Essential Quality Attributes of Culture Media Used as Substrates in the Sustainable Production of Pre-Basic Potato Seeds

Abstract

The sustainability of the primary sector is closely linked to meeting the demand for seeds using agro-industrial waste and bioresidues. Sustainability is a multidimensional concept focused on achieving environmental health, social justice, and economic viability. To this end, an experiment was designed based on a combination of biotechnological strategies accessible to many individuals. The first strategy involves the use of compost and vermicompost as cultivation substrates; the second is the in vitro acclimatization of potato plants to these substrates; and the third is the incorporation of Trichoderma asperellum into these substrates to determine the synergistic effect of both. The compost used in this work came from sewage sludge from an agri-food company (Cp); a dining room and pruning waste from a university campus (Cu); and vermicomposted coffee pulp waste (Cv). Each sample was mixed with coconut fiber (Fc) in proportions of 100, 75, 50, and 25%. In the resulting mixtures, María Bonita variety vitroplants were planted and placed in a greenhouse. The biometric response in the three cases indicated a dependence on the type of compost and the proportion of the coconut fiber mixture. The inoculation of Trichoderma asperellum with sewage sludge compost increased stem thickness (42.58%) and mini-tuber weight (6.74%). In contrast, uninoculated treatments showed the best performance in terms of the number of mini-tubers. A 50:50 mixture of sewage sludge compost with coconut fiber and without inoculation of Trichoderma asperellum was the best treatment for the production of pre-basic seeds of the María Bonita potato variety. The use of composted agricultural waste and bioresidues is shown as a valid and low-cost alternative for the sector, even independently of the incorporation of additional inoculants.

1. Introduction

The sustainable production of pre-basic potato seeds requires carefully formulated culture media that can support the growth and development of potato tissues while maintaining their genetic integrity. Potato seeds are crucial inputs for achieving self-sufficiency in food production, as they form the foundation for subsequent generations of potato crops. Potato is a strategic crop that can significantly contribute to mitigating hunger and poverty, which are crucial challenges currently faced globally [1]. Potatoes provide more food per unit of water input compared to rice, wheat, and corn, making them a valuable food source in areas with limited water resources [2].

In the context of world crop production volumes, the 370 million metric tons of potato produced per year rank fourth overall (FAO 2018) and are regularly consumed worldwide (47 kg/person per year, on average) [3]. Even so, the consumption of 19.2 kg per capita per year in Venezuela reveals a problem of scarce food supply and/or sustained undernourishment in the past decade [4]. The cultivated area of some 24,498 hectares, concentrated in the states of Mérida, Lara, Táchira, and Trujillo [5], demands approximately 2400 seeds/ha [6]. Unfortunately, the production of potato seeds in Venezuela is marginal, and farmers must regularly rely upon international suppliers [7]. The use of depleted or degenerated seeds has contributed to the contamination of soils with pests and diseases [8].

Parallel to the scenario described above, Latin America has witnessed important technological advances in the field of seed potato breeding [9]. These advances generally involve the use of large amounts of peat [10] and virgin mountain land as a substrate. Faced with these facts, an increase in waste generation is predicted to be proportional to the increase in food consumption, which is estimated to increase by 60% by 2050 [11]. These include agricultural waste, forestry waste, manure, sewage sludge, other materials classified as byproducts of the food industry that never become waste, and biowaste [1].

For the use of many types of organic waste, proper composting achieves multiple objectives since it stabilizes the material by eliminating phytotoxicity, pathogens, and weed seeds. Only stabilized products will generate the beneficial effects expected of soil fertilizers and/or cultivation substrates. Compost-enriched substrates, especially those with 20% or 45% compost, produce larger seedlings with thicker stems, greater fresh and dry weights, and a greater number of leaves [12,13,14,15,16]. Farmers highly value the nutrient-rich properties of compost and vermicompost, as they directly contribute to reduced production costs and increased yields in ornamental plants, vegetables, and fruits [17]. For instance, the incorporation of vermicompost has been shown to improve the pH, electrical conductivity, and levels of essential nutrients such as nitrogen, phosphorus, potassium, calcium, and magnesium. The use of vermicompost has also been found to enhance the growth and nodulation of Phaseolus plants [18], while the combination of vermicompost and rice husk improved the overall biometric response of tomato plants [19]. However, the benefits of compost and vermicompost extend beyond just their nutritional value. Their incorporation into the soil can lead to increases in humus-enzyme complexes in rice crops, which in turn enhances plant nutrition, quality, and yield [20]. Furthermore, vermicompost has been associated with improved tolerance to water stress [21] and reduced incidence of Phytophthora infestans [22]. This makes it a valuable tool in organic agriculture not only as a peat substitute in growing media, but also as a compost tea for foliar application as a biofertilizer and disease control agent.

The benefits of compost and vermicompost application in agriculture are multifaceted, with the biofertilization aspect being one of the most significant. Microbial communities, including plant growth-promoting microorganisms, play a crucial role in agroecosystems as they help restore and reinforce functions essential for their balance and health [23]. Biofertilizers, defined as products containing live microorganisms capable of colonizing the rhizosphere or plant tissues and inducing growth, have great potential to improve crop yields through environmentally friendly mechanisms [24]. Microorganisms connect soil, water and air, serve as the backbone of biogeochemical cycles, and maintain the flow of nutrients in terrestrial ecosystems. Microorganisms were the origin of the edaphic organic matter (OM) from which plants later developed. As microorganisms are microscopic, their importance in the global and vital processes of the planet, specifically in the nutrition of plants, was not appreciated until several decades ago. Microorganisms consolidate the particles in the soil, forming very important aggregates to hold soil particles together. Owing to the benefits provided by microorganisms to the ecosystem, they are collectively distinguished as “plant growth-promoting microorganisms” (PGPM). There are countless examples of the benefits that microorganisms provide to the soil and plants. Specifically, research has focused on the role of Trichoderma in accelerating the decomposition of OM and its humification by increasing the degradation rates of cellulose, hemicellulose, and lignin [25], which increases the availability of nutrients for plants [26,27,28,29,30], controls pathogens by inducing defense responses in plants [31,32,33,34], and producing phytohormones [35,36,37], siderophores [38,39], vitamins, protective enzymes, and antibiotics, further contributing to soil sustainability and optimal agricultural productivity. Trichoderma has been shown to use direct antagonism and competition in the rhizosphere, where it modulates its composition and interactions with other microorganisms [40,41]. During plant colonization, in the roots or as an endophyte [42], Trichoderma has developed the ability to communicate with plants and produce numerous multifaceted benefits for its host [43]; these benefits can be increased when Trichoderma is combined with composted OM fertilizer [44,45,46,47,48]. Compared with the absence of manure and Trichoderma, the presence of composted manure combined with Trichoderma significantly increased the growth and yield of shallot [49] cocoa [50], potato [51], oil palm [50], lettuce [51], and spinach [41], among others. The mechanisms by which Trichoderma improves the growth of tomato and pepper seedlings in nurseries and greenhouses are related to its ability to populate the substrate due to its high production of spores, its ability to produce siderophores that guarantee access to scarce nutrients, and its ability to produce indoleacetic acid, which is advantageous for plant cellular growth and phosphate (P) solubilization [52]. In previous tests, the quality of the composting process was evaluated, as was that of the compost itself, in terms of fertilization and growth-promoting effects on tomato, radish and cucumber lettuce seedlings [53]. Therefore, the aim of this work was to test the possible synergistic effect of inoculation with T. asperellum on plants by using composted and vermicomposted material as a culture medium.

