Grapefruit Extracts and Black Chokeberry Juice as Potential Antioxidant and Antifungal Agents for Carrot Seed Treatment

Abstract

Grapefruit extracts and black chokeberry juice have antioxidant and antimicrobial properties. The aim of the experiment was to evaluate the effect of grapefruit-based preparations, Biosept Active and Citrogrep, and black chokeberry juice on the germination, vigor, and health of carrot seeds. The seeds of two cultivars were soaked for 30 min in 0.25% grapefruit-based preparations and in 5 and 25% chokeberry juice. Standard ISTA methods were used to evaluate seed germination and health. The chemical composition of the applied preparations and juice was analyzed with ultra-high-performance liquid chromatography electrospray ionization mass spectrometry. The grapefruit-based preparations differed significantly in their chemical composition, qualitatively and quantitatively, but in both of them, flavanones and flavones prevailed. Biosept Active improved seed germination especially in the cultivar, which was characterized by a lower initial seed quality. The significant positive correlations between germination at the first and final counts, as well as the contents of flavanones and flavones, were identified in this cultivar. Moreover, the negative correlations between the percentages of diseased seedlings, dead seeds, the percentages of seed infested with Cladosporium spp., Epicoccum nigrum, Melanospora simplex, and Ulocladium spp., and the investigated compounds were found. Chokeberry juice, at the higher concentration, showed some antioxidant activity; however, it frequently stimulated the growth of the fungi.

1. Introduction

In modern carrot production, the farmers apply precise sowing and pay a lot of attention to seed quality, which depends on several factors, such as the seed development on the mother plant, climatic conditions, cultivation methods, harvesting and cleaning, as well as seed storage. Numerous abiotic stresses, occurring during seed production, processing, and storage, cause the deterioration of seed germination and vigor. They lead to the overproduction of reactive oxygen species (ROS), including free radicals (superoxide radicles, O₂^•−; hydroxyl radical, OH^•; perhydroxyradical, HO₂^•; alkoxy radicals, RO^•) and non-radical forms (hydrogen peroxide, H₂O₂; singlet oxygen, ¹O₂) in plants. Highly reactive and toxic ROS lead to the oxidation of essential molecules such as proteins, lipids, carbohydrates, and DNA, causing oxidative stress. Deteriorative processes lead to the destruction of cell membranes, enzyme inactivation, genome damage, and mitochondrial dysfunction [1,2].

Antioxidants are organic and inorganic compounds that effectively protect the cell membrane against oxidative damage, even if they are applied at low concentrations. They prevent the oxidative deterioration of lipids and maintain the structural and functional integrity of cells [1]. It has been proven that treating seeds with antioxidants may enhance their quality. Different antioxidants have been used for seed invigoration. Phenolic compounds, well known for their antioxidant properties, are produced by many plants [3]. Grapefruit [Citrus paradisi Macf., Rutaceae] and black chokeberry [Aronia melanocarpa (Michx., Rosaceae) Elliott] have been identified as a rich source of natural antioxidants. Citrus paradisi fruits contain many bioactive compounds, such as vitamins C, E, and A; phenolic compounds (flavanones, flavones, phenolic acids, and coumarins); and terpenic substances, including carotenoids and limonoids [4,5]. Aronia melanocarpa berries contain minerals, vitamins, carbohydrates, amino acids, organic acids, fats, aroma compounds, and a very high content of polyphenols, namely phenolic acids, proanthocyanins, anthocyanins, flavonols, and flavanones [6,7]. In addition to their antioxidant potential, the metabolites of grapefruit extract and black chokeberry juice also have been shown to have antibacterial and antifungal properties.

Infestation with seed-transmitted pathogenic fungi Alternaria dauci (J.G. Kühn) J.W. Groves & Skolko (Ascomycota, Pleosporaceae) and A. radicina Meier, Drechsler & E.D. Eddy (Ascomycota, Pleosporaceae), as well as Alternaria alternata (Fr.) Keissler (Ascomycota, Pleosporaceae), considered to be a weak pathogen of the carrot, is one of the main reasons for a low seed quality [8,9,10]. These fungi are causing severe carrot diseases (Alternaria blight, black root rot, damping off) and secrete mycotoxins, which can accelerate and deepen the process of seed deterioration [11,12]. The antimicrobial properties of grapefruit extract have been shown by many researchers, and numerous grapefruit-based preparations have been tested [13,14,15,16,17,18,19]. Treatment with a Biosept 33 SL preparation (a.i.33% grapefruit seed and pulp extract) decreased cabbage seed infestation in Alternaria alternata and Alternaria brassicicola, (Schwein.) Wiltshire (Ascomycota, Pleosporaceae) and Fusarium spp. (Ascomycota, Nectriaceae) reduced the presence of Alternaria zinniae M.B.Ellis (Ascomycota, Pleosporaceae) on zinnia seeds and the incidence of Botrytis allii Munn (Ascomycota, Sclerotiniaceae), B. cinerea Pers. (Ascomycota, Sclerotiniaceae), and Fusarium spp. on onion seeds. This treatment also improved the germination of cabbage and onion seeds at 10 °C and accelerated cabbage seed germination at 10 and 20 °C [13]. Biosept 33 SL reduced the in vitro growth of the bacteria Xanthomonas campestris pv. campestris (Pammel) Dowson (Proteobacteria, Xanthomonadaceae) and Clavibacter michiganensis subsp. michiganensis (Smith) Davis et al. (Actinobacteriota, Microbacteriaceae), as well as the fungi Alternaria dauci and Botrytis allii, isolated, respectively, from cabbage, tomato, carrot, and onion seeds, and eliminated more than 99% of the bacteria and most of the fungi from cabbage seeds [14]. The preparation limited the incidence of pathogenic Bipolaris sorokiniana Shoemaker (Ascomycota, Pleosporaceae), Drechslera teres (Sacc.) Shoemaker (Ascomycota, Pleosporaceae), and Fusarium spp. on barley grains [15]. Szopińska et al. [16] found that Biosept 33 SL improved carrot seed germination and decreased the occurrence of Alternaria radicina. The application of other preparations, like Citrosept (a.i.80% grapefruit extract) or Grevit 200 SL (a.i. 20% grapefruit extract), also frequently positively affected plants’ health and development [17,18,19]. There is no information in the literature about the effects of black chokeberry juice on seed quality; however, the antimicrobial potential of the extracts from A. melanocarpa fruits in relation to human pathogenic bacteria has been proven by some authors. Liepina et al. [20] and Kim et al. [21] observed that black chokeberry ethanol extracts inhibited the growth of the Gram-positive bacteria Bacillus cereus Frankland & Frankland (Firmicutes, Bacillaceae) and Staphylococcus aureus Rosenbach (Firmicutes, Staphylococcaceae). Valcheva-Kuzmanova and Belcheva [22] reported the in vitro bacteriostatic activity of black chokeberry fruit juice against Staphylococcus aureus and Escherichia coli (Migula) Castellani & Chalmers (Proteobacteria, Enterobacteriaceae).

