Composted Sludge and Trichoderma harzianum T-22 as a Dual Strategy to Enhance Wheat Growth and Soil Microbial Diversity

Abstract

This study evaluated the effects of Trichoderma harzianum strain T-22 on wheat (Triticum turgidum L. var. Durum, cv. Vitron) growth and soil microbial dynamics. Three inoculation levels (I0, I1, and I2) were applied to different soil substrates: Villacañas soil (V), Quero soil (Q), and composted sewage sludge (C) from Alcázar de San Juan. Over six months, soil physicochemical properties, fungal diversity, and plant development were analyzed. The results showed that Trichoderma significantly increased fungal diversity, particularly in compost-amended substrates. In treatments with composted sludge and Trichoderma (CVI2 and CQI2), Trichoderma colonization reached up to 112,000 propagules/g, enhancing microbial activity. Higher shoot biomass and spike weight were observed when combining compost with Trichoderma since it improved nutrient availability and plant growth. Additionally, Trichoderma inoculation reduced the presence of pathogenic fungi such as Helminthosporium and Fusarium, reinforcing its biocontrol potential. However, high salinity of the soil limited microbial proliferation and plant performance. In conclusion, composted sludge and Trichoderma improved soil microbiota, enhanced wheat growth, and increased resistance against pathogens. The results highlight the potential of Trichoderma as a sustainable alternative to chemical treatments in crop production. Further studies should further investigate field-scale applications to validate these findings under real agricultural conditions.

1. Introduction

Soil health is the continuous ability of a soil to function as an ecosystem that supports plants, animals, and humans. Therefore, sustainable agriculture requires effective management strategies to maintain and improve soil health and crop output [1]. The circular economy framework offers a sustainable approach to solid waste management by converting waste into valuable products, such as compost, biopesticides, biofertilizers, fungicides, compost tea, biofilters, and more. This approach not only incentivizes authorities and entrepreneurs to develop such commodities but also promotes environmentally responsible waste management [2]. The enrichment of compost with inorganic nutrients or nutrient-transforming microbial communities enhances its efficacy, yielding long-term benefits. Specifically, fortifying compost with selected microbial strains can expand its functionality. The development of nutrient-enriched compost-based products requires research beyond traditional composting methods. Identifying microbial communities capable of degrading and converting key nutrients, as well as microorganisms like plant growth-promoting rhizobacteria and ammonia-oxidizing bacteria, provides significant advantages for both farmers and agro-industries [3].

There exists a significant group of fungi and bacteria that exhibit antagonistic effects against other microorganisms, which can be harnessed for the biological control of plant pathogens. Among the most important microorganisms are bacteria from the genera Fusarium, Pseudomonas, and Bacillus, as well as fungi from the genera Gliocladium and Trichoderma.

The Trichoderma genus, proposed in 1794, includes over 200 species classified by their physiological and morphological traits. It is a fungus belonging to the class Hyphomycetes and is a natural inhabitant of soils. Its colonies range from green to yellow and have a coconut-like aroma. They are found near the rhizosphere, in decaying organic matter, and on tree bark. It produces three types of propagules (hyphae, chlamydospores, and conidia) with antagonistic activity against pathogens. It is resilient to extreme conditions, but its optimal growth temperature is 20–28 °C with 92% humidity [4].

Trichoderma is among the most used fungal antagonists of plant pathogenic fungi in modern agriculture. In a context of sustainable food production, without affecting the environment, the use of microorganisms for pest and disease control is a viable alternative to achieve significant increases in yields and crop quality and to reduce the negative impact of agrochemicals on the environment [5]. Some of its species produce enzymes and/or attack and inhibit phytopathogenic fungi, making it an excellent candidate for biological control. It is widely used to combat a significant group of soil-borne pathogens [6]. The primary effect of Trichoderma is hyperparasitism, although certain species and strains can also produce bioactive metabolites that enhance its action. Additionally, some isolates control nematodes. Trichoderma induces resistance in plants by stimulating natural defense mechanisms, leading to biochemical changes and the deposition of new layers in the epidermis. It also produces metabolites with antibiotic effects. Notably, it competes with Gliocladium for nutrients.

Trichoderma produces enzymes related to antibiosis and mycoparasitism, degrading the cell walls of host organisms during mycoparasitic interactions. Key enzymes include chitinases and glucanases. Additionally, Trichoderma generates secondary metabolites such as 6-pentyl-α-pyrone (6PAP) and toxins like pacibasins, trichodermin, gliotoxin, trichodermona, and viridiol.

Several Trichoderma species have been used in coupled fermentation systems on solid substrates or submerged cultures to degrade lignocellulosic residues and generate alternative energies, such as ethanol. Bioreactors, as fermentation systems, optimize cultivation conditions to enhance biomass and metabolite production [7]. Its effectiveness extends to both soil-borne and foliar fungal pathogens. The efficacy of Trichoderma as an antagonist against pathogenic fungi, such as R. solani and S. rolfsii, in combination with compost, has been tested for the management of soil-borne diseases in chickpea cultivation [8]. It has also been proven effective as a compost fortifier in tomato cultivation [9] with a lower cost in comparison to chemical pesticides. The inoculation of Trichoderma for seedling production in forest species also promotes several benefits to plant development, in addition to the low production cost [10].

One of the strategies used to improve the quality of pesticide-contaminated soils is the application of sewage sludge, a byproduct of wastewater treatment. Its efficacy in mitigating the effects of water stress by improving all studied growth and yield parameters in durum wheat has been proven [11]. Recycling sewage sludge to use as fertilizer in fields is desirable. Sludge application to agricultural soils is a strategy that enhances the physicochemical and biological characteristics of soil and serves as a substitute for chemical fertilizers [12]. As an organic substrate, it provides soil with key nutrients like nitrogen and phosphorus while also enriching it with organic matter, making it a beneficial fertilizer [13]. Because of its organic and nutrient content, sewage sludge can be used on agricultural grounds. The organic material in sewage sludge can improve the physicochemical features of soil, such as aggregation stability, aeration, water-holding capacity, and cation exchange capacity, and thereby promote the growth and performance of agricultural goods [14]. Despite its demonstrated advantages, the usage of sludge is still seen negatively by society because of where it forms and the potential for different toxins to appear. Additionally, using sludge as fertilizer carries the danger of contaminating water due to the potential for pollutants (such as heavy metals) and other soil constituents to leak into groundwater.

