Hop Waste Seed Coating (Pilling) as Circular Bioeconomic Alternative to Improve Seed Germination and Trichoderma Development

Abstract

This study investigates the use of hop cone residues as a sustainable alternative to peat in seed coating formulations for the delivery of biocontrol agents such as Trichoderma. Some native isolates, T. velutinum T029 and T. harzianum T019 and T059, were tested for their development on peat and hop residues using qPCR. The results showed significantly higher fungal growth in hop cones, indicating their value as a carbon-rich substrate. Seed germination tests on various species showed that hop-based coatings did not inhibit germination and in some cases improved it. Field trials confirmed that bean seeds coated with hops 24 h before sowing outperformed those coated with peat, particularly in integrated production systems, in terms of germination. The results of this study suggest a new area of research: using hop residues in sustainable seed treatments could promote the valorization of agricultural residues, while improving crop establishment and reducing the dependence on synthetic inputs.

1. Introduction

Seed treatment using a coating is a common practice in agriculture production. The coating is applied to the surface of the seed to protect the seed and improve planting conditions, for example, through surface treatment with pesticides or inoculants to combat diseases and insect pests [1,2,3]; or by means of agents to delay seed germination [4]; or to improve the ability of seeds to resist drought, heat [5], and soil salinity [6,7]. Also, it includes increasing the weight of the seed to facilitate handling [8].

When the coating clearly changes the size and shape of the seed, the type of treatment is known as pilling. Heretofore, the pelleted seed coating has comprised ingredients such as red clay, clay, perlite, fossil meal, calcium carbonate, talc, calcium hydroxide, dolomitic limestone, aluminum, hydroxide, or kaolin, with or without the addition of a binder or nutrients [1,9,10,11,12,13,14]. The coating of pelleted seeds usually also includes substances such as nutrients and pesticides, to favor the development of the seed. In these cases, the seeds are coated with an active agent in a crushed or wasted form, or the active agent is dissolved in a solvent and the obtained solution or suspension is used to treat the surface of the seeds [15]. Seeds with different dimensions, forms, textures, and germination types such as vegetables (garlic, artichoke, cress, tomato), cereals (rice, maize, sorghum), oilseeds (canola, cotton, sesame), and legumes (soybean, alfalfa, cowpea) have also been coated with beneficial microorganisms [16,17,18].

Biocontrol is a pest management strategy making use of living natural enemies, antagonists, or competitors, and other self-replicating biotic entities [19]. The use of biocontrol agents (BCAs) is a sustainable way to control phytopathogens because they have the capacity to reduce the population of the disease-causing agent or avoid its effects. There are formulations of BCAs formed by bacteria, such as Agrobacterium, Pseudomonas, Streptomyces, or Bacillus, and by fungi, such as Gliocladium, Trichoderma, Ampelomyces, Coniothyrium, etc. [1,20]. Different commercial products have been patented and marketed for the control of different plant diseases throughout the world, which are applied directly to the crop.

However, the action of BCAs can be variable, since it depends on the characteristics of the environment where it develops, the amount of carbon and/or organic matter available, and temperature, among other factors [21]. BCAs need a carbon source to develop properly and thus be able to protect plants. However, the biological resources used as carbon sources, such as peat [22,23], are finite and extracting them directly from our planet’s ecosystems may be unsustainable.

The isolates used in this study had been characterized previously. The Trichoderma harzianum T019 isolate was obtained from bean plant tissue samples, while Trichoderma velutinum T029 and T. harzianum T059 isolates were obtained from soil samples collected in fields where beans were part of the crop rotation [21]. These isolates demonstrated an inhibitory capacity against Rhizoctonia solani L. in in vitro assays [24]. Prior studies evaluated the development of T. harzianum T019 using qPCR when inoculated in substrates such as peat and vermiculite, as well as amendments including bentonite and cornmeal (the latter serving as a carbon source) [22]. T019 exhibited enhanced growth in peat supplemented with cornmeal and/or bentonite. Other investigations assessed the effects of T019, T029, and T059 on peat-coated bean seeds and observed increased germination and improved root and shoot development [21]. Furthermore, T029 and T059 exhibited superior growth in soils with high concentrations of organic matter, carbon, phosphorus, potassium, and other nutrients. The organic matter content, pH, and clay percentage were found to significantly influence their development [25].

Solutions are proposed by reducing the problem of ecosystem degradation, for example, using sustainable renewable resources available in the environment, such as the use of residues from human activity, which traditionally have not been valued or would be a pollutant. Consequently, the carbon source of the BCAs comes from residues or waste, to give them use without polluting the environment. However, BCAs can respond differently to different carbon sources, especially when they are part of complex mixtures of organic material. Also, the carbon source must not interfere with the growth of the plant.