The aim of the performed studies was to evaluate the effects of grapefruit-extract-based preparations (Biosept Active and Citrogrep) and black chokeberry juice on the germination, vigor and health of carrot seeds and to determine the correlations between the contents of active compounds detected in these preparations and black chokeberry juice and carrot seed quality parameters.

2. Materials and Methods

Two standard, commercially available, carrot seed samples cv. Berlikumer and cv. Karlena obtained, respectively, from the Seed Company TORSEED S.A. in Toruń, Poland, and the W. Legutko Company in Jutrosin, Poland, were used in the study. The samples differing in the initial seed quality (germination, vigor and infestation with fungi) were selected for the experiment. Preparations Biosept Active (a.i. 33% extract from grapefruit pulp and seeds; Cintamani Poland; Piaseczno, Poland) and Citrogrep (a.i. 33% grapefruit seed extract; Herb-pharma AG Schweiz, Zürich, Switzerland) and black chokeberry juice (Polska Róża, Poland) were applied for seed treatment. The seeds were soaked for 30 min in 0.25% aqueous solutions of Biosept Active and Citrogrep, and in chokeberry juice solutions at the concentrations of 5 and 25%. After treatments, the seeds were dried at 20 °C and 45% relative humidity for 48 h. The controls were the untreated seeds and the seeds treated with the fungicide Zaprawa Nasienna T 75 WS/DS (a.i. 75% thiram, produced by Zakłady Chemiczne Organica-Azot S.A., Jaworzno, Poland) at a dose 5 g kg⁻¹ of seeds.

Germination, vigor and health tests were performed for each combination. The seed germination was evaluated according to the ISTA Rules [23]. In each treatment, 300 seeds (6 replicates of 50) were assessed. The seeds were placed in 9 cm diameter Petri dishes on six layers of blotter paper moistened with distilled water, 50 seeds per dish, and incubated at 20 °C in darkness. Germination at the first count was evaluated after 7 days, whereas germination at the final count (the percentage of normal seedlings) and the percentages of abnormal diseased and deformed seedlings, as well as dead and fresh seeds, were determined after 14 days. Additionally, the total number of germinating seeds (G_max) was evaluated on the basis of a vigor test.

Six replicates of 50 seeds were used also to determine seed vigor. The seeds were incubated for 14 days in the same way as in the germination test. The seeds with a visible radicle (1–2 mm long) were counted daily and removed from Petri dishes. The SeedCalculator software (ver. 1.2) [24] was used for analyzing vigor parameters, i.e., T₁ (time to 1% of G_max), T₇₅ (time to 75% of G_max), MGT (mean gemination time) and U_75-25 (time from 25 to 75% of G_max).

The deep-freeze blotter test was applied to evaluate seed health according to the ISTA Rules [25,26]. Five replicates of 40 seeds were tested for each treatment. The seeds were placed on blotters soaked with distilled water in 9 cm diameter Petri dishes (20 seeds per plate). The seeds were kept at 20 °C in darkness for 24 h and then frozen at −20 °C for 24 h and incubated at 20 °C for eight days under an alternating cycle of NUV light and darkness (12 h of light and 12 h of darkness). After incubation, the fungi appearing on the seeds were identified on the basis of their growth and sporulation using a stereomicroscope and a compound microscope if necessary [27,28]. Additionally, the percentage of seeds free of fungi was calculated.

To establish the chemical composition of grapefruit extracts and chokeberry juice, reversed-phase (C18 column) ultra-high-performance liquid chromatography electrospray ionization mass spectrometry (RP-UHPLC-ESI-MS) analysis was applied. The analysis was performed using the Dionex UltiMate 3000 UHPLC (Thermo Fisher Scientific, Sunnyvale, CA, USA) coupled to the Bruker maXis impact ultra-high-resolution orthogonal quadrupole-time-of-flight accelerator (qTOF) equipped with an ESI source and operated in positive-ion mode (Bruker Daltonik, Bremen, Germany). The RP chromatographic separation was achieved with the Kinetex™ 1.7 µm C18 100 Å, LC column 100 × 2.1 mm (Phenomenex, Torrance, CA, USA). The ESI-MS settings were previously described by Mildner-Szkudlarz et al. [29]. The mobile phase was composed of water containing 1% formic acid (A) and acetonitrile containing 5% water and 1% formic acid (B). The flow rate was 0.2 mL/min with a gradient elution of 5%–95% B over 35 min and standing at 95% B for 20 min. The sample injection volume was 2 µL. The column temperature was set at 40 °C. The ESI-MS system was calibrated using sodium formate clusters [30] introduced by loop injection at the beginning of the LC-MS run. The LC-MS data were processed using Data Analysis 4.1 software (Bruker Daltonik, Bremen, Germany). Molecular ions [M+H]⁺ were extracted from full-scan chromatograms, and peak areas were integrated. The extraction window of individual ion chromatograms was ±0.005 m/z units. The compounds present in each sample were identified by comparing their retention times with those of standards and based on molecular mass and structural information from the MS detector. The limit of quantification (LOQ where S/N > 15) was determined for apigenin, caffeic acid, chlorogenic acid, p-coumaric acid, sinapic acid, quercetin, ferulic acid, and kaempferol, and it was not lower than 0.01 µg/mL.