The cultivation of durum wheat (Triticum durum) in semi-arid Mediterranean regions relies on two types of germplasm: (i) traditional local genotypes with a low yield potential and (ii) introduced genotypes with a high production potential. However, introduced varieties tend to disappear quickly due to their unstable yield performance over time and space. This is mainly due to poor adaptation and high sensitivity to climate variability, particularly in semi-arid regions with severe droughts and unfavorable soil conditions, such as soil salinity [11]. Research on the wheat genome plays a fundamental role in the development of research to improve the quantity and, above all, the quality of this crop of global importance for the food supply of the population [15]. Wheat root disease is likely to be initiated during the early developmental stages because of the high fungal diversity and the structural complexity of the rhizosphere microbial community. The presence of a pathogenic fungus is an essential prerequisite for wheat root rot disease. However, the abundance of pathogenic fungi is not always important [16].

An experiment was conducted in a controlled environment chamber (phytotron) using wheat pots (cultivar Vitrón) inoculated with the Trichoderma harzianum T-22 strain. This study was conducted under controlled conditions and focused on specific soil types and compost formulations, which may limit its extrapolation to broader field conditions. The main goal of the study was to investigate the impact of Trichoderma inoculation on wheat growth applied on two types of soil as growth substrates, to which composted sludge was added. Unlike previous studies, our work evaluates the combined effects of Trichoderma T-22 and composted sludge on both soil microbiota and plant development, under two contrasting soil salinity regimes, highlighting their interaction in semi-arid Mediterranean conditions. We hypothesize that the combined application of composted sewage sludge and Trichoderma harzianum T-22 will improve soil microbial diversity and wheat growth, particularly under saline conditions, due to enhanced nutrient availability and biological activity.

2. Materials and Methods

2.1. Experimental Design for Trichoderma Inoculation in Wheat (Triticum turgidum L. Var. Durum)

In our study, we used winter wheat (Triticum turgidum L. Var. Durum) as the experimental crop. The experimental region in Castilla-La Mancha has a semi-arid Mediterranean climate, with average annual precipitation of 350–400 mm, a mean annual temperature around 15 °C, and high evapotranspiration rates. Temperature conditions posed a challenge, as field planting occurs during winter with ambient temperatures occasionally near 0 °C, risking frost. Additionally, Trichoderma harzianum T-22 performs poorly below 10 °C. To counteract the adverse effects of low temperatures on fungal activity, we employed a controlled environment chamber (phytotron). The phytotron maintains a regulated atmosphere, allowing precise temperature control.

The effects of various substrates on the growth of wheat plants, in conjunction with Trichoderma application, have been investigated.

For substrate preparation, soils were sourced from two locations in the province of Toledo, Southeastern Spain: Villacañas and Quero. These locations are referenced as Villacañas (V) and Quero (Q), indicating their geographic origin rather than any soil classification.

According to the World Reference Base for Soil Resources (WRB 2022), the Villacañas soil corresponds to a Calcisol, while the Quero soil corresponds to a Solonchak, due to its high salinity and sodium accumulation. According to USDA Soil Taxonomy, the Villacañas soil can be classified as a Typic Calciorthid and the Quero soil as a Typic Aquisalids. The geographical reference (Villacañas and Quero) is retained only as a secondary identifier for location-based discussion.

Both soils were formed under a semi-arid Mediterranean climate with an average annual precipitation of 350–400 mm, an average annual temperature of 15 °C, and a high aridity index. These climatic conditions promote evapotranspiration exceeding precipitation, contributing to calcareous accumulation in Villacañas soils and salinization in Quero soils, respectively.

To prepare mixtures (

Table 1. Details of the composition of the studied treatments.

Control Treatments	Description
Q + I0	Quero soil without inoculum
V + I0	Villacañas soil without inoculum
CV + I0	1/3 composted sludge + 2/3 Villacañas soil without inoculum
CQ + I0	1/3 composted sludge + 2/3 Quero soil without inoculum
Studied Treatments	Description
V + I1	Villacañas soil with inoculum I1
V + I2	Villacañas soil with inoculum I2
CV + I1	1/3 composted sludge + 2/3 Villacañas soil with inoculum I1
CV + I2	1/3 composted sludge + 2/3 Villacañas soil with inoculum I2
Q + I1	Quero soil with inoculum I1
Q + I2	Quero soil with inoculum I2
CQ + I1	1/3 composted sludge + 2/3 Quero soil with inoculum I1
CQ + I2	1/3 composted sludge + 2/3 Quero soil with inoculum I2

), soil was used in its natural, non-sieved form to preserve its original structure and composition. In addition to unsieved V and Q soils, composted sewage sludge from the Alcazar de San Juan wastewater treatment plant (C) was used for the experiment. To produce the composted sewage sludge for agricultural application, cereal straw was incorporated into an aerated composting pile system. Both types of soils (V and Q) were merged separately with this sewage sludge compost (C) in a proportion of 1/3 by volume to prepare CV and CQ substrates, respectively.

The term “inoculum” refers to the biological preparation of Trichoderma harzianum T-22 used for soil treatment in this study. The formulations to apply Trichoderma as a bio-improving agent for different substrates are multiple [17]. In our study, the Trichoderma harzianum strain T-22 was used for the inoculum in two different formulations: (1) a granulated formulation mixed directly with the substrate and (2) a water-soluble formulation for irrigation. Three inoculation treatment groups were considered: I0: no inoculation as control treatment; I1: two applications of T-22: (a) initial application directly into the substrate using the granulated formulation at a rate of 750 g/m³ (by adding 2.6 g of T-22 per pot), and (b) second application 3 months later via irrigation water at a rate of 30 g/1000 plants; and I2: the same applications as I1 plus a third application 20 days after via irrigation water at a concentration of 30 g/1000 plants.

