2.41	*	n.s.	n.s.
K2O (mg L−1)	35.06 ± 5.11	35.97 ± 3.66	26.49 ± 2.91	38.31 ± 3.88	31.68 ± 4.55	27.53 ± 3.41	n.s.	n.s.	*
Ca2+ (mg L−1)	48.98 ± 12.29 ab	64.50 ± 8.37 a	25.55 ± 5.59 b	62.28 ± 11.40 a	42.33 ± 11.26 ab	34.42 ± 6.34 b	**	*	n.s.
Mg2+ (mg L−1)	8.27 ± 1.93 ab	11.54 ± 1.55 a	4.29 ± 2.38 b	13.84 ± 3.91	4.38 ± 1.14	6.60 ± 1.93	**	n.s.	*

Results are presented as means ± standard errors. Lowercase letters represent statistically significant differences between mean values for each main factor p ≤ 0.05 obtained by two-way ANOVA and the Tukey HSD test. First-order interactions (R × T) are shown, with significance: n.s., not significant; **, p ≤ 0.01; *, p ≤ 0.05.

show rootstock’s significant influence on EC and the concentration of NO₃⁻, P₂O_5, Ca²⁺ and Mg²⁺, while treatment influenced the concentration of NH₄⁺ and Ca²⁺ and mineral N content in the substrate. Furthermore, the results showed significant interactions between factors on EC and K₂O and Mg concentrations.

According to factor interaction, it can be noted that EC in the substrate was the highest among maidens on MaxMa 14 in T₁ and significantly differed from values of the substrate on rootstock SL 64 in T₁ and on rootstock Gisela 5 in T₁ and T₂ (Figure 2).

3.1.1. Ammonia, Nitrate and Total Mineral Nitrogen Content

Watering the maidens with AZOS suspension every two months had a negative effect on the NH₄⁺ content in the substrate, while there was no visible rootstock effect. The highest concentration of NH₄⁺ was recorded in the substrate at control (5.32 mg L⁻¹) and was not significantly different from the concentration in T₁ (4.43 mg L⁻¹), but was significantly different from the concentration in T₂ (2.69 mg L⁻¹). Furthermore, NO₃⁻ content did not differ significantly according to the treatment, but there was a significant difference between the rootstocks, where SL 64 and MaxMa 14 showed a higher concentration compared to Gisela 5. Likewise, significant differences in total mineral N were recorded among both factors. The highest content of mineral N was recorded in the C, but it was not different from T₂ values. The lowest N content was recorded in T₂ and it differed from C. Compared to the initial values of NH₄⁺, NO₃⁻ and total mineral N content in the substrate, the values of all N forms decreased after eight months in all treatments.

3.1.2. Phosphorus, Potassium, Calcium and Magnesium Content

Although there was no significant difference in P₂O₅ content between the treatments, between rootstocks there were dereferences (Figure 3). According to the post hoc test, it can be observed that maidens grown on SL 64 had the highest P₂O₅ concentration while those on Gisela 5 had the lowest P₂O₅ concentrations.

K₂O content did not differ significantly between treatments and rootstocks, but the interaction of these two factors was statistically significant, from which it follows that the highest K₂O values were recorded in the C treatment on SL 64 and T₁ on MaxMa 14, while the significantly lowest value was recorded in T₁ on Gisela 5.

Ca content in the substrate was influenced by both factors separately. Ca²⁺ was the highest in C treatment (62.28 mg L⁻¹), and was significantly different from the T₂ (34.42 mg L⁻¹). Furthermore, the highest concentration (64.50 mg L⁻¹) was recorded in the substrate with maidens on MaxMa 14, which was statistically different from the lowest concentration recorded in the substrate with maidens on Gisela 5 (25.55 mg L⁻¹).

Mg content did differ between rootstocks, with noted interaction differences between factors (Figure 4). The highest concentrations of Mg (15.51 mg L⁻¹) were recorded in the substrate of maidens on MaxMa 14, with the lowest concentrations recorded in the substrate of maidens on Gisela 5. However, differences were noted in the interaction of treatments with substrates. Factor interaction did not show significant differences between treatments for each rootstock. Furthermore, the highest Mg concentrations were recorded in the substrate of rootstock SL 64 in C (13.84 mg L⁻¹) and rootstock MaxMa 14 in T₁ (15.51 mg L⁻¹), while the statistically lowest concentrations of Mg (3.26 mg L⁻¹) were recorded in the substrate of rootstock Gisela 5 in T₁.

