New Trichoderma Strains Suppress Blue Mold in Oranges by Damaging the Cell Membrane of Penicillium italicum and Enhancing Both Enzymatic and Non-Enzymatic Defense Mechanisms in Orange Fruits

Abstract

Blue mold disease, caused by Penicillium italicum (P. italicum), presents a significant challenge to orange fruits (Citrus sinensis L.) and other citrus crops globally. Biological control, particularly Trichoderma species, offers a promising alternative to synthetic fungicides. Therefore, this study aimed to isolate, identify, and evaluate the antagonistic activities of two Trichoderma isolates against P. italicum. These isolates were molecularly identified and assigned accession numbers PP002254 and PP002272, respectively. Both isolates demonstrated significant antifungal activity in dual culture assays. Moreover, the culture filtrates (CFs) of Trichoderma longibrachiatum PP002254 and Trichoderma harzianum PP002272 suppressed the mycelial growth of P. italicum by 77.22% and 71.66%, respectively. Additionally, CFs reduced the severity of blue mold on orange fruits by 26.85% and 53.81%, compared to 100% in the control group. Scanning electron microscopy revealed that treated P. italicum hyphae were shrunken and disfigured. Enzyme activities (catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase) in treated oranges increased, along with total soluble phenolics and flavonoids. Conversely, malondialdehyde (MDA) levels decreased in treated fruits. These findings suggest that T. longibrachiatum PP002254 and T. harzianum PP002272 could be effective biocontrol agents for managing blue mold and other citrus postharvest diseases.

1. Introduction

Citrus fruits, including oranges (Citrus sinensis L.)are among the most commercially important fruits worldwide. Additionally, they are viewed as a good source of several biologically active compounds such as amino acids, vitamins, flavonoids, and essential oils, and are also rich in nutrients [1]. However, they are highly susceptible to postharvest diseases caused by various phytopathogenic fungi, including Penicillium italicum, Penicillium digitatum, Aspergillus niger, and Aspergillus flavus, which can lead to losses in terms of human health, the environment, and the economy [2].

Among these fungi, Penicillium italicum is considered the most prominent [3]. Blue mold resulting from Penicillium italicum is one of the most serious postharvest diseases and causes considerable economic losses for citrus fruits in all world regions [4]. Furthermore, P. italicum produces fungal toxins that present hazards to human health [5]. Generally, synthetic fungicides such as imazalil, fludioxonil, thiabendazole, prochloraz, and pyrimethanil are primarily used during waxing operations to minimize the economic losses caused by blue mold disease in citrus [6]. Nevertheless, the repeated and wide application of these synthetic fungicides has led to several issues, including the emergence of resistant P. italicum strains, environmental pollution, and harm to all organisms [7].

Biological control is considered one of the most promising methods to decrease the reliance on synthetic fungicides for managing postharvest diseases in citrus fruits [8]. Bioagents such as fungi, bacteria, and yeast are considered safer and non-toxic, and exhibit a broad spectrum of antagonistic activities [9]. Furthermore, these bioagents produce secondary metabolites with antifungal and effective properties to control postharvest diseases [10].

Trichoderma species are the most important fungal genera in controlling postharvest diseases [11]. They are found in many different types of ecosystem soil, such as rhizosphere [12] and plants [13]. Additionally, they have several beneficial effects on plant growth and defense mechanisms [14]. Moreover, they have been the focus of many research studies due to their wide distribution in diverse environments and ability to produce hundreds of bioactive compounds and growth-promoting chemicals [15,16].

Trichoderma fungus utilizes various mechanisms to suppress the growth of phytopathogens, including mycoparasitism, antibiosis, competition for space or nutrients, and the induction of host resistance [17]. Cell suspensions of Trichoderma species or their culture filtrates are acknowledged as biocontrol agents due to their ability to inhibit the growth of several phytopathogenic fungal species and effectively manage various plant diseases [18,19,20]. T. harzianum, for example, has been found to restrain the growth and development of P. griseofulvum on apple fruit [21], as well as inhibit Colletotrichum musae and C. gloeosporioides on bananas and mango, respectively [22,23]. It has also demonstrated effectiveness against a gray mold of grapes caused by Botrytis cinerea [24].

