Enhanced activity of Trichoderma asperellum introduced in solarized soil and its implications on the integrated control of strawberry-black root rot

Abstract

An effective method for maintaining the activity and longevity of microorganisms in adverse conditions is microencapsulation. In the present study, synthetic alginate pellets were developed as carriers for the biocontrol agent Trichoderma asperellum. In two field experiments, solarization was applied for three weeks to loamy clay soil that was naturally infested with strawberry-black root rot fungi (Fusarium solani, Rhizoctonia solani and Machrophomina phaseolina). Following solarization, T. asperellum-based alginate pellets or spore suspension were added to the soil. Data reveal that, three weeks solarization of irrigated soil increased its maximum temperature reached by 11–14.2 °C (1–10 cm depth), 11.6–13.1 °C (11–20 cm depth) and 10.1–12.2 °C (21–30 cm depth). In either trial, solarization also successfully lowers the vitality of strawberry-black root rot fungi directly after the solarization phase. When compared to controls, strawberry-black root rot was substantially less common in solarized plots. In two field trials, soil solarization followed by inoculation with alginate pellets based on T. asperellum led to the greatest reductions in black root rot incidence (59.3 and 74.1 %) and severity (72.5 and 75.2 %), as compared to un-solarized control plots. In vivo studies, this treatment dramatically increased the activity of defensive enzymes (peroxidase and chitinase) and strawberry yield (60.5 and 60.0 %, respectively), as compared to non-solarized control plots. In two field studies, the rhizosphere population of native Trichoderma spp. Developed more in solarized soils after the application of alginate pellets based on T. asperellum (86.5 and 83.6 %, respectively), compared to the non-solarized control.

1. Introduction

Strawberry (Fragaria x ananassa Duch.) is one of Egypt's most important horticulture crops, with an estimated 12,579 ha planted in 2021 and yielding 470,000 tones [1]. In Europe, Germany was ranked fourth after Spain, the Russian Federation and Poland in the same year, regarding the production of strawberries with about 144,000 tones on 13,200 ha [2]. It is a perennial herbaceous plant of the Rosaceae family grown for its fruit (or strawberry). Strawberry fruits are well-known for their nutritional value and several health advantages. They are the primary source of minerals and vitamins [3]. Under Egyptian conditions, strawberry production is challenged by a number of diseases that cause significant quantitative and qualitative losses in fruit output [4]. Yet, black root rot of strawberry demonstrates a complex and widespread diseases as several species of fungal pathogens are often inducing the disease. The main species that were recently recorded in Egypt comprise Fusarium solani, Macrophomina phaseolina, Pythium spp., Phytophthora spp. and Rhizoctonia spp [5,6]. In Europe, the disease affects plant growth which in consequence can result in losses at about 30–50 percent [7]. Strawberry plants with black root rot exhibit an overall lack of vigor, stunted runners and small fruit. Aboveground symptoms may be similar to those of other root disorders; thus the roots must be examined before the condition can be diagnosed [8]. Plants with black root rot have substantially smaller and less fibrous roots than healthy plants. The roots may contain black spots or be completely black. There are also fewer feeding roots. Plant injury is particularly noticeable in low or compacted strawberry fields with insufficient drainage. Wet soil deficient in organic materials promotes black root rot [8]. Progress of the infection gradually turns the roots into completely black with serious disorder in their functions where the outer bark rots and disintegrates from the vascular cylinder. Because the disease kills the feeder roots and deteriorates root functions, the whole plant vigor is substantially affected leading to serious reduction in the fruit production [9]. The diffusion and infection of such pathogens constitute a considerable impediment for producing strawberries with high qualitative and quantitative yields. With the frequent intensive culture in the same soil yearly, strawberry transplants and/or crop residues of the previous year represent sources of disease inocula which enhance the risk of root rot disease control failure for strawberry yield. On the other hand, growers and stakeholders must face the dilemma of restricting or banning many effective fumigant and chemical pesticides due to ecological pollution and hazards. This dilemma also materialized in maximum residue limits imposed by regulations to minimize pesticide residues especially for the export fruits. Thus, it is imperative to search for safe and reliable alternative techniques to suppress these pathogens and increased the yield.

In warm tropical regions, soil solarization provides a risk-free alternative to chemical fumigation of the soil to reduce soil-borne diseases, pests and weeds [10]. To increase heat accumulation, the soil surface is covered for a few weeks with a thin, clear plastic tarp. The consequent high soil temperatures partially disinfest the soil by inactivating some of the soil microorganisms [11]. Plant development and yield are typically improved when soil solarization is used to control soil-borne pests [12]. Solarization exclusively affects mesophyllic organisms, the majority of which are plant diseases and pests, while sparing helpful fungi and bacteria that encourage plant growth [10]. In solarized soil, soil-borne plant pathogens may grow weaker and more susceptible to antagonists or changes in soil environment even if they are not directly inactivated by heat [11]. Although soil solarization has a positive impact on pathogens in the topsoil layers, its effectiveness decreases as soil depth increases [13]. To increase the effectiveness of solarization, it is frequently required to include more suppressive components [13].