The obtained results were compared using the ANOVA analysis for the two-factor experiments in 5 or 6 replications in a completely randomized design. The averages were compared using the Newman–Keuls test at a significance level of α = 0.05. The percentages before statistical analysis were converted to Bliss degrees (y = arcsin) [31]. Parameters describing the rate and uniformity of seed germination were calculated using the SeedCalculator software (ver. 2.1) [24].

The principal component analysis (PCA) [32] and the Spearman’s rank correlation coefficient [33] were calculated using RStudio 2023.09.1+494 by Posit Software, PBC and R software (ver. 4.3.2) by the R Foundation for Statistical Computing. PCA analysis was performed using the FactoMineR library ver 2.6, while visualizations including variable contributions to the principal axes and color individuals by groups were performed in the Factoextra library ver. 1.0.7. The correlation analysis between the contents of active compounds in Biosept Active, Citrogrep, black chokeberry juice and the carrot seed quality parameters was performed.

The actual content of the active compounds used was calculated on the basis of the results of the chemical composition analysis and the concentration of the preparation in each combination. This calculated content was assigned to each treatment with the tested preparation, and zero values for the active compound content were assigned to the control. Data from five replicates (n = 25; 5 treatments × 5 replicates) with the exception of the fungicide treatment were used to calculate Spearman’s correlation coefficients. The same data were also used for the ANOVA analysis.

3. Results

The samples varied significantly in seed quality. Cultivar Berlikumer seeds were characterized by much lower germination at the first and final counts, the inferior vigor, and the higher infestation with fungi than cv. Karlena (

Table 1. The effect of the grapefruit-based preparations (Biosept Active, Citrogrep) and black chokeberry juice application on carrot seed germination.

Germination Parameter (%)	Cultivar	Treatment
		Con **		F		B		C		A5		A25
Gmax *	Berlikumer	85.0	a ***	88.0	a	82.0	a	84.0	a	86.7	a	87.3	a
	Karlena	85.7	a	81.7	a	82.0	a	81.3	a	84.7	a	82.0	a
Germination —1st count	Berlikumer	51.3	ab	56.3	a–c	66.3	cd	61.7	b–d	47.7	a	56.0	a–c
	Karlena	67.0	cd	67.0	cd	70.7	d	68.3	cd	61.7	b–d	71.7	d
Germination —final count	Berlikumer	55.7	ab	71.0	c–e	70.0	c–e	64.3	b–d	49.0	a	62.0	bc
	Karlena	72.3	c–e	79.3	e	73.3	c–e	73.3	c–e	65.3	b–d	75.3	de
Diseased seedlings	Berlikumer	27.0	e	9.3	ab	14.0	b–d	19.7	c–e	30.7	e	22.0	de
	Karlena	11.3	a–c	5.3	a	7.7	ab	8.7	ab	13.0	b–d	6.7	ab
Deformed seedlings	Berlikumer	1.7	a	3.7	a	1.0	a	2.3	a	1.3	a	1.0	a
	Karlena	1.0	a	0.3	a	0.3	a	0.7	a	2.0	a	0.3	a
Dead seeds	Berlikumer	15.0	d	6.3	c	12.0	d	12.0	d	17.0	d	14.7	d
	Karlena	5.3	c	0.3	a	1.7	ab	4.0	bc	2.7	bc	3.7	bc
Fresh seeds	Berlikumer	0.7	a	9.7	b	3.0	a	1.7	a	0.3	a	0.3	a
	Karlena	10.0	b	14.7	b	17.0	b	13.7	b	17.0	b	14.0	b

* G_max—the total percentage of germinating seeds. ** Con—control, untreated seeds; F—seeds treated with the fungicide Zaprawa Nasienna T 75 WS/DS at a dose 5 g kg⁻¹ of seeds; B—seeds treated with Biosept Active at the concentration of 0.25%; C—seeds treated with Citrogrep at the concentration of 0.25%; A5, A25—seeds treated with black chokeberry (Aronia melanocarpa) juice at the concentrations 5 and 25%, respectively. *** Values marked with the same letter are not statistically different according to the Newman–Keuls test at the level α = 0.05.

,

Table 2. The effect of the grapefruit-based preparations (Biosept Active, Citrogrep) and black chokeberry juice application on carrot seed vigor.

Vigor Parameter (Days)	Cultivar	Treatment
		Con		F		B		C		A5		A25
T1 *	Berlikumer	1.41	a	1.62	a–c	1.76	bc	1.71	bc	1.93	c	1.83	bc
	Karlena	1.54	ab	1.74	bc	1.76	bc	1.82	bc	1.87	bc	1.79	bc
T75	Berlikumer	3.57	cd	4.73	e	3.83	d	3.67	d	3.50	cd	3.60	cd
	Karlena	2.73	a	3.68	d	3.04	ab	3.20	bc	2.93	ab	2.89	ab
MGT	Berlikumer	3.16	cd	4.07	e	3.45	d	3.28	d	3.32	d	3.47	d
	Karlena	2.52	a	3.35	d	2.84	b	2.93	bc	2.84	b	2.79	b
U75-25	Berlikumer	1.31	b	1.80	c	1.23	b	1.10	b	1.00	ab	1.23	b
	Karlena	0.67	a	1.18	b	0.76	a	0.76	a	0.68	a	0.68	a

* T₁—time to 1% of the total percentage of germinating seeds (G_max); T75—time to 75% of G_max; MGT—mean germination time; U_75-25—time from 25 to 75% of G_max. For further explanations, see Table 1. Values marked with the same letter are not statistically different according to the Newman–Keuls test at the level α = 0.05.

and

Table 3. The effect of the grapefruit-based preparations (Biosept Active and Citrogrep) and black chokeberry juice application on carrot seed health.