Trichoderma species like T. harzianum, T. guizhouense, T. atroviride, and T. koningiopsis have shown antifungal activities against P. digitatum, the causative agent of green mold disease in oranges [25]. Furthermore, other species of Trichoderma are effective against phytopathogenic fungi that cause postharvest diseases; for example, T. longibrachiatum inhibited anthracnose growth on Papaya fruits [26] and red pepper [27].

Additionally, Trichoderma species culture filtrates significantly stopped various fungal diseases, such as chili anthracnose caused by C. gloeosporioides [28], and anthracnose of great millet caused by C. graminicola [29]. Although the antifungal potential of Trichoderma species has been demonstrated against several phytopathogens, including P. italicum [30], most of this research has been conducted under in vitro conditions. There remains a significant gap in the literature regarding the in vivo application of Trichoderma culture filtrates against P. italicum in citrus fruits, specifically focusing on their ability to enhance the fruit’s natural defense mechanisms. Addressing this gap is crucial for developing sustainable and effective biological control strategies in postharvest disease management.

This study aims to isolate and identify new Trichoderma strains and evaluate their antagonistic activity against P. italicum in both in vitro and in vivo conditions. Additionally, it investigates the effects of Trichoderma culture filtrates on enzymatic and non-enzymatic defense mechanisms in orange fruits, offering new insights into sustainable postharvest disease management.

2. Materials and Methods

2.1. Isolation and Identification of P. italicum

P. italicum was isolated from orange fruits exhibiting typical symptoms of blue mold disease. These orange fruits were collected from local markets in Tanta City, Egypt. Conidia were scraped from the infected fruits and cultured on potato dextrose agar (PDA) medium (39 g of commercial PDA powder added to one liter of distilled water then autoclaved at 121 °C for 20 min), followed by incubation at 25 ± 2 °C for seven days. The identification of P. italicum was confirmed through morphological and macroscopic analysis of the cultures and conidia, as referenced in [31]. A pathogenicity test was subsequently conducted to assess the virulence of the pathogen. A conidial suspension (1 × 10⁶ conidia per mL) was prepared from a seven-day-old culture at 25 °C in sterilized distilled water containing 0.01% Tween 80. Healthy, defect-free orange fruits were thoroughly washed with running water, superficially sterilized with 70% ethanol, and dried under sterilized conditions. Small wounds (3 × 3 mm) were made on the surfaces of the orange fruits using a cork borer, and 10 µL of the spore suspension was applied to each wound. Control fruits were inoculated with sterilized distilled water. The inoculated fruits were placed in plastic boxes lined with damp paper to maintain high humidity and incubated at 25 °C for ten days.

2.2. Isolation of Trichoderma Isolates

Six Trichoderma isolates were obtained from rhizosphere soil samples using the methods outlined in [32], collected from different agricultural ecosystems in Tanta City, Egypt, including the Faculty of Agriculture at Tanta University, Seberbay, and Mahalla Manouf. Briefly, soil samples weighing 1 g were placed into a 50 mL Falcon tube containing sterilized distilled water (SDW) and agitated for one hour at 180 rpm. The samples were serially diluted from 10⁻¹- to 10⁻⁵-fold (five-fold) with SDW, and subsequently 100 µL from the last dilution was spread on plates containing PDA. The Trichoderma colonies were purified through transfer to a fresh PDA medium and incubated at 28 ± 2 °C for 7 days. Additionally, the morphological identification of Trichoderma isolates was performed based on culture characteristics such as diameter, texture, color from both front and back views, and the presence or absence of pigment in the PDA medium as previously described by [33].