3.2. Vegetative Characteristics

The results of the post hoc analysis of maidens’ vegetative characteristics (

Table 3. Mean values of maiden height, rootstock and shoot circumferences, and leaf CCI of cv. Lapins grafted on different rootstocks (SL 64, MaxMa 14 and Gisela 5) and watered with AZOS suspension at different doses and intervals (C, T₁ and T₂) during the 2022 growing season.

Parameter	Rootstock			Treatment			R	T	R × T
	SL 64	MaxMa 14	Gisela 5	C	T1	T2	p	p	p
Maiden height (cm)	75.42 ± 0.76 b	80.48 ± 0.50 a	72.64 ± 0.20 c	74.58 ± 0.32	79.90 ± 0.55	74.47 ± 0.21	***	n.s.	n.s.
Rootstock circumference (mm)	37.59 ± 0.78 a	34.56 ± 0.39 b	31.42 ± 0.32 c	34.15 ± 0.61	33.12 ± 0.52	34.62 ± 0.66	***	n.s.	n.s.
Shoot circumference (mm)	21.21 ± 0.49 a	22.22 ± 0.39 a	18.36 ± 0.49 b	19.78 ± 0.56	20.74 ± 0.52	20.89 ± 0.56	***	n.s.	**
CCI	23.60 ± 1.21 a	23.61 ± 1.34 a	19.69 ± 1.01 b	17.51 ± 0.87 b	26.98 ± 1.40 a	22.01 ± 0.68 b	**	***	n.s.

Results are presented as means ± standard errors. Lowercase letters represent statistically significant differences between mean values for each main factor p ≤ 0.05 obtained by two-way ANOVA and the Tukey HSD test. First-order interactions (R × T) are shown, with significance: n.s., not significant; ***, p ≤ 0.001; **, p ≤ 0.01.

) showed that there were significant differences in plant height and trunk circumference among different rootstocks. Furthermore, there were no significant differences in growth between the treatments.

The results in Table 3 show a strong influence of rootstock on all measured characteristics, while bacterial treatment influenced only chlorophyl content index values. There was also an interaction between these two factors for shoot circumference. Maidens on MaxMa 14 showed the highest height values, while maidens on SL 64 had the highest rootstock circumference. Both rootstocks did not differ in shoot circumference but their values were significantly higher than the values of rootstock Gisela 5. Nevertheless, the interaction of both factors on shoot circumference showed significant differences in Gisela rootstock between C and T₂ maidens (Figure 5).

Both factors showed a significant influence on the CCI. Monthly watering with rhizobacteria (T₁) significantly increased CCI values (26.98) compared to T₂ (22.01) and C (17.51). Furthermore, mean CCI values were statistically higher on leaves from maidens on MaxMa 14 and SL 64 compared to those on Gisela 5.

Growth Dynamics

The results in

Table 4. Mean values of maiden shoot length, number of internodes and internode length of cv. Lapins maidens grafted on different rootstocks (SL 64, MaxMa 14 and Gisela 5) and watered with AZOS suspension at different doses and intervals (C, T₁ and T₂) during different growing stages in 2022.