2. Materials and Methods

The research was carried out in a greenhouse belonging to the Academic Unit of the National Experimental University of Táchira, UNET, located in La Pradera, Jáuregui municipality of Táchira state, Venezuela, at 8°11′21.5′ N/71°57′54.4′′ W and an altitude of 1750 m above sea level; its average temperature is 18 °C, and its average annual rainfall is between 1100 and 1200 mm. The average relative humidity is 73%, and the climate zone is classified according to Holdridge as a premontane humid forest (bh-pm) [54]. The Laboratory for the Recovery of Compostable Waste of the UNET was used to carry out the analyses described, unless otherwise indicated. This research was conceived as a continuation of the evaluation of the quality parameters of compost from three compostable mixtures, prepared from agro-industrial waste, biowaste, and agricultural waste [55]. The proportions of waste used to obtain compostable mixtures were calculated according to Richards for their optimization in terms of C/N and moisture content [56]. Therefore, the compost used in the test was wastewater sludge compost from the food industry (Cod. LER 02 01 03) (European Parliament 2014) [57], designated Cp; biowaste compost from the UNET University Campus (Cod. LER 20 01 08-20 02 01) [57], which comprises kitchen waste composted with garden pruning, designated Cu; and vermicompost comprising coffee pulp (Cod. LER 02 01 03) [57], designated Cv. The pulp is a byproduct of the wet processing of the coffee fruit and is composed of the mesocarp of the cherry or berry, which is located just below the pericarp or skin [58]. This product was purchased from the National Institute of Agricultural Research INIA located in the Junín municipality of the state of Táchira, Venezuela.

2.1. Laboratory Tests

2.1.1. Physical Assessment

The compost and coco fiber mixtures were analyzed for moisture content and solids in the fraction passing through a 4.5 mm sieve and less than 16 mm in size. The moisture and solids contents in the <16 mm fraction were subsequently determined. For this purpose, a 50 cm³ aliquot of sample prepared in a tared glass was weighed (accuracy of 0.001 g). The sample was dried at 70 ± 5 °C in a forced-air oven for 24 h. The mixture was subsequently allowed to cool in a desiccator for 30 min. The weight was determined again with an accuracy of 0.001 g. Total solids: TS (%) = b/a × 100, where b is the mass (g) of the dry sample at 70 ± 5 °C and a is the mass (g) of the wet sample [59]. The material was kept in the laboratory at an average temperature of 24 °C during the study [60] to measure the physical properties of the mixtures, including total porosity ($\mathrm{T}\mathrm{P}\mathrm{S}\left(\mathrm{\%}\right)=(\mathrm{V}\mathrm{a}+(\mathrm{P}\mathrm{H}-\mathrm{P}\mathrm{S})/\mathrm{P}\mathrm{a})/\mathrm{V}\mathrm{c}\times 100);$ aeration porosity (AP (%) = Va/Vc × 100%); apparent density (Bd (Mg·m⁻³) = PS/Vc); particle density (Pd (Mg·m⁻³) = Bd/(1 − PT/100)); and water retention capacity ($\mathrm{T}\mathrm{W}-\mathrm{H}\mathrm{C}\left(\mathrm{\%}\right)=(\mathrm{P}\mathrm{H}-\mathrm{P}\mathrm{S})/\mathrm{V}\mathrm{c}\times 100),$ where Va is the volume drained (cm ³), PH is the wet weight of the sample (g), PS is the dry weight of the sample (g), Pa is the specific weight of water (1 g cm⁻³), and Vc is the volume of the tube or cylinder (cm³).

For the determination of physical properties, porometers made of PVC tubes were prepared. Each had a diameter of 7.5 cm, of 15 cm, and a foot composed of the same material, which was sealed as a base and perforated on the side to allow water circulation. A ring was added as an extension of the cylinder to prevent sample loss due to expansion when it became wet. The samples were used to fill the porometers to their maximum capacity. Settling was promoted by dropping the porometers twice from a height of 7.5 cm and then again filling them to the upper edge. The cylinders with the samples and the upper rings were placed in a container with a water level just below the upper edge. This process forced moisture into the sample via the bottom holes, allowing the air to escape freely through the upper face. The samples were left in the water until the next day to standardize the saturation process. After 24 h, they were extracted from the container with water and levelled with a spatula. The samples were fitted with a cloth attached to a rubber band on top and completely immersed for 30 min. Rubber plugs were then placed in the bottom holes before removal from the water. Once removed, each porometer with its sample was placed on paths, the trays were removed to measure the volume of water drained in ten minutes, the wet sample was extracted from the porometers, and its dry weight was determined at 105 °C.

2.1.2. Chemical Evaluation: pH, Electrical Conductivity, Organic Matter, and Phosphorus

To measure the pH, the sample sieved at 16 mm moisture was mixed and stirred with water at a 1:5 ratio. The pH and electrical conductivity (EC) of the same extract were subsequently measured with a Hanna Instrument brand pH meter. The mass of the sample < 16 mm and wet (A) equivalent to 40 g of dry sample at 70 ± 5 °C and the volume of water (B) were calculated to achieve a ratio of 1:5 according to A = (40/ST) × 100 B = 200 − (A − 40), where A = mass in g of a sample < 16 mm and wet, ST = total solids in % for a wet sample, and B = volume in mL. For water, A = mass in g of a sample < 16 mm and wet [59]. To determine the OM content, 2 g of each dry sample was weighed at 36 °C and passed through a <16 mm sieve that was free of inert impurities. HCl (0.05 mol/L) was added to remove any carbonates that were present by adding an excess of HCl (0.05 mol/L) to the sample until bubbling ceased [61]. The mixture was then placed in an oven to dry at 70 ± 5 °C until reaching a constant mass. The sample was subsequently transferred to a tared crucible and incinerated in a muffle furnace at 550 °C for 2 h. The volatilized material was assumed to be the organic fraction, and the remaining ash was assumed to be the mineral fraction [59]. OM content was calculated according to OM (%) = (ab)/a × 100, where a is mass (g) of the dry sample at 70 ± 5 °C before calcination, and b is mass (g) of the sample calcined at 550 °C. The phosphorus content was determined by UV-vis spectroscopy via the molybdenum blue method [62].