2.3. Dual Culture Assay

In the beginning, the antifungal activities of six Trichoderma isolates against P. italicum were examined using a dual-culture assay, following the procedures outlined [34]. The findings revealed that only two isolates exhibited antifungal properties in the dual culture assay. Consequently, these two isolates were chosen for subsequent research, including genetic identification and species determination. Briefly, a 0.5 cm plug of Trichoderma longibrachiatum PP002254 and Trichoderma harzianum PP002272 individually was placed 1 cm away from the edge of the PDA plates and on the other side of the PDA plates, a plug of P. italicum (0.5 cm) was transferred. The plates were incubated at 25 ± 2 °C for 7 days. PDA plates containing P. italicum alone were used as a control. The percentage of inhibition was calculated as follows:

$$\mathrm{Inhibition} (\%)=\frac{R-R_1}{R}\times 100$$

R is the mycelia growth in the control treatment, while R₁ is the mycelia growth in the treatment.

2.4. Molecular Identification of Trichoderma Isolates

Furthermore, the molecular identification of T. longibrachiatum PP002254 and T. harzianum PP002272 was performed according to [35,36]. Briefly, T. longibrachiatum PP002254 and T. harzianum PP002272 were cultured on a sterilized potato dextrose broth (PDB) and kept at 28 ± 2 °C for 7 days. Subsequently, fungal mycelium and spores were collected, filtered through cheesecloth, washed twice with SDW, and dried using sterilized filter paper. Next, 100 mg of fungal mycelia were crushed into a fine powder using liquid nitrogen. The total DNA of T. longibrachiatum PP002254 and T. harzianum PP002272 was extracted and purified, and the region of the targeted sequence (internal transcribed spacer ITS) was PCR-amplified. After the PCR product was purified according to the manufacturer’s instructions using a Qiagen Gel extraction kit, it was forwarded to the AuGCT sequencing institution (Aoke Dingsheng Biotechnology Co., Beijing, China) for sequencing. Sanger sequencing was used to carry out the bidirectional sequencing of the ITS-5.8S rDNA sequence. DNABASER software (Heracle BioSoft S.R.L., Arges, Romania Version 4.10) was used to determine and construct consensus sequences. The Nucleotide–Nucleotide BLAST (BLAST n) algorithm was then used to match the consensus sequences to the most recent data available in GenBank and the National Center for Biotechnology Information (NBCI) database. Sequences were deposited in GenBank, NCBI (Accession No. PP002254 and PP002272).

2.5. Preparation and Evaluation of Trichoderma Culture Filtrate

The plugs of T. longibrachiatum PP002254 and T. harzianum PP002272, each with a diameter of 0.5 cm, were transferred to flasks containing 200 mL of PDB. The flasks were then incubated at 25 ± 2 °C for one week under shaking conditions. After the incubation period, the fungal mycelium was removed, and the filtrate was centrifuged at 12,000 rpm for 30 min at 4 °C. The resulting supernatant was sterilized using a 0.22 µm pore-size filter. Subsequently, the antagonistic properties of T. longibrachiatum PP002254 and T. harzianum PP002272 CFs against P. italicum mycelial growth were assessed according to the methods highlighted by [27]. Briefly, the culture filtrate CFs of T. longibrachiatum PP002254 and T. harzianum PP002272 (200 µL) were spread onto PDA plates. Plates without filtrate were used as a control. Then, 0.5 cm mycelial plugs from an active culture of P. italicum were placed into the center PDA plates. The plates were kept at 25 ± 2 °C for one week. The experiment was conducted twice with six replicate plates for each treatment. The inhibition percentage was calculated as follows.

$$\mathrm{Inhibition} (\%)=\frac{\left(Fungal growth\left(\mathrm{c}\mathrm{m}\right) in control-Fungal growth\left(\mathrm{c}\mathrm{m}\right) in treatment\right)}{Fungal growth\left(\mathrm{c}\mathrm{m}\right) in control}\times 100$$

