Effect of Selective Substrates on Germination of Pomegranate (Punica granatum) and Trifoliate Orange (Poncirus trifoliata) Seeds with and Without the Presence of Plant-Beneficial Microorganisms

Abstract

Insect-based (silkworm cocoons) and plant-based (cotton wool pads and gauzes) fiber substrates were used to support and ameliorate seed germination originating from trifoliate orange (Poncirus trifoliata) and pomegranate (Punica granatum) trees. Three different commercial formulations of beneficial microorganisms (Bacillus spp.-Azotobacter spp., Saccharomyces boulardii, and Saccharomyces cerevisiae) were administered to seeds in order to evaluate their contribution to germination and growth. The silkworm cocoons provided better germination rates for P. trifoliata seeds (83.33%) among the tested media without any microbial supplementation. These rates increased towards the absolute maximum (100%) when Bacillus spp.-Azotobacter spp., S. boulardii and S. cerevisiae were applied. Furthermore, inoculums of Bacillus spp.-Azotobacter spp. 2 mL and S. cerevisiae 3 g raised the pomegranate seed germination ability by 30–33.33% and 50–67.7%, respectively, on silkworm cocoon substrates when compared to plant-derived, cellulosic fiber substrates under the same biotic exposure. On increasing the size of applied microbial inoculums, seed germination moved from optimum to suboptimum for all germination media. Examination of multipartite pH compatibility (between seeds, microorganisms, and germination media) was beneficial and of functional value. In conclusion, the germination rates of both tree species can be raised using bacterial and yeast supplementation, including medical-grade S. boulardii, on environmentally friendly materials such as insect- and plant-based fiber substrates.

1. Introduction

Seed germination is one of the most important, real-time, biomechanical processes in a plant’s life cycle, occurring when conditions are suitable for the induction of the phenomenon [1]. Germination can take place in (a) soil, (b) soil mixtures with organic or inorganic additives enhancing fertility, (c) partially decayed vegetation or organic matter, (d) specialized germination media such as paper and rockwool, and (e) naturally derived germination media solely from natural fibers [2,3,4,5,6,7]. Furthermore, germination can be enhanced by a wide range of beneficial soil microorganisms such as bacteria and fungi [8]. In terms of bacteria, the Bacillus and Azotobacter species have been examined in terms of their plant growth-promoting attributes; however, documentation of tree species used as rootstocks in commercial agriculture and low-commercial-demand tree species for cultivation remain limited [9,10,11,12,13]. These bacteria can modulate nutrient availability, produce phytohormones, and mitigate abiotic and biotic stresses, potentially complementing the effect of selective germination [14,15,16]. Similarly to plant growth-promoting growth bacteria, benefits have been recorded in the presence of non-pathogenic fungi such as Saccharomyces genus yeasts [17,18]. Cucumber seedlings treated with S. cerevisiae promoted growth characteristics, amino acid content, and hormonal activity [19]. Being taxonomically close to S. cerevisiae yeast, S. boulardii was recently engineered to produce plant-related abscisic acid for nutraceutical use in animals; however, even in its native state, the availability of information on S. boulardii is limited in terms of its potential bioactive roles while interacting with tree species [20,21].

Cultivating pomegranate trees is a highly profitable activity that is smoothly integrated with vital societal sectors: (a) the retail sector for raw food; (b) the agro-industrial sector for juice, oil, other food-related secondary byproducts and fire-resistant extracts; and (c) the pharmaceutical sector for medical-grade products formulated for therapeutic use [22,23,24,25,26]. Pomegranate seeds have been studied in depth in terms of their size and anatomy and have been shown to exhibit relatively robust germination potential under suitable conditions (natural dormancy breaks with no human intervention, stratification, scarification, and exogenous hormone management), making them a perfect model for experimentation on conditional germination [27,28,29,30]. Fruit arils (60% of total fruit weight), which enclose pomegranate seeds, are 80% liquid content (i.e., a watery juice rich in glucose and fructose, pectins, and medically bioactive molecules) and 20% seeds [31]. Domestication of this tree species approximately 5000 years ago resulted in beneficial utilization of its seeds in Chinese and global medicine to cure gastrointestinal, oncological, and diabetic diseases due to their unique synthesis of bioactive compounds [32,33,34]. Apart from medically important substrates, pomegranate seeds can also synthesize chitinases, which are important defense enzymes against pathogenic fungi [35]. Further, trifoliate orange, a hardy rootstock widely used in citrus cultivation, presents unique seed germination challenges for process standardization and environmental compliance [36]. Understanding the factors that optimize germination in these species has practical implications for agriculture and tree conservation [37,38].

Natural, insect-based fibers reflect ectodermal secretions that are cell/tissue-soluble in pre-stored forms and thereafter polymerized, resulting in non-soluble fibers as they are excreted from the body cavity; specialized grands are involved in the process. Their mechanical strength is comparable to plant-based and synthetic fibers, with thermostable properties that are quite resilient, making them suitable for environmental uses [39,40]. Dissection of a silkworm fiber reveals a leaflet structure, microfibrils, fibril bundles, fibroin strands, sericin, and raw silk filament [41]. From a chemical synthesis viewpoint, silkworm fiber is formed of linear polymerized complexes of fibroin-heavy chains (fibroin H; 350–500 kDa), fibroin-light chains (fibroin L; 25 kDa), sericin (150–250 kDa) glycoproteins, sugars, waxes, inorganic substrates, and dyes [42,43].

The production of silkworm fibroins differs from that of other insects due to the triple formation complex of fibroin-light and -heavy chains via disulfide linkages in the presence of glycoproteins, which permits structural changes in the final macromolecular fiber product [44]. Silkworm fibroin is composed of glycine (43%), alanine (30%), serine (12%), tyrosine (5%), and valine (3%) and can be found in three different forms: (a) spiral (silk type I), (b) crystalline (silk type II), and (c) unstable (silk type III) [45,46,47]. The Βombyx mori Ν-terminal of fibroin molecules is responsible for fiber formation when pH declines [48]. Fibroin has been used in animal and human tissue regeneration; in aquatic environments, fibroin provides porous networks of microtubules, nanospheres and 3D thin-film surfaces, sponges, and nano-sieves, which are widely applicable in science and industry [49,50]. In the agri-food sector, silk fibroin has been integrated into (a) polymer complexes for antimicrobial seed coating, (b) trehalose complexes coupled with phage and growth-promoting bacteria to fight tomato soilborne diseases and saline soil stress, and (c) fructose and other edible complexes for food packaging purposes [51,52,53,54].

Sericin, the latter major macrocomponent of silk fiber, consists of serine (40%) and to a lesser extent glycine, aspartic acid, glutamic acid, threonine, and tyrosine, whereas hydrogen bonds among the hydroxyl groups of amino acids secure adhesive agglomerations of molecules [50,55]. Other molecules that contribute to the porous formation of silkworm cocoons are also secreted from adrenal secretions via the induction of Ser1,2,3, MSGS-3, MSGS-4, and MSGS-5 B. mori genes [45,56,57,58]. Silkworm sericin is already used in the cosmetic industry as a hydrative medium because of its high content of serine and glycine, especially when administered in collagen-formulated mixtures [59,60]. Due to its coagulant and water viscosity modification properties, sericin has become an important polymeric material for hydrogels used in smart water management farming [61,62]. Progressive adaptation of these technologies to seed coating (e.g., cross-linked polyvinyl alcohol (PVA) with silk sericin under various protocols) resulted in hydrophilic conditions for seeds with higher germination rates due to enhanced imbibition capacity and water vapor uptake [63].

Cellulosic plant-based fibers are used as seed germination media, with a high preference for cotton-based ones, wherein seeds from tree species have been reported to perform well [64,65,66]. Cotton fibers are the dried cell walls of formerly living cells with up to 95% cellulose; their non-cellulosic content consists of proteins, amino acids and other N-containing molecules, waxes, pectic substances, organic acids, sugars, inorganic salts, and very small amounts of pigments [67]. Cotton fiber technology has produced a series of cotton woven and non-woven dry gauzes which are also frequently used as tree seed germination media [68,69,70].

Herein, two tree species, trifoliate orange (Poncirus trifoliata) and pomegranate (Punica granatum), were evaluated for their germination and growth characteristics using insect (silk cocoons) and plant-based (cotton wool pads and cotton gauzes) germination media, supporting a more green, circular, recyclable, low-carbon-footprint and resilient way to sustain nursery propagation and tree breeding selection practices.