2.2. Greenhouse Test

2.2.1. Design of the Experiment

For this test, we worked with plants of the Maria Bonita variety (CIP No. 388676-1) generated in vitro. This promising variety, which was selected and launched in Peru in 1995, is resistant to PVY and late blight, is tolerant of drought and hot climates, and is adapted to low-lying and arid lands. The tubers have an oblong shape with cream-colored meat and are appropriate for industrial production [63]. Given its desirable characteristics and adaptation [64] in Venezuela, it has been propagated by the Biotechnological Center for Training in the Production of Agamic Seeds (Cebisa), which is located in Mucuchíes, Mérida State, and was launched by a network of producers, Páramo Integral [65]. The T. asperellum strain was obtained from the microbiological control laboratory of the National Experimental University of Táchira, Venezuela. A completely random factorial experimental design was adopted with three factors, which corresponded to the compost, the mixing proportions of the compost with coconut fiber (Fc), and inoculation with T. asperellum. Four repetitions were included, and a tray with three plants generated in vitro was used as the experimental unit.

2.2.2. Test Establishment

First, moist heat was applied to the mixtures (for sterilization. To do so, 40 L portions were transferred to clean nylon bags that were then placed in a 150 L capacity metal container with a double bottom grid to form a water bath at a height of 0.25 m; the water level was adjusted to maintain this level when necessary. The treatment lasted for two hours after boiling began and was repeated twice. Propylene trays with a 1 liter capacity were labelled, perforated and filled with the substrates (mixtures). Perforations were opened at the base of the trays to guarantee drainage. Next, three in vitro-grown plants were planted in each tray and then randomly placed on counters within the greenhouse. The moisture of the mixtures in the trays was maintained at field capacity with a microsprinkler irrigation system two or three times per week, depending on the temperature and relative humidity conditions present. The potato plants generated in vitro were inoculated with T. asperellum at a density of 1.5·10⁷ spores m·L⁻¹ at the time of transplantation, and the application was repeated 15 days later at a rate of 2 g·L⁻¹ water, 1000 µL/plant, following the manufacturer’s instructions.

2.2.3. Agronomic Evaluation

The survival of the in vitro-generated plants was determined 15 days after sowing. Reseeding was undertaken to reestablish the plants generated in vitro lost due to a lack of attachment. Thirty days after sowing, the height of the plants, the number of leaves and the number of stems per plant were measured. After 90 days of growth, the number and weight of the mini tubers per tray were determined. We wanted to determine the best proportion and combination of Fc compost mixtures to produce pre-basic seeds of the potato variety María Bonita [66] from plants generated in vitro and the effectiveness of using T. asperellum as a growth promoter.

2.2.4. In Vitro Evaluation of the Growth-Promoting Activity of T. asperellum

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In vitro evaluation of IAA production by T. asperellum

The objective of this test was to determine the capacity of T. asperellum to produce indole-3-acetic acid (IAA) in glucose peptone broth (GPB, 99% Himedia, India) modified with or without L-tryptophan (L-TRP, 99% Merck, Germany). The flasks (100 mL), each with 20 mL of sterile GPB, were modified with 5 mL of L-TRP (5%) [67]. To prepare the inoculum of T. asperellum, 4 mL of an aqueous solution of 0.89% NaCl + 80% tween (0.1%) (Sigma-Aldrich, Germany) was placed in a GPB petri dish with the mycelia of seven-day-old T. asperellum, and the surface was scraped with a glass rod to loosen the spores. One milliliter of that suspension was successively withdrawn from 9 mL of sterile water four times. The chamber was loaded to reach a concentration of 10⁸ spores mL⁻¹, which was considered the 10⁻¹ dilution. The flasks were inoculated with 1 mL of this dilution, covered with aluminum foil, and incubated on a shaker at 150 rpm at 25 °C in the dark for 7 days. Uninoculated flasks with and without L-tryptophan served as controls. After incubation, the suspension in each flask was centrifuged for 30 min at 12,000× g. The supernatant was filtered through sterile Millipore membranes (pore size 0.22 µm, Sigma-Aldrich, Germany) and collected in sterile tubes. The culture supernatants (1 mL) were transferred to test tubes, and 2 mL of Salkowski’s reagent (7.9 M H₂SO₄ (99.9% Eisen-Golden Laboratories; USA) in 40 mM FeCl₃ (98% SRL) [9] was added for each mL of microbial culture [68,69]. The mixture was incubated for 30 min to develop a red color, after which the optical density was measured at 530 nm [70] via a spectrophotometer (6405 UV-vis brand Jenway).

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In vitro phosphate dissolution test

For this test, two isolates, T. asperellum and Penicillium rugulosum IR-94MF1, which were provided by the Biofertilizers Laboratory of the UNET, were used as positive controls. The plants were subsequently grown in National Botanical Research Institute phosphate (NBRIP) media [71] or modified media (MM) [72]. The NBRIP medium contained the following components: 10.0 g L⁻¹ glucose (Merck); 2.7 g L⁻¹ hydroxyapatite (97% Alfa Chemical, China); 5.0 g L⁻¹ MgCl_2·6H₂O (99% SRL); 0.25 g L⁻¹ MgSO_4·7H₂O (99.9% EISEN-GOLDEN); 0.2 g L⁻¹ KCl (99.9% Ciencia CA); and 0.1 g L⁻¹ (NH₄)₂SO₄ (99% Eisen Golden Laboratories; USA). The MM contained double distilled water; 0.4 g L⁻¹ NH₄Cl (99.5% SRL); 0.78 g L⁻¹ KNO₃ (99% Merck); 0.1 g L⁻¹ NaCl (99.9% Merck); 0.5 g L⁻¹ MgSO_4·7H₂O; 0.1 g L⁻¹ CaCl_{2 ·}2H₂O (99% Merck); 0.5 mg L⁻¹ FeSO_4·7H₂O(99.9% Merck); 1.56 mg L⁻¹ MnSO_4·H₂O (98% SRL); 1.40 mg L⁻¹ ZnSO_4·7H₂O (99% SRL); and 10 g L⁻¹ glucose, plus 20 g agar (Merck). In both cases, controls were prepared with KH₂PO₄ as a source of phosphorus. The pH was measured before inoculation of the media. The quantitative estimation of phosphate solubilization was carried out using Erlenmeyer flasks (125 mL) containing 50 mL of medium inoculated in triplicate with 1 mL of conidia collected from Petri dishes flooded with 9 mL of sterile NaCl solution at 0.89%. The concentration of the conidial suspension was 1·10 ⁷ spores mL⁻¹. The mixture was incubated at 25 °C in an incubator shaker (New Brunswick Scientific, I26) at 120 rpm for 96 h in the dark at room temperature. The experiments were carried out in triplicate; the values are expressed as the means. An aliquot of each sample was transferred to a Falcon tube (10 mL) and then centrifuged at 7000 rpm (Damon/IEC, Needham Heights, MA, USA) for 10 min, after which the pH and phosphate concentration of the supernatant of each culture were analyzed. The phosphate in the culture supernatant was estimated using the following techniques of Murphy and Riley [62] adapted by Mateus-Rodríguez et al. [73] and is expressed as phosphate equivalents (µg mL⁻¹). One milliliter of the supernatant was transferred to 10 mL volumetric flasks, to which 0.8 mL of mixed reagent was added after waiting approximately 10 to 30 min for the color to develop. The optical density was measured at 882 nm [62].