The circular bioeconomy can be defined as “a production and consumption model which involves reusing, repairing, refurbishing and recycling existing materials and products to keep materials within the economy wherever possible. A circular economy implies that waste will itself become a resource, consequently minimizing the actual amount of waste. It is generally opposed to a traditional, linear economic model, which is based on a ‘take-make-consume-throw away’ pattern” [26]. Agriculture is not an activity free of residues that have the possibility of being reused. To ensure that crop growers fully benefit from emerging biotechnological advances, it is imperative to develop novel formulations with an extended shelf life, enhanced soil persistence, slow-release dynamics, and high adaptability across diverse temperature ranges [27]. Such formulations must also demonstrate resilience to local climatic variability and provide consistent, broad-spectrum efficacy under field conditions. The incorporation of functional materials into these formulations can offer dual advantages: protection against pathogens and the delivery of essential nutrients, thereby reducing reliance on synthetic fertilizers and fungicides and enhancing plant resilience to environmental stressors. Hops are a crop that generates residues that can be given a second life as a carbon source for these microorganisms. Humulus lupulus L. is a plant whose female flowers are used for the brewing and pharmaceutical industry because they secrete lupulin, which is where the main active ingredients of hops accumulate. Carbon-rich materials may be employed to nourish both plants and beneficial soil microorganisms. In this context, hop-derived materials offer promising potential. Hop cones are composed primarily of terpenoids and prenylated phenolic compounds, including acylphloroglucinols—commonly known as bitter acids (α- and β-acids)—and prenylflavonoids, all of which exhibit well-documented antimicrobial properties, encompassing antibacterial, antiviral, antiparasitic, and antifungal activities [27,28,29,30]. While the bioactive composition of hop cones has been extensively studied, the potential of hop by-products such as leaves and stems—often treated as agricultural waste—remains largely unexplored. Notably, these by-products represent approximately 75% of the total hop biomass and could be valorized within a circular economy framework to enhance sustainability in agricultural systems [31]. During the hop harvest, leftover biomass from plants or cones that have not passed quality control is left behind. There are no published official figures detailing the exact amount of organic hop waste generated per campaign in Spain or internationally. However, it is possible to make an estimate based on the widely accepted sectoral proportion. For every kilogram of hop cones harvested, which are the part used by the brewing industry, approximately 3.5 kg of organic waste in the form of stems, leaves, and roots are generated [32]. According to data from Spain’s Ministry of Agriculture, Fisheries and Food, the annual production of dried cones is estimated at 1100 tons [33,34]. Therefore, according to the ratio of 1:3.5 (cones to waste), the organic waste generated per campaign in Spain would be approximately between 3500 to 4200 tons of organic hop waste, considering the average production and the sector’s standard ratio. The hops sector estimates that around 15 tons of residual biomass is generated per hectare. This includes plant residues, and the synthetic polypropylene twine (PP) used to support hop plants during growth [33]. However, these are highly valuable compounds as a source of organic matter and nutrients that can be used again for other applications such as a carbon source for BCAs.

The aims of this study are to evaluate the development of Trichoderma isolates in hop waste using the qPCR technique, to check the effect of pelleting with hop waste on the germination of different seeds, to evaluate the effect of pelleting the seeds with a biocontrol agent and hop waste in pots, and finally, to study the effects on germination in bean seeds pelleted with hops and Trichoderma under field conditions.

2. Materials and Methods

2.1. Trichoderma Isolates

The present study was conducted with native Trichoderma isolates collected from the production area of the Protected Geographical Indication (PGI), called “Alubia La Bañeza—León” (EC Reg. n.256/2010 published on 26 March 2010, OJEU L880/17), without any genetic manipulation (

Table 1. Trichoderma strains used in this study.

Isolate	Culture Collection	Trichoderma spp.	References
T019	PAULET38	T. harzianum	[21,24,35]
T029	PAULET44	T. velutinum
T059	PAULET74	T. harzianum

). The isolates were stored in the collections of the Research Group of Engineering and Sustainable Agriculture (University of León, León, Spain).

Trichoderma isolates used in all studies were inoculated on potato-dextrose-agar (PDA, Sigma-Aldrich, St. Louis, MO, USA) plates and grown in dark conditions (25 °C) for one week.

2.2. In Vitro Evaluation of Trichoderma Strains in Hop Waste

All substrates (leftover hop leaves and leftover hop cones) were ground to a particle size of less than 500 μm (gringer Black Coffee Grinder, Samsparty, Toledo, Spain) and autoclaved at 121 °C for 20 min (autoclave Presoclave II, J.P. Selecta, Abrera, Barcelona, Spain) to remove any organisms. A total of 5 g of each substrate was weighed in Petri dishes 60 mm in diameter and 5 mL of autoclaved distilled water (121 °C for 20 min) was added. A total of 1 mL solution of spores of each Trichoderma isolate (Table 1), (2·10⁷ spores/mL) was added to each Petri dish. Plates were sealed with Parafilm and incubated for 15 days at 25 °C, in dark conditions and relative humidity around 60% (Bacteriological and culture ovens, Incubat, J.P. Selecta, Abrera, Barcelona, Spain). Three repetitions were made per treatment.

After this period, they were stored at −80 °C until processed (Ultracold freezer Thermo Scientific™ Forma™ serie 900, Madrid, Spain). DNA isolation was carried out as previously described [22]. For total DNA extraction, 250 mg of each substrate was used. The FavorPrep Soil DNA Isolation Kit (Favorgen Biotech Corporation, Ping Tung, Taiwan) was used, following the manufacturer’s instructions. After the extractions, the samples were kept at −20 °C until use.

The standard curves for the Trichoderma isolates and the α-actin gene used in this research were previously described by Mayo-Prieto et al. [23,25].

Each measurement was conducted in triplicate. The qPCR reactions were carried out using Step One Plus (Applied Biosystems, Foster City, CA, USA). The reactions were performed with 10 μL of Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 0.4 μL of 10 μM Forward Primer, 0.4 μL of 10 μM Reverse Primer, 5 μL of cDNA, and diluted with H₂O to 20 μL [23,25].