Stage	Parameter	Rootstock			Treatment			R	T	R × T
		SL 64	MaxMa 14	Gisela 5	C	T1	T2	p	p	p
BBCH 34	Shoot length (cm)	7.01 ± 1.28 b	13.00 ± 1.65 a	14.81 ± 1.69 a	9.21 ± 1.34 b	15.01 ± 1.93 a	13.24 ± 1.82 ab	**	*	n.s.
	Internode n°	2.13 ± 0.31 b	3.78 ± 0.41 a	4.05 ± 0.40 a	2.97 ± 0.34	3.97 ± 0.46	3.65 ± 0.45	**	n.s.	n.s.
	Internode length (cm)	2.91 ± 0.26	3.02 ± 0.19	3.22 ± 0.19	2.60 ± 0.20 b	3.22 ± 0.19 ab	3.49 ± 0.20 a	n.s.	*	n.s.
BBCH 39	Shoot length (cm)	60.50 ± 3.09	62.18 ± 2.90	59.22 ± 3.22	57.12 ± 2.85	66.02 ± 3.43	59.42 ± 3.05	n.s.	n.s.	n.s.
	Internode n°	17.54 ± 0.57 ab	18.59 ± 0.60 a	16.07 ± 0.66 b	17.27 ± 0.58	17.87 ± 0.83	16.91 ± 0.60	*	n.s.	n.s.
	Internode length (cm)	3.41 ± 0.10	3.29 ± 0.10	3.62 ± 0.09	3.25 ± 0.11 b	3.46 ± 0.09 ab	3.68 ± 0.06 a	n.s.	*	n.s.
BBCH 91	Shoot length (cm)	66.42 ± 2.65	68.91 ± 1.96	61.23 ± 2.81	63.26 ± 2.11	69.16 ± 2.92	63.82 ± 2.76	n.s.	n.s.	n.s.
	Internode n°	18.79 ± 0.60 a	20.32 ± 0.46 a	16.59 ± 0.54 b	18.73 ± 0.56	18.55 ± 0.68	18.09 ± 0.57	***	n.s.	n.s.
	Internode length (cm)	3.53 ± 0.08	3.40 ± 0.07	3.64 ± 0.09	3.39 ± 0.08 b	3.50 ± 0.08 ab	3.71 ± 0.08 a	n.s.	*	n.s.

Results are presented as means ± standard errors. Lowercase letters represent statistically significant differences between the mean values for each main factor p ≤ 0.05 obtained by two-way ANOVA and the Tukey HSD test. First-order interactions (R × T) are shown, with significance: n.s., not significant; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05.

show that both factors significantly influenced the shoot length at BBCH 34. Moreover, rootstock significantly influenced the internode number in all three stages while treatment influenced the mean internode length in all three stages. After the measurement at BBCH 34, there were no significant differences in the average shoot length between the rootstocks in the latter two growing stages (BBCH 39 and 91), and it is evident that shoot growth stopped in July. Furthermore, after BBCH 34 the average internode number began to change between the rootstocks, and in BBCH 39 the highest internode number was recorded in maidens on MaxMa 14 rootstock (18.59) and was significantly different from the internode number recorded in maidens on Gisela 5 rootstock (16.07). The same trend continued in BBCH 91, after which vegetative growth ceased.

3.3. Leaf Mineral Content

Table 5. Mean values of leaf dry matter content (LDMC) and mineral content of cv. Lapins maidens grafted on different cherry rootstocks (SL 64, MaxMa 14 and Gisela 5) watered with AZOS suspension at different doses and intervals (C, T₁ and T₂) during the 2022 growing season.

Parameter	Rootstock			Treatment			R	T	R × T
	SL 64	MaxMa 14	Gisela 5	C	T1	T2	p	p	p
LDMC (%)	40.57 ± 0.44	38.81 ± 1.58	40.32 ± 0.84	38.88 ± 1.46	40.50 ± 1.85	40.31 ± 1.90	n.s.	n.s.	n.s.
N	1.66 ± 0.07	1.77 ± 0.11	1.87 ± 0.05	1.57 ± 0.05 b	1.96 ± 0.07 a	1.77 ± 0.07 ab	n.s.	**	n.s.
P	0.21 ± 0.02	0.20 ± 0.01	0.21 ± 0.02	0.24 ± 0.05	0.20 ± 0.04	0.20 ± 0.04	n.s.	n.s.	*
K	1.66 ± 0.07 b	1.42 ± 0.06 b	2.01 ± 0.07 a	1.81 ± 0.09	1.65 ± 0.09	1.64 ± 0.13	***	n.s.	n.s.
Ca	0.87 ± 0.04 b	1.15 ± 0.05 a	0.93 ± 0.05 b	0.89 ± 0.05 b	1.01 ± 0.06 ab	1.05 ± 0.07 a	***	*	n.s.
Mg	0.47 ± 0.03 b	0.60 ± 0.04 a	0.32 ± 0.02 c	0.40 ± 0.05 b	0.50 ± 0.05 a	0.49 ± 0.05 ab	***	*	n.s.

Results are presented as means ± standard errors. Lowercase letters represent statistically significant differences between the mean values for each main factor p ≤ 0.05 obtained by two-way ANOVA and the Tukey HSD test. First-order interactions (R × T) are shown, with significance: n.s., not significant; ***, p ≤ 0.001; **, p ≤ 0.01; *, p ≤ 0.05. Mineral content is displayed as the percentage in dry matter.

shows the treatment influence on leaf N, Ca and Mg content and rootstock influence on leaf K, Ca and Mg content. There was also a significant factor interaction in leaf P content.