2. Materials and Methods

2.1. Collection and Processing of Trifoliate Orange and Pomegranate Seeds

Mature fruits from trifoliate orange trees were collected in December 2023 from the Department of Agriculture, University of Patras (GR) orchard. Trifoliate orange seeds were removed from fruits with mechanical pressure, washed several times with distilled water, and sterilized with 2% sodium hypochlorite (NaClO) for 5 min. Thereafter, the seeds were soaked in 500 ppm gibberellic acid (GA₃) solution for 12 h (modified protocol from Khan et al. [71] and Sharaf et al. [72]).

Pomegranate fruits cv. Wonderful were procured from a local market in December 2023. For seeds, juice was removed using a pomegranate manual and hand cold-press juicer (Acer 10-234-002, Nava SA, Thessaloniki, Greece), and the solid residue was immersed in a container with water for pomace–seed separation. The collected seeds were washed with distilled water and disinfected with sodium hypochlorite (NaClO) 2% for 5 min. To break seed dormancy, a modified protocol from Monteiro et al. [30] was applied; initially, seeds were immersed in 500 ppm gibberellic acid (GA₃) solution for 12 h and then placed in a box with wet cotton as a substrate and kept at a low temperature (4 °C) for 60 days.

2.2. Seed Germination Substrates

In order to investigate the effect of substrates on seed germination, cotton wool pads, cotton gauze, and silkworm cocoon materials were selected. Cotton and gauze were placed in Petri dishes, and distilled water was added until saturation. Silkworm cocoons were soaked in distilled water for 24 h before being placed in transparent 30 mL cups of water.

2.3. Beneficial Microorganisms

Commercial microbial products with active substances—(a) Bacillus subtilis, Bacillus pumillus, Bacillus licheniformis, Bacillus megaterium, and Azotobacter spp. at 1 × 10¹¹ cfu/L (enriched with NPK 0.6-1.2-3+0.3% CaO, 0.1% MgO, 0.1%) [commercial name RIZOBAC produced by Humofert S.A., Metamorfosi, Athens, Greece], (b) medical-grade Saccharomyces boulardii CNCM I-745 [commercial name ULTRA-LEVURE, produced by Petsiavas S.A., Kifisia, Athens, Greece], and (c) Saccharomyces cerevisiae strain LAS 117 (cell walls 94.1% w/w) [commercial name ROMEO produced by Hellafarm S.A., Paiania Attikis, Greece]—were used for the biopriming of seeds. From each microbial product, three solutions with different concentrations were prepared: (a) Bacillus spp.-Azotobacter spp. 1 mL, 2 mL, and 3 mL per 100 mL of distilled water; (b) Saccharomyces boulardii 1000 mg, 1250 mg, and 1500 mg per 100 mL of distilled water; and (c) Saccharomyces cerevisiae 2.5 g, 3 g, and 5 g per 200 mL of distilled water.

2.4. Treatments

Seeds of trifoliate orange (P. trifoliata) and pomegranate (P. granatum) were soaked in microbial solutions or distilled water (control) for 10 min and then placed in Petri dishes or transparent cups. In each Petri dish with cotton or gauze substrate, 10 seeds were placed, consisting of one replicate. Respecting the natural three-dimensional structure of silkworm cocoon and applying the most minimally invasive technique, 2 seeds were placed inside each thimble in transparent cups; a group of 5 cups (10 seeds in total) represented one replicate. Each treatment had five replicates, with a total of 50 seeds per treatment. Seeds were transferred to a germination chamber with a 20 °C temperature and 12 h photoperiod. They were kept hydrated by adding distilled water when needed.

2.5. Seedling Germination and Growth Parameters

Every two days, for both tree species, the number of germinated seeds and the shoot and radicle length of seedlings were recorded. At the end of the germination process, the seedlings were divided into roots and shoots, and their fresh weights were measured. Root and shoot dry weights were determined after shade drying, until their weights remained constant. In addition, the first and last days of seed germination were recorded.

From measurements made during the germination period, the following parameters were estimated: (a) germination rate, (b) mean germination time, (c) germination index, (d) coefficient of the velocity of germination, (e) time spread of germination, and (f) shoot–root weight ratio (

Table 1. Calculation of germination parameters.

Germination Rate (% G.R.): number of seeds that germinated × 100

Germination Rate Index (%⁄day), calculated according to the equation below: $\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\mathrm{G}.\mathrm{R}.\mathrm{I}({\displaystyle \raisebox{1ex}{$\%$}\!\left/ \!\raisebox{-1ex}{$day$}\right.})={\displaystyle \frac{\Sigma 1}{1}}+{\displaystyle \frac{\Sigma 2}{2}}+\dots {\displaystyle \frac{\Sigma x}{x}}$ where Σ1 = Germination rate × 100 on the first day after sowing Σ2 = Germination rate × 100 on the second day after sowing Σx = Germination rate × 100 at x day after sowing

Time Spread of Germination (T.S.G.): the time in days between the first and last day of germination.

Coefficient of velocity of germination (% C.V.G.), calculated according to the following equation: $\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\%C.\mathrm{V}.G.=100\times {\displaystyle \frac{\sum {\rm N}i}{\sum NiTi}}$ where Ni: The number of seeds that germinated per day Ti: Days in the experimental period

Mean Germination Time (M.G.T), calculated according to the following equation: $\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}\hspace{1em}M.G.T.={\displaystyle \frac{\sum {\rm N}iTi}{\sum Ni}}$ where Ni: the number of seeds that germinated per day Ti: Days in the experimental period

), [73,74].

2.6. Statistical Analysis

A completely randomized factorial design with 28 treatments per tree species—3 growth substrates, 3 microbes, 3 microbe concentrations and 1 control (water-treated seeds)—was used in this study. A minimum of five replicates were used for each value.

Data were analyzed using the 95% confidence limits overlap protocol of Sokal and Rohlf [75]. Tables are presented as means ± standard error of the mean. An α level of 0.05 was chosen. Prism 8.0 (GraphPad) was used for data analysis.

3. Results

Seeds from P. trifoliata (Figure 1) and P. granatum (Figure 2) germinated in both cotton- and silk fiber-based substrates with or without microbial presence. For both plant species, data and their analysis for seed germination rate, mean germination time, coefficient of velocity of germination, germination rate index, time spread of germination, shoot and root fresh and dry weights, and seedling shoot and root lengths are cited below.

3.1. Seed Germination Rate

The germination rate of trifoliate orange seeds was affected by the substrates used, with the seeds in silkworm cocoons having the highest germination percentage (83%) without microbial supplementation. Cotton was also an effective substrate, allowing 43% of the seeds to germinate. Lower but comparable to cotton was the ability of seeds to germinate on gauze substrate (30%) (

Table 2. Germination parameters of trifoliate orange seeds treated with beneficial microorganisms.