The statistical analysis of the fungal development in the different substrates was performed using an analysis of variance (one-way ANOVA) and the mean was completely randomized. All analyses were performed using the IBM SPSS Statistics for Windows Version 26.0 (IBM Corp Armonk, New York, USA).).

2.3. Effect of Hop Waste Seed Coating on Seed Germination

2.3.1. Selected Seeds to Carry out the Pilling with Hop Waste

Different types of seeds were used to evaluate the effects of the coating immediately before sowing and one month after the pilling. This last evaluation was carried out to study whether the hop waste could harden and affect seed germination. For this reason, seeds of different shapes and sizes were evaluated:

• Small seeds, <3 mm: broccoli, rapeseed, and alfalfa;
• Medium seeds, 3–10 mm: lentil and wheat;
• Large seeds, >10 mm: corn, bean, chickpea, sunflower, and melon.

2.3.2. Seed Pelleting Process

The germination evaluation was carried out in laboratory conditions (25 °C, in natural light conditions, and relative humidity around 60%). Seeds were disinfected using a 20 min cleaning cycle in 10% sodium hypochlorite solution and 3 washes in autoclaved distilled water (121 °C 20 min). The seeds were allowed to dry in a laminar flow hood for 20 min.

The coating process was (Figure 1) as follows: (1) the seeds were placed in a glass container with a capacity of 200 mL; (2) a percentage of the weight of the seeds of gum Arabic was added, (3) it was stirred until the seeds were coated; (4) a percentage of the weight of the seeds of the waste of hop cones, H. lupulus, was added; and (5) it was shaken until the seeds were coated. The percentages used were as follows: for small seeds (<3 mm), 60–30% (hop-gum Arabic) of the seed weight; for medium seeds (3–10 mm), 20–15% (hop-gum Arabic), and for big seeds (>10 mm), 7–3% (hop-gum Arabic). Seeds without a coating were used as a control.

Germination was assessed in two batches: seeds were sown on the same day and one month after coating (stored at 4 °C until use).

Three repetitions were carried out for each species and each batch. A total of 10 seeds were used in the case of melon and 20 seeds were used for the rest of species; these were distributed homogeneously in Petri dishes (80–100 mm in diameter depending on the size of the seed) on two qualitative filter papers (grade 600, VWR International) by adding 1–15 mL of autoclaved distilled water (121 °C 20 min), depending on the type of seed. The Petri plates were sealed with Parafilm^®, kept in the dark for 12 days at 22 ± 1 °C in a previously described culture chamber, and germination was checked daily.

2.3.3. Evaluation of Seeds Pelleted with Hop Waste

Seed germination was recorded when the radicles were larger than 3 mm. Seed germination data, including the length of the root of the emerging seedlings as well as the fresh weight of the emerged radicles, were measured when 95% of the seeds had germinated, or when no new germinations had occurred for 3 consecutive days [36]. Also, the following indexes were calculated [37]:

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{g}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)={\displaystyle \frac{\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s} \left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)}{\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{d} \mathrm{s}\mathrm{e}\mathrm{e}\mathrm{d}\mathrm{s} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}\times 100$$

(1)

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{b}\mathrm{i}\mathrm{o}\mathrm{m}\mathrm{a}\mathrm{s}\mathrm{s}\left(\%\right)={\displaystyle \frac{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)}{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n}\mathrm{f}\mathrm{r}\mathrm{e}\mathrm{s}\mathrm{h} \mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}\times 100$$

(2)

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h}\left(\%\right)={\displaystyle \frac{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n} \mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)}{\mathrm{M}\mathrm{e}\mathrm{a}\mathrm{n} \mathrm{r}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}\times 100$$

(3)

$$\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}\left(\%\right)={\displaystyle \frac{\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)}{\mathrm{G}\mathrm{e}\mathrm{r}\mathrm{m}\mathrm{i}\mathrm{n}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}\times {\displaystyle \frac{\mathrm{R}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \left(\mathrm{t}\mathrm{r}\mathrm{e}\mathrm{a}\mathrm{t}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\right)}{\mathrm{R}\mathrm{o}\mathrm{o}\mathrm{t} \mathrm{l}\mathrm{e}\mathrm{n}\mathrm{g}\mathrm{t}\mathrm{h} \left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}\times 100$$

(4)

The statistical analysis of seed development was performed using an analysis of variance (one-way ANOVA) and the mean was completely randomized for each seed. All analyses were performed using the IBM SPSS Statistics for Windows Version 26.0 (Armonk, NY: IBM Corp.).

The same procedure and evaluation were followed for the seeds that were sown one month after coating.

2.4. Effect of Hop Waste and Trichoderma Coating on Seed Germination in Pot

Different types of seeds were used for the evaluation of pilling, considering their shape and size. The effect of carrying out pilling before sowing was evaluated. Rapeseed was selected as the small seed, wheat as the medium seed (3–10 mm) and bean as the large seed (>10 mm). They were disinfected following the procedure previously described in the previous section. Subsequently, seeds were impregnated with a solution consisting of 75% v/v of fixing agent (gum Arabic) and 25% v/v solution of Trichoderma spores (T. harzianum T019) (8·10⁷ spores/mL). They were then covered with hop cone waste. Control seeds were not coated.