For the management of soil borne diseases, biological control using invasive hostile microorganisms offers another appealing and environmentally benign option. The proliferation and colonization of biocontrol agents may ideally be aided by the combination of biological control and solarization, which would then result in soil suppressiveness to plant diseases [14]. According to earlier research, solarization and the use of Trichoderma spp. can effectively manage diseases that are transmitted through the soil [15]. Numerous Trichoderma spp. have been discovered in soil and plant roots. Research has shown that Trichoderma spp. uses a variety of antagonistic, antibiotic, competitive and nutrient-scavenging mechanisms, as well as the creation of systemic plant resistance, to achieve biological control [16]. However, the successful implementation and field applications of biological control are dependent on the efficient transport of these agents in their most functionally active form to the target site [17]. Encapsulation is one breakthrough technology in current formulations that has opened up new opportunities for generating environmentally acceptable and effective biopesticides to manage plant diseases [18]. This process is classified as macro-, micro-, or nanoencapsulation according to the size of the particles formed, which ranges from a few nanometers to a few millimeters [18]. Encapsulation in macroparticles (1–4 mm in diameter) is a potential technology, particularly in developing nations, because it does not require any specific equipment or resources and the necessary processes are widely available [18]. Fungal encapsulation is a realistic technology that protects Trichoderma spp. spores and ensures their long-term survival by gradually releasing them into the soil. Encapsulated Trichoderma spp. are thus more likely than free-living spores to be successful at controlling plant diseases due to their extended survival under adverse environmental circumstances [19,20]. Microencapsulation creates a protective barrier around the biological control organism, ensuring that harmful environmental elements such as pH, humidity, and UV light cannot interfere with its action [21,22]. Due to its biocompatibility, low cost, low toxicity and easy synthesis (by adding divalent cations like Ca⁺²), alginate, a natural copolymer, has gained a lot of interest in the food, medical and agricultural industries [23]. The sodium alginate solution is dripped via a syringe needle or nozzle into a CaCl₂ solution in the classic extrusion generation approach to create spherical and homogeneous calcium alginate hydrogel beads. Because of their uniform shape, tiny size and effective encapsulation properties, microcapsules are the most often used formulation [19]. This is because encapsulated microorganisms have a relatively high survivability. Many biocontrol agents, including T. asperellum, have been produced into microcapsules using extrusion methods to date [17,24]. However, there are no data on the field efficacy of combining T. asperellum macrocapsules and solarization. As a result, major breakthroughs are required to fully realize the potential of this advantageous combination in disease management and to enable the commercialization of farmer-friendly solutions. Therefore, the current study attempted to evaluate the efficacy of soil solarization followed by the introduction of T. asperellum-based alginate beads for the treatment of black root rot in strawberries under field conditions.

2. Material and methods

2.1. Strawberry-black root rot pathogens

The soil borne fungi, R. solani, F. solani and M. phaseolina, the pathogens causing strawberry-black root rot, were used in this investigation. These pathogens were isolated from strawberry plants exhibiting black root rot symptoms. Plants with infected roots were cleaned with running tap water, their root cut into small pieces (1.0 cm) and surface sterilized for 2 min in 1 percent aqueous solution of sodium hypochlorite, rinsed twice in sterile distilled water and dried in a laminar flow-cabinet. The sterilized root segments were placed on 1.5 percent water agar amended with streptomycin sulfate. After three days incubation, hyphal tips of isolates were transferred into potato dextrose agar medium (PDA Difco Laboratories, Detroit, MI, EUA) plates, which were incubated in the laboratory at room temperature (25±2°C). The fungal isolates were purified following hyphal tip technique, identified using standard key and stored in PDA slants in test tubes at 5°C [25]. Pathogenic ability of these strains toward strawberry plants was confirmed in a previous study [26].

2.2. Trichoderma asperellum strain

The current study used the strain of Trichoderma asperellum (accession number ON764312.1 (https://www.ncbi.nlm.nih.gov/nuccore/ON764312.1/). This strain was isolated from the rhizosphere of healthy strawberry plants, purified and identified using morphological characteristics and based on sequencing the internal transcribed spacer (ITS) and tef1 gen regions of ribosomal DNA in a previous study [27]. This strain had strong antagonistic activity against strawberry-black root rot pathogens in vitro and in vivo, according to a prior study [28].

2.3. Laboratory experiments

2.3.1. Preparation of alginate pellets based- T. asperellum

Trichoderma asperellum alginate pellets were prepared as described by Locatelli et al. [29]. Trichoderma asperellum was grown in 1-liter sterile Erlenmeyer flasks with 500 mL of sterile potato dextrose broth (PDB, Difco Laboratories, Detroit, MI, EUA). For two weeks, inoculated flasks were incubated on a Lab-Line Orbit Environ-shaker at 150 rpm and 28 ± 2 °C. The shake culture suspension was gently mixed for 20 s in a coffee mixer on low speed, and then filtered through a sterilized double-layered muslin cloth. The spore population was adjusted to 10⁸ CFU mL⁻¹ and the resulting spore suspension was utilized to prepare a lignite-based formulation [30]. Trichoderma asperellum alginate pellets were prepare by adding 50 mL of the spore suspension (10⁸ spore/mL) with 20 g of starch to 100 mL of 2 percent (wt/vol) aqueous sodium alginate (Sigma Chemical Company, St. Louis, Mo.) and thoroughly mixing. The mixture was dropped into 0.25 M aqueous CaCl₂. To get typical solid beads, the beads were kept in the CaCl₂ solution at room temperature for an additional 1–2 h. After removing the CaCl₂ solution, the produced pellets were placed on waxed paper to dry at 22–25 °C. Before testing, the new pellets were stored in sealed flasks at 5 °C for two days. Before the in vitro test and before exposure to soil in field experiments, the viable population size of T. asperellum in the pellets was determined by disintegrating the pellets in sterile water in a sterile mortar and assaying for colony-forming units by dilution plate method using Glucose-Czapek's medium containing Rose-Bengal at a concentration of 1:5000 (wt/vol) [30].

2.3.2. Antagonistic activity of alginate pellets based- T. asperellum

Alginate pellets based- T. asperellum were examined in vitro for their ability to inhibit R. solani, F. solani and M. phaseolina. Each pathogenic fungal species was cultivated individually on potato dextrose agar (PDA, Difco Laboratories, Detroit, MI, EUA) medium for 7 days at 25 ± 2 °C before experiments. In vitro tests were carried out using the dual culture technique [31] to evaluate the biocontrol ability of alginate pellets based- T. asperellum on the growth of each fungus (F. solani, R. solani and M. phaseolina). One T. asperellum pellet and one disc (5-mm diameter) of the target fungus species were placed on opposite sides of a 9-cm-diameter Petri plates with PDA medium and were incubated at 25 ± 2 °C for 10 days. Each test used five plates (replicates) and was repeated once. On each fungal growth region, the percentage reduction apparently influenced by T. asperellum pellet was determined.