Infestation with (%)	Cultivar	Treatment
		Con		F		B		C		A5		A25
Alternaria alternata	Berlikumer	99.0	e	41.5	d	94.5	e	96.5	e	99.0	e	97.0	e
	Karlena	17.0	bc	0.0	a	11.5	b	16.0	bc	25.0	c	18.0	bc
Alternaria dauci	Berlikumer	3.5	c	3.5	c	0.0	a	2.0	bc	0.5	a	1.0	ab
	Karlena	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a
Alternaria radicina	Berlikumer	6.0	d	2.0	bc	2.0	a–c	2.5	bc	2.5	bc	4.5	cd
	Karlena	0.5	ab	0.0	a	1.0	ab	0.5	ab	0.0	a	0.0	a
Cladosporium spp.	Berlikumer	1.5	a	1.5	a	0.0	a	1.5	a	8.5	b	11.5	b
	Karlena	2.0	a	0.0	a	0.5	a	2.5	a	1.5	a	2.5	a
Epicoccum nigrum	Berlikumer	6.5	b	0.5	a	1.0	a	1.5	a	9.5	bc	14.5	c
	Karlena	0.0	a	0.0	a	0.0	a	2.5	a	0.5	a	0.5	a
Fusarium spp.	Berlikumer	4.0	bc	0.5	a	13.5	d	8.5	cd	10.5	d	4.0	b
	Karlena	2.0	ab	0.0	a	0.0	a	0.0	a	1.0	a	0.5	a
Melanospora simplex	Berlikumer	3.0	b	0.0	a	2.0	ab	0.5	a	6.5	c	1.5	ab
	Karlena	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a
Penicillium spp.	Berlikumer	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a	1.0	b
	Karlena	0.0	a	0.0	a	0.0	a	0.0	a	1.5	b	3.5	c
Phoma sp.	Berlikumer	0.0	a	0.0	a	0.0	a	0.0	a	0.0	a	1.0	a
	Karlena	0.0	a	0.0	a	0.0	a	0.0	a	16.0	b	0.0	a
Stemphylium spp.	Berlikumer	3.0	bc	1.0	ab	1.0	ab	6.5	c	5.0	bc	4.0	bc
	Karlena	0.0	a	0.0	a	0.0	a	0.0	a	0.5	ab	0.5	ab
Ulocladium spp.	Berlikumer	44.0	cd	2.5	b	35.5	c	40.5	cd	48.0	de	56.5	e
	Karlena	0.0	a	0.0	a	0.0	a	1.0	ab	0.5	ab	0.0	a
Non-sporulating fungi	Berlikumer	10.0	cd	5.5	cd	1.5	ab	5.0	cd	3.5	bc	3.5	bc
	Karlena	22.0	e	0.0	a	7.5	cd	13.0	d	12.0	d	3.0	bc
Seeds free of fungi	Berlikumer	0.5	a	48.5	b	0.5	a	1.0	a	0.0	a	1.5	a
	Karlena	55.5	b	100.0	e	78.5	d	66.0	c	46.0	b	71.0	c

For explanations, see Table 1. Values marked with the same letter are not statistically different according to the Newman–Keuls test at the level α = 0.05.

).

Soaking seeds in Biosept Active solution proved to be the most effective of the tested treatments. This treatment resulted in the improvement of the seed germination in cv. Berlikumer. The significant increase in seed germination at the first and final counts and the decrease in the percentage of abnormal diseased seedlings were observed. In the case of cv. Karlena, the application of Biosept Active limited only the number of dead seeds. Other treatments had no effects on the germination parameters (Table 1).

None of the applied treatments improved seed vigor. Moreover, all treatments negatively affected the earliest stages of seed germination (T₁) in both cultivars and mean germination time in the case of cv. Karlena (Table 2).

The fungi Alternaria alternata and Ulocladium spp. (Ascomycota, Pleosporaceae) prevailed on the seeds of cv. Berlikumer (99 and 44%, respectively), and the applied treatments, except the fungicide, did not affect their occurrence. The pathogenic fungi Alternaria dauci and A. radicina infested only 3.5 and 6.0% of these seeds, respectively. Treatments with Biosept Active and black chokeberry juice at both concentrations limited significantly the percentage of seeds infested with A. dauci in this cultivar, whereas both grapefruit-extract-based preparations and black chokeberry juice at a lower concentration effectively reduced the number of seeds infested with A. radicina. Treatments with Biosept Active and Citrogrep led also to the decrease in seed infestation with Epicoccum nigrum Link (Ascomycota, Didymellaceae), and additionally Biosept Active treatment reduced the percentage of seeds infested with non-sporulating fungi in relation to the control and the fungicide treatment. Cultivar Karlena seeds were generally much healthier than those of cv. Berlikumer. The fungi occurring on the seeds barely grew and did not sporulate. Nevertheless, all applied treatments reduced significantly the percentage of these fungi and, except for the treatment with black chokeberry juice at 5% concentration, increased significantly the percentage of seeds free of fungi. Black chokeberry juice frequently stimulated the growth of the fungi, causing the increase in the seed infestation with Cladosporium spp. (Ascomycota, Cladosporiaceae) (at both concentrations), E. nigrum (at 25% concentration), Fusarium spp. (at 5% concentration), Melanospora simplex (Corda) D.Hawksw. (Ascomycota, Ceratostomataceae) (at 5% concentration), Penicillium spp. (Ascomycota, Aspergillaceae) (at 25% concentration), and Ulocladium spp. (at 25% concentration) in cv. Berlikumer. In the case of cv. Karlena, after the chokeberry juice treatment, the excessive growth of Penicillium spp. (at both concentrations) and Phoma sp. (Ascomycota, Didymellaceae) (at 5% concentration) was found. Additionally, the increase in seed infestation with Fusarium spp. was observed when seeds of cv. Berlikumer were treated with Biosept Active (Table 3).