Three repetitions of 10 seeds of each type were distributed homogeneously in polypropylene pots with a 250 mL capacity with peat (TYPical, Brill Substrate GmbH & Co, Georgsdorf, Germany). Each pot was watered prior to sowing until it reached the field capacity. They were kept in a culture chamber at 22 ± 1 °C and watered every 2 days with 25 mL of water.

The statistical analysis of seed development have been described in the previous section.

2.5. Evaluation of Hop Waste and Trichoderma Coating Seed in Bean Fields

The trials were carried out in four environments: two plots with integrated management production (field 1: Bustillo del Páramo, León, Spain, Area 201-Plot: 15; field 2: Bustillo del Páramo, León, Spain, Area 207-Plot: 46) and another two with organic management production (field 3: Matalobos del Páramo, León, Spain, Area 401-Plot: 45; field 4: Matalobos del Páramo, León, Spain, Area 401-Plot: 43) (Figure 2). All plots were sprinkler-irrigated and phytosanitary treatments were sprayed according to the type of management. Soil samples were taken from each farm for the analysis of their physicochemical properties. These analyses were carried out by Biome Makers Inc. (Valladolid, Spain). Soil samples were taken from each farm for the analysis of their physicochemical properties. These analyses were carried out by Biome Makers Inc. (https://biomemakers.com/es/inicio, accessed on 4 June 2025).

The soil temperature at the time of germination was measured using a G1700 thermometer (Greisinger, Regenstauf, Germany).

Bean seeds of the “Riñón” variety were used in this evaluation. They were washed with tap water. Subsequently, they were disinfected in 1% bleach for 3 min and washed in autoclaved distilled water (121 °C 20 min) for 6 min. The seeds were allowed to dry in a laminar flow hood for 20 min.

The coating process followed was described in the Section 2.4. Seeds were impregnated with a solution consisting of 75% v/v of gum Arabic and 25% v/v solution of T. velutinum T029 and T. harzianum T059 spores (2·10⁷ spores/mL). They were then covered with hop cone waste.

In the case of the control seeds, peat (TYPical, Brill Substrate GmbH & Co, Georgsdorf, Germany) was ground and autoclaved at 121 °C for 20 min and used for coating without Trichoderma isolates, with the same method described above.

Conventional tillage was used for land preparation, including moldboard ploughing and vibrating tine cultivation to prepare a suitable seedbed. The experimental plot was 32.5 m² for organic management and 36.3 m² for integrated management, with rows 0.55 m apart (5 rows per plot in organic management and 7 rows in integrated management), 100 seeds per row, and a space between seeds of 14.8 cm for organic management and 11 cm for integrated management. Sowing was carried out with a single-seed pneumatic precision planter owned by the farmer. The experiment was carried out following a statistical pattern of split-split plots according to a randomized complete block design, with three replications. The germination was evaluated at 6, 12, 19, and 28 days after the sowing, and it was counted in the 10 m centered on the furrow.

Means and error of the recorded data were calculated to evaluate bean germination. These bean parameters were analyzed by Kolmogorov–Smirnov’s and Levene’s tests and compared by an analysis of variance (two-way ANOVA) for a completely randomized design, including the main effects of Trichoderma isolates with two levels (T. harzianum T059 and T. velutinum T029) and effects of the coating with two levels (peat and hop waste). Finally, a post hoc analysis of Tukey’s test was performed.

3. Results

3.1. In Vitro Evaluation of Trichoderma Strains in Hop Waste

The results (

Table 2. DNA concentration obtained in the different hop waste.

Substrate	DNA Concentration (µg/mL)
	T. harzianum T019	T. harzianum T059	T. velutinum T029
Leftover leaves	5.274	10.947	0.003
Remains of cones or flowers	3358.898	73.386	2.492

) show that the hop cones where Trichoderma isolates have been growing are much higher in hop cones than in leftover leaves. Hop cones have promoted the development of these BCAs. T. harzianum T019 reached 3358 µg/mL in hop cones. In the case of the T. harzianum T059 isolate, it was inferior to T019. The leftover hop leaves did not promote the development of any Trichoderma isolate used.

3.2. Effect of Hop Waste Seed Coating on Seed Germination

Observing the results regarding seed coating with hop waste and subsequent sowing (

Table 3. Germination of the different seeds coated (mean ± standard error) with hop waste and their controls being pelleted and sown on the same day. Different letters indicate statistically significant differences according to Tukey’s test, p < 0.05. (n = 10 seeds for melon; n = 20 for other species with three repetitions for each seed).