2.6. Microscopic Examination of P. italicum Hyphae Treated with Culture Filtrate of T. longibrachiatum PP002254 and T. harzianum PP002272 by Scanning Electron Microscope

The morphological characteristics of P. italicum hyphae were examined using SEM as described by [37]. Hyphae of P. italicum treated with the culture filtrate of T. longibrachiatum PP002254 and T. harzianum PP002272 were fixed with glutaraldehyde (2.5%) in phosphate buffer (0.1 M, pH 7.2) for 2 h, followed by post-fixation in osmium tetroxide (1%) for 2 h. After washing with phosphate buffer (0.1 M) three times for 20 min each, the samples were dehydrated with a series of ethanol (ranging from 30% to 100%) for 15 min each. Subsequently, the samples were critically dried with liquid carbon dioxide (in EMS 850, Hatfield, PA, USA) and then plated with gold using a sputter coater (SPI Module, West Chester, PA, USA). Finally, observation was conducted using a JSM-5500LV scanning electron microscope (JEOL, Tokyo, Japan) at a 15 kV accelerating voltage.

2.7. In Vivo Treatments

Commercially ripe and defect-free Washington variety orange fruits were acquired from a local market. Initially, the orange fruits were washed with tap water, immersed in a sodium hypochlorite solution (2%) for two minutes, and then rinsed with sterilized distilled water before being left to air dry at room temperature. On the external peel of each fruit, two holes of 0.3 cm in both diameter and depth were created using a sterilized borer. Into these holes, 10 μL of a P. italicum conidial suspension (1 × 10⁶ conidia per mL) was added. Four hours later, at 25 °C, 20 μL of culture filtrates (CFs) of T. longibrachiatum PP002254 and T. harzianum PP002272 were introduced into each hole, and the control fruits received 20 μL of sterilized potato dextrose broth (PDB). The inoculated fruits were stored for one week at 25 ± 2 °C in plastic boxes containing moist cotton to maintain 95–100% humidity. The experiment was replicated twice with the CFs of T. longibrachiatum PP002254 and T. harzianum PP002272, each having three replicates and each replicate containing 10 fruits. Disease severity was assessed using the formula provided by [8].

$$\mathrm{Disease} \mathrm{severity} (\%)=\frac{average lesion diameter of treatment \left(\mathrm{c}\mathrm{m}\right)}{average lesion diameter of control \left(\mathrm{c}\mathrm{m}\right)}\times 100$$

2.8. Effect of T. longibrachiatum PP002254 and T. harzianum PP002272 Culture Filtrate on Physiological and Biochemical Changes in Orange Fruits

2.8.1. The Activity of Defense-Related Enzymes

Orange fruits were collected at 24, 48, 72, and 96 h post-treatment (hpt) with T. longibrachiatum PP002254 and T. harzianum PP002272 CFs to assay the defense-related enzymes, including catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL). Samples were taken from the peel and inside the wounds on the fruits. For CAT, POD, and PPO, 500 mg of orange samples were mixed with 3 mL of 50 mM Tris buffer (pH 7.8) containing 7.5% polyvinylpyrrolidone (PVP) and 1 mM ethylenediaminetetraacetic acid (EDTA-Na2). The homogenate was centrifuged for 30 min at 12,000 rpm at 4 °C and the supernatant was collected for enzyme assay according to the methods described by [38]. The CAT enzyme activity was assessed using the modified methodology of [38,39], by mixing 75 μL of crude enzyme extract, 150 μL of hydrogen peroxide H₂O₂ (269 mM), and 3 mL of sodium phosphate buffer (0.1 mM, pH 6.5). The H₂O₂ extinction coefficient was 0.040 mM⁻¹ cm⁻¹, and the activity of CAT was measured by observing the degradation of H₂O₂ at 240 nm in a quartz cuvette.