2.4. Field experiments

During the growing season of 2021–2022, two field experiments were undertaken on clay-loam soil at two sites in Met Kanana village, Qallubia governorate, Egypt, which had previously been grown with strawberry and had a history of strawberry-black root rot. The soil had 7.6 mEq/L Ca⁺⁺, 5.2 mEq/L Mg⁺⁺, 7.3 mEq/L Na+, 0.37 mEq/L K⁺, 1.7 mEq/L HCO⁻₃, 12.0 mEq/L Cl−, 9.88 mEq/L SO₄, 143.4 ppm available nitrogen (N), 27.5 ppm available phosphors (P), 317.8 ppm available potassium (K), 4.7 ppm iron, 8.0 ppm manganese, 1.5 ppm zinc, pH (1:2:5) 7.52, and EC (dS/m) (1:5) 1.29. The field was fully pulverized and raised nursery plots measuring 4.0 × 8.0 m with a buffer space of 0.6 m were prepared. Plots were set up in a randomized block design using a factorial scheme (2 × 3), with a mix of soil solarization (solarized and non-solarized soils) and the application of chemical or biological treatments, as well as controls. Each treatment was carried out three times. Prior to solarization, plots were irrigated up to field capacity. A clear, UV-stabilized polyethylene tarp (200 μm thick) was laid as a tarp and exposed to sunshine during three weeks (from 2021 to 07-24 to 2021-08-14). A soil thermometer (Elite Scientific Corp., Ind.) was used to continuously measure soil temperature at 10, 20 and 30 cm depths. After solarization, the tarp was removed and T. asperellum inocula (spore suspension or alginate pellets) were incorporated into soil at a rate of 300 g pellets (10⁶ CFU/g fresh pellet) or 300 mL (10⁶ CFU mL⁻¹) spore suspension/m length of the planted row. Strawberry transplants (California cv.) were transplanted at 7 cm depth in rows on September 1st (32 holes/row, a seedling was put in each hole). Strawberry transplants were soaked in the fungicide Actamyl (70 % Thiophanate-methyl, Arrest Life Science Company, France) solution (3 g WP/L water) before transplantation for comparison. Production practices were followed during the growing season [9]. The treatments used in the two field experiments were outlined in Table (1).

Table 1.

Treatments applied to soil before strawberry transplants sown under two field locations in Qalubia governorate during 2021/2022 growing season.

Treatment	Description	Method of application
T1	Spore suspension-based T. asperellum application	Immediately before strawberry transplants were transplanting, soil was treated with spore suspension-based T. asperellum by the rate of 300 mL (106 CFU mL−1) spore suspension/m length of the planted row.
T2	Alginate pellet-based T. asperellum application	Immediately before strawberry transplants were transplanting, soil was treated with alginate pellet-based T. asperellum by the rate of 300 g pellets (106 CFU/mg fresh pellet)/m length of the planted row.
T3	Fungicide application	At sowing, strawberry transplants were soaked in Actamyl fungicide by the rate of 3 g/L.
T4	Short time solarization	Soil was irrigated to field capacity and immediately covered with 200 μm thick transparent polyethylene sheets for three weeks.
T5	Short time solarization followed by spore suspension-based T. asperellum application	Soil was irrigated to field capacity and immediately covered with 200 μm thick transparent polyethylene sheets for three weeks. Thereafter, soil was treated with spore suspension-based T. asperellum by the rate of 300 mL (106 CFU mL−1) spore suspension/m length of the planted row.
T6	Short time solarization followed by alginate pellet-based T. asperellum application	Soil was irrigated to field capacity and immediately covered with 200 μm thick transparent polyethylene sheets for three weeks. Thereafter, soil was treated with alginate pellet-based T. asperellum by the rate of 300 g pellets (106 CFU/mg fresh pellet)/m length of the planted row.
T7	Short time solarization followed by fungicide application	Soil was irrigated to field capacity and immediately covered with 200 μm thick transparent polyethylene sheets for three weeks. At sowing, strawberry seedlings were soaked in Actamyl fungicide by the rate of 3 g/L.
T8	Untreated control	Soil was irrigated to field capacity three weeks before sowing.

2.5. Viability of black root rot pathogens in solarized and non-solarized soil

The cultures of F. solani, R. solani and M. phaseolina used in this work were kept in PDA culture medium, which had previously been shown to be aggressive in causing strawberry-black root rot disease [26]. The F. solani inoculum was obtained in accordance with the instructions provided by Silva et al. [32]. A spore suspension was made from cultures cultivated in 90 mm Petri plates at 25 ± 1 °C for 10 days on (PDA, Difco Laboratories, Detroit, MI, EUA). The colonies were treated with sterile deionized water and spores were dislodged with a sterile glass rod. Using a haemocytometer (American Optical Company, USA), the spore suspensions were suspended in sterile deionized water and the concentration was adjusted to 10⁸ conidia mL⁻¹. The isolates of R. solani and M. phaseolina were cultivated in solid PDA medium until they covered the 9 mm-diameter Petri dish. One dish's contents were crushed in 100 mL of sterile distilled water. It was possible to obtain a mycelial suspension with 13,000 CFU mL⁻¹. The clay-loam soil was autoclaved at 120°C for 1 h on two consecutive days before being inoculated with 1 mL of each F. solani, R. solani and M. phaseolina suspension per 1 g of soil (initial fresh weight, water content 40 %). The inoculated soil was placed in small nylon bags holding 100 g of soil (initial fresh weight, water content 40 %) and buried at 10, 20 and 30 cm soil depth in three points in each of the three replicate plots per treatment. The vitality of pathogen propagules was assessed using the serial dilutions method at the end of the solarization treatment (3 weeks). Soil suspensions from F. solani and R. solani were placed in five Petri dishes (90 mm) with PDA medium. The Mihail and Alcorn [33] approach was utilized to identify M. phaseolina. Total CFU/g of dry soil relative to its original count were measured for each fungus in a bag soil to assess fungal decrease as follows:

$$\text{Reduction}\text{in}\text{viability}(\%)=\frac{\text{PA}‐\text{PB}}{\text{PA}}\times 100$$

Whereas PA, Pathogen population (CFU/g of dry soil) after solarization and PB, Pathogen population (CFU/g of dry soil) before solarization.