Treatments	Seed Germination (%)	Mean Germination Time (MGT)	Coefficient of Velocity of Germination (CVG)	Germination Rate Index (%/day)	Time Spread of Germination (TSG)
Water
Cotton	43.33 ± 12.02 a,a	7.15 ± 0.56 a,a	14.13 ± 1.02 a,b	33.42 ± 10.68 a,a	4.00 ± 1.73 a,a
Gauze	30.00 ± 5.77 a,a	11.89 ± 2.06 a,ab	8.97 ± 1.65 a,a	11.23 ± 4.35 a,a	10.33 ± 3.28 a,a
Cocoon	83.33 ± 16.67 a,b	14.42 ± 1.44 a,b	7.07 ± 0.66 ab,a	23.56 ± 15.02 a,a	6.00 ± 4.51 a,a
Bacillus spp. + Azotobacter (1 mL)
Cotton	53.33 ± 3.33 a,a	9.50 ± 0.56 a,ab	10.60 ± 0.66 a,ab	29.75 ± 3.35 a,a	11.33 ± 0.33 a,a
Gauze	25.67 ± 2.69 a,b	14.49 ± 1.95 a,a	7.30 ± 0.98 a,a	3.01 ± 0.20 a,b	8.00 ± 2.65 a,ab
Cocoon	66.67 ± 16.67 a,a	8.53 ± 0.85 a,b	11.97 ± 1.22 a,b	23.33 ± 7.26 a,a	2.00 ± 1.00 a,b
Bacillus spp. + Azotobacter (2 mL)
Cotton	43.33 ± 8.82 a,a	14.15 ± 0.75 a,ab	7.1 ± 0.36 a,a	10.49 ± 4.61 a,a	15.00 ± 3.00 a,a
Gauze	26.67 ± 3.33 a,a	10.90 ± 1.99 a,a	9.77 ± 1.65 a,a	7.50 ± 2.36 a,a	6.33 ± 3.93 a,a
Cocoon	100.00 ± 0.00 a,b	17.67 ± 1.67 a,b	5.63 ± 0.52 b,a	30.31 ± 13.68 a,a	13.00 ± 2.89 a,a
Bacillus spp. + Azotobacter (3 mL)
Cotton	20.00 ± 5.77 a,a	12.67 ± 3.33 a,a	9.70 ± 3.45 a,a	6.14 ± 2.29 a,a	9.00 ± 5.19 a,a
Gauze	30.00 ± 0.00 a,a	19.60 ± 0.00 a,a	5.10 ± 0.00 a,a	2.70 ± 0.22 a,a	13.00 ± 1.15 a,a
Cocoon	83.33 ± 16.67 a,b	14.99 ± 1.02 a,a	6.73 ± 0.47 ab,a	8.94 ± 1.40 a,a	5.33 ± 2.18 a,a
S. boulardii (1000 mg)
Cotton	53.33 ± 12.02 a,a	10.10 ± 1.62 a,a	10.53 ± 1.99 a,a	32.40 ± 9.87 a,a	11.33 ± 4.05 a,a
Gauze	33.33 ± 3.33 a,a	13.70 ± 3.35 a,a	8.33 ± 2.19 a,a	12.16 ± 5.56 a,a	9.33 ± 2.40 a,a
Cocoon	83.33 ± 16.67 a,b	13.56 ± 2.42 a,a	7.83 ± 1.30 ab,a	11.05 ± 1.17 a,a	5.33 ± 3.84 a,a
S. boulardii (1250 mg)
Cotton	46.67 ± 8.82 a,a	11.68 ± 0.92 a,a	8.67 ± 0.71 a,a	20.21 ± 5.66 a,a	12.00 ± 3.46 a,a
Gauze	36.67 ± 3.33 a,a	13.96 ± 2.68 a,a	7.80 ± 1.69 a,a	10.22 ± 6.32 a,a	10.67 ± 2.60 a,a
Cocoon	100.00 ± 0.00 a,b	17.00 ± 4.50 a,a	7.27 ± 2.62 ab,a	24.22 ± 17.89 a,a	5.67 ± 0.88 a,a
S. boulardii (1500 mg)
Cotton	33.33 ± 3.33 a,a	10.20 ± 2.11 a,a	10.97 ± 2.82 a,a	16.51 ± 6.84 a,a	10.00 ± 5.29 a,a
Gauze	40.00 ± 0.00 a,a	13.51 ± 0.00 a,a	7.40 ± 0.00 a,a	10.44 ± 3.22 a,a	13.67 ± 2.60 a,a
Cocoon	66.67 ± 16.67 a,a	14.14 ± 1.58 a,a	7.23 ± 0.74 ab,a	7.24 ± 0.60 a,a	1.00 ± 0.00 a,b
S. cerevisiae (2.5 g)
Cotton	46.67 ± 12.02 a,a	10.12 ± 0.48 a,a	9.92 ± 0.49 a,a	31.75 ± 6.79 a,a	14.33 ± 2.03 a,a
Gauze	50.00 ± 10.00 a,a	16.52 ± 2.91 a,b	6.40 ± 1.00 a,b	19.17 ± 11.77 a,a	13.67 ± 4.33 a,a
Cocoon	66.67 ± 16.67 a,a	19.08 ± 0.92 a,b	5.43 ± 0.23 b,b	5.91 ± 1.44 a,a	1.00 ± 0.00 a,b
S. cerevisiae (3 g)
Cotton	30.00 ± 11.55 a,a	7.35 ± 1.33 a,a	14.40 ± 2.20 a,b	24.03 ± 7.04 a,a	4.67 ± 4.67 a,a
Gauze	33.33 ± 12.02 a,a	10.96 ± 1.97 a,a	9.90 ± 2.16 a,ab	11.39 ± 3.19 a,a	10.33 ± 4.98 a,a
Cocoon	100.00 ± 0.00 a,b	15.66 ± 1.58 a,b	6.53 ± 0.73 ab,a	16.60 ± 6.24 a,a	9.33 ± 4.26 a,a
S. cerevisiae (5 g)
Cotton	30.00 ± 5.77 a,a	11.51 ± 3.44 a,a	10.52 ± 3.23 a,a	10.76 ± 4.64 a,a	7.00 ± 5.56 a,a
Gauze	26.67 ± 3.33 a,a	15.59 ± 2.79 a,a	6.87 ± 1.29 a,a	5.49 ± 3.52 a,a	6.67 ± 1.33 a,a
Cocoon	50.00 ± 0.00 a,b	12.61 ± 0.32 a,a	7.96 ± 0.20 ab,a	6.75 ± 0.26 a,a	1.00 ± 0.00 a,a

The different letters indicate a significant (p < 0.05) difference; the first letter demonstrates differences between water and microbe treatments and the second one among substrates. Data are presented as the mean ± SE of five replicates.

). In pomegranate, no differences were observed between the substrates concerning the seed germination rate (

Table 3. Germination parameters of pomegranate seeds treated with beneficial microorganisms.