All these substrates were distributed across 36 pots (3 replicates per treatment), which were disinfected using sodium hypochlorite (Figure 1). Subsequently, 7–8 wheat seeds were sown per pot (replicate) and placed in two trays (1.25 × 0.58 m) within the controlled germination chamber. Each tray held 3 rows of 6 pots, allowing for a total of 36 pots in the chamber. Finally, the phytotron was programmed for 12 h of daylight and 12 h of darkness, with a maximum temperature of 15 °C, a minimum temperature of 10 °C, and a humidity of 70%.

To avoid the shading of the trays, the order of placement of the pots inside the chamber was periodically reversed. Moisture was maintained with manual watering.

Sixty days after planting, the pots were moved to an outdoor bench inside the laboratory to the end of the experiment to allow better growth and the development of spikes.

The inoculation, applied 190 days after seeding, involved adding 400 mL of a T-22 water-soluble preparation in irrigation water to the I1 and I2 treatment groups. Additionally, 400 mL of water were applied to the I0 group as a control irrigation. A second inoculation, applied 215 days after sowing, was administered exclusively to the I2 group following the same procedure. Final sampling of the soil/substrate and plant material was conducted six months after sowing to prepare for subsequent laboratory analysis.

2.2. Sampling and Measurement

2.2.1. Crop

The aerial portion and roots were evaluated separately as follows.

Aerial Part: For each of the 36 pots, all plants were cut at the base, and the number of plants per pot, as well as the total weight of the biomass, were recorded. Subsequently, the ears were separated, and both the total weight of the ears and the number of ears per pot were reported.

Fifty days after planting, the first ANOVA was carried out to find a relationship between the number of plants per pot and the type of substrate where they had germinated. The statistical program used was Statgraphics Plus version 5.1.

Roots: After collecting aerial samples, each pot was carefully emptied onto a tray to recover the entire root system. The roots were gently dry-cleaned and weighed. They were then placed on blotting paper and stored in labeled plastic bags, which were left partially open to prevent rot. This process was repeated for each of the three replicates per treatment, and the roots were grouped and labeled for subsequent mycoflora analysis in the laboratory. In total, 12 samples were prepared for mycoflora analysis, representing each treatment combination: VI0, VI1, VI2, QI0, QI1, QI2, CVI0, CVI1, CVI2, CQI0, CQI1, and CQI2. After removing the roots, the three replicates of each treatment were pooled for final mycoflora analysis.

2.2.2. Characterization of Substrates

Determination of Physicochemical Parameters

The soils are derived from the sedimentary parent materials typical of the Central Iberian Plateau, predominantly composed of calcareous sediments and alluvial formations that influence their texture and chemical properties. The texture of the Villacañas soil (V) was sandy loam (sand: 72%; silt: 18%; clay: 11%). There was a very basic pH (8.60), which was slightly saline according to electrical conductivity (520 µS cm⁻¹). The C:N ratio of 11 suggests balanced organic matter decomposition and a favorable nitrogen mineralization potential. Iron, zinc, and copper contents were medium, total nitrogen and magnesium levels were low, and the Ca:Mg ratio possibly had Mg deficiencies. Finally, the assimilable P, K, Na, and Mn levels were high.

The analyzed Quero soil (Q) had a sandy loam texture, with a very basic pH (9.15) and high salinity (electrical conductivity: 13,650 µS cm⁻¹), which may affect nutrient availability and plant productivity. The organic matter is medium (2.22%), but the total nitrogen is low (0.10%), indicating a low nitrogen release capacity. Assimilable phosphorus is scarce (9 mg/kg), while potassium is high, though the high magnesium content (17.74 meq/100 g) and the low K:Mg ratio could lead to magnesium deficiencies. The cation exchange capacity (CEC) is adequate (10 meq/100 g), but the high sodium levels (3.80 meq/100 g) may cause soil structure issues. Micronutrients such as iron (0.35 mg/kg) are low, potentially limiting crop growth. Concerning heavy metals, values are within the established limits, indicating no significant contamination.

Composted sewage sludge was analyzed to determine its fertilizing value and contribution to the mixtures with soils. It is shown in

Table 2. N, P₂O₅, K₂O, and organic matter content of the sewage sludge composted applied in the mixtures. Note: the values refer to dry weight.

N (%)	P2O5 (%)	K2O (%)	Organic Matter (%)
3.57	3.12	0.89	6.10

.

The compositions of the CV and CQ mixtures are shown in

Table 3. Physicochemical composition of CV and CQ substrates. * Refers to the active calcium carbonate fraction, commonly used in agronomic evaluation of calcareous soils.

		CV		CQ
Parameter	Units	Result	Remarks	Result	Remarks
Sand	%	47.4	Loam texture (USDA)	65.3	Loam texture (USDA)
Loam	%	34.1		19.0
Clay	%	19.0		16.0
pH (extract 1:2.5)		7.8	Basic	7.9	Basic
Electrical Conductivity: EC (saturated paste)	µS cm−1	7600	Saline	9.150	Very saline
Chlorides	ppm	48		46
Sulfates	mg gypsum/100 g soil	503		592
Organic matter *	%	3.03	High	4.22	High
Total Nitrogen	%	0.43	Very high	0.37	High
C/N ratio		4.1	High nitrogen release	6.6	High nitrogen release
Nitric nitrogen	ppm	1.0	Very low	62.0	Moderate
Assimilable phosphorus	ppm	346.0	Very high	449.0	Very high
Total carbonates	%	44.3	Very high	21.9	High
Active limestone *	%	13.3	Potential nutritional problems	5.7
Assimilable potassium	meq/100 g	2.53	Very high	1.72	Very high
Assimilable sodium	meq/100 g	2.39	Very high	2.13	Very high
Assimilable calcium	meq/100 g	16.11	Very high	14.31	Very high
Assimilable magnesium	meq/100 g	2.23	Normal	2.03	Very high
K/mg ratio		1.1	Possible Mg deficiencies	0.8	Possible Mg deficiencies
Ca/mg ratio		7.224		7.05
Cation Exchange Capacity (CEC)	meq/100 g	17.14	Medium–normal	12.12	Moderate–normal
Assimilable iron	ppm	14.81		12.62
Assimilable zinc	ppm	13.71		13.43
Assimilable copper	ppm	2.48		2.61
Assimilable manganese	ppm	28.27		22.26
Assimilable boron	ppm	2.06		4.15