Moreover, the POD activity was conducted according to [40], with minor modifications as described by [38]. Briefly, the reaction mixture consisted of 2.2 mL of sodium phosphate buffer (100 mM, pH 6.0), 100 µL of H₂O₂ (12 mM), 100 µL of guaiacol, and 100 µL of crude enzyme extract. The increase in absorbance at 436 nm indicated the production of the conjugate, using an extinction value of 26.6 mM⁻¹ cm⁻¹. The PPO activity was measured using the methods mentioned by Malik and Singh [41]. The assay was performed by mixing 3 mL of buffered catechol solution (0.01 M), newly prepared in 0. 1 M phosphate buffer (pH 6.0), and 100 µL of crude enzyme extract. PPO activity was determined by measuring the absorbance changes at 495 nm every 30 s for three minutes.

Phenylalanine ammonia-lyase (PAL) was extracted by 5 mL of boric acid buffer (100 mM, pH 8.8) containing 4% (w/v) PVP, 1 mM ethylenediaminetetraacetic acid (EDTA), and 50 mM β-mercaptoethanol. The extracts were then centrifuged at 12,000 rpm for 30 min at 4 °C [42], and the supernatants were used for the enzyme assays. PAL activity was assayed as described by the method of [43] with some modifications. A total of 500 µL of enzyme extract was incubated with 500 µL of 20 mM l-phenylalanine and 2000 µL of 50 mM borate buffer (pH 8.8) at 37 °C for 1 h. The reaction was ended with 100 µL 6M HCl. The augmentation in absorbance at 290 nm due to the production of trans-cinnamate was measured by a spectrophotometer. PAL activity was expressed as U290, where U290 = 0.01 A290 mg⁻¹ protein h⁻¹.

2.8.2. Total Soluble Phenolic Compounds

The Folin–Ciocalteu reagent was utilized to quantify the total soluble phenolic compounds, following the methods described by Kähkönen [44]. To extract the total soluble phenolic compounds, treated orange fruits were collected at 24, 48, 72, and 96 hpt. A total of 100 mg of orange tissue was homogenized and mixed with 20 mL of 80% methanol for one day and then filtrated. For total soluble phenolics assessment, 0.2 mL of methanolic extract was mixed with 1 mL of Folin–Ciocalteu reagent (10%) and vortexed for 30 s, then 0.8 mL of sodium carbonates (7.5% w/v) was added. Subsequently, the mixture was incubated for half an hour at room temperature. Finally, the spectrophotometer was used to measure the absorbance at 765 nm. A standard curve of known concentrations of Gallic acid was used for the quantification of total soluble phenolics, which was expressed as mg Gallic acid equivalents per gram fresh weight (mg GAE g⁻¹ FW).

2.8.3. Total Soluble Flavonoid Compounds

The amount of total soluble flavonoids was evaluated according to the methodology described by [45] with minor modifications. Briefly, a specific volume (1 mL) of a methanolic extract extracted from 100 mg of orange tissue was homogenized and mixed with 20 mL of 80% methanol for one day, mixed with the same volume (1 mL) of aluminum chloride solution (2% in methanol), strongly mixed, and then incubated for a quarter-hour at room temperature. A spectrophotometer was used to measure the absorbance at 430 nm. Subsequently, the total soluble flavonoids were expressed as milligrams of Rutin equivalents per gram of fresh weight (mg RE g⁻¹ FW).

2.8.4. Assessment of Malondialdehyde as an Indication of Lipid Peroxidation

Malondialdehyde (MDA) was determined as described by Du and Bramlage [46] with a slight modification. Briefly, MDA was extracted from 1 g of homogenized fresh orange tissue using 20% trichloroacetic acid (TCA) containing 0.01% butyl hydroxyl toluene (BHT), heated to 95 °C, then centrifuged at 12,000 rpm for 10 min. The MDA concentration was determined in the supernatant by measuring absorbance at 532 and 600 nm with a spectrophotometer, and the results were expressed as nmol per gram of fresh weight (FW).