Treatments	Seed Germination (%)	Mean Germination Time (MGT)	Coefficient of Velocity of Germination (CVG)	Germination Rate Index (%/day)	Time Spread of Germination (TSG)
Water
Cotton	46.67 ± 17.64 a,a	18.67 ± 1.19 a,a	5.40 ± 0.36 a,a	4.34 ± 1.69 a,a	10.33 ± 4.98 a,ab
Gauze	46.67 ± 6.67 a,a	18.94 ± 1.16 a,a	5.32 ± 0.34 a,a	3.95 ± 0.46 a,a	14.00 ± 0.58 a,a
Cocoon	50.00 ± 0.00 a,a	21.63 ± 1.50 a,a	4.67 ± 0.32 a,a	4.16 ± 0.48 ab,a	1.00 ± 0.00 a,b
Bacillus spp. + Azotobacter (1 mL)
Cotton	33.33 ± 8.82 a,a	17.84 ± 1.72 a,a	5.70 ± 0.50 a,a	2.99 ± 0.95 a,a	8.67 ± 2.19 a,ab
Gauze	36.67 ± 8.82 a,a	19.95 ± 0.95 a,a	5.03 ± 0.23 a,a	3.52 ± 1.11 a,a	13.67 ± 1.67 a,a
Cocoon	66.67 ± 16.67 a,a	16.97 ± 1.31 a,a	6.00 ± 0.46 a,a	15.71 ± 2.09 ab,b	2.67 ± 0.88 ab,b
Bacillus spp. + Azotobacter (2 mL)
Cotton	16.67 ± 3.33 a,a	20.78 ± 3.06 a,a	5.07 ± 0.87 a,a	1.27 ± 0.47 a,a	4.67 ± 2.33 a,a
Gauze	43.33 ± 6.67 a,a	20.50 ± 1.56 a,a	4.93 ± 0.38 a,a	2.92 ± 0.24 a,b	8.67 ± 0.33 a,a
Cocoon	66.67 ± 16.67 a,b	16.02 ± 0.13 a,a	6.25 ± 0.06 a,a	17.43 ± 0.33 ab,c	7.00 ± 0.58 bc,a
Bacillus spp. + Azotobacter (3 mL)
Cotton	50.00 ± 15.28 a,a	17.00 ± 2.80 a,a	6.23 ± 1.09 a,a	9.53 ± 1.42 b,a	11.00 ± 2.00 a,a
Gauze	53.33 ± 12.02 a,a	17.16 ± 1.32 a,a	5.90 ± 0.47 a,a	4.95 ± 1.08 a,a	10.67 ± 2.96 a,a
Cocoon	50.00 ± 0.00 a,a	22.19 ± 3.60 a,a	4.80 ± 0.91 a,a	4.81 ± 1.77 ab,a	1.00 ± 0.00 a,b
S. boulardii (1000 mg)
Cotton	53.33 ± 3.33 a,a	17.64 ± 0.98 a,a	5.70 ± 0.30 a,a	4.50 ± 0.56 a,a	8.00 ± 1.00 a,a
Gauze	60.00 ± 0.00 a,a	19.53 ± 2.44 a,a	5.27 ± 0.59 a,a	4.92 ± 0.87 a,a	11.67 ± 1.76 a,a
Cocoon	66.67 ± 16.67 a,a	18.18 ± 0.03 a,a	5.63 ± 0.03 a,a	12.49 ± 0.26 ab,b	7.00 ± 0.58 bc,a
S. boulardii (1250 mg)
Cotton	36.67 ± 8.82 a,a	19.49 ± 0.26 a,a	5.13 ± 0.07 a,a	2.79 ± 0.63 a,a	10.33 ± 0.67 a,a
Gauze	26.67 ± 6.67 a,a	19.79 ± 1.35 a,a	5.10 ± 0.35 a,a	2.29 ± 0.89 a,a	12.00 ± 1.53 a,a
Cocoon	66.67 ± 16.67 a,a	17.30 ± 0.99 a,a	5.82 ± 0.32 a,a	9.05 ± 3.04 ab,a	2.67 ± 1.67 ab,b
S. boulardii (1500 mg)
Cotton	26.67 ± 3.33 a,a	17.92 ± 2.46 a,a	5.80 ± 0.81 a,a	2.38 ± 0.64 a,a	5.67 ± 2.67 a,a
Gauze	30.00 ± 5.77 a,a	20.69 ± 1.22 a,a	4.87 ± 0.29 a,a	2.12 ± 0.63 a,a	8.00 ± 3.06 a,a
Cocoon	66.67 ± 16.67 a,a	21.32 ± 3.29 a,a	5.07 ± 0.78 a,a	35.13 ± 9.66 c,b	6.00 ± 2.89 bc,a
S. cerevisiae (2.5 g)
Cotton	43.33 ± 18.56 a,a	20.47 ± 3.36 a,a	5.13 ± 0.76 a,a	4.69 ± 0.99 a,a	9.67 ± 4.67 a,a
Gauze	33.33 ± 8.82 a,a	14.85 ± 1.73 a,a	6.93 ± 0.86 a,a	3.76 ± 0.31 a,a	8.67 ± 2.60 a,a
Cocoon	50.00 ± 0.00 a,a	24.42 ± 2.60 a,a	4.20 ± 0.50 a,a	3.51 ± 0.74 a,a	1.00 ± 0.00 a,a
S. cerevisiae (3 g)
Cotton	33.33 ± 8.82 a,a	19.14 ± 2.36 a,a	5.40 ± 0.72 a,a	2.61 ± 0.60 a,a	9.33 ± 3.18 a,a
Gauze	50.00 ± 0.00 a,a	12.65 ± 6.38 a,a	3.53 ± 1.78 a,a	4.10 ± 0.26 a,a	13.67 ± 0.88 a,a
Cocoon	100.00 ± 0.00 a,b	17.19 ± 2.27 a,a	6.00 ± 0.70 a,a	20.68 ± 2.30 bc,b	8.00 ± 2.65 c,a
S. cerevisiae (5 g)
Cotton	43.33 ± 6.67 a,a	17.75 ± 1.30 a,a	5.70 ± 0.45 a,a	3.62 ± 0.31 a,a	7.67 ± 2.33 a,ab
Gauze	50.00 ± 10.00 a,a	17.82 ± 2.22 a,a	5.80 ± 0.78 a,a	3.59 ± 0.80 a,a	15.67 ± 2.73 a,a
Cocoon	50.00 ± 0.00 a,a	16.02 ± 0.89 a,a	6.53 ± 0.36 a,a	7.15 ± 0.55 ab,b	1.00 ± 0.00 a,b

The different letters indicate a significant (p < 0.05) difference; the first letter demonstrates differences between water and microbe treatments and the second one among substrates. Data are presented as the mean ± SE of five replicates.

). The effect of substrates tested on germination rate for trifoliate orange and pomegranate seeds revealed a close relationship between plant species and substrates in successful seed germination and growth. Seeds treated with beneficial microorganisms increased their germination ability. Trifoliate orange seeds with Bacillus spp.-Azotobacter spp. 2 mL, S. boulardii 1250 mg and S. cerevisiae 5 g had the highest germination rate (100%) in silkworm cocoon substrate (Table 2). A similar effect was observed with the use of beneficial microorganisms on pomegranate seeds placed in the same media. Seeds treated with Bacillus spp.-Azotobacter spp. 2 mL and S. cerevisiae 3 g increased their germination ability by 30–33.33% and 50–67.7%, respectively, in the silkworm cocoon substrate compared to cotton and gauze (Table 3).

3.2. Mean Germination Time (MGT)

In trifoliate orange, water-treated seeds germinated faster in cotton (7.15 days) than in gauze (11.89 days) and silkworm cocoon substrate (14.42 days). The MGT was the same between seeds treated with water and those treated with beneficial microorganisms in the same substrate. However, the application of Bacillus spp.-Azotobacter spp. 1 mL decreased the MGT to 8.53 days in seeds placed in silkworm cocoons, while the application of S. cerevisiae 2.5 g and 3 g increased the time to 19.08 and 15.66 days, respectively, on the same substrate. Seeds treated with these microorganisms required a shorter period of time to germinate on cotton and gauze substrates (Table 2). No difference in MGT was observed between the substrates and beneficial microorganisms for pomegranate seeds (Table 3).

3.3. Coefficient of Velocity of Germination (CVG)

The coefficient of velocity of germination values of trifoliate orange seeds were similar between treatments on cotton and gauze substrates. On the silkworm cocoon substrate, the application of Bacillus spp.-Azotobacter spp. 1 mL raised seed germination speed; in contrast, Bacillus spp.-Azotobacter spp. 2 mL and S. cerevisiae 2.5 g delayed seed germination, as these microbes duplicated the time needed for root formation compared to Bacillus spp.-Azotobacter spp. 1 mL treatment. Near CVG values were observed in all other treatments on the silkworm cocoon substrate. Among the substrates, seeds treated with water and S. cerevisiae 2.5 g and 5 g had greater CVG values in cotton than in gauze and cocoon (Table 2). In pomegranate, growth substrates and microorganisms had the same effect on CVG values (Table 3).

3.4. Germination Rate Index

Water-treated and microbe-treated trifoliate orange seeds had similar germination rate indices when placed in cotton, gauze and silkworm cocoon growth media. Differentiation between gauze and the other two substrates was observed in seeds treated with Bacillus spp.-Azotobacter spp. 1 mL, where the GRI was severely reduced (Table 2).

In pomegranate, seeds treated with Bacillus spp.-Azotobacter spp. 3 mL had higher and faster germination (greater GRI) when placed on cotton substrate compared to other treatments in the same vegetation media. Concerning silkworm cocoon substrate, the application of S. boulardii 1500 mg and S. cerevisiae 3 g increased seeds’ GRI. No differences in GRI were recorded between water-treated and microbe-treated seeds on gauze substrate (Table 3). Among the seed germination substrates, it seems that the silkworm cocoon promotes seeds’ germination when they have previously been treated with a beneficial microorganism (Bacillus spp.-Azotobacter spp. 1 mL, 2 mL, S. boulardii 1000 mg and 1250 mg and S. cerevisiae 3 g and 5 g).

3.5. Time Spread of Germination (TSG)

There were no differences in the time spread of trifoliate orange seed germination between treatments in each vegetation medium. In the cotton substrate, the TSG index ranged from 4 days for water-treated seeds to 15 days for Bacillus spp.-Azotobacter spp. 1 mL treated seeds. In gauze substrate, seeds needed more time for germination with the TSG index ranging from 6.33 days (Bacillus spp.-Azotobacter spp. 2 mL) to 13 days (S. cerevisiae 2.5 g). In silkworm cocoons, seeds treated with S. boulardii 1500 mg and S. cerevisiae 3 g and 5 g completed their germination in one day, with the TSG index ranging from 1 to 13 days (Table 2). The substrate type and pre-sowing seed treatments regulated the spread time of germination. In the silkworm cocoon, seeds treated with Bacillus spp.- Azotobacter spp. 1 mL and S. cerevisiae 2.5 g completed their germination in 1 or 2 days; however, in cotton and gauze growth media, seeds treated with the same microbes required 8–10 days to complete the germination process (Table 2).

In pomegranate, the time spread of germination was similar between treatments on cotton and gauze substrates. The TSG index ranged from 4.67 to 11 days for cotton and from 8 to 15.67 days for gauze substrates. In the silkworm cocoon, the TSG index was affected by pre-sowing seed treatment. Seeds treated with water, Bacillus spp.-Azotobacter spp. 3 mL, and S. cerevisiae 2.5 g and 5 g completed germination rapidly (TSG 1 day), while when seeds were treated with Bacillus spp.-Azotobacter spp. 2 mL, S. cerevisiae 3 g, and S. boulardii 1000 mg, the germination process was delayed (TSG 7–8 days).