A significant difference in leaf N content was observed between C (1.57% DW) and T₁ (1.95% DW).

In the leaves of maidens on MaxMa 14 and Gisela 5, the P₂O₅ content did not change significantly according to the treatments. However, there were significant differences between T₁ and T₂ compared with C in maidens on SL 64, where the highest P content was recorded (0.28%) (Figure 6).

Furthermore, treatment with rhizobacteria did not significantly affect leaf K content; however, it was influenced by the rootstock. The highest K content was determined in maiden leaves on Gisela 5 (2.01% DW), while the leaves on the other two tested rootstocks had a similar content.

Compared to C treatment (0.89% DW), leaf Ca content increased significantly in maidens in T₂ (1.05% DW), while it did not differ significantly between C and T₁. Furthermore, significant differences were also noted between rootstocks. The highest leaf Ca content was noted in maidens on MaxMa 14 (1.15% DW), and the lowest content was noted in maidens on SL 64 (0.87% DW).

The obtained results showed that both rootstock and watering with rhizobacteria significantly influenced leaf Mg content, which in T₁ was 0.50% DW, and was significantly different from the C treatment (0.40% ST). However, there were no statistical differences between the deferent inoculation intervals. It was also determined that the highest value was recorded in maiden leaves on MaxMa 14 (0.60% DW), and the lowest value was recorded in maidens on Gisela 5 (0.32% DW).

4. Discussion

The rhizosphere is a complex soil zone both affected by and affecting roots, and there is growing evidence of the altered characteristics of it, including changes in the pH, and the release of substances that promote the growth of beneficial microorganisms and enhance nutrient availability [77,78]. The pH value as an indicator of substrate fertility can determine nutrient availability for the plant [79]. Values of the substrate pH of all plants did not differ significantly between rootstocks and treatments and all pH values in this experiment were slightly below the optimum value of 6.5 recommended for cherry orchard soils [80].

Preliminary macronutrients analysis of the substrate used in this study has showed that it has a lower nutrient content adequate only for initial seedling growth, while the content of N in the form of NO₃⁻ is deficient compared to the optimal levels in the soil according to the literature [81]. All observations could be related to possible denitrification occurring in peat substrates [82]. Neilsen and Kappel [83] proposed the possibility that inadequate initial soil nutrition could cause weaker growth of cherry trees. Furthermore, N as the main component in plant metabolism is usually a growth-limiting factor in unfavorable conditions [84].

In the results of this study, a lower EC and concentrations of NO₃⁻, P₂O₅, Ca and Mg ions with a lower mineral N content were found in cherry maidens on Gisela 5 rootstock. It can be assumed that the reason for the lower nutrient content in the substrate of Gisela 5 trees is the increased nutrient uptake. Furthermore, final substrate analyses revealed that its characteristics include low levels of the macronutrients N, P and K, and elevated levels of Ca and Mg, which could be related to the quality of irrigation water, containing high concentrations of these minerals in the Zadar County watershed [85]. Therefore, substrate quality is not optimized for long-term cherry seedling production; however, it could be used for easier detection of microbial influence. On the other hand, substrate producers could put in the substrate viable spores of beneficial microbes that would infect seedling roots.

On the other hand, the significantly lowest values of NH₄⁺, Ca²⁺ and mineral N content were found in trees watered with AZOS every two months (T₂). Gallart et al. [86] have reported that substrate inoculation with rhizobacteria did not affect the N uptake into macadamia seedlings [86]. In the same research, the presence of rhizobacteria increased the leaching of NH₄⁺ from the substrate, which could also explain the case of lower concentrations in T₂. The presence of microorganisms in the rhizosphere can also stimulate the release of root exudates and influence the activities that support the uptake of nutrients from the soil solution [87,88].

The results of maiden tree height and rootstock circumference showed that there were statistical differences between rootstocks, while there were no differences in shoot circumferences of cv. Lapins between rootstocks SL 64 and MaxMa 14, with maiden trees having a higher shoot circumference compared to those on rootstock Gisela 5. These results agree with the results of other studies [89,90]. However, in those studies trees have been cultivated directly in the soil. It is known that growing maiden trees in a container limits root growth, and consequently slows down the growth of the aerial part of the plant [91] and ultimately affects its overall development [92]. Akova et al. [93] recommend containers with a volume of 10 L for the cultivation of grafted sweet cherry seedlings, which is somewhat consistent with the container volume of 12 L used in this study.