. The physicochemical parameters were determined as follows: the texture was measured by the Bouyoucos hydrometer method; pH was measured in a 1:2.5 soil–water extract using potentiometry; and electrical conductivity (EC) in saturated paste extract was determined by conductimetry. Chlorides were quantified by argentometry and sulfates using turbidimetry. Organic matter content was measured using the Walkley–Black method, total nitrogen via Kjeldahl digestion, and the C/N ratio was calculated arithmetically. Available phosphorus was analyzed by the Olsen method. Cation concentrations (Ca, Mg, K, and Na) were determined by atomic absorption spectrophotometry (AAS). Micronutrients (Fe, Zn, Cu, Mn, and B) were also measured via AAS. Carbonate content was assessed by Bernard’s calcimeter method and active lime by volumetric gasometry. The cation exchange capacity (CEC) was measured through ammonium acetate extraction. UV-VIS spectrophotometry was used for nitric nitrogen analysis.

The CV substrate, composed of one-third composted sludge and two-thirds soil from the Villacañas test plot, had a loamy texture with a basic pH. Based on its electrical conductivity, it was classified as a saline substrate. This substrate contains a high level of organic matter, and its C:N ratio suggests a high potential for nitrogen release. However, the percentage of active limestone could lead to potential nutritional issues, while its cation exchange capacity (CEC) is medium–normal. Levels of assimilable total nitrogen, phosphorus, potassium, sodium, and calcium were very high. Although the assimilable magnesium level was normal, the K/Mg ratio indicates potential magnesium deficiencies depending on the crop requirements.

In contrast, the CQ substrate, which consists of one-third composted sludge and two-thirds soil from the Quero trial plot, had a sandy loam texture, also with a basic pH and high salinity. This substrate featured high organic matter content, total nitrogen, and total carbonates. Its C:N ratio similarly indicated a high potential for nitrogen release, and the CEC is considered medium–normal. Levels of assimilable phosphorus, potassium, sodium, calcium, and magnesium are very high. As in the CV substrate, the K/Mg ratio suggests that magnesium deficiencies may occur.

Observation of Table 3 suggests that both substrates are characterized by high levels of organic matter and nutrients. The primary distinction between them lies in salinity; the CQ substrate exhibits high salinity, likely influenced by soil conditions at the Quero test plot. This factor should be considered when assessing future crop outcomes, as elevated salinity is often a limiting factor for optimal plant growth and development.

2.2.3. Determination of Mycoflora of Substrates Before Sowing

Many studies confirm the positive effect of sewage sludge on the growth of oligo- and macrotrophic bacteria and soil fungi, such as Geotrichum, Fusarium, Mucor, Penicillium, Mortierelta, Verlicillium, and Trichoderma after the direct application of sludge [13].

Each of the substrates used (Q, V, CQ, and CV), both with and without inoculum, was analyzed to determine its original total mycoflora. A total of eight samples were analyzed. The number of propagules per gram of soil for each fungal genus identified, along with counts of other microorganisms, is presented in

Table 4. Initial analysis of mycoflora in soil/substrates. Results expressed in number of propagules per gram of soil. Q: Quero soil; V: Villacañas soil; CQ: 1/3 composted sludge + 2/3 Quero soil; CV: 1/3 composted sludge + 2/3 Villacañas soil; QI: Quero soil with T-22 inoculum; VI: soil of Villacañas with inoculum of T-22; CQI: 1/3 composted sludge + 2/3 of Quero soil with T-22 inoculum; CVI: 1/3 composted sludge + 2/3 soil from Villacañas with T-22 inoculum.

Microorganism	Q	V	CQ	CV	QI	VI	CQI	CVI
Alternaria	0	0	2000	0	0	0	0	2000
Aspergillus	14,000	4000	40,000	0	0	2000	54,000	0
Candida	0	8000	4000	4000	0	0	0	0
Chirosporium	0	0	2000	4000	0	0	0	0
Cladosporium	0	2000	0	0	0	0	0	0
Fusarium type 1	4000	0	38,000	4000	0	0	4000	0
Fusarium type 2	0	0	0	4000	0	0	0	4000
Gliocadium	0	0	0	2000	0	0	4000	0
Helminthosporium	0	0	0	0	0	0	2000	0
Yeast	0	0	2000	0	0	0	0	0
Mucor	0	26,000	2000	10,000	2000	4000	0	4000
Oomycetus type 1	6000	4000	2000	10,000	4000	4000	0	0
Oomycetus type 2	4000	6000	0	4000	4000	16,000	0	8000
Other oomycetes distinct from each other	0	0	0	0	0	6000	2000	0
Paecylomices	0	2000	0	0	0	0	2000	0
Penicillium	16,000	10,000	22,000	80,000	0	6000	8000	2000
Periconiella	0	0	0	2000	0	0	0	2000
Phymatotrichum	0	0	24,000	12,000	0	0	10,000	0
Rhizopus	26,000	10,000	0	24,000	92,000	12,000	0	8000
Stigmine	0	6000	0	0	0	0	0	0
Thielaviopsis	0	2000	0	0	0	0	0	0
Tieghemiomyces	2000	12,000	10,000	24,000	0	4000	4000	8000
Non-sporulating type 1	10,000	0	16,000	0	4000	0	12,000	0
Non-sporulating type 2	0	4000	52,000	26,000	12,000	18,000	10,000	26,000
Non-sporulating other fungi	0	2000	26,000	4000	0	0	16,000	4000
Total Fungi (not including Trichoderma)	82,000	98,000	242,000	214,000	118,000	74,000	128,000	68,000
Trichoderma	0	0	0	6000	6000	30,000	0	108,000
Total Fungi (including Trichoderma)	82,000	98,000	242,000	220,000	124,000	104,000	128,000	176,000
Bacteria	2000	8000	0	0	0	2000	2000	2000
Total Microorganism	84,000	106,000	242,000	220,000	124,000	106,000	130,000	178,000

.