The type of substrate had a critical role in the completion of the seed germination process. On cotton and gauze substrates, seeds had a longer germination period compared to the silkworm cocoon. Water-treated seeds needed 10.33–14 days for germination on cotton and gauze but only 1 day on silkworm cocoon media (Table 3).

3.6. Shoot and Root Fresh Weight

The shoot fresh weight of trifoliate orange seedlings treated and non-treated with microbes was similar, independently of the germination media used.

However, in Bacillus spp.-Azotobacter spp. 2 mL-treated seeds, the average shoot fresh weight was greater in gauze (0.34 g) than in cotton and silkworm cocoons (0.26 g). Concerning root fresh weight, all P. trifoliata seedlings had similar values, independent of growth medium or beneficial microorganism seed treatment (

Table 4. Trifoliate orange seedlings’ growth parameters when treated with beneficial microorganisms.

Treatments	Shoot Fresh Weight (g)	Root Fresh Weight (g)	Shoot Dry Weight (g)	Root Dry Weight (g)	Shoot Length (cm)	Root Length (cm)
Water
Cotton	0.35 ± 0.02 a,a	0.023 ± 0.003 a,a	0.137 ± 0.014 a,a	0.003 ± 0.0010 a,a	1.85 ± 0.24 a,a	1.46 ± 0.04 a,a
Gauze	0.31 ± 0.01 a,a	0.023 ± 0.003 a,a	0.115 ± 0.005 a,a	0.002 ± 0.0008 a,a	1.00 ± 0.50 a,a	0.96 ± 0.17 a,a
Cocoon	0.30 ± 0.03 a,a	0.020 ± 0.006 a,a	0.134 ± 0.013 ab,a	0.002 ± 0.0000 a,a	1.50 ± 0.58 a,a	1.27 ± 0.12 a,a
Bacillus spp. + Azotobacter (1 mL)
Cotton	0.35 ± 0.02 a,a	0.017 ± 0.003 a,a	0.129 ± 0.004 a,a	0.006 ± 0.0003 a,a	1.64 ± 0.15 a,a	1.33 ± 0.05 bc,a
Gauze	0.32 ± 0.03 a,a	0.040 ± 0.000 b,b	0.131 ± 0.007 a,a	0.004 ± 0.0005 a,a	0.10 ± 0.06 a,b	1.06 ± 0.17 a,a
Cocoon	0.31 ± 0.01 a,a	0.017 ± 0.007 a,a	0.118 ± 0.014 ab,a	0.003 ± 0.0008 a,a	0.75 ± 0.52 a,a	1.07 ± 0.52 a,a
Bacillus spp. + Azotobacter (2 mL)
Cotton	0.26 ± 0.02 a,a	0.020 ± 0.006 a,a	0.129 ± 0.014 a,a	0.004 ± 0.0003 a,a	1.17 ± 0.17 a,a	1.04 ± 0.07 a,a
Gauze	0.34 ± 0.02 a,b	0.023 ± 0.003 a,a	0.128 ± 0.006 a,a	0.002 ± 0.0006 a,a	0.50 ± 0.50 a,a	1.03 ± 0.24 a,a
Cocoon	0.26 ± 0.09 a,a	0.027 ± 0.003 a,a	0.120 ± 0.0003 ab,a	0.003 ± 0.0000 a,a	0.00 ± 0.00 a,a	0.98 ± 0.23 a,a
Bacillus spp. + Azotobacter (3 mL)
Cotton	0.25 ± 0.02 a,a	0.027 ± 0.003 a,a	0.103 ± 0.005 a,a	0.004 ± 0.0019 a,a	0.45 ± 0.29 a,a	1.07 ± 0.08 a,a
Gauze	0.21 ± 0.01 a,a	0.023 ± 0.003 a,a	0.107 ± 0.023 a,a	0.001 ± 0.0003 a,a	0.67 ± 0.44 a,a	0.77 ± 0.21 a,a
Cocoon	0.22 ± 0.02 a,a	0.027 ± 0.003 a,a	0.089 ± 0.004 a,b	0.002 ± 0.0009 a,a	0.83 ± 0.44 a,a	1.23 ± 0.23 a,a
S. boulardii (1000 mg)
Cotton	0.35 ± 0.03 a,a	0.027 ± 0.003 a,a	0.137 ± 0.010 a,a	0.007 ± 0.0009 a,a	1.74 ± 0.23 a,a	1.31 ± 0.02 a,a
Gauze	0.31 ± 0.05 a,a	0.027 ± 0.003 ab,a	0.136 ± 0.011 a,a	0.004 ± 0.0009 a,a	1.57 ± 0.79 a,a	1.18 ± 0.20 a,a
Cocoon	0.31 ± 0.03 a,a	0.020 ± 0.010 a,a	0.155 ± 0.009 b,a	0.006 ± 0.0035 a,a	1.07 ± 0.53 a,a	1.87 ± 1.08 a,a
S. boulardii (1250 mg)
Cotton	0.34 ± 0.01 a,a	0.030 ± 0.000 a,a	0.133 ± 0.021 a,a	0.004 ± 0.0006 a,a	1.67 ± 0.88 a,a	1.19 ± 0.09 a,a
Gauze	0.33 ± 0.01 a,a	0.023 ± 0.003 a,a	0.137 ± 0.007 a,a	0.003 ± 0.0012 a,a	1.06 ± 0.63 a,a	1.08 ± 0.15 a,a
Cocoon	0.27 ± 0.04 a,a	0.017 ± 0.007 a,a	0.136 ± 0.017 ab,a	0.002 ± 0.0006 a,a	0.63 ± 0.63 a,a	0.77 ± 0.37 a,a
S. boulardii (1500 mg)
Cotton	0.31 ± 0.02 a,a	0.027 ± 0.007 a,a	0.125 ± 0.008 a,a	0.005 ± 0.0018 a,a	2.00 ± 0.38 a,a	1.35 ± 0.12 a,a
Gauze	0.25 ± 0.01 a,a	0.030 ± 0.000 ab,a	0.104 ± 0.001 a,a	0.002 ± 0.0000 a,a	0.67 ± 0.33 a,a	0.93 ± 0.33 a,a
Cocoon	0.29 ± 0.04 a,a	0.030 ± 0.006 a,a	0.109 ± 0.017 ab,a	0.003 ± 0.0012 a,a	1.67 ± 0.44 a,a	1.67 ± 0.44 a,a
S. cerevisiae (2.5 g)
Cotton	0.37 ± 0.04 a,a	0.027 ± 0.003 a,a	0.142 ± 0.009 a,a	0.006 ± 0.0009 a,a	1.85 ± 0.28 a,a	1.16 ± 0.08 a,a
Gauze	0.32 ± 0.04 a,a	0.027 ± 0.003 ab,a	0.126 ± 0.014 a,a	0.003 ± 0.0009 a,a	1.30 ± 0.70 a,a	0.87 ± 0.20 a,a
Cocoon	0.22 ± 0.03 a,a	0.010 ± 0.006 a,b	0.097 ± 0.011 a,a	0.003 ± 0.0006 a,a	0.20 ± 0.20 a,b	1.27 ± 0.39 a,a
S. cerevisiae (3 g)
Cotton	0.37 ± 0.08 a,a	0.023 ± 0.003 a,a	0.138 ± 0.007 a,a	0.005 ± 0.0007 a,a	1.33 ± 0.67 a,a	1.31 ± 0.10 a,a
Gauze	0.29 ± 0.04 a,a	0.020 ± 0.000 a,a	0.123 ± 0.003 a,ab	0.002 ± 0.0000 a,b	0.33 ± 0.33 a,a	0.83 ± 0.03 a,a
Cocoon	0.28 ± 0.03 a,a	0.027 ± 0.003 a,a	0.110 ± 0.006 ab,b	0.002 ± 0.0007 a,b	0.67 ± 0.44 a,a	0.88 ± 0.19 a,a
S. cerevisiae (5 g)
Cotton	0.30 ± 0.04 a,a	0.020 ± 0.000 a,a	0.124 ± 0.006 a,a	0.004 ± 0.0003 a,a	1.97 ± 0.54 a,a	1.09 ± 0.12 a,a
Gauze	0.22 ± 0.01 a,a	0.023 ± 0.003 a,a	0.101 ± 0.011 a,a	0.003 ± 0.0003 a,a	0.33 ± 0.33 a,b	0.85 ± 0.27 a,a
Cocoon	0.23 ± 0.02 a,a	0.023 ± 0.003 a,a	0.095 ± 0.008 a,a	0.002 ± 0.0006 a,a	0.63 ± 0.20 a,b	0.73 ± 0.15 a,a

The different letters indicate a significant (p < 0.05) difference; the first letter demonstrates differences between microbes’ treatments and the second one among substrates. Data are presented as the mean ± SE of five replicates.