2.6. Determination of strawberry-black root rot disease

Symptoms of strawberry-black root rot are usually most prominent in the last few weeks before harvest. Plants affected by black root rot are characterized by stunted growth and wilt. The leaves of affected plants are typically smaller and few runners are produced. Therefore, 100 days after transplantation, the symptomatology of the aerial part was monitored weekly until the end of experiment. Strawberry-black root rot incidence (%) was determined as the proportion of plants having the above-mentioned symptoms on the areal parts compared to the total number of plants, using the following formula:

$$\text{Strawberry}‐\text{black}\text{root}\text{rot}(\%)=\frac{\text{Number}\text{of}\text{infected}\text{plants}}{\text{Total}\text{number}\text{of}\text{plants}}\times 100.$$

At the end of the experiment, 150 days after transplanting, all strawberry plants were removed from the soil. Black root rot are seen showing blackened brittle and dead roots. The symptomatology of the aerial and root parts was scored based on severity of symptoms according to a scale from 0 to 5 described by Abd-El-Kareem et al. [5], as follow: 0 = Plant well developed, no disease symptoms. 1 = no visible symptoms on above ground parts, 25 percent of roots discolored. 2 = Plant slightly stunted, black necrosis on petiole bases, 26–50 percent of roots discolored. 3 = Plant stunted, black necrosis on petiole bases, yellowing and death of outer leaves, 51–75 percent of roots discolored. 4 = Plant severely stunted, outer leaves collapsed, younger leaves bluish green and wilting, >75 percent of roots discolored. 5 = Plant dead. Strawberry-black root rot severity (%) was estimated using the following formula:

$$\text{Disease}\text{severity}(\%)=\frac{\mathrm{\Sigma }\left(\text{Disease}\text{grade}\times \text{Number}\text{of}\text{plants}\text{in}\text{each}\text{grade}\right)}{\text{Total}\text{number}\text{of}\text{plants}\times \text{Highest}\text{disease}\text{grade}}\times 100\text{.}$$

In order to confirm that the symptoms observed were a consequence of the black root rot pathogens re-isolates on PDA medium, from crown and root tissues, plating pieces of approximately 1.0 cm, from visibly infected roots were performed.

2.7. Determination of enzyme activities

Plant leaves taken 120 days after planting were homogenized with 0.1M sodium phosphate buffer (pH 7.1) at the rate of 1/3 w/v. For 15 min, the homogenate was centrifuged at 3000 rpm. The enzymatic activities were measured in the supernatant. Peroxidase activity was measured by incubating 0.1 mL of enzyme extract with 4 mL of guaicol solution for 1 min at 25 °C and absorbance at 470 nm [34]. Its activity is defined as a rise in absorbance at 470 nm/g fresh weight/1 min measured with a spectrophotometer (Spectronic 20-D). Chitinase activity was determined using the method described by Monreal and Reese [35]. Spectrophotometer (Spectronic 20-D) was used to detect optical density at 540 nm. Its activity was measured in terms of mM N-acetylglucose amine equivalent released/g fresh weight tissue/60 min.

2.8. Quantification of native Trichoderma spp. in rhizosphere soil and strawberry yield

At the end of the experiments, strawberry plants were carefully uprooted (10 plants per plot). Taping the roots carefully removed the additional soil that had adhered to the roots. Pooled root samples (5 g) containing soil were transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterile distilled water. To assess the population of Trichoderma spp., aliquots of soil dilutions were plated into Trichoderma-special-medium (TSM) [36], supplemented with streptomycin (30 μg mL⁻¹). Petri plates were incubated at 28 °C for 5 days and the colonies grown were counted. The cumulative strawberry yield (Ton/feddan) for each treatment was calculated.

2.9. Statistical analysis

Prior to statistical analysis, data were verified for normality and variance homogeneity. Percentage data were changed using an arcsine square root transformation to improve variance homogeneity; nonetheless, untransformed data were presented. The field experiment was designed in a randomized block design and repeated twice, with results analyzed individually. All data were examined for significant differences using analysis of variance (ANOVA) with means separation using the least significant difference (LSD) (p = 0.05) by CoStat6303software, which calculated Duncan's multiple range test.

3. Results

3.1. Laboratory experiments

In this study, T. asperellum strain was cultured for seven days before being encapsulated with sodium alginate (Fig. 1 A). Trichoderma asperellum alginate pellets were prepared by combining 50 mL of the spore suspension (10⁸ spore/mL) with 20 g of starch in 100 mL of 2 percent (wt/vol) aqueous sodium alginate (Fig. 1 B). After drying, the granules have an average diameter of 1.8–2.0 mm. A single gram granule comprises about 6.0 granules. The moisture percentage of granules is 42.0 percent. Each dry gram granule contains 10⁶ CFU of T. asperellum. The results presented in Table 2 and Fig. 2 show the inhibitory effect of alginate pellets based- T. asperellum against the strawberry-black root rot fungi; i.e. R. solani, F. solani and M. phaseolina. The Dual plate confrontation of alginate pellets based- T. asperellum and the strawberry-black root rot pathogens showed 90.9, 91.5 and 89.6 percent radial growth inhibition for R. solani, F. solani and M. phaseolina, respectively (Table 2). Overgrowth of strawberry-black root rot pathogens by T. asperellum was observed and T. asperellum also produced competitive growth, i.e. the antagonist grew faster than the pathogen and gained space and nutrients (Fig. 2A and B and C).