Mycoflora analysis was carried out using traditional isolation and culture techniques as a diagnostic method. Soil and substrate samples were analyzed to determine their total mycoflora. This analysis involved performing serial dilutions, followed by inoculation onto a culture medium.

From Table 4, it should be noted that the only genus that is pathogenic in cereals is Helminthosporium. The Fusarium obtained is ruled out. They can be Fusarium graminearum, a pathogenic fungus also found in cereals and detected in this service. Phymatotrichum is a fungus that can cause, according to the literature, root rot in some plants. However, it is not cited as a pathogen in cereals. On the other hand, it should be considered that very fast-growing fungi have been detected, such as Rhizopus spp., which, although they have been eliminated as soon as they have been detected and pricked, have been able to prevent the detection of some nearby colonies.

2.2.4. Statistical Procedure

Data were subjected to statistical processing by an analysis of variance. All the statistical calculations were performed with Statgraphic Plus 5.1. An ANOVA was conducted to analyze treatment effects, and significant differences were assessed using the least significant difference (LSD) test at p < 0.05.

3. Results and Discussion

3.1. Results in Substrates

Soil–crop interactions influenced by additives such as composted sludge or the introduction of microorganisms like Trichoderma are challenging to evaluate. Their study must be conducted on a case-by-case basis, considering the specific doses of each component used [18].

In our study, the analysis of the samples was carried out by serial dilutions, and then, the sowing was conducted in a culture medium. The number of propagules per gram of soil for each of the fungal genera obtained, as well as other microorganisms, is presented in Figure 2. From these results, it should be noted that sporulated species that have not been identified are not suspected of being pathogenic in plants. The Fusarium obtained is ruled out to be Fusarium graminearum, a pathogenic fungus in cereals. One of the most prevalent soilborne fungi, Fusarium spp., can infect a wide variety of plant species across a range of climates and are the cause of mycotoxin formation in plant-based products that are preserved [19]. In this sense, two significant wheat diseases are Fusarium crown and root rot (FCRR) and Fusarium head blight (FHB) [20]. It is known thanks to genomic studies on diseases of bread wheat [21].

On the other hand, it should be considered that the presence of Trichoderma, a very fast-growing fungus, may have prevented the detection of some nearby colonies. This fungus was chopped and eliminated as soon as it was detected, but in some cases, it invaded nearby colonies.

The results in Figure 2 show significant differences in the diversity and abundance of fungi across the substrate treatments from V soil. Several key trends can be observed.

Sewage sludge is used in different ways to improve soil quality. For example, it is used to produce biochar and improve soil acidity and the production of crops such as wheat or spinach, depending on the dose used [22]. Changes in the activity and diversity of the soil microbiome were also predicted after the application of composted sewage sludge [13].

According to reports, microorganisms found in sludge are unable to fully inhabit the soil environment and die quickly, becoming an extra source of organic matter for native bacteria [23,24]. In our study, composted sludge clearly influenced microbial dynamics, as treatments containing it (CVI0, CVI1, CVI2) exhibit a higher total microbial count compared to soils without compost (VI0, VI1, VI2). Hence, Penicillium, Trichoderma, and Fusarium notably increase in presence when composted sludge is applied. Moreover, the influence of inoculation is remarkable. In this sense, Trichoderma was particularly abundant in inoculated treatments (VI2, CVI1, and CVI2), reaching up to 112,000 propagules/g in CVI2. In contrast, in non-inoculated soils (VI0, CVI0), Trichoderma is absent, indicating that its presence is directly related to inoculation and was not naturally occurring in the substrates used.

In soils without compost (VI0, VI1, VI2), Fusarium is absent, but it appears in the composted sludge treatments (CVI0, CVI1, CVI2), suggesting that sludge composting may favor its development. However, the high presence of Trichoderma in these same treatments could counteract this effect, given its biocontrol potential. Concerning total mycoflora, treatments with composted sludge (CVI0, CVI1, CVI2) exhibit a higher fungal load compared to soils without compost. In conclusion, CVI1 and CVI2 showed the greatest fungal diversity, indicating that the combination of compost and inoculation can substantially modify soil microbial composition.

Figure 3 illustrates the comparison of microbial composition in V-substrates (graph on the left) and CV-substrates (graph on the right) based on the number of propagules per gram of soil. The total microbial count was consistently higher in CV substrates compared to V substrates, with values exceeding 600,000 propagules/g in certain treatments (CVI0 and CVI1). This suggests that composted sludges significantly enhance microbial proliferation, likely due to increased organic matter availability.

In the V substrates, the fungal community (excluding Trichoderma) fluctuates, showing a moderate increase in VI1 and VI2. On the other hand, in CV substrates, the total fungal population remains relatively stable, indicating that composted sludge may support fungal diversity but does not drastically increase non-Trichoderma fungal populations. Considering the total fungi (including Trichoderma) in V substrates, Trichoderma introduction in VI1 and VI2 leads to a marked increase in total fungal propagules, reaching the highest values in VI2. In CV substrates, the total fungi (including Trichoderma) remains high across all treatments, reinforcing the idea that composted sludge provides a favorable environment for fungal colonization.

Considering only Trichoderma propagules, in V substrates, Trichoderma is barely present in the control (V and VI0) but increases substantially in VI1 and VI2 due to inoculation. In the CV substrates, the Trichoderma propagules were also low in CV and CVI0 but raised significantly in CVI1 and CVI2, suggesting successful colonization in the presence of composted sludge. The peak in Trichoderma population in CVI2 (~100,000 propagules/g) confirms that combining composted sludge with inoculation enhances its establishment.

Figure 4 presents the fungal and bacterial composition in the substrate treatments from Q soil at the end of the experiment, with the results expressed in propagules per gram of soil.