2.9. Statistical Analysis

All experiments were conducted in duplicate, and the average results are presented. Data were analyzed using one-way analysis of variance (ANOVA), with mean comparisons performed using Tukey’s HSD test at a significance level of (p < 0.05). Statistical evaluations were carried out using JMP data analysis software, version 14.

3. Results

3.1. Morphological and Molecular Identification of T. longibrachiatum PP002254 and T. harzianum PP002272 Isolates

T. longibrachiatum PP002254 and T. harzianum PP002272, which exhibited the highest inhibition rates in dual culture tests, were selected for comprehensive morphological and molecular identification due to their strong antagonistic effects on P. italicum (Figure 1a–d). The fungal growth of these two isolates appeared light green on the upper side of the plate (Figure 1a,b), while the reverse side showed yellow and light beige colors on the PDA medium after seven days of incubation (Figure 1c,d). These isolates were then subjected to genetic analysis. A phylogenetic tree was constructed using the ITS sequences of the Trichoderma strains/isolates and sequences obtained from GenBank. Genetic analysis indicated that the sequences of the obtained isolates were highly similar to Trichoderma longibrachiatum strain CBS 130685 (GenBank accession number MH877290.1; Figure 1e) and T. harzianum strain ADEL 101 (GenBank accession number MN877923.1; Figure 1f). The new sequences were uploaded to the NCBI database and named T. longibrachiatum Isolate AE 2023 (GenBank accession number PP002254; Figure 1e) and T. harzianum Isolate AE 2023 (GenBank accession number PP002272; Figure 1f).

3.2. Dual Culture Assay: Antifungal Activity of Trichoderma Isolates

The antifungal properties of T. longibrachiatum PP002254 and T. harzianum PP002272 against P. italicum were investigated in vitro using a dual culture method. T. longibrachiatum PP002254 and T. harzianum PP002272 exhibited high inhibition of P. italicum mycelial growth. The radial growth of P. italicum was significantly reduced by both isolates compared to the control (9.00 ± 0.00 cm). T. longibrachiatum PP002254 reduced growth to 3.38 ± 0.18 cm (62.41 ± 2.04% inhibition), while T. harzianum PP002272 reduced it to 3.02 ± 0.23 cm (66.48 ± 2.53% inhibition). Both values were statistically significant compared to the control group (p < 0.05) (

Table 1. Antifungal activity of T. longibrachiatum PP002254 and T. harzianum PP002272 against P. italicum in dual culture assay.

Treatment	Radial Mycelial Growth (cm)	% Inhibition
Control	9.00 ± 0.00 a	0.00 ± 0.00 b
T. longibrachiatum PP0022542	3.38 ± 0.18 b	62.41 ± 2.04 a
T. harzianum PP002272	3.02 ± 0.23 b	66.48 ± 2.53 a

Values are mean ± standard error (SE). This means values within each column followed by different letters are significantly different according to Tukey’s HSD test at p < 0.05.

, Figure 2).

3.3. Culture Filtrate of T. longibrachiatum PP002254 and T. harzianum PP002272 and Its Effect on P. italicum Growth

The culture filtrates (CFs) of T. longibrachiatum PP002254 and T. harzianum PP002272 were tested for antifungal properties. Both CFs significantly suppressed P. italicum growth compared to the control (9.00 ± 0.00 cm). T. longibrachiatum PP002254 CF inhibited mycelial growth by 77.22 ± 0.38% (2.05 ± 0.03 cm), while T. harzianum PP002272 CF achieved 71.66 ± 1.10% inhibition (2.55 ± 0.09 cm), both of which were significantly different from the control (p < 0.05) (

Table 2. Effect of T. longibrachiatum PP002254 and T. harzianum PP002272 culture filtrates (CFs) on the radial growth of P. italicum.