).

No differences were observed in the shoot fresh weight of pomegranate seedlings between water- and microbe-treated seeds when they were placed in cotton, gauze and silkworm cocoon growth media. However, cotton and silkworm cocoon substrates contributed to the formation of heavier shoots when seeds were treated with Bacillus spp.-Azotobacter spp. 1 mL and 3 mL; S. boulardii 1000 mg, 1250 mg, and 1500 mg; and S. cerevisiae 2.5 g and 5 g (

Table 5. Pomegranate seedlings’ growth parameters when treated with beneficial microorganisms.

Treatments	Shoot Fresh Weight (g)	Root Fresh Weight (g)	Shoot Dry Weight (g)	Root Dry Weight (g)	Shoot Length (cm)	Root Length (cm)
Water
Cotton	0.034 ± 0.003 a,a	0.010 ± 0.000 ab,a	0.018 ± 0.001 a,a	0.0004 ± 0.0001 a,a	1.16 ± 0.24 a,a	0.63 ± 0.12 a,a
Gauze	0.030 ± 0.001 a,a	0.008 ± 0.003 a,ab	0.017 ± 0.003 a,a	0.0006 ± 0.0001 a,a	1.14 ± 0.26 a,a	0.44 ± 0.07 a,a
Cocoon	0.048 ± 0.011 a,a	0.002 ± 0.001 a,b	0.019 ± 0.006 a,a	0.0003 ± 0.0001 a,a	0.40 ± 0.40 a,a	0.43 ± 0.28 ab,a
Bacillus spp. + Azotobacter (1 mL)
Cotton	0.045 ± 0.003 a,a	0.017 ± 0.001 b,a	0.012 ± 0.001 a,a	0.0009 ± 0.0001 a,a	1.69 ± 0.21 a,a	1.01 ± 0.02 a,a
Gauze	0.033 ± 0.000 a,b	0.011 ± 0.002 a,b	0.014 ± 0.002 a,a	0.0007 ± 0.0000 a,b	1.35 ± 0.22 a,a	0.84 ± 0.06 b,a
Cocoon	0.048 ± 0.001 a,a	0.003 ± 0.000 a,c	0.021 ± 0.001 a,a	0.0003 ± 0.0000 a,c	0.00 ± 0.00 a,b	0.15 ± 0.03 a,b
Bacillus spp. + Azotobacter (2 mL)
Cotton	0.035 ± 0.008 a,a	0.012 ± 0.002 b,a	0.013 ± 0.005 a,a	0.0008 ± 0.0004 a,a	0.85 ± 0.48 a,a	0.58 ± 0.33 a,a
Gauze	0.031 ± 0.001 a,a	0.007 ± 0.001 a,ab	0.012 ± 0.002 a,a	0.0005 ± 0.0001 a,a	0.86 ± 0.07 a,a	0.51 ± 0.10 a,a
Cocoon	0.048 ± 0.001 a,a	0.002 ± 0.001 a,b	0.015 ± 0.000 a,a	0.0005 ± 0.0001 a,a	0.50 ± 0.06 a,a	0.90 ± 0.03 b,a
Bacillus spp. + Azotobacter (3 mL)
Cotton	0.041 ± 0.004 a,ab	0.012 ± 0.003 b,a	0.014 ± 0.002 a,a	0.0008 ± 0.0004 a,a	1.35 ± 0.36 a,a	0.75 ± 0.22 a,a
Gauze	0.034 ± 0.003 a,b	0.010 ± 0.001 a,ab	0.012 ± 0.002 a,a	0.0006 ± 0.0001 a,a	1.03 ± 0.06 a,a	0.52 ± 0.04 a,a
Cocoon	0.055 ± 0.003 a,a	0.002 ± 0.000 a,b	0.017 ± 0.004 a,a	0.0003 ± 0.0001 a,a	0.50 ± 0.50 a,a	0.50 ± 0.21 ab,a
S. boulardii (1000 mg)
Cotton	0.048 ± 0.006 a,a	0.006 ± 0.001 a,a	0.013 ± 0.002 a,a	0.0004 ± 0.0000 a,a	1.19 ± 0.10 a,a	0.64 ± 0.03 a,a
Gauze	0.031 ± 0.002 a,b	0.009 ± 0.000 a,a	0.016 ± 0.000 a,a	0.0005 ± 0.0001 a,a	0.90 ± 0.13 a,a	0.38 ± 0.06 a,b
Cocoon	0.044 ± 0.001 a,ab	0.002 ± 0.000 a,b	0.020 ± 0.000 a,a	0.0003 ± 0.0000 a,a	0.00 ± 0.00 a,b	0.17 ± 0.02 a,c
S. boulardii (1250 mg)
Cotton	0.058 ± 0.001 a,a	0.004 ± 0.002 a,a	0.017 ± 0.001 a,a	0.0004 ± 0.0001 a,a	1.40 ± 0.23 a,a	0.58 ± 0.11 a,a
Gauze	0.031 ± 0.003 a,b	0.006 ± 0.001 a,a	0.016 ± 0.003 a,a	0.0004 ± 0.0001 a,a	0.98 ± 0.08 a,a	0.36 ± 0.03 a,ab
Cocoon	0.050 ± 0.008 a,ab	0.002 ± 0.001 a,a	0.020 ± 0.001 a,a	0.0003 ± 0.0000 a,a	0.13 ± 0.13 a,b	0.17 ± 0.03 a,b
S. boulardii (1500 mg)
Cotton	0.045 ± 0.006 a,ab	0.006 ± 0.001 a,a	0.012 ± 0.003 a,a	0.0007 ± 0.0002 a,a	1.46 ± 0.13 a,a	0.75 ± 0.09 a,a
Gauze	0.033 ± 0.001 a,a	0.007 ± 0.001 a,a	0.016 ± 0.002 a,a	0.0005 ± 0.0001 a,a	0.98 ± 0.27 a,a	0.36 ± 0.06 a,a
Cocoon	0.049 ± 0.000 a,b	0.002 ± 0.001 a,b	0.017 ± 0.004 a,a	0.0004 ± 0.0001 a,a	0.65 ± 0.38 a,a	0.50 ± 0.13 ab,a
S. cerevisiae (2.5 g)
Cotton	0.044 ± 0.003 a,a	0.004 ± 0.001 a,a	0.014 ± 0.003 a,a	0.0004 ± 0.0001 a,a	0.88 ± 0.46 a,a	0.39 ± 0.10 a,ab
Gauze	0.031 ± 0.001 a,b	0.009 ± 0.001 a,b	0.016 ± 0.002 a,a	0.0004 ± 0.0000 a,a	1.15 ± 0.23 a,a	0.57 ± 0.09 ab,a
Cocoon	0.049 ± 0.000 a,a	0.001 ± 0.000 a,a	0.021 ± 0.002 a,a	0.0003 ± 0.0001 a,a	0.10 ± 0.10 a,a	0.13 ± 0.03 a,b
S. cerevisiae (3 g)
Cotton	0.053 ± 0.008 a,a	0.006 ± 0.000 ab,a	0.016 ± 0.003 a,a	0.0006 ± 0.0001 a,a	1.47 ± 0.23 a,a	0.60 ± 0.13 a,a
Gauze	0.029 ± 0.001 a,a	0.009 ± 0.001 a,a	0.015 ± 0.002 a,a	0.0004 ± 0.0000 a,a	1.17 ± 0.08 a,a	0.41 ± 0.01 a,a
Cocoon	0.048 ± 0.009 a,a	0.002 ± 0.001 a,b	0.016 ± 0.005 a,a	0.0006 ± 0.0002 a,a	0.58 ± 0.30 a,a	0.48 ± 0.17 ab,a
S. cerevisiae (5 g)
Cotton	0.046 ± 0.007 a,a	0.006 ± 0.001 a,a	0.010 ± 0.004 a,a	0.0005 ± 0.0000 a,ab	1.35 ± 0.08 a,a	0.78 ± 0.06 a,a
Gauze	0.026 ± 0.002 a,b	0.011 ± 0.000 a,b	0.012 ± 0.001 a,a	0.0006 ± 0.0001 a,b	1.49 ± 0.12 a,a	0.53 ± 0.06 ab,a
Cocoon	0.039 ± 0.000 a,ab	0.001 ± 0.000 a,c	0.020 ± 0.000 a,b	0.0003 ± 0.0001 a,a	0.40 ± 0.06 a,b	0.20 ± 0.06 a,b

The different letters indicate a significant (p < 0.05) difference; the first letter demonstrates differences between microbes’ treatments and the second one among substrates. Data are presented as the mean ± SE of five replicates.