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Evaluation of phosphate dissolution by metabolic extracts of T. asperellum.

The fungal strains T. asperellum and Penicillium rugulosum IR94-MF1 were obtained from Microbiological Control and Biofertilizer Laboratories, respectively, of the National Experimental University of Táchira UNET in Venezuela. The strains were cultured in solid NBRIP (T. asperellum) and MM (P. rugulosum) in Petri dishes for seven days. Nine milliliters of sterile NaCl were dispensed into Petri dishes to scrape off the spores with an inoculation loop (Figure 1). Then, 1 mL was transferred to a test tube with 9 mL of NaCl solution (0.89%). The tube was vortexed, and the number of spores was counted using a hemocytometer. A total of 1000 µL of each isolate was dispensed with 10⁸ spores in different media in sterile conical flasks containing autoclaved NBRIP and MM. The isolates were grown at room temperature on a shaker at 120 rpm for 7 days. The cultures of T. asperellum and P. rugulosum IR94-MF1 were filtered through filter paper, and the filtrates were stored at 2 °C. The filtered culture broth (100 mL each) was extracted with EtOAc (ethyl acetate, ARVI). The organic fraction was dried (Na₂SO₄, (99% Eisen-Golden Laboratories) and evaporated in vacuo at 38–40 °C. The recovered residue was dissolved in 2 mL of dimethylsulfoxide (DMSO, 99.5% Merck) [74]. A test was developed to verify the presence of solubilizing substances in the extracts obtained from the isolates of T. asperellum and P. rugulosum IR-94MF1 grown in both hydroxyapatite (HA) and KH₂PO₄ media to verify the solubilization of HA from the treatment process in the middle of the KH₂PO₄ medium, which indicates that the solubilizing agent is not induced by the presence of poorly soluble inorganic phosphates.

Petri dishes were prepared with a 2 mm thick layer of agar containing HA-MM (7.5 mM P) [72]. Dilutions of the metabolic extracts were prepared at concentrations of 100, 200, and 300 µg mL⁻¹, while the active substances (e.g., gluconic acid and 6-pentyl-alpha-pyrone) were present in the culture medium at concentrations ranging from 2000 to 7000 µg mL⁻¹ [72,75] and were applied to aliquots of 25 µL of dilutions of organic extracts on the plates inside 5 mm wells, which had been previously opened with a punch under axenic conditions. The dissolution halos were measured a few minutes after the extracts were poured into the wells [72] (Figure 2).

2.3. Data Analysis

Prior to the statistical analyses, compliance with the assumptions of normality and homoscedasticity was verified in the data. These variables were normally distributed; most showed uniform variances (p > 0.05). Principal component analysis, multivariate variance, univariate variance, and multiple linear regression were performed. Principal component analysis was carried out to determine the variables that contributed the most to the data. The analysis of variance allowed us to determine the effects of the treatments on the variable weight of the mini tubers. A linear regression analysis between the variable that best explained the biometric response of preference when producing potato seeds and the variable that presented the highest correlation in the analysis was performed. All the data were processed with the statistical package InfoStat v. 2022 [76].

3. Results

3.1. Evaluation of the Physical and Chemical Properties of Compost and Its Mixtures Used as Substrates

To protect environmental and human health, current legislation in Spain requires the type of material used for co-composting to be considered. On the basis of its origin, certain treatments may be needed. Similarly, recently approved regulations specify the need to monitor and demonstrate the existence of a thermophilic phase during the composting process. Therefore, for composting plants, measurements and documentation of the necessary temperatures for the minimum time required to eliminate pathogens and viable seeds must be conducted. Regulation (EU) 2019/1009 of the European Parliament and of the Council of 5 June 2019 [77], which establishes provisions for placing EU fertilizer products on the market, dictates that composting is as follows:

“The controlled process of aerobic and thermophilic biological transformation of biodegradable organic materials that gives rise to the types of fertilizers or organic amendments, whose characteristics are detailed in Groups 2 and 6 of annex I”.

Additionally, this document indicates that the treatment guidelines for composting must comply with these regulations. Therefore, the compost used in this study fell into Group 6 of annex 1 and Group 6.04. 6.03 and 6.05 for Cp, Cu and Cv, respectively. The verification of compliance with the requirements revealed that pH and EC (Table 1) were the only parameters used for labelling. The compost tested was suitable because it lacks improper parameters. The high P contents were also notable, but above all, phytotoxic compounds were absent. The mandatory requirements for proper composting can be found in the European Union regulation 2019/1009 [77].

The compost described in Table 1 has ideal characteristics for use not only as a natural organic fertilizer but also as a growing substrate. The comparative advantages of one or the other depend on the joint expression of their physical, chemical, and biological characteristics. In accordance with the above, the results of the initial analysis of pH, EC, apparent density, and OM (Figure 3) showed notable differences. Both the compost alone and their mixtures presented values close to those suitable for the development of seedlings, especially for potatoes. For example, if the pH is 75–50% in mixtures with Fc, values closer to what is appropriate are obtained (dashed line). The pH of the mixtures in general was adjusted to favorable limits between 5.3 and 6.5. However, the mixtures with 75%, 50%, and 25% compost provided the best conditions according to this variable, generating an environment conducive to better nutrient availability. Both the compost and the Fc had EC values greater than the maximum recommended 0.5 dS m⁻¹. Excepting Cu, whose values were the closest to the recommended value (Figure 3), the EC was high in all the mixtures, with values that exceeded the maximum catalogued as adequate (<0.5 dS m⁻¹). The expression of the biological response was informative in this sense. The Bd values indicated a favorable contribution of the Fc in the resulting mixtures, especially in the case of Cp but also in those of Cu and Cv when mixed in equal parts. In the first case, the value decreased, and in the other two cases, it increased, bringing it closer to the desired value. A similar situation occurred with OM, since the mixture with Fc led to an increase in Bd; although still low in Cp and Cu, the Bd values became increasingly close to the optimum of <0.4 T m⁻³ [78], as indicated by the dotted line.