Seed		Germination (%)	RG 1 (%)	Biomass (mg)	RB 2 (%)	Root Length (mm)	RRL 3 (%)	GR 4 (%)
Small size
Alfalfa	C 5	58.33 ± 3.33 a	105.71	2.49 ± 0.14 a	114.62	24.72 ± 1.81 b	134.05	141.71
	T 6	61.67 ± 10.93 a		2.86 ± 0.17 a		33.14 ± 2.82 a
Broccoli	C	65.00 ± 5.00 a	43.59	3.67 ± 0.23 b	269.48	36.73 ± 3.03 a	47.70	43.59
	T	28.33 ± 3.33 b		9.89 ± 3.53 a		17.52 ± 2.93 b
Rapeseed	C	90.00 ± 10.00 a	55.56	4.13 ± 0.24 a	97.66	54.79 ± 3.93 a	77.65	55.56
	T	50.00 ± 2.89 b		4.03 ± 0.26 a		42.55 ± 4.07 b
Medium size
Lentil	C	88.33 ± 4.41 a	94.34	7.47 ± 0.13 a	99.17	13.60 ± 0.61 a	84.94	94.34
	T	83.33 ± 7.26 a		7.41 ± 0.17 a		11.55 ± 0.54 b
Wheat	C	100.00 ± 0.00 a	96.67	7.29 ± 0.16 a	91.64	17.43 ± 0.63 a	85.54	96.67
	T	96.67 ± 1.67 a		6.68 ± 0.16 b		14.91 ± 0.86 b
Big size
Chickpea	C	98.33 ± 1.67 a	77.97	74.84 ± 0.78 a	97.64	13.96 ± 0.67 a	37.49	77.97
	T	76.67 ± 3.33 b		73.07 ± 0.77 a		5.23 ± 0.28 b
Sunflower	C	95.00 ± 0.00 a	85.96	17.91 ± 0.32 a	98.92	21.16 ± 1.02 a	66.82	85.96
	T	81.67 ± 6.01 b		17.71 ± 0.40 a		14.14 ± 1.27 b
Bean	C	98.33 ± 1.67 a	94.92	114.72 ± 2.63 a	99.13	43.07 ± 3.44 a	73.53	94.92
	T	93.33 ± 3.33 a		113.72 ± 2.80 a		31.67 ± 2.59 b
Corn	C	95.00 ± 0.00 a	94.74	48.80 ± 0.96 a	100.07	21.12 ± 1.40 a	81.19	94.74
	T	90.00 ± 2.89 b		48.83 ± 1.13 a		17.15 ± 1.38 b
Melon	C	73.33 ± 14.53 a	54.55	12.68 ± 0.53 b	118.19	41.77 ± 4.50 b	139.81	54.55
	T	40.00 ± 5.77 b		14.99 ± 0.66 a		58.39 ± 5.75 a

¹ Relative germination, ² relative biomass, ³ relative root length, ⁴ germination rate, ⁵ control (C), ⁶ hop treatment (T).

), germination was not inhibited in most species. However, a significant reduction in the germination percentage was observed in broccoli, rapeseed, chickpea, sunflower, maize, and melon when seeds were pelletized, with values notably lower than those of the untreated control. This suggests that the pelleting process itself may have a limiting effect on germination in certain species, beyond any potential influence of the hop coating material. Regarding biomass production (Table 3), any seed reduced their development, highlighting that in the broccoli and melon, the development was greater with respect to the control. So, the relative biomass parameter was better in alfalfa (increase of 14.62% respect to control plant), broccoli (increase of 169.48% respect to control plant), and melon (increase of 18.19% respect to control plant).

As for the root length (Table 3), the application of the pellet caused an increase in some seeds, such as alfalfa and melon. The relative root length in alfalfa was 134–04% and in melon it was 139.81%. For the other seeds, root development was significantly reduced by pilling.

Regarding the germination rate (Table 3), the seeds presented high values, except for broccoli and melon.

One month after the pelleting process (

Table 4. Germination of the different seeds coated (mean ± standard error) with hop waste and their controls, being sown one month after the coating. Different letters indicate statistically significant differences according to Tukey’s test, p < 0.05. (n = 10 seeds for melon; n = 20 for other species with three repetition for each seed).

Seed		Germination (%)	RG 1 (%)	Biomass (mg)	RB 2 (%)	Root Length (mm)	RRL 3 (%)	GR 4 (%)
Small size
Alfalfa	C 5	36.67 ± 8.82 a	145.45	4.71 ± 0.69 a	67.25	45.94 ± 6.97 a	98.19	142.82
	T 6	53.33 ± 16.67 a		3.17 ± 0.36 b		45.11 ± 5.97 a
Broccoli	C	73.33 ± 6.67 a	90.91	3.05 ± 0.37 a	82.32	28.44 ± 5.52 a	95.57	86.89
	T	66.67 ± 6.67 a		2.51 ± 0.35 a		27.18 ± 4.76 a
Rapeseed	C	63.33 ± 17.64 a	68.42	3.21 ± 0.37 a	87.05	56.98 ± 13.76 a	112.63	77.06
	T	43.33 ± 8.82 a		2.80 ± 0.46 a		64.17 ± 13.93 a
Medium size
Lentil	C	79.26 ± 0.74 a	113.55	10.73 ± 0.39 a	88.28	18.69 ± 1.45 a	71.10	80.73
	T	90.00 ± 10.00 a		9.47 ± 0.32 b		13.29 ± 1.00 b
Wheat	C	93.33 ± 3.33 a	100.00	9.88 ± 0.41 a	93.73	22.97 ± 1.63 a	101.08	101.08
	T	93.33 ± 3.33 a		9.26 ± 0.31 a		23.22 ± 1.29 a
Big size
Chickpea	C	83.33 ± 3.33 b	120.00	86.34 ± 1.90 a	94.42	14.44 ± 1.77 a	88.66	106.39
	T	100.00 ± 0.00 a		81.52 ± 1.24 b		12.80 ± 1.27 a
Sunflower	C	80.00 ± 10.00 a	66.67	34.14 ± 1.21 a	108.34	28.03 ± 2.57 a	135.79	90.53
	T	53.33 ± 12.02 b		36.99 ± 3.93 a		38.06 ± 6.13 a
Bean	C	93.33 ± 3.33 a	85.71	113.64 ± 7.00 a	104.12	25.13 ± 3.41 a	82.13	70.39
	T	80.00 ± 0.00 b		118.32 ± 5.97 a		20.64 ± 2.40 a
Corn	C	100.00 ± 0.00 a	90.00	52.46 ± 1.27 a	104.12	29.91 ± 2.47 a	68.11	61.29
	T	90.00 ± 5.77 b		54.62 ± 1.55 a		20.37 ± 1.89 b
Melon	C	20.00 ± 10.00 b	250.00	24.12 ± 0.91 a	74.77	28.74 ± 7.90 b	172.56	431.40
	T	50.00 ± 0.00 a		18.04 ± 1.05 b		49.59 ± 4.47 a