Fruit tree characteristics such as height and diameter can be influenced by the vigor of certain rootstocks and cultivated varieties [94], the type of soil and its quality and the application of growth-promoting microorganisms [54]. De Salvador et al. [23] described the effect of different rootstocks on cv. Lapins at three different growing locations. In another study, there were no significant differences in trunk width between SL 64 and MaxMa 14 rootstocks [89]. Moreover, Aglar and Yıldız [90] determined that in the years after planting were no differences in cv. 0090 Ziraat tree height between trees grafted on SL 64 and MaxMa 14 rootstocks. The same authors reported that in the first four growing seasons the rootstock and cultivar trunk width including shoot length did not significantly differentiate between trees grafted on MaxMa 14 and Gisela 5 rootstocks [90]. In the research of Domozetova and Radomirska [95] different varieties were cultivated on the same rootstocks under different conditions and sweet cherry trees on MaxMa 14 showed a greater vigor compared to trees on Gisela 5, agreeing with the results of this study. Due to tree vigor variability, Lang [96] recommends to test rootstocks for their specific growing area. According to the previous literature, Gisela 5 belongs to dwarf rootstocks [3,7,18], decreasing the tree diameter and its overall vegetative growth, which ultimately reduces tree vigor [14]. Overall, in this study Gisela 5 shoot circumference was smaller in maiden trees from C and T₁ treatment, and when compared to maidens of MaxMa 14 in the same treatments those differences were significant. Nevertheless, maiden trees grafted on Gisela 5 in the control treatment had a significantly lower shoot circumference than trees in T₂. According to the literature, certain rhizobacteria can affect plant physiological processes, such as increasing the efficiency of photosynthesis and increasing the production of carbohydrates, which in turn promotes vegetative growth [97]. There are several literature examples of the effective use of rhizobacteria in fruit growing. For example, De Silva et al. [98] reported that the application of Pseudomonas fluorescens increased the leaf area and stem diameter of highbush blueberry (Vaccinium corymbosum L.). Likewise, Orhan et al. [99] reported the increased growth and development of raspberry (Rubus spp.) seedlings fertilized with higher concentrations of macronutrients or inoculated with rhizobacteria. Some authors also investigated the use of A. brasilense in fruit species, and confirmed its influence on strawberries [38,40] and on Prunus spp. rootstocks GF 677 and Mr.S 2/5 [57,63]. On strawberries, the application of rhizobacteria improved the vegetative growth and yield, while on rooted rootstock cuttings influenced the stem length, the number of nodes and the root growth.

The action mechanisms in plants provided by microbial biofertilizers are not completely understood yet; however, PGPR can upregulate the expression of genes related to cell expansion and shoot growth, photosynthesis and biomass production, improving chlorophyll and phytohormone levels in stressful environments [100].

These findings are in accordance with the results of the initial shoot growth and internode length of treated maidens in this study. Shoot growth dynamics showed that maiden trees responded better in the first three inoculation periods when conditions of field temperatures and humidity were more suitable for the growth of the bacterial population. However, during hot summer days the population was possibly endangered by extremely high temperatures and plant growth was in halt. In order to obtain healthier plants that will meet stressful periods more prepared, nursery growers need to correctly use biofertilizers before and after. PGPR will need to be reinoculated to restore their population in the rhizosphere.