Table 1. Results of the analytical tests for the quality variables of the starting compost.

Parameter	Cp	Cu	Cv	Ref. Decree 506/2013 [79]
Group	6.04	6.03	6.05
Treatment	Sanitized and stabilized. Obtained by aerobic biological decomposition (including thermophilic phase) under controlled conditions of biodegradable organic materials of annex IV. Collected separately.	Sanitized and stabilized. Obtained by aerobic biological decomposition (including thermophilic phase) exclusively of leaves, cut grass, and plant debris or pruning under controlled conditions.	Stabilized product obtained from organic materials by digestion via worms under controlled conditions.
OM (%)	22.81.4 ± 0.52	35.24 ± 2.62	79.98 ± 2.25	35
C/N	9.05	17.69	17.56	<20
pH	6.87 ± 0.15	6.5 ± 0.1	4.9 ± 0.1
EC (dS m−1)	6.72 ± 0.35	1.7 ± 0.1	3.8 ± 0.1
Improper	Exempt	Exempt	Exempt	<2.5 stones <1.5% metals, glass, plastics
N (%)	1.4 ± 0.2	0.5 ± 0.01 3	2.53 ± 1.1	1 1
P2O5 (%)	10.23 ± 0.34	4.26 ± 0.44	6.09 ± 0.27	1 1
Bd (t m−1)	0.7 ± 0.1	0.35 ± 0.02	0.2 ± 0.1	1 1
Granulometry (mm)	<4.8	<4.8	<4.8	400–700
Phytotoxicity 2 (%)	140	154	124	>90

¹ Declare if it exceeds 1%. ² Determined according to Thomson [61] or ³ Villalba [80].

Table 1. Results of the analytical tests for the quality variables of the starting compost.

Parameter	Cp	Cu	Cv	Ref. Decree 506/2013 [79]
Group	6.04	6.03	6.05
Treatment	Sanitized and stabilized. Obtained by aerobic biological decomposition (including thermophilic phase) under controlled conditions of biodegradable organic materials of annex IV. Collected separately.	Sanitized and stabilized. Obtained by aerobic biological decomposition (including thermophilic phase) exclusively of leaves, cut grass, and plant debris or pruning under controlled conditions.	Stabilized product obtained from organic materials by digestion via worms under controlled conditions.
OM (%)	22.81.4 ± 0.52	35.24 ± 2.62	79.98 ± 2.25	35
C/N	9.05	17.69	17.56	<20
pH	6.87 ± 0.15	6.5 ± 0.1	4.9 ± 0.1
EC (dS m−1)	6.72 ± 0.35	1.7 ± 0.1	3.8 ± 0.1
Improper	Exempt	Exempt	Exempt	<2.5 stones <1.5% metals, glass, plastics
N (%)	1.4 ± 0.2	0.5 ± 0.01 3	2.53 ± 1.1	1 1
P2O5 (%)	10.23 ± 0.34	4.26 ± 0.44	6.09 ± 0.27	1 1
Bd (t m−1)	0.7 ± 0.1	0.35 ± 0.02	0.2 ± 0.1	1 1
Granulometry (mm)	<4.8	<4.8	<4.8	400–700
Phytotoxicity 2 (%)	140	154	124	>90

¹ Declare if it exceeds 1%. ² Determined according to Thomson [61] or ³ Villalba [80].

The OM content of the Cv compost mixed with Fc in all proportions theoretically presented a concentration of carbon similar to that of the desirable OM levels. The OM contents of Cp and Cu increased as the Fc content increased. Therefore, the 75:25 mixture of the three composts had the greatest difference in terms of OM content (Figure 3).

The characteristics of each composted material in each of the proportions generated an effect that was measured in the biometric response of the María Bonita variety potato plants. The traits of this variety of potato have been described in previous studies [63,64], but the incidence of P. infestans or viruses has not been reported. Owing to the availability of more than one response variable in this study, a multivariate analysis was carried out with the intention of globally capturing the effects of the treatments (Wilks, Pillai, and Lawley-Hotelling). The results revealed significant differences in the effects of compost type (p < 0.0001), Fc proportion (p < 0.0001), and treatment with T. asperellum (p < 0.0001). The analysis also revealed interactions between the following factors: type of compost, mixing ratio, and biological treatment with T. asperellum. For this reason, the factors should not be analyzed individually; in contrast, their interactions must be studied. However, the study of a triple interaction would require more detailed statistical techniques that are unfortunately not possible in this work.

The interactions among the three factors were noteworthy; thus, a joint analysis of the characteristics measured in the compost and its mixtures is needed to determine their effects on the biometric variables. We aimed to determine the best configuration of factors; namely, the type of compost, mixing ratio of Fc and treatment with T. asperellum, in terms of their effects on the following biometric variables: number of leaves, plant height, thickness of the stem, and number and weight of the mini tubers. The Hotelling test to discriminate the best treatment in a multivariate manner (

Table 2. Hotelling test results for the multivariate mean contrast for the factors compost type, mixing ratio, and treatment with T. asperellum in the production of mini tubers via potato var. Maria Bonita plants generated in vitro.