The PCA analysis of health data in cv. Berlikumer showed that two principal components Dim1 and Dim2 explained 32.2 and 16.8% of variability, respectively. The clear distinction was observed among the fungicide and other treatments. It was related to the high percentage of seeds free of fungi. The Biosept Active treatment partially covered the control, whereas the Citrogrep treatment covered it completely. The distinct activity of Biosept Active from the control was connected mainly with the incidence of A. alternata and Ulocladium spp. The chokeberry juice treatment at the concentration of 25% was almost completely distinct from the control, as only small areas of the ellipses, which represented both groups, were shared. However, the effect was different from the fungicide. The activity of 25% chokeberry juice was connected mainly with the incidence of Penicillium spp., Phoma sp. and Cladosporium spp. On the other hand, the effect of 5% chokeberry juice was related to the presence of Ulocladium spp., A. alternata and E. nigrum (Figure 1a,b).

In cv. Karlena, which was characterized by much lower seed infestation with fungi, two principal components, Dim1 and Dim2, explained, respectively, 26.8 and 16.7% of variability. There were not very clear differences among the treatments. The distinct activity of Biosept Active and Citrogrep from the control was related mainly to the percentage of seeds free of fungi and additionally, in the case of the latter, to the incidence of E. nigrum and the non-sporulating fungi. The distinctiveness of 5% chokeberry juice treatment was connected mainly with its influence on seed infestation with A. alternata, the non-sporulating fungi, E. nigrum, Phoma sp. and Stemphylium spp., whereas in the case of 25% chokeberry juice treatment, it was related mostly to the percentage of seeds free of fungi and the presence of Stemphylium spp. (Figure 1c,d).

When all germination, vigor and health data were taken under consideration in cv. Berlikumer, the PCA analysis revealed that two principal components, Dim1 and Dim2, explained, respectively, 34 and 12% of variability. The clear distinction was observed for the fungicide treatment compared to the other ones. It was related mainly to the percentage of seeds free of fungi, seed germination at the final count, the percentage of fresh seeds and vigor parameters T₇₅, MGT and U₇₅₋₂₅. The Biosept Active treatment was also separated from the control; only small areas of the ellipses, which represented both groups, were shared. The distinctiveness of the Biosept Active treatment was connected mainly with its influence on seed germination at the first and final counts. The distinct activity of Citrogrep from the control was also related to seed germination at the first and final and the incidence of A. alternata and Ulocladium spp. The black chokeberry juice treatment at the concentration of 25% covered completely the control, whereas the ellipse, which represented 5% chokeberry treatment, included most of the control. The effect of the latter treatment was related mainly to the percentages of diseased seedlings and dead seeds (Figure 2a,b).

In cv. Karlena, two principal components, Dim1 and Dim2, explained, respectively, 27.1 and 14.4% of variability. The clear distinction was found between the fungicide treatment and the control. It was related mainly to the percentage of seeds free of fungi, seed germination at the final count, and vigor parameters T₇₅, MGT and U₇₅₋₂₅. The Biosept Active and Citrogrep treatments covered the control to some extent. The distinctiveness of these treatments from the control was connected mostly with their influence on seed germination at the first and final counts and the percentage of fresh seeds. The black chokeberry juice treatments had quite large parts in common with the control. The effect of 5% treatment was related mainly to the percentages of fresh seeds and diseased seedlings as well as the incidence of A. alternata and Phoma sp. The distinct activity of 25% chokeberry juice from the control was involved with the percentage of seeds free of fungi and seed germination at the first and final counts (Figure 2c,d).

The preparations based on the grapefruit extract differed significantly in their chemical composition, both qualitatively and quantitatively, but in both of them, flavanones and flavones prevailed. Naringenin and narirutin were dominant components of Biosept Active, while nobiletin and narirutin followed by hesperidin, naringenin and sinensetin prevailed in Citrogrep. Moreover, Biosept Active contained apigenin, didymin-poncirin, heptamethoxyflavone, nobiletin and sinensetin. All compounds detected in Biosept Active were also found in Citrogrep but in a much higher amount. Furthermore, the latter contained several others metabolites belonging to coumarins, phenolic acids, flavonols and limonoids. In black chokeberry juice, the majority of the detected metabolites belonged to phenolic acids, anthocyanins and flavonols, but also some amount of catechin (flavanols), procyanidin B2 and procyanidin B5 (tannins) was found. Caffeic acid, cyanidin 3-galactoside/glucoside, quercetin 3-galactoside/glucoside and quercetin-glucoside were detected in chokeberry juice in the highest amount (

Table 4. The chemical composition of grapefruit-based preparations (Biosept Active and Citrogrep) and black chokeberry juice determined using LC-MS.