Fig. 1.

Encapsulation of T. asperellum ON764312.1: (A) Seven days old culture of T. asperellum strain grown on potato dextrose agar medium. (B) Formation of alginate pellets based- T. asperellum in 0.25 M CaCl₂.

Table 2.

Effect of alginate pellet-based T. asperellum on growth area of strawberry black-root rot pathogens under laboratory conditions.

Treatment	Black-root rot pathogen growth area (cm2)
	F. solani	R. solani	M. phaseolina
Alginate pellet-based T. asperellum	13.0 ± 1.63 b	11.8 ± 1.91 b	13.8 ± 1.91 b
Control	63.6 ± 0.00 a	63.6 ± 0.00 a	63.6 ± 0.00 a

Values are mean of four replications for each treatment. Means ± standard errors within a column followed by the same letter are not significantly different according to Duncan'smultiple range test at P = 0.05.

Fig. 2.

Dual culture technique of the interactions between alginate pellets based- T. asperellum ON764312.1 and strawberry black-rot pathogens in potato dextrose agar (PDA) culture medium. (A) Fusarium solani, (B) Rhizoctonia solani and (C) Machrophomina phaseolina. The two fungi (each pathogen and T. asperellum) were planted opposite to each other, dual culture with T. asperellum on the left and the control (the pathogen only) on the right. Images were taken at 7 days of growth.

3.2. Field experiments

3.2.1. Soil temperature and viability of black root rot pathogens

When solarized soil treatments were compared to the control, soil temperature increased significantly, particularly in the top layers of the soil (Table 3). Soil temperature decreased as soil depth increased (p = 0.05). Data presented in Fig. 3 shows the effect of solarization on soil temperatures at different depths. The effects of solarization on soil temperatures were comparable throughout our two field studies. In field trial I, the average maximum soil temperature in solarized plots was 53.3, 49.1, and 42.1 °C at 10, 20, and 30 cm depths, respectively, as opposed to 39.1, 36.0, and 32.0 °C in non-solarized plots (Fig. 3). In field trial II, the average maximum soil temperature in solarized plots was 51.7, 48.8, and 43.5 °C at 10, 20, and 30 cm depths, respectively, as compared with 40.7, 37.2, and 31.3 °C in non-solarized plots (Fig. 3). In two field trials, three weeks of solarization increased the maximum temperature obtained by 11–14.2 °C (1–10 cm depth), 11.6–13.1 °C (11–20 cm depth) and 10.1–12.2 °C (21–30 cm depth). Solarization had a strong and consistent effect on the vitality of black-root rot fungi immediately after removal of the polyethylene film in both field trials of our investigation (Table 3). In field trial I, solarization significantly reduced the viability of F. solani, R. solani and M. phaseolina in soil by 70.0, 65.0 & 65.0 percent; 60.0, 63.3 & 63.3 percent and 40.0, 46.0 & 48.0 percent at 10, 20 and 30 cm depths respectively, when compared to 22.0, 20.0 & 18.0 percent; 12.0, 11.0 & 11.0 percent and 10.0, 9.0 & 9.0 percent at those depths in non-solarized plots (Fig. 4).

Table 3.

Analysis of variance for maximum and minimum soil temperature (^oC) attained at 10, 20 and 30 cm during two months and percent reduction in viability of strawberry black-root rot pathogens under solarized and non-solarized soil.

Source of variation	df	Mean Square
		Soil temperature
		Trial I			Trial II
		Maximum		Minimum	Maximum		Minimum
Blocks	2	0.5605556 ns		0.2538889 ns	0.1205556 ns		0.1538889 ns
Main Effects
Solarization (S)	1	696.88889***		698.13389***	606.68056***		1011.0006***
Depth (D)	2	127.17389***		66.162222***	119.76722***		38.277222***
Interaction
S × A	2	6.6938889***		4.7622222***	0.5105556***		3.9238889***
Error	10	0.1418889		0.0978889	0.0892222		0.2025556
Source of variation	df	Mean Square
		Reduction in viability
		Trial I			Trial II
		F. solani	R. solani	M. phaseolina	F. solani	R. solani	M. phaseolina
Blocks	2	0.6666667 ns	0.2222222 ns	1.5555556 ns	0.22222 ns	6.88888 ns	0.7222222 ns
Main Effects
Solarization (S)	1	7938***	9022.72***	9568.0556***	8320.5***	7896.0***	9022.7222***
Depth (D)	2	662***	346.888***	262.88889***	640.72***	369.05***	305.05556***
Interaction
S × A	2	162***	88.2222***	67.555556***	192.16***	28.388***	84.388889 ***
Error	10	1.0666667	5.8222222	5.5555556	1.08888	10.0888	2.1222222

Ns, not significant; ***P = 0.05.

Fig. 3.

Maximum and minimum soil temperature (^oC) attained at 10, 20 and 30 cm as influenced by soil solarization during 3 weeks period under two field locations (trial I and trial II) at Qalibia governorate. Solarized soil, 10 cm-depth (T1), solarized soil, 20 cm-depth (T2), solarized soil, 30 cm-depth (T3), un-solarized soil, 10 cm-depth (T4), un-solarized soil, 20 cm-depth (T5) and un-solarized soil, 30 cm-depth (T6). Values are means of three replications. Bars with the same letters within each variable indicate that the means ± standard errors are not significantly different at P = 0.05, according to Duncan's multiple range tests.

Fig. 4.

Reduction in inocula viability of strawberry black-root rot pathogen buried at 10, 20 and 30 cm depth as influenced by soil solarization during 3 weeks period under two field locations (trial I and trial II) at Qalibia governorate. Solarized soil, 10 cm-depth (T1), solarized soil, 20 cm-depth (T2), solarized soil, 30 cm-depth (T3), un-solarized soil, 10 cm-depth (T4), un-solarized soil, 20 cm-depth (T5) and un-solarized soil, 30 cm-depth (T6). Values are means of three replications. Bars with the same letters within each variable indicate that the means ± standard errors are not significantly different at P = 0.05, according to Duncan's multiple range tests.