).

The root fresh weight of pomegranate seedlings was significantly higher on cotton and gauze substrates. Seedlings treated with Bacillus spp.-Azotobacter spp. 1 mL, 2 mL, and 3 mL and S. boulardii 1000 mg and 1500 mg formed the heavier root systems of all treatments in these substrates. Seedlings in silkworm cocoons formed a weak root system with recorded fresh weights of 0.001–0.002 g (Table 5).

3.7. Shoot and Root Dry Weight

Cotton and gauze growth media did not affect the shoot dry weights of trifoliate orange seedlings developed from water- and microbe-treated seeds. In the silkworm cocoon substrate, the microbe treatments produced the maximum and minimum shoot dry weight values. Seeds treated with S. boulardii 1000 mg had the greatest shoot dry weight (0.115 g) but when treated with Bacillus spp.-Azotobacter spp. 3 mL and S. cerevisiae 2.5 g and 5 g had the lowest shoot dry weight values (0.089 g, 0.095 g, and 0.099 g, respectively). Growth media and beneficial microorganisms did not affect the root dry weight of trifoliate orange seedlings (Table 4). Neither substrates nor microbes affected the shoot–root weight ratio.

In pomegranate, treatments with Bacillus spp.-Azotobacter spp. 1 mL and S. cerevisiae 5 g exhibited higher shoot dry weights on the silkworm cocoon substrate than in cotton and gauze growth media. The shoot dry weights of the seedlings were 0.010–0.018 g in cotton, 0.012–0.017 g in gauze, and 0.015–0.021 g in silkworm cocoon substrates. There was no effect of growth media or microbe application on seedling root weight (Table 5). The shoot–root weight ratio was similar between treatments.

3.8. Seedling Shoot and Root Length

The substrates allowed shoot and root growth of trifoliate orange seedlings. Shoot and root elongation values in water-treated seeds were 1–1.85 cm and 0.97–1.46 cm, respectively. Seeds treated with Bacillus spp.-Azotobacter spp. 1 mL and S. cerevisiae 5 g formed shorter shoots in the gauze substrate, while S. cerevisiae 2.5 g treatment significantly restricted shoot growth in cocoons (Table 4).

In pomegranate, the combination of cocoon substrate and microbes had a negative effect on seedling shoot and root growth. Shoots and roots formed by seeds treated with Bacillus spp.-Azotobacter spp. 1 mL, S. boulardii 1000 mg and 1250 mL, and S. cerevisiae 5 g showed significantly lower development in cocoons than on cotton or gauze substrates (Table 5).

4. Discussion

Several factors such as temperature, water availability, oxygen, light, and substrate type are involved in the seed germination process [27,36,38,76,77,78]. For instance, temperature affects the enzymatic activities necessary for breaking dormancy and initiating metabolic processes. Studies have shown that each plant species has an optimal temperature range for germination [79], while water imbibition is the first step in germination, reactivating metabolic pathways, since oxygen availability is crucial for cellular respiration [80]. Knowledge of the biochemical and molecular mechanisms during seed germination and an understanding of external factors involved in the process are essential for optimizing agricultural practices, enhancing ecological restoration efforts, and creating more resilient, sustainable tree fruit production [81,82,83].

The highest germination rates of Poncirus trifoliata seeds without microbial presence were exhibited on silk cocoons (83%); similar rates were recorded when (a) seeds of this species were set on a mixed soil medium enriched with sand, leaf mold, and rice husk in a 1:1:1:1:1 ratio; (b) seeds were extracted from fresh and stored fruits and germinated on germination paper; and (c) seeds were extracted from fresh fruits and germinated on wet rolled towel [36,84,85]. On cotton, equivalent low germination rates of orange trifoliate seeds were observed when the seeds were exposed to the above mixed soil media (a) enriched with poultry manure and sawdust [36]. The contribution of gauze media to seed germination was found to be extremely low under environmentally controlled and/or field conditions [36,84].

In this work, pomegranate cv. Wonderful seed germination rates remained unaffected by the type of germination medium exposure (46.67–50.00%); these rates were higher than those recorded in fresh seeds of Kashgar Akeqishiliu, Yecheng Suanshiliu, Hotan CeLe1#shiliu, and Turpan Suanshiliu varieties (16–20%) when germinated on moist filter paper or additionally exposed to gibberellic acid (22–35%) [27]. In contrast, these varieties showed higher germination rates after exposure to chemical scarification with H₂SO₄ and/or cold stratification for 2 months [27].

Trifoliate orange seeds treated with beneficial microorganisms showed increased germination ability. P. trifoliata seeds with Bacillus spp.-Azotobacter spp. 2 mL, S. boulardii 1250 mg and S. cerevisiae 5 g achieved the maximum germination rate (100%) on the silkworm cocoon substrate. This positive outcome was formed through a complex interaction (cocoon–microorganisms–P. trifoliata seed germination), where pH compatibility from all parts was technically assured. Formation of silk fiber from the silk gland of B. mori larvae takes place segmentally by gland divisions, where each one has its own pH standards with values ranging from 4.8 to 6.9 [86]. P. trifoliata seeds germinate at pH 5.7 [87]; most of the Bacillus spp. are capable of growth within the pH range 4–9.5 [88]. Furthermore, the yeasts S. cerevisiae and S. boulardii preferably grow at pH values of 4–6 and 2–8 (with the optimal being between 4.5 and 6.5), respectively [89,90,91,92]. All these overlapping pH value ranges suggest a clear multipartite functionality for the benefit of P. trifoliata seed germination.

In nature, the seeds of many Citrus species have been found to carry Bacillus species to their microbiome with auxin-inducing properties [93]. Bacillus subtilis OSU-142 has been reported to enhance root formation in P. trifoliata rootstocks; however, this microbial enhancement of orange trifoliate plant growth is not directly comparable to the physiological stage of the seed, where information is extremely limited [94]. Furthermore, mycorrhizal inoculation of P. trifoliata seeds enhances root system development of the seedlings; however, it may not be classified as a direct interaction between seeds and fungi, since it requires a two-month period for initial observation in a potted environment [95]. In our work, the effects of microbial and germination media for both plant species were measured close to the time of seed microbial inoculation and deposition on germination media; late-stage evaluations of S. cerevisiae in P. trifoliata seeds (7 months after sowing) also concluded that the presence of yeasts positively contributed to the growth and development of this Citrus species [96].

Similarly, microbial inoculums positively affected pomegranate seed germination (30–67.7%) when treated with Bacillus spp.-Azotobacter spp. 2 mL and S. cerevisiae 3 g in silkworm cocoons. The applied microbial inoculums reached optimal and thereafter suboptimal germination rates; the suboptimum formed conditions may due to (a) excessive microbial metabolites and/or induction of seed germination mechanisms under stressful conditions, (b) saturated microbial presence on germination media where porosity and other physical and chemical attributes resulted in such conditions, (c) aberrant facilitating germination mechanisms (e.g., solubilization nutritive elements and activation or mobilization of bioactive compounds) from the typical physiological functionality of the seed, (d) permeability alterations on seed tissues due to excessive microbial presence, and (e) induction of non-affordable oxidative stress between microorganisms (interspecific/intraspecific) and/or seed tissues [97,98,99]. Furthermore, the pH compatibility of P. granatum seeds with germination media and microorganisms revealed a smaller overlapping pH value range due to less acidic and more alkaline plant preferences for germination (5.5–7.2) [100].