¹ Relative germination, ² relative biomass, ³ relative root length, ⁴ germination rate, ⁵ control (C), ⁶ hop treatment (T).

), the hop-based coating did not inhibit germination in most species. In fact, germination was significantly enhanced in chickpea and melon, with relative germination in coated seeds of alfalfa, lentil, chickpea, and melon increasing between 13% (lentil) and 150% (melon) compared to the control. In contrast, germination in sunflower, maize, and common bean remained significantly lower than in the control group. Notably, no significant differences were observed in broccoli and rapeseed, suggesting that the inhibitory effect detected earlier had diminished over time. These findings highlight species-specific responses to hop pelleting over extended periods.

Regarding biomass (Table 4), it was significantly lower in some seeds, such as alfalfa, lentil, and melon, but in the rest, they were not reduced in any studied seed with respect to the control.

In the case of root length (Table 4), it was not decreased in any seeds, except for lentils, corn, and melon, where there were some significant differences. Melon seeds had an increase in root development. The relative root length was increased in melon seed, reaching 172.56%.

In the germination rate (Table 4), melon seeds produced a notable increase in this value, i.e., 431.4%.

3.3. Effect of Hop Waste and Trichoderma Coating on Seed Germination in Pot

Regarding the effect of seed pelleting with Trichoderma and hop waste (

Table 5. Germination of the different seeds covered with hop waste and Trichoderma sp. in pot. Different letters indicate statistically significant differences according to Tukey’s test, p < 0.05. (n = 10 seeds with three replicates).

Seed		Germination (%)	RG 1 (%)	Biomass (mg)	RB 2 (%)	Root Length (mm)	RRL 3 (%)	GR 4 (%)
Small size
Rapeseed	C 5	23.33 ± 3.33 b	271.43	34.76 ± 7.53 a	99.47	46.11 ± 9.00 a	64.86	176.04
	T 6	63.33 ± 17.64 a		34.57 ± 10.27 a		29.91 ± 9.38 a
Medium size
Wheat	C	23.33 ± 12.02 b	414.29	130.84 ± 12.55 b	159.47	100.43 ± 8.07 b	157.21	651.31
	T	96.67 ± 3.33 a		208.65 ± 9.99 a		157.89 ± 3.14 a
Big size
Bean	C	90.00 ± 10.00 a	103.70	1990.51 ± 136.04 b	152.61	117.93 ± 8.19 b	137.04	142.12
	T	93.33 ± 3.33 a		3037.73 ± 158.27 a		161.62 ± 4.20 a

¹ Relative germination, ² relative biomass, ³ relative root length, ⁴ germination rate, ⁵ control (C), ⁶ hop treatment (T).

), no significant differences were observed in the data according to the Kolmogorov–Smirnov and Levene’s tests in the three types of seeds. Germination presented very high values, just as biomass production and the length of the root system. Colza was not significantly different in any parameter studied with respect to the control. However, the relative germination and germination rate reached values higher than 100%. The opposite case was observed in wheat, where all parameters were significant if they were coated with hop waste, reaching 414.9% in relative germination and 651.31% in germination rate. For bean seeds, the combination between hops and Trichoderma was significant in biomass production and root system length, with a value of 142.1% in the germination rate.

3.4. Evaluation of Hop Waste and Trichoderma Coating Seed in Bean Fields

The soil analysis of the trial plots revealed that the moisture content at planting varied between cultivated land. Fields 1 and 4 had moisture contents ranging from 3.5% to 2.6%, whereas fields 2 and 3 had contents ranging from 5.6% to 6.1%. The pH ranged from slightly acidic to neutral across all fields. In terms of nutrient content, all plots had a high carbon content, low nitrogen and phosphorus content, and medium potassium content. The soil texture in all fields is a sandy loam texture (unpublished data).

In Kolmogorov–Smirnov’s test and Levene’s test, the data did not present significant differences. There were no significant differences between Trichoderma isolates (T029 and T059), but between coating products (peat and hop waste), there were significantly differences (

Table 6. Mean squares of two-way ANOVA (Trichoderma strains and coating) for bean germination.

Source of Variation	df 1	Day 28
Trichoderma strains (Ts)	2	84.028
Coating (C)	1	1220.083 *
Ts x C	1	5.333
Error	55	225.989
Total	60

¹ Degrees of freedom. * Significant at p < 0.05.

). In the case of the interactions of Trichoderma strains and coatings, neither were significant with respect to control.