Treatment	Height (cm)	Stem Diameter (mm)	Minitubers/ Tray	Minitubers/ m2	Minitubers Weight (g)	Group
Cp50A	15.96 ± 2.21	3.57 ± 0.48	17 ± 2.83	568.56	12.9 ± 1.11	a
Cp50P	16.89 ± 1.94	5.09 ± 0.46	14 ± 6.58	468.23	13.77 ± 1.36	b
Cv50P	16.7 ± 1.38	5.02 ± 0.29	12.75 ± 4.79	426.42	11.45 ± 1.59	bd
Cp25P	12.62 ± 2.13	4.86 ± 0.64	13 ± 3.83	434.78	12.71 ± 1.23	bd
Cp75P	25.88 ± 2.75	4.49 ± 0.7	15 ± 0	501.67	12.57 ± 0	c
Cu50A	12.41 ± 2.88	3.93 ± 0.25	11.5 ± 2.65	384.62	11.56 ± 1.45	d
Cu75A	13.47 ± 1.92	4.46 ± 0.15	16.25 ± 0.96	543.48	10.37 ± 1.29	dej
Cu100P	15.64 ± 2.97	4.18 ± 0.35	14.25 ± 2.99	476.59	9.52 ± 1.16	e
Cu100A	13.51 ± 1.09	4.1 ± 0.37	16.25 ± 4.5	543.48	8.5 ± 0.94	ehj
Cp25A	11.79 ± 0.95	4.26 ± 0.15	13.75 ± 5.68	459.87	8.05 ± 1.32	ej
Cu50P	14.76 ± 1.88	4 ± 0.58	13.5 ± 2.38	451.51	8.93 ± 1.26	ej
Cv50A	23.15 ± 1.75	4.33 ± 0.53	12.75 ± 3.86	426.42	10.07 ± 0.98	f
Cv75P	23.88 ± 2.39	5.59 ± 0.2	11.5 ± 4.51	384.62	6.02 ± 0.75	g
Cv25A	14.69 ± 1.65	4.95 ± 0.37	14 ± 7.35	468.23	9.68 ± 1.5	h
Cv25P	11.53 ± 2.11	4.52 ± 0.22	15.75 ± 3.77	526.76	8.05 ± 0.57	h
Cp100P	15.97 ± 0.26	3.85 ± 0.5	8 ± 1.63	267.56	10.22 ± 0.79	i
Cp75A	14.1 ± 0.9	2.99 ± 0.64	6 ± 0	200.67	9.49 ± 0	i
Cu75P	9.75 ± 1.99	3.9 ± 0.59	12.75 ± 1.89	426.42	9.57 ± 0.76	j
Cu25P	3.52 ± 0.83	2.43 ± 0.21	7.25 ± 1.71	242.47	5.96 ± 1.96	k
Cp100A	13.75 ± 1.9	0.35 ± 0.2	6.75 ± 1.26	225.75	7.28 ± 0.03	l
Cu25A	9.74 ± 0.3	2.38 ± 0.27	6 ± 2.45	200.67	6.83 ± 1.38	m
Cv75A	0 ± 0	0 ± 0	16 ± 3.37	535.12	4.3 ± 0.54	n
Cv100A	0 ± 0	0 ± 0	11 ± 4.97	367.89	3.84 ± 1.02	n
Cv100P	0 ± 0	0 ± 0	9 ± 1.63	301	3.72 ± 0.83	n
Fc100A	0	0	5.75 ± 3.77	192.31	0.79 ± 0.22	o
Fc100p	0	0	0 ± 0	0	0 ± 0	o

Note: Hotelling test. Means with one letter in common are not significantly different (p > 0.05). The number of mini tubers per tray with three plants was counted. The weight (g/mini tuber) is the average of the weights of the trays with three plants.

) accounts for the highest averages of the different variables and (depending on their standard deviation) groups and prioritizes the most important variables among statistically different treatments. Therefore, the greatest effect of the treatments on the globality of the biometric variables was achieved with 50% Cp compost combined with the absence of T. asperellum. Similar to the overall effect, the best quantity of seeds produced and number of mini tubers per tray with three plants were also obtained with the Cp treatment in the 50% proportion without T. asperellum.

3.2. Effects of the Properties of the Compost Mixtures and Inoculated with Trichoderma on the Biometric Variables of Maria Bonita Variety Potato Plants Generated In Vitro in the Greenhouse

The PCA yielded 15 main components applicable to the variables measured in the test, and the performance variables yielded 15 main components (corresponding to the 15 variables) that explained 100% of the variability provided by the compost mixtures. The first three components accounted for 80% of the variability. The individual weight of component 1 (CP1) accounted for 42%, that of component 2 (CP2) accounted for 25%, and that of component 3 accounted for 14%. With the help of eigenvectors, the parameters that best contributed to the construction of the first two components were selected because of their higher values. The variables pH, apparent density, total porosity, and OM stood out in CP1 along with all biometric variables; EC, Superv% of the plants generated in vitro, and P stood out in CP2. Similarly, all the variables presented correlations greater than 0.7 with the original variables, 0.96 of which were cophenetic correlations.

In the graph or biplot of the main components (Figure 4), the interpretation given above can be expanded. The angles formed by the mentioned vectors indicate a positive correlation (angle less than 90°), indicating that the P content may mediate a better response in potato plants. For the C1 component of the biplot, which accounted for 42% of the variability in the analysis, the closeness between the corresponding vectors and the stem thickness, the number and weight of the mini tubers, and the height of the plant were observed. This proximity indicates a positive correlation between these parameters and the P content, pH, and Bd of the analyzed mixtures. For the C2 component, the survival of the seedlings coincided with that of the EC but had the opposite trend, indicating that the best mixing configuration had a harmful effect on the plants generated in vitro at the time of transplantation. C3 demonstrated the importance of aeration capacity for stem thickness. However, for practical purposes, only C1 and C2 were considered. The P content in the samples must be considered because it is essential for understanding the biometric response. When the effects on the weight and number of potato mini tubers in the Cp treatment were visualized, we observed a high content of these elements in the mixtures used as substrates, with an association that became stronger in the treatments with T. asperellum.

Continuing with the biplot (Figure 4), we observed that in the +/+ quadrant, the yield and growth variables congregated together mainly with the Cp, and in the +/- quadrant, the highest yield of potato seeds was associated with Cu. In contrast, the treatments with the worst conditions were in the +/− and −/− quadrants, with a predominance of high EC and Pd together with low pH. Together with the contrasting effects of each compost on the biometric variables, this first impression revealed a marked effect in response to the different proportions of mixing with Fc.

The relationships between the properties measured in the mixtures and the biometric response indicate that the use value of a mixture is determined by the joint effect of physical, chemical, and biological variables, as shown in the next analysis. Moreover, with a multivariate approach, three factors under study were considered: (a) the three types of compost, Cp, Cv, and Cu; proportions of 100–75–50–25 and 0; and treatment with Trichoderma to determine the effects of its presence and absence on the biometric variables measured in potato plants. The PCA provided a rough approximation of the relationships among the biometric variables, in contrast to the physical, chemical, and biological variables. The relationships between the yield variables and the growth variables were of interest to us because of their predictive value preceding the actual harvest. Multiple linear regression analyses of the biometric variables revealed a positive linear relationship between the following variables: (a) plant height was correlated with stem thickness (p < 0.01) and mini-tuber weight (p < 0.01); (b) stem thickness was correlated with plant height (p < 0.01), the number of leaves (p < 0.01), the number of mini-tubers (p < 0.05), and the weight of the mini-tubers (p < 0.01); (c) the number of leaves was correlated with stem thicknesses (p < 0.01); (d) the number of the mini-tubers was associated with stem thickness (p < 0.01); (e) the weight of the mini-tubers was correlated with stem thickness (p < 0.01) and plant height (p < 0.01).

The stem thickness was strongly related to the other variables, indicating a pattern in the biometric behavior of potato plants. For example, considering that the number of mini tubers is the most important variable in potato seed production, we can infer that, in potato plants with greater thickness, the greatest number of mini tubers can be obtained. The weight of the mini tubers and the height of the plant depend on the stem thickness, as described by the following equations:

Mini tuber weight = 2.99 + 2.92 − 0.29 X² With R² = 0.63; p < 0.0001

Plant height = 3.15 X + 1.81     With R² = 0.65; p < 0.0001

The representation of the biometric variables by the stem thickness is useful for understanding what property of the composition of the mixtures is mediated by the treatments. For example, for the P content, the presence of Trichoderma Cv 75 resulted in the greatest increase in stem thickness and a better general response to all the treatments, which disappeared when Cp, Cv, and Fc were used alone; i.e., without mixing with Fc. The fact that Cu responded best in Cu100 may be associated with the use of appropriate values (Burés) for Bd. The presence of Trichoderma may compensate for the deficiencies in the properties of the mixture in more than one situation.