3.2.2. Strawberry-black root rot disease and enzymatic activities

The analysis of variance revealed that significant mean effects for soil solarization, biological application and soil solarization × biological application were obtained in the studies (Table 4). In both trials, the incidence and severity of strawberry-black root rot were high in untreated controls (54.0, 40.0 and 58.0, 43.5 % in trials I and II, respectively; Fig. 5). Soil solarization reduced black root rot incidence and severity by 24.0, 20.0 and 28.0, 23.0 percent in trials I and II, respectively (Fig. 5). Solarization combined with biocontrol agent soil inoculation resulted in more effective strawberry-black root rot control than individual treatments. Alginate pellets based- T. asperellum was more effective than spore suspension based- T. asperellum in suppressing the disease. In trials I and II, soil inoculation of biocontrol agents without solarization resulted in significant disease reductions of 19.0, 18.0 and 21.0, 18.2 percent for alginate pellets based- T. asperellum and 28.0, 23.0 and 28.0, 25.0 percent for spore suspension based- T. asperellum. In trials I and II, the highest level of disease reduction was observed when soil solarization was combined with inoculation with alginate pellets based- T. asperellum (13.0, 15.0 % for incidence and 11.0, 10.8 % for severity) or combined with application of Actamyl fungicide (13.0, 17.0 % for incidence and 13.0, 12.5 % for severity). Re-isolation procedure revealed that black root rot pathogens i.e., R. solani, F. solani and M. phaseolina were obtained from naturally infected roots of the strawberry plants. All treatments significantly (p = 0.05) increased the activity of peroxidase and chitinase enzymes in comparison to the controls (Fig. 5). Interactions between soil solarization and treatments, on the other hand, were significant (Table 4). In both field trials, enzyme activity followed a similar pattern across treatments. The application of T. asperellum treatments in conjunction with soil solarization resulted in a considerable increase in enzymatic activities in both field trials (Fig. 6). When soil solarization was combined with inoculation with alginate pellets based- T. asperellum, the highest level of enzyme increase over control was reported (60.6, 64.8 % for peroxidase and 67.9, 74.2 % for chitinase, in trials I and II, respectively). It was then followed by a soil solarization treatment along with the application of spore suspension based- T. asperellum, which resulted in enzyme increases over control of 58.0, 61.8 percent for peroxidase and 67.5, 69.9 percent for chitinase in trials I and II, respectively.

Table 4.

Analysis of variance for effect of soil solarization for three weeks followed by spore suspension- or alginate pellet-based T. asperellum application on strawberry black-root rot disease, enzymatic activity, rhizosphere population of Trichoderma spp. and fruits yield.

Source of variation	df	Mean Square
		Strawberry black-root rot disease
		Trial I		Trial II
		Incidence	Severity	Incidence	Severity
Blocks	2	1.125 ns	1.625 ns	0.875 ns	5.5629167 ns
Main Effects
Solarization (S)	1	1350 ***	864 ***	1261.5 ***	891.82042 ***
Treatment (T)	3	622.5***	265 ***	723 ***	380.45042 ***
Interaction
S × T	3	163***	49 ***	172.5 ***	59.540417 ***
Error	14	1.4107143	2.1964286	3.5892857	2.5200595
Source of variation	df	Mean Square
		Enzymatic activity
		Trial I		Trial II
		Peroxidase	Chitinase	Peroxidase	Chitinase
Blocks	2	0.0029167 ns	0.1066667 ns	0.0116667 ns	0.0204167 ns
Main Effects
Solarization (S)	1	3.4504167 ***	39.270417 ***	4.9504167 ***	58.281667 ***
Treatment (T)	3	1.7270833***	9.79375 ***	2.2581944 ***	14.111667 ***
Interaction
S × T	3	0.14375 ***	2.2770833 ***	0.0948611***	6.2227778 ***
Error	14	0.0100595	0.4409524	0.012619	0.2108929
Source of variation	df	Mean Square
		Population of Trichoderma spp. and fruits yield
		Trial I		Trial II
		Trichoderma spp.	Yield	Trichoderma spp.	Yield
Blocks	2	2.375 ns	0.5254167	3.875 ns	0.5416667 ns
Main Effects
Solarization (S)	1	253.5 ***	225.70667	234.375 ***	220.82667 ***
Treatment (T)	3	117.61111***	40.723333	78.152778 ***	28.816667 ***
Interaction
S × T	3	25.388889 ***	4.1566667	24.486111***	1.0166667 ***
Error	14	0.9464286	1.9254167	0.9702381	1.2559524

Ns, not significant; ***P = 0.05.

Fig. 5.

Effect of solarization and soil inoculation of T. asperellum on strawberry black-root rot under two field locations (trial I and trial II) at Qalibia governorate. Spore suspension-based T. asperellum application (T1), alginate pellet-based T. asperellum application (T2), fungicide application (T3), Short time solarization, three weeks (T4), solarization followed by spore suspension-based T. asperellum application (T5) solarization followed by alginate pellet-based T. asperellum application (T6), solarization followed by fungicide application (T7) and untreated control (T8). Values are means of three replications. Bars with the same letters within each variable indicate that the means ± standard errors are not significantly different at P = 0.05, according to Duncan's multiple range tests. Percentages data were transformed into arcsine square-root transformation for analyses of variance, however untransformed data are presented.

Fig. 6.

Effect of solarization and soil inoculation of T. asperellum on peroxidase and chitinase activities in strawberry leaves, 120 days after transplanting, grown under two field locations (trial I and trial II) at Qalibia governorate. Spore suspension-based T. asperellum application (T1), alginate pellet-based T. asperellum application (T2), fungicide application (T3), Short time solarization, three weeks (T4), solarization followed by spore suspension-based T. asperellum application (T5) solarization followed by alginate pellet-based T. asperellum application (T6), solarization followed by fungicide application (T7) and untreated control (T8). Values are means of three replications. Bars with the same letters within each variable indicate that the means ± standard errors are not significantly different at P = 0.05, according to Duncan's multiple range tests. Peroxidase activity was expressed as the increase in absorbance at 470 nm/g fresh weight/minute. Chitinase activity was expressed as mM N-acetylglucosamine equivalent released/gram fresh weight/60 min at 540 nm.