In P. trifoliata, water-treated seeds germinated faster in cotton than on gauze and silkworm cocoon substrates without microbial supplementation; in the case of Bacillus spp.-Azotobacter spp. supplementation, cocoons provided mean germination times that were close to those of cotton. In most cases, with all germination media and/or microbial supplementation, P. trifoliata exhibited lower mean germination times in comparison to Citrus limetta seeds when exposed to sand and paper roll media [101]. It should be mentioned that the mean germination time does not reveal the synchronicity of the phenomenon in time, and it must be evaluated jointly with other germination indices [102]. For all other cases where administration of microorganisms increased the mean germination time, suboptimal plant cellular signaling, hormonal status, and environmental sensing of the seed could provide potential explanations [103,104]. In contrast, the mean germination time in pomegranate seeds was found to be independent of microbial presence and germination media; this may be due to different chronobiological cascade signaling and sensing [105]. No differences in seed germination rate in the presence of microorganisms have been recorded for plant growth-promoting Bacillus species on the parasitic weed Cuscuta campestris [106].

The CVG parameter in P. trifoliata germination tests was found to be independent of germination media when it was not supplemented with microorganisms; however, microbial inoculations increased germination speed, as in the case of Bacillus spp.-Azotobacter spp. 1 mL inoculum when seeds were set on silkworm cocoons. Similarly, Bacillus subtilis and Pseudomonas aeruginosa provided higher CVG rates when supplemented with Pisum sativum seeds grown in 1% NaCl [107]. In pomegranate, growth substrates and microorganisms had the same effect on CVG values; a similar lack of effect was found in non-drought-stressed Secale montanum seeds when supplemented with Bacillus cereus, Pseudomonas aeruginosa, Azospirillum lipoferm, and Azotobacter chroococcum [108]. Among substrates, seeds treated with water and S. cerevisiae 2.5 g and 5 g had greater CVG values in cotton than in gauze and cocoon. Medical-grade absorbent cotton and cotton-based gauze exhibit a pH range of 5.5–7.5; however, this attribute is related to higher CVG values [109]. Other factors should also be taken into consideration.

The germination rate index was independent of germination media for P. trifoliata; in contrast, pomegranate’s GRI was cotton and microbial-specific in order to advance parametrical values. TSG value specificity was also recorded for both plant species when exposed to different germination media and microorganisms.

Microorganisms induced greater root and shoot fresh weights in P. trifoliata and P. granatum germinated seed lines; germination media (silk cocoon, cotton, and gauze) did not significantly contribute to the developmental process of these two plant species. However, based on our data, the recorded low significance of germination media is converted to great, highly sensitive importance when microorganisms are hosted in these volumes. The structural and physicochemical properties of germination materials are able to enhance the 3D topological presence of microorganisms, which optimally provides bioactive compounds and/or signaling molecules to seeds for plant growth [110,111,112,113,114]. There are many plant species in which bacteria and yeasts have been reported to support shoot and root growth; however, most of them do not refer to agricultural tree species and/or describe the effects long after germination [14,115,116,117,118,119].

In all experimental sets for both plant species, the shoot–root weight ratios were found to be similar, independently of the germination media and/or biotic exposure. Plant growth-promoting bacteria and yeasts are able to (a) induce hormone effects in seeds and plantlets and (b) alter the shoot–root ratio under stress conditions [10,17,120,121]. It is also possible that evaluation at a later physiological stage (e.g., young plantlets) may reveal other chronologically based effects of beneficial microbe–plant interactions [122,123]. Studies at a later stage advance the ability of germinated seeds and young plantlets to release larger amounts of secretions; these conditions can solidify further forms of communication between microorganisms and plants [124].

Raw silkworm cocoon is characterized by mean thickness, high porosity (61.9%), high water absorption ability, and thermal properties [125], which can create an environment suitable for seed germination. In addition, sericin, a hydrophilic molecule produced by B. mori, can be used as a nitrogen source for plant growth [126]. Adding 2% silkworm sericin to the polymer seed coating film increased the germination percentage in Lablab beans [63]. Similarly, silk fibroin seed coating enhanced wheat seedling development and root elongation at low temperatures [51]. Herein, P. trifoliata seeds had a greater germination rate and well-developed shoot and root systems in silkworm cocoons, revealing the positive effect of sericin and silkworm cocoon structure on seedling emergence. However, these effects seemed to be plant species-dependent as no differences in germination and seedling growth were observed in pomegranate seeds between the substrates used. The different behavior in germination profiles of P. trifoliata and P. granatum seeds on the same substrate revealed the specific needs of these plant species in order to propagate.

The physical properties of the substrates could create suitable conditions for enhancing the percentage of seed germination, MGT, CVG, GI, and seedling growth. Listea glutinosa seeds germinated faster in vermiculate medium than in sand or on filter paper [127]. In this work, the CVG, MGT, and GRI indices of trifoliate orange were 30–50% higher than those of cotton. In contrast, in pomegranate seeds, germination was completed in 18–20 days in all substrates, but in cocoon, the TSG was only 1 day.

Biopriming seeds has a positive effect on seed germination and plantlet growth [8,128,129]. Corn and lettuce plants increased their biomass, chlorophyll content, and nutrient uptake ability when inoculated with Saccharomyces cerevisiae. Similarly, foliar application or seed treatment of dried S. cerevisiae stimulated plant growth [130]. The Bacillus subtilis GIBI 200 strain promoted tomato seedling growth by increasing plant length and shoot and root fresh weights [131]. Seeds of legumes, vegetables, cereals and oilseed plants exhibited higher and faster germination when treated with probiotics (L. plantarum 14917 and S. boulardii CNCM I-745) in the imbibition phase [20]. These results are consistent with our findings when silkworm cocoon substrates were used in the presence of S. cerevisiae, S. boulardii, and Bacillus spp.-Azotobacter spp., which promoted trifoliate orange and pomegranate seed germination.

The combination of substrate, beneficial microbes, and biopriming solution concentration could affect specific germination parameters and seedling growth. In silkworm cocoons, trifoliate orange seeds treated with Bacillus spp.-Azotobacter spp. 1 mL germinated faster, while pomegranate seeds primed with S. boulardii 1250 mg had greater and faster germination ability. In cotton, trifoliate orange seedlings formed longer shoots, particularly when the seeds were treated with S. cerevisiae 2.5 g and 3 g. Similar growth patterns were observed in pomegranate seedlings; Bacillus spp.-Azotobacter spp. 1 mL, S. boulardii 1000 mg and 1250 mL, and S. cerevisiae 5 g promoted increased shoot and root lengths on cotton and gauze substrates.

This work has revealed unique information about bacterial and yeast microecosystemic contributions to different types of natural fiber substrates, which can benefit P. trifoliata and P. granatum. Both fiber types and microorganisms are promising tools for further advancement of seed technology for these species (coating, novel mixed fiber and microbial delivery systems on seeds, and formulation of agrochemical products to enhance germination and growth) with minor residual effects on the environment [132]. Examination of microbial signaling, exudation of metabolites, interaction of the outer microbial surface with tree seeds, and (on the contrary) induced alterations of the outer surface of seeds to benefit microorganisms are needed if novel practices for Citrus and pomegranate propagation, in relation to their commercial development, are to emerge from this microscale mechanistic practice.

5. Conclusions

Seeds from two tree species exposed to different microbial and germination media were found to be capable of achieving up to 100% germination rates [orange trifoliate (Poncirus trifoliata) on cocoon substrate with 2 mL Bacillus spp.-Azotobacter spp. and pomegranate (Punica granatum) on cocoon substrate with 3 g S. cerevisiae exposure, respectively], thus solving problems that may be encountered in the conservation, cultivation, and sexual propagation of these species using sustainable techniques for resilient agriculture.

Bacterial complexes and yeast supplementation revealed advancement in germination rates and synchronicity; the presence of microorganisms on insect- and plant-based fiber materials provides specific types of interactions for tree species seed germination. Even at the initial physiological stage of the plant life cycle (seed germination) where plant secretions are not excessive, interactions are established for the benefit of plants. Examination of the physicochemical properties of multipartite interactions such as pH (pH for seed germination; pH of silk fiber; pH of medical cotton and cotton gauze) revealed a valid range of values, where interaction is not stressful for either plant or microorganisms. In this work, the uses of Saccharomyces boulardii have been expanded to include tree growth supplementation in addition to its previously known contributions to medicine.

Further work is needed to (a) understand the functionality of silk and cotton fiber in seed germination based on their physicochemical properties, (b) reveal the quantitative and qualitative role of plant exudates in the signaling of plant growth-promoting bacteria and yeasts for mutual or plant-sided benefits, (c) examine the timing of chemotactic interactions between this multipartite co-existence, and (d) investigate whether potential alterations in tissue formation patterning occur while these microorganisms affect early plant growth stages.