Relationships between Physical and Chemical Properties Measured for the Mixtures Used as Substrates and Biometric Variables

The variables measured in the mixtures influenced potato plant growth and seed production. Of all those that were measured, four showed a clearer effect on the biological response. PCA allowed us to determine the relationships between the properties of the compost and the OM content, phosphorus content, pH, and EC of the compost mixture. Multiple regression analysis with the biometric variable plant height was performed. The number of leaves and stem thickness were measured 60 days after transplantation, the weight of the mini tubers and the number of mini tubers were measured 90 days after transplantation, and the relationships between pH and all biometric variables were determined (p < 0.01) such that high values contributed to better plant development. This is explained by the fact that the Cp of the compost presented the highest pH values (Figure 5) and therefore the best results compared with those of Cv and Fc.

3.3. Phosphorus-Dissolving Activity and In Vitro IAA Production of T. asperellum

In this experiment, P. rugulosum (IR-94MF1) was included [72] as a positive control, and another method was selected for the quantification of phosphate in two different liquid media so that each antagonist grew in its optimal medium. As shown in the results (

Table 3. IAA production (mg mL⁻¹) by T. asperellum in the presence (+Trp) and absence (- Trp) of tryptophan in the culture medium and of calcium phosphate (HA) in contrast to IR- as a positive control.

Treatment	+Trp	−Trp	P (ppm) *	ΔpH	Halo ** (300)	Halo (200)	Halo (100)
p-value	0.0171	0.0118	0.0097	0.0001	0.05	0.05	0.05
T. asperellum	11.95 ± 2.75	4.19 ± 0.80	0.78 ± 0.12 b	1.23 ± 0.15 b	12.25± 2.99 a	10.25 ± 1.50 b	5.75 ± 0.50 c
P. rugulosum	nd	nd	1.41± 0.32 a	2.88 ± 0.13 a	13.75 ± 1.41 a	9.25 ± 0.96 b	5.75 ± 1.26 c

ΔpH: decrease in pH; * P-ppm: phosphorus in ppm. ** Hydroxyapatite halo.

), although T. asperellum showed some phosphate solubilization capacity, it was surpassed by P. rugulosum, which showed a greater capacity to dissolve HA in association with the evident decrease in pH (Table 3). The acidification of the growth medium by P. rugulosum indicated that organic acids were produced. As expected, tryptophan influences the production of indole-3-acetic acid by T. asperellum, increasing its production up to 3-fold.

4. Discussion

There is a desire and an environmental sustainability commitment to exploit biowaste and byproducts generated by agro-industrial activity so that they are no longer wasted. This can be achieved by properly composting these materials. However, even when the well-known and feasible technique of composting is carried out, products lacking in total quality, i.e., minimum conditions for their safe use, can result. A high-quality compost is stable, free of pathogens and weed seeds, rich in humic substances similar to those of the soil, not attractive to insects or vectors, and beneficial to both the soil and the growth of plants [81]. Compost must meet certain quality parameters, such as an OM content of approximately 35%, a maximum moisture content of less than 40%, a carbon/nitrogen (C/N) ratio of less than 15 or 20 depending on the original material, the absence of impurities such as plastics or other unwanted materials, the absence of Salmonella and Escherichia coli, and the presence of essential nutrients for the growth and health of plants [74]. Although quality criteria have become important for its use as a fertilizer, they are vital for its use as a growing substrate. The quality criteria suggested that the compost used as a substrate in horticulture be applied before being mixed with other substrates rather than to the final growing medium; furthermore, additional quality criteria may be required depending on the intended use. These desirable criteria take into account the importance of EC, stability, and plant response [82].

The type of compost and the proportion of compost mixed in the compost used to prepare the substrate were notable (both p < 0.001) (Table 2). The highest statistical significance detected (p< 0.001) in the univariate analysis of variance for the variable weight of minitubers was in the proportion of compost added to the mixture (p < 0.001). Next in importance is the interaction type of compost * proportion used in the mixture and between the proportion of compost and inoculation with T asperellum as well as between the three factors involved (p < 0.05).

Therefore, each type of compost will produce a different response depending on the proportion in which it is used and whether the biological treatment is carried out. The response is based on the interactions of these three factors and thus must be interpreted. To determine the optimal type of compost and compost proportion and the effects of the biological treatment, we performed a multivariate contrast test. According to the results of this analysis, the best compost for many of the mini tubers was Cp compost in a mixture of equal volumes with Fc and in the presence of T. asperellum (Figure 6). In depleted mushroom compost inoculated with T. longibrachiatum and Phanerochaete chrysosporium, there was a remarkable lignocellulosic and moisturizing effect that was correlated with greater stability and quality of the compost [25].

Endophytic colonization of T. asperellum in consortium with Bacillus sp. has been reported in banana seedlings [27], as reflected by the dissolution of insoluble phosphates, the production of IAA, and the greater accumulation of biomass, which reduced the growth of plants by only 13% with the application of 100% lawn compost, obtained from garden mowing residue, chopped banana stems, cattle manure, and poultry at a 1:3:1:3 ratio. An improvement in nutrient assimilation induced by Trichoderma has been reported. In general, Trichoderma affects the assimilation of phosphorus. In the case of beans [29], the effect of Trichoderma was reflected in better assimilation of Mg. However, it was surpassed by Beauveria bassiana and Metarhizium anisopliae. Nevertheless, the effects of this promoter are multifaceted. For example, in tomato, T. asperellum CHF 78 promoted growth via the dissolution of phosphates and the production of siderophores and IAA through endophytic mechanisms that decreased the severity of Fusarium infection [22]. Sometimes, the beneficial effect of T. asperellum is reflected simultaneously in both the biocontrol of pathogens and better lettuce growth due to the production of many volatile compounds related to these aspects [30]. However, what other aspects can Trichoderma inoculation affect? Changes in the rhizosphere microbiome can lead to the biocontrol of P. nicotianae [32]. In other crops of great importance, Trichoderma has been shown to have beneficial effects, as measured by the greater presence of antioxidants in the grape and greater yield due to the effect of harzic acid [34]. In cucumber, the beneficial effect has also been demonstrated through the production of IAA even in a saline environment with the dissolution of insoluble phosphates, thus providing a better example of its potential for use [36]. The spore concentration used in biological treatments for plant growth promotion can have a significant impact on their effectiveness. Previous studies have reported variable results with different spore concentrations and found that a spore concentrationand of 10⁸ CFU was effective in promoting the growth of plants. Sánchez-Montesinos et al. [52] demonstrated the effectiveness of a 10⁶ CFU spore concentration in enhancing plant growth, suggesting that the lower concentration used in the current study may still be sufficient. In our study, we also obtained beneficial results at an intermediate concentration to the indicated levels.