The highest total microbial count was observed in CQI0 (306,000 propagules/g), indicating that composted sludge without inoculation fosters a highly diverse microbial community. Quero soils (QI0, QI1, and QI2) show lower microbial counts compared to their composted sludge counterparts (CQI0, CQI1, and CQI2), suggesting that sludge amendment enhances microbial proliferation. The lowest values appear in QI0 (164,000 propagules/g) and CQI1 (140,000 propagules/g), indicating possible inhibitory effects or competition among microbial groups.

Considering total fungi (without Trichoderma), Quero soils (QI0, QI1, and QI2) displayed a fungal population ranging between 114,000 and 164,000 propagules/g, with QI0 showing the highest fungal diversity. Composted sludge treatments (CQI0, CQI1, and CQI2) exhibit a significant increase in total fungi, with the highest value in CQI0 (306,000 propagules/g), reinforcing the idea that organic matter amendments stimulate fungal colonization. However, in CQI1 and CQI2, fungal propagules decrease, possibly due to competitive interactions with Trichoderma.

Concerning key fungal groups, it was observed that Aspergillus and Penicillium were among the most abundant fungi across all treatments, highlighting their competitive advantage in different soil conditions. On the other hand, Fusarium populations were moderate in all treatments, with slight reductions in CQI substrates, potentially due to the antagonistic effects of Trichoderma. Finally, non-sporulated fungi were prominent, especially QI2 with 80,000 propagules/g, but there was a significant decrease in the composted sludge treatments, indicating a shift in fungal community dynamics.

It can be stated that composted sludge enhances total microbial and fungal diversity, especially in CQI0. Moreover, Trichoderma proliferates significantly when both composted sludge and inoculation are applied (CQI2), demonstrating its adaptability to organic amendments. Finally, fungal communities shift in response to sludge amendment and inoculation, with non-sporulated fungi decreasing and beneficial fungi (Trichoderma, Penicillium, and Aspergillus) becoming more dominant.

Figure 5 presents the microbial composition of Q-substrates (left) and CQ-substrates (right) at the end of the experiment.

Concerning total fungi (without Trichoderma), the population of total fungi varied from 82,000 (Q) to 164,000 (QI0), followed by a decrease to 114,000 (QI1) and a moderate increase to 156,000 (QI2). In contrast, the fungal population was much higher across all composted treatments, starting at 242,000 (CQ), increasing to 306,000 (CQI0), and then decreasing to 106,000 (CQI1) and 88,000 (CQI2). Consequently, the total fungal population (without Trichoderma) was consistently higher when compost was applied to the substrates (242,000–306,000) compared to those without compost (82,000–164,000). This confirms that the combination of composted sludge and inoculation strongly favors Trichoderma colonization, likely due to improved nutrient availability.

Considering Trichoderma propagules, Trichoderma is absent in QI0 but reaches 32,000 propagules/g in QI1 and 18,000 propagules/g in QI2, showing that inoculation is effective. In CQI substrates, Trichoderma follows an increasing trend, with CQI0 having no Trichoderma, CQI1 reaching 34,000 propagules/g, and CQI2 displaying the highest concentration (134,000 propagules/g).

To sum up the comparison among all substrates and number of microorganisms, it is worth noting that the number of microorganisms existing in any of the substrates with only Quero soil (Figure 5) was considerably lower than the substrates already seen with the Villacañas soil (Figure 3). One of the reasons related to this fact could be the high salt content of the Quero soil, which generally inhibits microbial activity. There were hardly any differences in the number of microorganisms between the inoculum and non-inoculum treatments.

With a more pronounced difference than in the case of Quero soil without compost, the CQI1 treatment is the one that contained the lowest number of total microorganisms. The differences between the treatments, with respect to the initial substrate, are smaller than in the case of the initial soil of Villacañas. It seems that the composition of this type of very saline substrate hinders microbial activity despite inoculations.

3.2. Results in Plant Material

3.2.1. Physical Parameters

The number of plants per pot containing uninoculated compost fifty days after seeding (CVI0 and CQI0) was statistically significantly less than the rest (Figure 6).

According to the p-value obtained for the additive effect (

Table 5. p values for additive (A): composted/non-composted; inoculum (B): I₀, I; soil (C): Q/V and interactions (A)-(B), (A)-(C), (B)-(C), and (A)-(B)-(C) in number of plants per pot. (*) Denotes significant differences with a confidence level greater than 95%, since p-value is less than 0.05 for the least significant difference (LSD) test.

	p-Values
Additive (A)	0.0306 *
Inoculum (B)	0.7515
Soil (C)	0.4863
Interactions
AB	0.4026
AC	0.4863
BC	0.9038
ABC	0.2416

), the number of plants up to this time of the experiment was mainly influenced by the addition or not of compost to the soil.

Although low C:N ratios can sometimes be associated with compost immaturity or phytotoxicity, no adverse effects on seed germination were observed in our experiment. In fact, emergence was enhanced in treatments with compost and Trichoderma. Those seeds grown on these same media with Trichoderma inoculum (CVI and CQI) grew a greater number of plants (Figure 6). It should be noted that plants with the I2 treatment were not yet available at this time.

At the end of the experiment (215 days after sowing), the parameters shown in

Table 6. Average value of the physical parameters measured in the plants according to the different substrates used. Different letters indicate significant differences.