Groups of Metabolites	Sub-Group	Compound	Mean Value Calculated as Chlorogenic Acid (μg mL−1)			Relative Standard Deviation RSD (%)
			Biosept Active	Citrogrep	Chokeberry Juice	Biosept Active	Citrogrep	Chokeberry Juice
Coumarins		geranylcoumarin	-	1.73	-	-	3.1	-
		Total		1.73
Phenolic acids		caffeic acid	-	80.65	467.32	-	1.3	6.1
		chlorogenic acid	-	-	120.18	-	-	2.0
		ferulic acid	-	2.43	1.19	-	0.4	0.4
		neochlorogenic acid	-	-	64.59	-	-	16.8
		p-coumaric acid	-	0.78	-	-	9.0	-
		Total		83.87	653.29
Flavonoids	Anthocyanins	cyanidin 3-arabinoside	-	-	98.96	-	-	1.7
		cyanidin 3-galactoside/glucoside	-	-	277.46	-	-	1.7
		cyanidin 3-xyloside	-	-	16.06	-	-	2.2
		Total			392.48
	Flavanols	catechin	-	-	5.09	-	-	2.7
		Total			5.09
	Flavanones	didymin-poncirin	8.91	596.94	-	1.6	0.9	-
		hesperidin	-	1227.78	-	-	1.6	-
		naringenin	466.73	1072.00	-	4.9	2.3	-
		narirutin	947.96	1669.02	-	7.1	0.8	-
		Total	1423.60	4565.73
	Flavones	apigenin	8.11	104.84	-	1.9	5.5	-
		heptamethoxyflavone	11.11	299.74	-	8.0	1.1	-
		nobiletin	12.03	1783.07	-	1.2	2.8	-
		sinensetin	4.56	1387.31	-	5.3	5.2	-
		Total	35.82	3574.96
	Flavonols	kaempferol	-	3.06	-	-	7.6	-
		quercetin	-	-	32.73	-	-	6.5
		quercetin 3-galactoside/glucoside	-	-	209.45	-	-	4.3
		quercetin 3-rutinoside	-	-	67.23	-	-	5.2
		quercetin 3-vicianoside	-	-	13.05	-	-	12.4
		quercetin-glucoside	-	-	208.13	-	-	5.3
		Total		3.06	530.60
Tannins	Proanthocyanidins	procyanidin B2	-	-	1.19	-	-	0.4
		procyanidin B5	-	-	6.95	-	-	9.1
		Total			8.14
Triterpenoids	Limonoids	deacetyl-nomilin	-	33.47	-	-	3.7
		nomilin	-	8.46	-	-	2.7	-
		Total		41.93

).

In cv. Berlikumer, significant and positive correlations were found between G_max and the contents of phenolic acids (except p-coumaric acid), anthocyanins, catechin, flavonols (except kaempferol) and proanthocyanidins. The same relationships were observed for vigor parameter T₁. The negative correlations, on the other hand, were noted between the percentage of fresh seeds and the mentioned compounds. Germination at the first and final counts was positively correlated with the contents of flavanones (except hesperidin) and flavones, whereas the negative correlations with these compounds were found in the case of diseased seedlings and dead seeds. The effect of particular compounds on the incidence of individual fungi varied. Significant correlations were observed for Cladosporium spp., E. nigrum, M. simplex, Penicillium spp. and Ulocladium spp. The percentages of seeds infested with Cladosporium spp., E. nigrum, Penicillium spp. and Ulocladium spp. were positively correlated with the contents of phenolic acids (except p-coumaric acid), anthocyanins, catechin, flavonols (except kaempferol) and proanthocyanidins. The negative correlations, on the other hand, were observed between the percentages of seeds infested with Cladosporium spp. and Ulocladium spp. and the contents of flavanones (except hesperidin) and flavones. In the case of E. nigrum and M. simplex, the negative correlations were found for geranylcoumarin, p-coumaric acid, flavanones, flavones, kaempferol and limonoids. The percentage of seeds infested with Fusarium spp. was positively correlated with the contents of flavanones (except hesperidin) and flavones (

Table 5. Spearman’s rank correlation coefficients between the contents of active substances in the grapefruit-based preparations (Biosept Active, Citrogrep) and black chokeberry juice, and the seed germination, vigor and health parameters (cv. Berlikumer).

Compound	Gmax	Germination 1st Count	Germination Final Count	Diseased Seedlings	Fresh Seeds	Dead Seeds	T1	Cladosporium spp.	Epicoccum nigrum	Fusarium spp.	Melanospora simplex	Penicillium spp.	Ulocladium spp.
geranylcoumarin	- *	-	-	-	-	-	-	-	−0.479 **	-	−0.412	-	-
Total Coumarins	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-
caffeic acid	0.452	-	-	-	−0.406	-	0.530	0.742	0.610	-	-	0.428	0.587
chlorogenic acid	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
ferulic acid	0.452	-	-	-	−0.406	-	0.530	0.742	0.610	-	-	0.428	0.587
neochlorogenic acid	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
p-coumaric acid	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-
Total Phenolic Acids	0.452	-	-	-	−0.406	-	0.530	0.742	0.610	-	-	0.428	0.587
cyanidin 3-arabinoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
cyanidin 3-galactoside/glucoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
cyanidin 3-xyloside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
Total Anthocyanins	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
catechin	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
Total Flavanols	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
didymin-poncirin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
hesperidin	-	-	-		-	-	-	-	−0.479	-	−0.412	-	-
naringenin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
narirutin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
Total Flavanones	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
apigenin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
heptamethoxyflavone	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
nobiletin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
sinensetin	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
Total Flavones	-	0.573	0.530	−0.495	-	−0.420	-	−0.513	−0.797	0.403	−0.493	-	−0.515
kaempferol	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-
quercetin	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
quercetin 3-galactoside/glucoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
quercetin 3-rutinoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
quercetin 3-vicianoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
quercetin-glucoside	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
Total Flavonols	0.452	-	-	-	−0.406	-	0.530	0.742	0.610	-	-	0.428	0.587
Total Flavonoids	-	-	-	-	-	-	0.597	0.687	0.500	-	-	0.417	0.518
procyanidin B2	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
procyanidin B5	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
Total Proanthocyanidins	0.465	-	-	-	−0.420	-	0.480	0.768	0.751	-	-	0.466	0.641
deacetyl-nomilin	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-
nomilin	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-
Total Limonoids	-	-	-	-	-	-	-	-	−0.479	-	−0.412	-	-

* not significant; ** significant at the α = 0.05 level; For further explanations see Table 1 and Table 2.