3.2.3. Strawberry yield and rhizosphere population of Trichoderma spp.

Strawberry fruit yield increased as a result of soil solarization. In trials I and II, the mean fruits yield in solarized soil was 11.5 and 12.5 compared to 7.7 and 7.4 (ton/feddan) in the non-solarized control, respectively (Fig. 7). In trials I and II, the highest mean fruits yield (19.5 and 18.5 ton/feddan) was observed in solarized and alginate pellets based- T. asperellum applied plots, followed by solarized and spore suspension based- T. asperellum applied plots (15.0 and 16.7 ton/feddan), respectively (Fig. 7). Furthermore, soil solarization increased the number of Trichoderma spp. propagules in the strawberry rhizosphere. At the end of the season, the populations of Trichoderma spp. in solarized soils were 8.3 and 8.0 ( × 10³ CFU g⁻¹), but in non-solarized controls, the populations were 7 and 3.0 ( × 10³ CFU g⁻¹) in trial I and II, respectively (Fig. 7). Strawberry plants obtained from T. asperellum inoculated solarized/non-solarized soil demonstrated efficient rhizosphere colonization of native Trichoderma spp. (Fig. 7). Soil solarization, on the other hand, increased the number of Trichoderma spp. in the rhizosphere. When soil solarization was combined with the application of alginate pellets based- T. asperellum, the largest population of Trichoderma spp. was observed (20.0 × 10³ CFU g⁻¹ for trial I and 18.3 × 10³ CFU g⁻¹ for trial II).

Fig. 7.

Effect of solarization and soil inoculation of T. asperellum on strawberry fruits yield and rhizosphere population of native Trichoderma spp., under two field locations (trial I and trial II) at Qalibia governorate. Spore suspension-based T. asperellum application (T1), alginate pellet-based T. asperellum application (T2), fungicide application (T3), Short time solarization, three weeks (T4), solarization followed by spore suspension-based T. asperellum application (T5) solarization followed by alginate pellet-based T. asperellum application (T6), solarization followed by fungicide application (T7) and untreated control (T8). Values are means of three replications. Bars with the same letters within each variable indicate that the means ± standard errors are not significantly different at P = 0.05, according to Duncan's multiple range tests.

4. Discussion

The antagonistic effect of alginate pellets based- T. asperellum on the development area of strawberry black-root rot pathogenic fungi i.e., F. solani, R. solani and M. phaseolina was determined in vitro in the current study. The antagonistic test is regarded as a straightforward method for comprehending a limited sector of biological role in disease prevention. Alginate pellets based- T. asperellum restricted the growth on dual culture plates and subsequently overgrew the mycelium of the strawberry black-root rot pathogenic strains on these plates. Within 6–7 days of incubation, the pathogens growth and all the available surface of the Petri dish involved was covered by sporulating hyphae of T. asperellum. Productions of volatile and non-volatile antibiotics, as well as nutritional competition, have been implicated [37]. Furthermore, mycoparasitism is thought to play a significant role in Trichoderma spp. antagonistic activity [38]. It is possible to conclude that one or more of the preceding elements play an essential role in T. asperellum's antagonistic action against strawberry black-root rot fungi. Many studies stated that, T. asperellum's potential as a bioagent of soil-borne plant diseases. According to Wu et al. [16], T. asperellum GDFS1009 mycelium has a high growth rate, a high sporulation rate and strong inhibitory effects against pathogens of cucumber fusarium wilt and maize stalk rot caused by F. oxysporum f. sp. cucumerinum Owen and F. graminearum, respectively. Trichoderma asperellum IMI 393899 was found to have mycoparasitic activity against F. graminearum, Penicillium commune and Aspergillus parasiticus as reported by Stracquadanio et al. [39]. Yadav et al. [40] found that, out of the 21 Trichoderma isolates tested, three isolates named T. koningiopsis, T. reesei and T. asperellum, inhibited the growth of F. oxysporum f. sp. cumini by 62.65, 79.85 and 84.31 percent, respectively. Sharma et al. [41] discovered that T. asperellum isolate UCRD5 suppressed many plant fungal pathogens such as Fusarium oxysporum, Sclerotinia sclerotiorum, Alternaria solani and Phytophthora sp. Mirsam et al. [42] reported that the five T. asperellum isolates tested were able to inhibit the growth of R. solani in vitro on PDA medium with an inhibition percentage of ≥50 percent. According to these scientists the mycoparasitic activity of the T. asperellum strains involved both direct penetration and enveloping the hyphae of the pathogen; in the latter instance, a high number of hydrolytic enzymes are produced prior to hyphae destruction.

Under field conditions, a significant reduction in the viability of strawberry black-root rot fungi i.e., F. solani, R. solani and M. phaseolina was seen shortly after the polyethylene sheets were removed during the 3 weeks of summer solarization (Fig. 4). The mean maximum temperatures measured in solarized soil matched the reductions measured at the three soil depths. The results of the present findings support the previous work of Dwivedi and Dwivedi [43], who observed that solarization increased the temperature in moistened field soil covered with transparent polythene sheet, particularly in the top layer of soil, i.e. 0-5 cm depth. We discovered that temperature differences at different soil depths had a significant impact on the survival of black-root rot fungi in soil. These temperatures, however, were high enough to account for a decrease in inoculum densities of black-root rot pathogens examined to a depth of 20 cm, where strawberry roots develop primarily [13]. These findings are consistent with those obtained by Barakat and AL-Masri [44]. They found that soil solarization increased mean maximum soil temperatures by 8.8–14.1°C and reduced the population of Fusarium oxysporum f. sp. lycopersici by 86 percent. Dwivedi and Dwivedi [43] discovered that solarized plots had much lower population dynamics of Fusarium solani and Fusarium oxysporum than non-solarized plots. Tyagi and Raj [45] found that soil solarization for 40 days reduced the viability of R. solani sclerotia by 88.7 percent as compared to non-solarized soil.