Leaves can reflect plant health in different environmental conditions. Leaf chlorophyll content is an important indicator of leaf development and is often used to test the lack of leaf nutrients and changes in chlorophyll [101]. Both rootstock and rhizobacteria significantly affected the chlorophyll content index in leaves. This effect could be attributed to the influence of a higher amount of N in chlorophyll synthesis [102]. Authors El-Naby et al. [103] found that application of plant growth regulators increases CCI values in apricot leaves, and Rueda et al. [43], combining Azospirillum spp. with deficit N concentrations, reported elevated values of chlorophyll content in strawberry leaves. Sweet cherry maidens inoculated with A. brasilense showed significantly higher chlorophyll values compared to the control. CCI values were significantly lower in leaves on Gisela 5 compared to the other rootstocks. The ability of bacteria to produce phytohormones auxin and gibberellin affects the growth of shoots, and by increasing the total leaf area photosynthetic activity also increases. In general, chlorophyll concentration in sweet cherry leaves sampled in July was not generally high, which was influenced by the lower initial concentration of N in the substrate. The relationship between the field chlorophyll reading and leaf N content is also influenced by environmental factors, leaf characteristics and plant species [102]. Moreno et al. [104] found that sweet cherry trees cultivated on SL 64 showed a lower concentration of leaf chlorophyll compared to other rootstocks, which is not in accordance with the results of this study in which limiting the volume of the substrate for root growth could affect nutrient redistribution to upper parts. Jiménez et al. [105], comparing the concentration of chlorophyll in trees on different rootstocks, found no significant differences in chlorophyll concentration between the varieties, which agrees with the results of SL 64 and MaxMa 14. However, Pérez et al. [106] reported that the same variety grown on different rootstocks showed different photosynthetic activity, which is in accordance with the results of the CCI from maiden tree leaves on Gisela 5 rootstock. These results of the CCI helped to provide an initial insight into photosynthetic activity in sweet cherry leaves. Additional testing of chlorophyll fluorescence in future research would more precisely determine the impact of rhizobacteria on photosynthetic activity under the stressful conditions of pot-grown plants. Improving substrate characteristics by adding biofertilizers will possibly maintain or increase growth of nursery crops, helping them to extend the period of durability in pots.

According to the results of leaf mineral analysis, rootstock had significantly influenced leaf K, Ca and Mg content while bacterial treatment significantly influenced leaf N, Ca and Mg content (Table 5). At the same time, leaf P content was affected by the interaction of those two factors. Maidens on Gisela 5 rootstock had the significantly lowest Mg content, which is in accordance with the results of previous research [105]. On the other hand, Gisela 5 maidens had the significantly highest K content when compared to the other two rootstocks. Also, an antagonism between K and Mg is observed on MaxMa 14 and Gisela 5 rootstocks. Furthermore, leaf Ca content was significantly higher on MaxMa 14 compared to the other two rootstocks. In previous studies, the leaf N content in sweet cherry maidens was higher than the results of this study, while the content of other macronutrients has varied, which shows that besides rootstock and inoculation, pedoclimatic conditions, genotype and growing season could also play an important role in the leaf mineral composition of sweet cherry maidens [107,108,109]. The leaf N content in maiden trees on SL 64 rootstock was deficient, while on the other two rootstocks the values were below normal. At the same time, the control trees had deficient N levels, while both treated groups had N levels below normal for cherry growth [110]. Leaf K content in MaxMa 14 rootstock was below the normal reference values, while all rootstocks have showed Ca levels below normal [110]. In certain rootstocks such as Colt and SL 64, higher leaf Ca levels can occur [104,105], which was not the case in this study. Furthermore, Ca deficiency can be triggered by unfavorable growing conditions, e.g., a drought, which affect the reduced nutrient movement in soil suspension [111]. However, an addition of rhizobacteria in T₂ significantly improved leaf Ca levels. These are also possible occurrences of Mg deficit in leaves on different cherry rootstocks [109,112], which is not the case in our study. In previous studies, it was determined that the chlorophyll concentration in cherry and peach leaves was negatively correlated with Ca [89,113], which coincides with the data of this study. In the leaves of treated plants, the increase in N content resulted in an increase in Mg content. The influence of N on the increase in leaf Mg content was also found in other studies [112,114] but the difference between plants in the control and those fertilized with N was not always significant. Moreover, Jiménez et al. [105] reported a positive correlation between the concentration of chlorophyll and the N content, which also agrees with the results of this study. It has been previously established that rhizobacterial activity undergoes complex processes in the soil and depends on bacterial strains and populations, the combination of bacterial strains and plant genotype and above all environmental conditions [115,116]. Thus, in apples inoculation contributed to an increase in vegetative growth and mineral content, but according to the authors this strongly depended on the tested varieties and bacterial strains [35].