).

In cv. Karlena, there were no significant correlations between the germination and vigor parameters and the contents of different groups of metabolites; however, the percentages of seeds infested with Fusarium spp. and Penicillium spp. were negatively correlated with the contents of flavanones (except hesperidin) and flavones. In the case of non-sporulating fungi, the negative correlations were found for phenolic acids (except p-coumaric acid), anthocyanins, catechin, flavonols (except kaempferol) and proanthocyanidins (

Table 6. Spearman’s rank correlation coefficients between the contents of active substances in the grapefruit-based preparations (Biosept Active, Citrogrep) and black chokeberry juice, and the seed health parameters (cv. Karlena).

Compound	Fusarium spp.	Penicillium spp.	Non-Sporulating Fungi
geranylcoumarin	- *	-	-
Total Coumarins	-	-	-
caffeic acid	-	-	−0.507 **
chlorogenic acid	-	-	−0.538
ferulic acid	-	-	−0.507
neochlorogenic acid	-	-	−0.538
p-coumaric acid	-	-	-
Total Phenolic Acids	-	-	−0.507
cyanidin 3-arabinoside	-	-	−0.538
cyanidin 3-galactoside/glucoside	-	-	−0.538
cyanidin 3-xyloside	-	-	−0.538
Total Anthocyanins	-	-	−0.538
catechin	-	-	−0.538
Total Flavanols	-	-	−0.538
didymin-poncirin	−0.402	−0.487	-
hesperidin	-	-	-
naringenin	−0.402	−0.487	-
narirutin	−0.402	−0.487	-
Total Flavanones	−0.402	−0.487	-
apigenin	−0.402	−0.487	-
heptamethoxyflavone	−0.402	−0.487	-
nobiletin	−0.402	−0.487	-
sinensetin	−0.402	−0.487	-
Total Flavones	−0.402	−0.487	-
kaempferol	-	-	-
quercetin	-	-	−0.538
quercetin 3-galactoside/glucoside	-	-	−0.538
quercetin 3-rutinoside	-	-	−0.538
quercetin 3-vicianoside	-	-	−0.538
quercetin-glucoside	-	-	−0.538
Total Flavonols	-	-	−0.507
Total Flavonoids	-	-	−0.624
procyanidin B2	-	-	−0.538
procyanidin B5	-	-	−0.538
Total Proanthocyanidins	-	-	−0.538
deacetyl-nomilin	-	-	-
nomilin	-	-	-
Total Limonoids	-	-	-

* not significant; ** significant at the α = 0.05 level; For further explanations, see Table 1 and Table 2.

).

4. Discussion

The grapefruit-based preparations, applied in the experiment, varied significantly in their influence on the seed quality parameters, although both of them included 33% of the fruit extract. The chemical analysis revealed that the amount of polyphenols in Citrogrep was much higher than in Biosept Active. The content of polyphenols in fruits may be affected by numerous factors, such as variety, ripeness at the time of harvest, environmental conditions (soil type, sun exposure, rainfall, the way of culture, fruit yield per tree etc.), processing, and storage [3]. Nevertheless, from among the phenolic compounds in both preparations, flavanones dominated. They represented 97.5% of the polyphenols in Biosept Active and 55.5% of those in Citrogrep. Flavones constituted 2.5% and 43.5% of the total polyphenols content in these preparations, respectively. Citrogrep contained also a small amount of phenolic acids, flavonols, coumarins and limonoids. In Biosept Active, the highest contents of narirutin and naringenin were observed, whereas in Citrogrep, nobiletin prevailed, which was followed by narirutin, sinensetin, hesperidin and naringenin. The results of our experiment confirmed the findings of other authors. Agudelo et al. [4] reported that flavanones were the dominant polyphenols in all grapefruit samples, representing about 93% of the total flavonoids, with naringin and narirutin being the predominant compounds. Sicari et al. [5] observed that naringin was a major flavonoid in grapefruit juice, which was followed by narirutin and poncirin. Xi et al. [34], who evaluated the chemical composition of different C. paradise fruit parts, found that naringin, nobiletin, and gallic acid were the major phenolic compounds.

Despite the differences in the chemical composition of both grapefruit-based preparations, the results of the variance and PCA analyses showed their beneficial influence on seed germination at the first and final counts, which was connected with the decrease in the percentage of diseased seedlings. It was especially apparent in the case of cv. Berlikumer, which was characterized by the lower seed quality. Biosept Active was more effective than Citrogrep. Similar findings were reported by Dorna et al. [35]. The authors observed the favorable effects of the Biosept Active treatment on carrot seed germination in the case of a lower-quality sample. The significant positive correlations between germination at the first and final counts and the contents of flavanones (except hesperidin) and flavones, and the negative correlations between the percentage of diseased seedlings and dead seeds and these compounds, were found only in the case of cv. Berlikumer. The low initial quality of these seeds was probably related to more advanced deteriorative processes and the higher infestation with fungi. It may be the reason why in this cultivar, the higher effectiveness of antioxidants, present in grapefruit extracts, was observed. The antioxidant activity of flavonoids, as electron or hydrogen donors, relates to the reduction potentials and reactivity of the substituent reactive groups. Jayaprakasha et al. [36] found the high correlation between the total polyphenol content in the grapefruit extract and the radical scavenging activity by the DPPH method. The studies of Sicari et al. [5] revealed the significant correlation between the content of flavonoids and the antioxidant capacities. Agudelo et al. [4] reported that in the DPPH scavenging activity, didymin, followed by naringin, narirutin, poncirin, and hesperidin, presented the best correlation. The analysis of variance showed that treating seeds with Biosept Active and Citrogrep did not influence considerably the incidence of Alternaria alternata in the examined cultivars, whereas both preparations decreased cv. Berlikumer seed infestation with Alternaria radicina. Moreover, there were no significant correlations between particular chemical compounds and the percentage of seeds infected with these fungi. The negative correlations between the percentages of seeds infested with Cladosporium spp., Epicoccum nigrum, Melanospora simplex, Ulocladium spp. and the contents of flavanones and flavones have been found in cv. Belikumer. The impact of these compounds on the presence of Fusarium spp. is unclear, because the positive correlations were observed in cv. Berlikumer and the negative ones were observed in cv. Karlena. This discrepancy could be explained by the different species composition of the Fusarium genus in both cultivars.