However, significant differences were observed among the experimental plots (Figure 3). It was found that plots under integrated production systems exhibited higher germination rates compared to those under organic production. Regarding the treatments studied, seeds coated with hop residues and containing Trichoderma spores showed significantly higher germination rates than those coated with peat. In the case of the trial conducted in field 2, no significant differences were observed among the treatments or compared to the control. However, in the remaining fields, it was found that the peat coating reduced the germination of bean seeds. As for field 3, control seeds germinated slightly better than those pelleted with Trichoderma isolates, though there were no significant differences with those coated with hop waste.

4. Discussion

In modern agriculture, it is a common strategy to cover seeds with inert material, with phytosanitary compounds such as insecticides, fungicides, etc. In this way, it is possible to facilitate their handling and protect them in the first stages of development in the presence of microorganisms or other factors that cause them biotic and abiotic stress. The seed treatment with fungicides or insecticides is often necessary to prevent crop establishment failure caused by seed- or soil-borne plant pathogens or insects. The seed application of microbial antagonists to soil-borne pathogens is an ideal delivery system, as it introduces the inoculum into the rhizosphere where plant pathogens such as Pythium or Rhizoctonia are active, causing seed rot and seedling damping-off [24,38]. Some bacterial and fungal antagonists have been used experimentally and commercially for this purpose [17,22,23,39,40,41], but their use in seed treatments is less common [42].

The germination of seeds of different sizes and shapes was studied to assess whether hop pelleting affected their germination. In one aspect, the coating of the seed is carried out in a single step by bringing a mixture of all the coating components into contact with the seed. However, it is also possible to add the coating components sequentially until the coated seed of the present invention is formed. In the coating used in this study, a two-step process is employed: first, a mixture of the fixative and biocontrol agent is brought into contact with the seed surface; then, the seed is brought into contact with the remaining component, i.e., the hop waste, to produce a seed coated with hop cones and the biocontrol agent. In a further aspect, the contacting is accompanied by mechanical agitation to improve the efficiency and/or speed of the coating.

The components of the formulation form a thick outer layer which, when exposed to dry habitats, delays germination and radicle emergence. This could be because a thicker outer layer affects permeability, limiting water uptake and oxygen transfer, which affects seed embryo development and consequently the germination rate [41]. In this study, the coating layer formed around the seeds was not thick enough to prevent radicle emergence. Chickpea seeds coated with hop cone residues had significantly higher germination rates than uncoated seeds.

Another possible reason for the differences in germination could be the inherent viability of the seeds. The loss of vitality in ageing seeds can reduce the total number of germinated seeds. This reduction may be due to storage conditions such as relative humidity and temperature. The decline in vigor, germination, and viability is closely related to the deterioration process, as it affects the seed quality. Several studies have observed that pea seeds stored at 5 °C and 22 °C for one year showed different responses in terms of seedling vigor and germination [43,44,45]. In this study, the germination of seeds coated for one month before sowing was not affected, except in the case of melon, where the reduction in germination was more pronounced in uncoated seeds.

Seed coating is a technique in which an active ingredient, such as a microbial inoculant, is applied to the seed surface using a binder, such as natural gum, and, in some cases, a filler that acts as a carrier. In this study, Arabic gum was added as an adhesive to bind the hop cone waste to the seed. This product has been used in other studies, where it has been found to have no effect on the germination of seeds [3,46,47,48,49,50]. As in these studies, the use of Arabic gum as a natural adhesive to fix the hop waste to the seed did not affect the germination of the seeds studied.

Seed coating has been proposed as a promising tool for inoculating various crop seeds, due to its ability to apply small amounts of inoculants with precision [51]. Currently, numerous studies are investigating the potential use of biological macromolecules, particularly biomass materials, as seed coating substrates [52,53,54,55].

Peat is a standard carrier material for dry formulations in seed coating [56]. Peatlands are the most significant terrestrial ecosystems for carbon storage and climate regulation. Their critical role in biodiversity conservation at both species and ecosystem levels has been well documented. Additionally, peatlands play a key role in water resource management and provide essential resources and livelihoods for millions of people worldwide. However, the current status and use of peatlands are unsustainable [57]. Other compounds, such as chitosan, possess inherent antifungal properties that can reduce pesticide use when applied as a seed coating [58,59]. Similarly, alginate [60], silk fibroin [61], and cellulose and lignocellulose [62,63] function as coating substrates or active ingredients by adhering to the seed surface and naturally degrading after fulfilling their function, without causing environmental pollution. The use of biomass-based materials in seed coatings presents a promising approach for sustainable and eco-friendly agricultural practices. Among these materials, lignocellulose and its components, such as cellulose and lignin, stand out due to their advantages, including cost-effectiveness, abundance, and low carbon footprint [62,64].