Treatment	Number of Plants Per Pot	Number of Spikes	Total Weight of Spikes (g)	Average Weight of a Spike (g)	Root Weight (g)	Shoot Weight (g)	Shoot/Root Ratio
VI0	9.0 ab	2.7 ab	0.15 a	0.04 ab	0.61 c	1.30 ab	3.30 abc
VI1	5.0 ab	5.0 b	0.37 a	0.07 abc	0.16 abc	0.76 ab	4.70 abc
VI2	8.7 ab	3.3 ab	0.22 a	0.04 ab	0.50 abc	1.20 ab	2.58 ab
CVI0	9.0 ab	2.3 ab	0.20 a	0.07 abc	0.24 abc	1.87 b	7.37 cde
CVI1	4.7 a	3.3 ab	0.30 a	0.09 bc	0.14 ab	1.50 ab	10.37 of
CVI2	6.0 ab	1.3 a	0.07 a	0.03 ab	0.08 a	0.92 ab	11.47 e
QI0	5.0 a	4.3 ab	0.31 a	0.08 bc	0.48 bc	0.92 ab	2.09 a
QI1	7.3 ab	3.3 ab	0.21 a	0.07 abc	0.40 bc	1.07 ab	2.76 ab
QI2	9.6 b	3.3 ab	0.27 a	0.04 ab	0.42 abc	1.26 ab	3.03 ab
CQI0	5.0 a	1.5 a	0.05 a	0.02 a	0.07 a	0.48 a	6.36 abcd
CQI1	5.3 ab	1.3 a	0.06 a	0.03 ab	0.21 ab	0.77 ab	3.46 abc
CQI2	6.3 ab	3.0 ab	0.31 a	0.10 c	0.27 abc	1.80 b	6.78 bcd

were recorded, and a new factorial ANOVA was conducted to statistically assess the results (

Table 7. p values for additive (A): composted/non-composted; inoculum (B): I₀, I1, I2; soil (C): Q/V and interactions (A)-(B), (A)-(C), (B)-(C), and (A)-(B)-(C) in number of plants per pot, total root weight, number of spikes per pot, total spikes weight, total weight of the spikes, and shoot/root ratio. (*) It denotes significant differences with a confidence level greater than 95%, since p-value is less than 0.05 for the least significant difference (LSD) test.

	Number of Plants Per Pot	Total Root Weight	Number of Spikes Per Pot	Total Shoot Weight	Total Spikes Weight	Shoot/Root Ratio
Additive (A)	0.1273	0.0010 *	0.00261 *	0.5766	0.2004	0.0000 *
Inoculum (B)	0.2125	0.5401	0.6626	0.6309	0.7584	0.5154
Soil (C)	0.4731	0.9658	0.6395	0.3156	0.7858	0.0106 *
Interactions
AB	0.4197	0.4476	0.8776	0.9798	0.9354	0.4281
AC	0.7187	0.7111	0.7546	0.3930	0.6117	0.0447 *
BC	0.0515	0.3521	0.2474	0.0700	0.1334	0.4243
ABC	0.9568	0.5904	0.4031	0.1586	0.3467	0.2907

).

Regarding the final number of plants per pot, the highest values (more than eight plants per pot) were obtained with treatments VI₀, VI2, CVI0, and QI2. The statistical analysis determines that the highest number of plants per pot was obtained significantly compared to the rest of the treatments with the QI2 substrate (Quero soil with the second dose of inoculum). However, there are no significant differences in the number of plants per pot between the CVI₁, QI0, and CQI0 treatments, nor between VIO, VI1, VI2, CVI0, CVI2, QI1, CQI1, and CQI2.

Studies on wheat have shown how the addition of sewage sludge to limestone soil affects dry matter and heavy metal content [25], and it was found that the root restriction for heavy metals translocation depended on the type of metal and affected the spikes. Regarding the number of spikes per pot, the highest value was obtained, significantly compared to the rest of the treatments with VI1. With the CVI2, CQI0, and CQI1 substrates, there were no differences between the rest: VI0, VI2, CVI0, CVI1, QI0, QI1, QI2, and CQI2.

Statistically, no differences were observed regarding the total weight of the spikes between the treatments. They did occur with the rest of the weights taken in the trial. Thus, both the average weight of a spike and the weight of the aerial part were significantly higher with the CQI₂ treatment, i.e., Quero soil with compost and three applications of T-22, although it should also be noted that a similar value for the aerial weight was also obtained with CVI0 (substrate that also contains composted sludge). Likewise, the significantly lower values of these weights, which, in this case, were given with CQI0, coincide with the same treatment.

Finally, the root system with the lowest weight occurred in the CVI2 and CQI0 treatments. On the other hand, the root biomass with the highest weight occurred significantly with substrate VI₀.

On the other hand, a factorial ANOVA (Table 7) shows that the effect of the additive, i.e., the presence of compost in the soil, had a significant effect on total root weight, number of spikes per pot, and shoot/root ratio. Moreover, the shoot/root ratio depended on the soil and the interaction between the additive and the soil. The interaction between the application of manure with or without Trichoderma has been studied [26], and it has been seen how the inoculation improves the growth parameters (leaf, root, dry weight, etc.). And it was also found that Trichoderma-reinforced compost with poultry manure was very beneficial for lowering preemergence and post-emergence seedling mortality, disease incidence, and disease severity of chickpea in the field [27]. The combination of Trichoderma harzianum with manure has also proven useful in promoting plant growth and enhancing photosynthetic efficiency in the cultivation of Cordia americana [28]. These results are consistent with our findings, which show that adding Trichoderma to composted sludge substrates improves plant physical characteristics.

Although the emergence of seedlings was enhanced in treatments containing compost and Trichoderma harzianum, we recognize that seedling count alone may not comprehensively reflect the potential phytotoxicity of the composted substrate. Therefore, future studies should include a germination index (GI) test as a standardized and sensitive method to evaluate phytotoxicity and seed vigor. The GI could also serve as a consistent metric to compare the effects of different soil amendments under various treatment conditions.

3.2.2. Mycoflora

It is important to analyze the rhizosphere for pathogens such as Fusarium. It shows the importance of the soil microbiome to compete against this fungus and other pathogens, since soil health and the inhibition of soil-borne pathogens were significantly improved by the high fungal diversity [29]. A fungal analysis of the samples was conducted on the neck and roots of the plants to diagnose the presence of fungi. The diagnostic method consisted of isolation and obtaining a pure culture. In each sample, fungal isolation was carried out from both the neck and the roots.

Five explants were used. Two corresponded to the neck and five to the roots.