Our studies showed that the Biosept Active treatment was more beneficial for carrot seeds than the Citrogrep treatment, although the second preparation contained more antioxidant compounds and at much higher concentrations. It is probable that the concentrations of flavanones and flavones, and the ratio of particular compounds in Biosept Active, were more adequate. Moreover, extraction techniques can affect the bioactivities of the extracts [37,38].

Phenolic acids were the dominating polyphenols in chokeberry juice (41.1%) followed by flavonols (33.4%) and anthocyanins (24.7%). Moreover, small amounts of flavanols (0.3%) and proanthocyanidins (0.5%) were detected. Up to now, many researchers analyzed the phytochemical composition of the chokeberry fruits and products, and their results show big differences in the content of polyphenols [39,40,41]. Tasinov et al. [39], who examined the composition of three chokeberry juices, found that the content of phenolic acids ranged from 45.5% to 51.5% of identified polyphenols, which was followed by the proanthocyanidins (29.2–34.4%), anthocyanins (8.1–19.9%), and flavonols (3.0–3.3%). In the experiment of Oziembłowski et al. [40], on the other hand, the most important group of the identified polyphenolic compounds in six tested juice samples was anthocyanins, which accounted for about 40% of the total polyphenols. The predominant compound was cyanidin-3-O-galactoside. In our studies, it was also revealed that this compound occurred at the largest extent among the detected anthocyanins. The results obtained by Oszmiański and Wojdylo [41] showed that the largest group of polyphenols in chokeberry fruits was presented by polymeric proanthocyanins (66%).

The chemical composition of chokeberry fruits depends on many factors, including climate conditions, soil composition, berry maturity, harvest methods and storage conditions [6,7,40]. Moreover, the polyphenol content in the extract/juice depended on the extraction procedure [42].

The results of our experiment showed that 5% black chokeberry juice had the adverse influence on the carrot seed germination and decreased the percentage of seeds free of fungi compared to the control. Chokeberry juice applied at the concentration of 25%, on the other hand, increased seed germination at the first and final counts in both cultivars, although the differences were not statistically significant compared to the control. This treatment increased also the percentage of seeds free of fungi. The PCA analysis confirmed the positive influence of 25% black chokeberry juice on the germination of cv. Karlena seeds. The significant positive correlations were found in cv. Berlikumer between G_max and the contents of phenolic acids (except p-coumaric acid), anthocyanins, catechin, flavonols (except kaempferol) and proanthocyanidins, occurring in the chokeberry juice. The contents of these compounds were positively correlated with the percentages of seeds infested with Cladosporium spp., E. nigrum, Penicillium spp. and Ulocladium spp., which indicates their beneficial impact on the growth of the mentioned fungi. Some advantageous influence of black chokeberry juice on carrot seed germination could be explained by the antioxidant activity of detected polyphenolic compounds. According to Denev et al. [43], almost 40% of the antioxidant activity of chokeberries is attributed to procyanidins. Skupien and Oszmianski [44] also demonstrated that 40% of the in vitro antioxidant activity of these fruits is related to the contents of proanthocyanidins followed by anthocyanins (24%), hydroxycinnamic acid (18%) and epicatechin (11%). The black chokeberry juice used in our studies contained a very low amount of proanthocyanidins and did not include hydroxycinnamic acid and epicatechin. This was likely a reason why the antioxidant activity of this juice was so weak. It must be also taken into consideration that the polyphenolic compounds detected in black chokeberry juice did not show antimicrobial properties, and they even increased seed infestation with some fungi.

It should be pointed out that for both cultivars, the quite low percentage of variability in the PCA analysis was explained by the incidence of fungi (Dim1 values 32.2% and 26.8%) and was likely connected to the differential responses of individual fungi to the applied treatments. Microorganisms present on seeds create a complex community and can interact with each other. The removal any of the organisms may result in the excessive development of the other ones. Microbes interact with seeds at all stages of plant development. These interactions may be casual or intimate, and they all contribute to varying degrees to an evolving seed microbiome. Microbial interactions taking place in, on, and around seeds have a significant impact on seed health, and they also further plant health and productivity [45].

The seed samples available on the market differ significantly in quality depending on the cultivation procedures, environmental conditions, seed harvest, and processing and storage methods. Our study showed that treating seeds with the grapefruit extract before sowing may improve their germination and vigor, especially in the case of the samples characterized by a low initial seed quality. Additionally, the antifungal properties of this extract have been proven. The pre-storage application of the grapefruit extract could also retard the deterioration processes in seeds and reduce their infestation with storage fungi, thus improving seed storability.

5. Conclusions

The grapefruit-based preparations, Biosept Active and Citrogrep, differed significantly in their chemical composition, qualitatively and quantitatively, but in both of them, flavanones and flavones prevailed. Biosept Active improved seed germination especially in cv. Berlikumer, which was characterized by a lower initial seed quality. The significant positive correlations between germination at the first and final counts and the contents of flavanones (except hesperidin) and flavones were identified in this cultivar. Moreover, the negative correlations between the percentages of diseased seedlings, dead seeds, the percentages of seed infested with Cladosporium spp., Epicoccum nigrum, Melanospora simplex, and Ulocladium spp., and the investigated compounds were found. Black chokeberry juice at the higher concentration showed some antioxidant activity; however, it frequently stimulated the growth of the fungi on carrot seeds.