Other commonly used materials include inorganic carriers (e.g., charcoal, clays), organic carriers (e.g., biochar, wheat/soy/oat bran, grape bagasse, vermicompost, animal manure), and inert carriers (e.g., perlite, vermiculite, bentonite) [2,3,18]. Several patents have proposed the use of various carrier materials. For instance, patent ES2221357 [65] describes a plant protection product based on plant-derived preparations that protect treated plants. Patent ES2041605 [66] utilizes seaweed-derived compounds as fertilizers or seed coatings. Patent ES2680899 [67] describes a seed coating composed of a fine dry powder waste of micronutrients mixed with a dispersant. However, these patents do not consider the use of biological control agents or strategies to optimize their growth. Although patent CN103039439B [68] incorporates Trichoderma along with polyethylene glycol, xanthan gum, and methyl cellulose to attach spores to seeds, it does not use any organic substrate to promote microbial growth

Various formulation strategies have been explored to protect and enhance the biological activity of Trichoderma sp. conidia. These include encapsulation within carboxyl cellulose and vermiculite [69]; the use of different powders, such as kaolin, chitosan, vermiculite, ethyl cellulose, biochar, peat, and Arabic gum [70]; talc-based formulations [71]; alginate capsules [72,73]; microencapsulation with molasses and glycerol [74]; invert emulsions [75]; liquid formulations based on starch supplemented with copper [76]; dispersible granules [77]; and seed coatings [78], among others. However, these approaches only partially address key limitations, such as preserving biological activity, preventing desiccation, ensuring the controlled release of the active ingredient, and maintaining stability during storage.

In this study, an environmentally friendly alternative for seed coating formulations was evaluated. Specifically, residues from hop harvesting, including leaves and cones that did not meet quality standards, were used as potential substrates. When Trichoderma isolates were inoculated on hop leaves, their development was lower than when they were in contact with hop cones. In a previous study [22], the T. harzianum T109 isolate was inoculated on peat and vermiculite together with various additives such as cornmeal and bentonite, and it was found that development was lower in the presence of peat and/or vermiculite than when it was inoculated on hop waste. When flour was added to the peat, the development of T. harzianum T109 increased, but it was not as great as when it was inoculated into hop cones. In a previous study, the addition of cornmeal to a substrate as a carbon source increased the development of the biocontrol agent; in this study, the use of hop cone residues led to a significant increase in the growth of Trichoderma isolates. In another study, winery wastes such as grape marc and wine lees, obtained after the removal of grape seeds and ethanol, were used. An isolate of T. viride was inoculated and its development was assessed. It was observed that spore production increased over time, as the content of these products in the mixture increased [79].

So, hop wastes are a very good carbon source for the development of this biocontrol agent. Several studies have investigated the composition of hop cones [80,81], particularly for their antifungal properties. Research has evaluated the control capacity of hop cone extracts, which have been shown to inhibit the development of pathogens belonging to the genera Alternaria, Epicoccum, Fusarium, Aspergillus, and Botrytis, with inhibition rates exceeding 67% and, in some cases, reaching 100% [82,83,84]. However, dried hop cones were found to contain various sugars, with glucose and fructose being the most abundant, followed by maltose, galactose, and xylose/mannose. The presence of myo-inositol and lactate has also been reported, the latter being an energy substrate that regulates lipolysis and fatty acid oxidation. These compounds may have contributed to the development of Trichoderma in the present study, as they can serve as a carbon source necessary for fungal metabolism. Another study analyzed the compounds produced by bean plants in response to Trichoderma [85]. The results showed a significant increase in sucrose concentration in plants exposed to the fungus, which was subsequently used by Trichoderma to promote its development in the rhizosphere. Therefore, while this plant may be beneficial for the development of Trichoderma, it can be detrimental to germination, as has been observed with broccoli, chickpeas, and melons. In this case, the germination percentage of some seeds was reduced when they were coated with hops compared to the control. One possible cause is that a compound in the hops inhibits their development. One way to avoid this would be to conduct further research into the composition of hops to identify the compounds that influence seed germination. In various studies, the compounds present in hops are evaluated, as well as their antimicrobial capacity and other effects such as antibacterial, antiviral, antiparasitic, and antifungal activities [28,30,81]. Germination seeds have also been affected by the conditions of the medium. In the field, it was observed that the control beans had germinated more than the beans coated with hops and Trichoderma in one of the test plots (field 3), but this difference was not significant. One possible cause of this may be the moisture content at the time of sowing. In this study, the moisture content of this plot was higher than that of the others, which may have affected germination. Soil texture is another soil characteristic that can affect germination. In this study, however, all the plots had a sandy loam texture, so this would not be a factor affecting germination.

5. Conclusions

This study demonstrated the potential of hop cone residues as an effective substrate for the development and application of Trichoderma spp., as BCAs in vitro analyses exhibited significantly greater growth when inoculated on hop cone residues compared to hop leaves, suggesting that the cone material serves as a more suitable carbon source. Seed coating with hop powder did not adversely affect germination for most species tested. When hop powder was combined with Trichoderma isolates in seed coatings, the performance of several seed species was improved, particularly under field conditions. In bean field trials, hop-based coatings consistently outperformed peat-based formulations, particularly in integrated production systems. These results highlight the agronomic potential of hop waste as a sustainable carrier for biocontrol inoculants in seed coating applications. These results suggest a new area of research: using hop residues in sustainable seed treatments could promote the valorization of agricultural residues, while improving crop establishment and reducing dependence on synthetic inputs. Future research will focus on evaluating the compounds produced by hops that influence the development of biocontrol agents, such as Trichoderma, as well as their effect on seeds and future plants.

6. Patents

The seed coating herein presented has been patented with the number ES 2872599 B2 in the Spanish Office of Patents and Trademarks and submitted with the international PCT number PCT/ES2021/070288 and with the international publication number WO 2021/219915.