For the weight of individual mini tubers, the global growth variables were the best without T. asperellum, in contrast to the multivariate means for Cp at a 50:50 ratio with Fc. However, for the number of mini tubers, the best treatment was Cp at a 50:50 ratio with Fc in the presence of Trichoderma. The presence of chicken manure and wastewater sludge from the agri-food industry was reflected in the composition of this treatment. It is rich in nutrients, is deductible from the measured EC of 1.55 ± 0.29, and has a phosphorus content of 4.29 ± 0.05% on a dry basis. The EC that corresponds to the best compost in the treatments of this test coincides with values reported for the Agata and Asterix varieties of 2.1 and 1.7 dS m⁻¹ [83] in substrates with composted materials as the ingredient and the Agria variety potato of 2.37 dS m⁻¹ [84] on the Tezontle substrate.

The compost chosen for the formulation of the mixtures used in this test met the requirements described above but required adjustments for their use as substrates. The acclimatization of plants generated in vitro is specialized. In potato, biological evaluation is needed to clarify the relationships between the properties of the mixtures and the biometric response [85,86]. The effects of the type of compost and the proportion of compost in the mixture on the response of the potato plants generated in vitro were decisive. In theory, a pH between 5.3 and 6.5, an apparent density close to 0.4 t⁻³, and mainly OM content close to 20% would generate the best results [78,87,88]. This study aimed to determine the effects of various proportions of mixtures of compost and vermicompost with Fc on both the survival, growth, and yield of potato plants generated in vitro. This effect can be illustrated in principle via PCA (Figure 7 followed by linear discriminant analysis (Figure 7A,B). With the PCA, the variables with the greatest impact on the biological variables were filtered—pH, EC, MO, and Bd—and then, with the ADL discriminant analysis, groups were formed using appropriate ranges of these variables. The analysis of the main components revealed that a strong negative effect on the survival of the plants generated in vitro was associated with high values of EC. For the growth and weight of the mini tubers, the negative effect was associated with a high content of OM.

Table 1, which presents ranges of values of the properties measured in the mixtures, was used for the interpretation of the discriminant analysis results (Figure 7A). A high influence of the EC (angle < 90°) on the survival of the plants generated in vitro was observed, with the best performance occurring for the Cu compost in all mixing proportions along with Fc alone (Fc100). The corresponding ECs were between 0.21 and 0.65 dS m⁻¹ (

Table 4. Range of values for the properties measured in the mixtures within the classifications obtained via linear discriminant analysis.

	Bd	EC	OM	pH	P
a_sup	0.18–0.35	0.21–0.31	35.24–53.48	7.38–7.58	1.86–3.77
a_prod	0.17–0.65	0.21–4.57	22.81–84.2	7.35–8.45	1.86–4.29
b_sup	0.13–0.34	0.85–1.55	32.52–79.98	7.35–8.45	1.84–4.29
b_prod	0.17–0.19	1.84–2.93	79.98–82.95	6.05–6.38	2.33–2.66
c_sup	0.50–0.65	3.49–4.57	22.81–26.33	7.03–7.23	4.17–4.47
c_prod	0.20	0.65	85.14	6.78	0.67

Bd: apparent density; EC: electrical conductivity; OM: organic matter; P: phosphorus content.

). The response changed in the growth and production phases according to the discriminant analysis (DA) (Figure 7B). The best response, measured as the weight of the mini tubers, was observed for all the proportions of the Cp and Cu composts and for the Cv25 and Cv50 composts that were included in classification a. This response was positively associated with the P content and the pH of the mixtures according to their location, as shown in Figure 7A. The worst results in terms of growth and yield variables were obtained for Cv100, Cv75, and Fc. According to the arrangement of the vectors in Figure 7B, with the exception of the number of mini tubers, the biometric variables responded in a unified manner to the physical and chemical properties of the mixtures, indicating a positive response associated with the pH and P content in the mixture [89]. On the other hand, the number of mini tubers separated from the general group of biometric variables indicated a response associated with the P content in the mixture as well as the stem thickness.

The results of the biometric tests indicated that EC was positively associated with the performance variable but negatively associated with the establishment or survival of the plants generated in vitro. The negative effect of the EC in the establishment stage was expected because, as mentioned, the ceiling of desirable values is 0.5 dS m⁻¹ [78] and 0.195 according to the specifications for the use of quality compost in ECN culture media [90]. Focusing on potato seed production in 0.1 dS m⁻¹ zeolite substrate [91] yielded 7.05 mini tubers per plant [92]. However, when the effect of EC on the yield of mini tubers in aeroponics was studied, the best ECs were 2.1 dS m⁻¹ for potato var. Agate and 1.7 for potato var. Asterix [83], with 21.9 and 16.3 mini tubers per plant, respectively. For potato var. Agria, the yields measured in the mixtures with composted materials with EC = 2.37 dS m⁻¹ and 12.10% OM, and vermicomposted materials with EC = 2.34 dS m⁻¹ and 11.77% OM as ingredients were 52.75 and 55.55 mini tubers, respectively [84].

The maturation and stability of compost are crucial factors in determining its effectiveness as a biocontrol and growth-promoting agent. Compost quality is often unstable due to the variable nature of the raw materials and fermentation conditions used in its production. As a result, the interactions between compost, native soil microbiota, and the target plants are not well understood. Careful management of the composting process, including monitoring of organic carbon, nitrogen, phosphorus, and potassium levels, as well as the microbial community composition, can help ensure the stability and consistency of the final compost product.

5. Conclusions

In this research, we highlight the value of compost and vermicompost waste from various sources, including water sludge from the food industry, agricultural waste and biowaste, which are collected separately as suitable materials for use as substrates in the production of potato seeds. Incorporating Fc into the mixture was necessary to improve the chemical and physical properties. The best results, as indicated by the highest yield and quality of the mini tubers, were obtained when the pH was greater than 7.23, the OM content was <84%, and the EC was <3.49 dS m⁻¹. Although there were notable differences among the composts, all three were suitable for the production of pre-basic potato seeds, but this was dependent on the proportion of Fc added. Inoculation with T. asperellum had a favorable effect on the biometric variables measured, especially on the stem thickness and, to some extent, on the weight of the mini tubers. The possible dissolution of phosphates carried out by T. asperellum could cause conflict due to the high concentration of phosphorus in the compost evaluated, which globally hindered the promotion of growth in the inoculated treatments.