In general, rootstock affects the nutrient concentration in leaves and shoots [4]. According to Hrotkó [117], rootstock is responsible for the absorption of mineral substances, and has the ability to selectively absorb and transport them into the plant, which results in different concentrations reaching the leaves. Leaves of cv. Lapins budded on Gisela 5 rootstock showed the best nutritional balance, as in the work of Hrotkó [117]. Jiménez et al. [105] reported that dwarfing cherry rootstocks show a higher uptake sensitivity when exposed to limited soil conditions. In contrast, vigorous rootstock SL 64 showed concentrations of individual mineral nutrients in leaves below normal levels, most likely as a result of poor adaptation to unfavorable soil conditions [117]. The beneficial rhizobacteria can be used to alleviate this issue and achieve sustainable agriculture by supporting crop and soil health in a number of ways, for instance to support the use of lower concentrations of mineral N.

Inoculation treatments had a positive influence on leaf N, Ca and Mg content while the interaction of both factors affected the content of leaf P. Leaf N, Ca and Mg content were significantly increased in cv. Lapins maidens treated with rhizobacteria.

These data are in accordance with research conducted on apples [118]. On the contrary, in the study of Arikan and Pirlak [47], concentrations of N, K and Mg in sour cherry leaves were not significantly changed after the application of Bacillus spp. Leaf P content was significantly lower in both treatments when compared to the control, but only in maidens on SL 64 rootstock. These results could indicate a possible effect of microbial phosphorus immobilization; however, it is unclear why the differences are visible only in the case of one rootstock [119,120].

In general, N in the plant reaches highest levels during flowering and fruit growth, while the lowest level occurs after harvest [121]. Leaf Ca content was significantly lower in C treatment compared to T₂, while Mg content was highest in T₁. P, K and Mg content in all treatments was at optimal levels; only Ca in all treatments was at an insufficient level according to reference values for sweet cherry leaves [110]. Many previous studies have determined the influence of PGPR on foliar plant nutrition [33,35,46,49,99,118,122]. Eşitken et al. [49] have found that the application of bacteria affects N, P, K, Ca and Mg content in apricot leaves while Shirkot and Sharma [33] have found the same effect on N, P and K content in apple leaves. Furthermore, Eşitken et al. [46] found that rhizobacteria treatments increased N, P and K content in cherry leaves, while Ca and Mg content did not increase. The lack of Ca in the leaf can be explained by its very low level of mobility in the plant, while the lack of Mg is generally caused by antagonism with macronutrients such as Ca and K [123]. However, higher Ca leaf levels in treated maidens could indicate a plant’s better resistance to heat and drought stresses, as previously noted [124,125].

The results of this study confirmed the different vegetative activity between the rootstocks, which was expected. In general, rootstock had a strong influence on maiden tree growth in height and width. By measuring the CCI, the different photosynthetic activity among rootstocks was also determined. Furthermore, the presence of A. brasilense in the substrate affected initial shoot growth regardless of the fact that no significant difference was recorded in late season growth. Also, the effect of rhizobacteria was recorded in the content of NH₄⁺, Ca and mineral N in the substrate, the average internode length, the CCI and leaf N, Ca and Mg content. These changes could be indicators that A. brasilense affected the growth of maiden trees.

The interaction effect of rootstock and treatment was determined for EC, as well as the K₂O and Mg content in the substrate, shoot circumference and leaf P content. It can be assumed that, in addition to rootstock type and the presence of rhizobacteria, different mineral compositions in the substrate and leaves could have been influenced by other factors that were not measured, such as water quality, type of substrate, organic matter content in the substrate, microbiological activity in the substrate during the growing season and nutrient seasonal movement.

5. Conclusions

Rootstock significantly influenced EC, concentrations of NO₃⁻, P₂O₅, Ca and Mg and mineral N content in the substrate. Also, there were significant differences in the height and circumference, shoot length at the BBCH 34 stage and the internode number at all measuring stages, including leaf CCI. The results of leaf mineral composition showed significant differences in K, Ca and Mg content.

Furthermore, the results showed the influence of rhizobacteria on NH₄⁺ and Ca content and the amount of mineral N in the substrate, on the CCI, on the average internode length and on leaf N, Ca and Mg content.

The rootstock and treatment interaction were determined for EC, as well as the concentrations of K₂O and Mg in the substrate, shoot circumference and P content in the leaves.

This study confirmed that use of microbiological products such as inoculant in nursery production conditions has a conceptual and targeted need for application in sustainable agricultural production. However, it is challenging to predict the response of a microorganism in in vivo conditions. This concept is not viable if it is not implemented in accordance with the rules of microbial biotechnology as well as in accordance with the climatic features where this study was conducted. Microclimatic features on which the experiment was set up provide relevant data for future scientific research.