The results were as follows:

• VI0:
• Neck: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one Penicillium sp.
• Roots: five isolated fungi: one non-sporulated fungus (suspected of Fusarium sp.), two non-sporulated fungi distinct from each other, and two isolates identified as possible Dendryphiopsis.
• VI1:
• Neck: three isolated fungi: one non-sporulated fungus (suspected of Fusarium sp.), one Mucor sp., and one Helminthosporium sp.
• Roots: eight isolated fungi: one non-sporular (suspected of Fusarium sp.), one non-sporular, one Fusarium sp., one Penicillium sp., three isolates identified as possible Dendryphiopsis, and one unidentified sporulated isolate.
• VI2:
• Neck: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one Helminthosporum sp.
• Roots: six isolated fungi: three non-sporulated fungi (suspected of Fusarium sp.), two non-sporulated fungi, and one possible Dendryphiopsis.
• CVI0:
• Neck: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.), and one Helminthosporium sp.
• Roots: three isolated fungi: three non-sporulated fungi (suspected of Fusarium sp.).
• CVI1:
• Neck: two isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.).
• Roots: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one possible Dendryphiopsis.
• CVI2:
• Neck: three isolated fungi: two non-sporular (suspected of Fusarium sp.) and one non-sporular.
• Roots: four isolated fungi: one non-sporular (suspected of Fusarium sp.), one non-sporular, two Fusarium sp.
• QI0:
• Neck: two isolated fungi: two Fusarium sp. (different from each other).
• Roots: six isolated fungi: one non-sporulated fungus (suspected of Fusarium sp.), two different non-sporulated fungi, and three equal non-sporulated fungi.
• QI1:
• Neck: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one Helminthosporum sp.
• Roots: five isolated fungi: four non-sporular (suspected of Fusarium sp.) and one Fusarium sp.
• QI2:
• Neck: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one Rhizopus sp.
• Roots: five isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.), one Fusarium sp., one Penicillium sp., and one non-sporulated fungi.
• CQI0:
• Neck: three isolated fungi: three non-sporulated fungi (suspected of Fusarium sp.).
• Roots: four isolated fungi: four non-sporulated fungi (suspected of Fusarium sp.).
• CQI1:
• Neck: two isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.).
• Roots: three isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.) and one Penicillium sp.
• CQI2:
• Neck: two isolated fungi: two non-sporulated fungi (suspected of Fusarium sp.).
• Roots: four isolated fungi: two non-sporular (suspected of Fusarium sp.), one Fusarium sp., one non-sporular.

From these results, it should be noted that the only genus that has been detected and is known as a pathogen in cereals is Helminthosporum. The Fusarium obtained is ruled out to be Fusarium graminearum, a pathogenic fungus in cereals. The fungi identified as non-sporulated fungi (suspected of Fusarium sp.) are similar to each other. By microscopic preparation, some of these isolates were identified as Fusarium. Several species of Fusarium, apart from F. Gramineearum, are cited as cereal pathogens. However, Fusarium sp. is a soil fungus that is frequently detected in the underground parts of plants without symptoms. In general, pathogenicity testing is necessary for complete identification. Fungi identified as possible Dendryphiopsis have not been recognized as pathogens in cereals. Notably, fungi that have not shown sporulation have not been identified. Hence, the analysis of treatments based on the fungal flora isolated from wheat roots and root collars revealed the following key differences. The presence of Fusarium sp. is mainly in its non-sporulating form and is the predominant fungus across all treatments. It is found in both the root collar and root but is more frequent in roots. Regarding the fungal diversity in the roots vs. the root collar, it is remarkable that the roots generally exhibited greater fungal diversity compared to the root collar. In treatments VI1 and QI2, there was higher species variability in roots, including Dendryphiopsis, Penicillium, and Rhizopus. In contrast, the root collar typically contains two to three isolates with lower diversity. With regard to differences among the treatments, VI showed relatively high diversity in the roots, with the presence of Fusarium, Dendryphiopsis, Penicillium, and other genera. CVI exhibited lower diversity compared to VI, with Fusarium predominating in both roots and the root collar. QI maintained diversity similar to VI but differed in the fungi present in the root collar (e.g., Rhizopus in QI2). Finally, CQI displayed the lowest diversity, with nearly exclusive dominance of Fusarium in both plant parts.

4. Conclusions

This study demonstrates that the combined application of Trichoderma harzianum strain T-22 and composted sewage sludge significantly influences wheat growth and soil microbial composition, The analysis of the physical parameters of plant material shows that the presence of composted sewage sludge in the substrates affects seed emergence and significantly modifies the total root weight, the number of spikes, and the shoot-to-root ratio. A greater number of plants were observed in Villacañas soils, while the average weight of the spikes and the aerial part was higher in treatments with composted sludge (CQI2 and CVI0). Regarding fungal analysis, Helminthosporium was identified as the only pathogen in cereals, while Fusarium sp. predominated in the controls, suggesting that experimental treatments promote greater fungal diversity. In the substrates, there was a general increase in microorganisms after cultivation, except in CQI1, with lower microbial activity in the saline soils of Quero. High salinity in Quero soil negatively impacted microbial activity and wheat growth, highlighting the need to consider salinity specifically when applying organic amendments in semi-arid environments. The high salinity observed in Quero soil likely originates from its sedimentary parent material and natural soil development under semi-arid conditions, where evapotranspiration exceeds precipitation. Nevertheless, the addition of composted sewage sludge increased fungal diversity and abundance, including Trichoderma, potentially benefiting the biological control of pathogens. These findings support the hypothesis that organic amendments and microbial inoculation can enhance soil microbiota and improve plant resilience. In conclusion, the synergy between Trichoderma and composted sludge offers a promising sustainable alternative to chemical fertilizers and fungicides, improving soil health and plant resilience.

Furthermore, we recommend incorporating the germination index (GI) in future experiments to better assess the potential phytotoxicity of the composted amendments. The GI would provide an additional layer of validation and facilitate standardized comparisons across treatments.

One major limitation of this study was the controlled environment. Therefore, future research should focus on large-scale field experiments to validate these findings and refine the application strategies tailored to different soil conditions and climatic regions.