The application of T. asperellum (alginate pellets- or spore suspension-based formulations) to solarized soils resulted in the greatest disease suppression and plants receiving the aforementioned treatments produced more fruits than the control. Furthermore, the population of native Trichoderma grew significantly in the plant rhizosphere. The natural suppressiveness of certain plant diseases in solarized soils has been related to an increase in the activity of fungal antagonists such as Trichoderma spp [10]. The effectiveness of this natural phenomenon can be increased by artificially introducing Trichoderma spp. into solarized soil, as attempted in this study. Soil solarization would have ideally reduced the number of viable pathogenic propagules and the remaining propagules after solarization may have been weakened at the end of the procedure [45]. Soil inoculation of antagonistic propagules shortly after solarization provides a highly opportune setting for them to grow quickly and colonize the vacuum area created by solarization [14]. It also promotes antagonist colonization in the plant rhizosphere [15]. High soil temperatures during solarization reduced the survival of black root rot-propagules in the current investigation and the addition of T. asperellum increased the population level of native Trichoderma. Alginate pellets based- T. asperellum outperformed spore suspension based- T. asperellum. These experiments revealed that microcapsules made of alginate pellets derived from T. asperellum had great efficiency, most likely due to the encapsulating material forming a protective shell over the conidia [46]. Release is a key measure of microcapsule durability and sustained release [47]. The release procedure is divided into two stages: rapid release and gradual release [48]. Ultra-violet irradiation is another major factor influencing T. asperellum biocontrol. When administered via soil application, microcapsules can protect microorganisms against the impacts of soil moisture, pH and UV in terms of microbial survival and lifespan [27,49,50]. Previous research has shown that this strategy can be used to control a variety of crop diseases [29,51]. The combination of solarization and an introduced beneficial organism may thus improve the solarized soil's long-term suppressiveness [13,14]. The disease incidence in untreated plots in the current field experiments was as high as 54.0, 40.0 and 58.0, 43.5 percent in trial I and II, respectively. At such a high inoculum pressure, it is not unexpected that biocontrol agents fail to operate adequately when used alone. Multiple disease control factors, such as solarization, are expected to be more successful than individual treatment under such conditions, as demonstrated by the current study. Previous research has shown that this strategy can be used to control a variety of crop diseases. The combined treatments of five weeks of soil solarization and soil application of T. harzianum resulted in entire loss of Armillaria spp. inoculum viability, which was like 10 weeks of solarization without administration of the antagonist [52]. According to Porras et al. [53], soil solarization in combination with Trichoderma applications, improved strawberry production. Ibrahim and Abdelaziz [54] discovered that combining soil solarization with antagonistic fungal inocula (Chaetomium, Gliocladium & Trichoderma), improved the efficacy of suppressing lupine-fusarium-root rot.

All treatments significantly increased the specific activity of defense-related enzymes such as peroxidase and chitinase in strawberry leaves 120 days after transplanting in the current study. The maximum activity was reported when soil solarization was combined with alginate pellets based- T. asperellum inoculation. Increased peroxidase activity may enhance oxidative stress due to increase H₂O₂ generation [55]. Many microbial pathogens are hazardous to H₂O₂ and other free radicals [56]. During plant-pathogen interactions, the oxidative potential of H₂O₂ contributes to the formation of lignin via peroxidase-mediated crosslinking of proline-rich structural proteins and phytoalexin biosynthesis, as well as the conversion of O-dihydroxyphenols to toxic o-quinones via polyphenoloxidase [57]. Chitinase enzymes, on the other hand, aid in plant defense against fungus by hydrolyzing cell walls. Because chitin is a fundamental structural component of many pathogenic fungi's cell walls, their numbers increase considerably, and they play a vital part in fungal pathogen defense by destroying cell walls. Previous research has shown that solarization and Trichoderma spp. can systematically produce disease resistance in a variety of plants [58].

4.1. Future aspects

In the present study, using alginate pellets based- T. asperellum formulation in combination with short-time soil solarization may therefore represent a new step in the biological control of black-root rot disease in strawberry. The sodium alginate encapsulation preserved the viability and protected the bio-efficacy of T. asperellum in field trials. However, future studies about the long-term efficacy and safety of the alginate pellets based- T. asperellum and investigating its potential for combination therapy with other antimicrobials are essential for evaluating the effectiveness and stability of the microencapsulated biocontrol agent are needed. Further, environmental impact of the integrated control strategy, including its effects on soil microbial diversity, non-target organisms, and ecosystem services, would ensure sustainable agricultural practices.

5. Conclusions

In this study, alginate pellets based- T. asperellum were prepared. This preparation efficiently controlled strawberry black-root rot fungi in vitro, including F. solani, R. solani and M. phaseolina. Under field conditions, the combination of alginate pellets based- T. asperellum and soil solarization resulted in considerable disease control of strawberry black-root rot. The maximum yield improvement and highest levels of Trichoderrna spp. collected from strawberry plant rhizosphere soil were seen in plots when alginate pellets based- T. asperellum was administered in conjunction with soil solarization. These findings suggested that T. asperellum microcapsules combined with soil solarization are effective and environmentally benign microbial agents for the field management of strawberry black-root rot disease.

Funding statement

This work was supported by funds of National Research Centre Centre; In-House project No. 13050112.

Data availability statement

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

CRediT authorship contribution statement

Ibrahim Elshahawy: Methodology. Nehal Saied: Writing – original draft. Farid Abd-El-Kareem: Methodology. Mahfouz Abd-Elgawad: Writing – review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.