Treatment	Radial Mycelial Growth (cm)	% Inhibition
Control	9.00 ± 0.00 a	0.00 ± 0.00 c
T. longibrachiatum PP0022542	2.05 ± 0.03 c	77.22 ± 0.38 a
T. harzianum PP002272	2.55 ± 0.09 b	66.48 ± 1.10 b

Values are mean ± standard error (SE). This means values within each column followed by different letters are significantly different according to Tukey’s HSD test at p < 0.05.

, Figure 3). The consistency of the inhibition across assays further supports the effectiveness of the CFs as potential bio-control agents.

3.4. Scanning Electron Microscopy (SEM) of P. italicum Hyphae Treated with Culture Filtrate

To assess the impact of T. longibrachiatum PP002254 and T. harzianum PP002272 CF treatments on the growth of P. italicum, we observed changes in hyphal morphology using SEM. The SEM images revealed that P. italicum mycelia in the control group were regularly shaped, thick, elongated, and uniform (Figure 4). Additionally, the conidiospores exhibited a consistent shape. However, P. italicum mycelia treated with T. longibrachiatum PP002254 and T. harzianum PP002272 CFs displayed considerable damage, characterized by irregular shrinkage, cell deformation, and cellular collapse (Figure 4).

3.5. In Vivo Biocontrol Activity of T. longibrachiatum PP002254 and T. harzianum PP002272

To assess in vivo efficacy, oranges inoculated with P. italicum were treated with T. longibrachiatum PP002254 and T. harzianum PP002272 CFs. After 7 days, the CF from T. longibrachiatum PP002254 led to the smallest lesion size (12.67 ± 1.61 mm) and lowest disease severity (26.85 ± 3.88%), followed by T. harzianum PP002272 (25.67 ± 1.57 mm of lesion size, and 53.81 ± 3.57% of disease severity). Both treatments significantly reduced lesion size and disease severity compared to the untreated control, which exhibited 100% disease severity (p < 0.05) (Figure 5). These results suggest that T. longibrachiatum PP002254 is more effective in controlling blue mold in oranges in vivo.

3.6. Impact of T. longibrachiatum PP002254 and T. harzianum PP002272 Culture Filtrates on Enzyme Activity in Orange Fruits

In general, treatment with CFs of T. longibrachiatum PP002254 and T. harzianum PP002272 resulted in a considerable rise in the profile of studied enzymes, including CAT (Figure 6a), POD (Figure 6b), PPO (Figure 6c), and PAL (Figure 6d), in comparison with the control treatment (Figure 6). Overall, the activity of the investigated enzymes changed after the treatment with T. longibrachiatum PP002254 and T. harzianum PP002272 filtrates. Briefly, CAT activity saw a sharp increase at 24 h post-treatment (hpt) with T. longibrachiatum PP002254 filtrate (30.75 ± 2.79 µm H₂O₂ g⁻¹FW min⁻¹), while T. harzianum PP002272 filtrate reached its peak at 72 hpt (40.30 ± 1.81 µm H₂O₂ g⁻¹FW min⁻¹) (Figure 6a). Similar to this, POD activity peaked at 24 hpt with T. longibrachiatum PP002254 filtrate (3.12 ± 0.16 μM Tetraguaiacol g⁻¹ FW min⁻¹), but T. harzianum PP002272 filtrate took longer, reaching its maximum at 96 hpt (8.21 ± 0.44 μM Tetraguaiacol g⁻¹ FW min⁻¹) (Figure 6b). Additionally, Figure 6c illustrates that the peak levels of polyphenol oxidase (PPO) enzymatic activity were observed at 24 and 48 h post-treatment with T. longibrachiatum PP002254 filtrates. Conversely, T. harzianum PP002272 exhibited its highest activity at 72 and 96 h post-treatment. Notably, the control group showed a higher PPO activity than T. longibrachiatum PP002254 at 72 h post-treatment. Moreover, we studied the profile of PAL enzymatic activity and observed that the treatment with T. longibrachiatum PP002254 and T. harzianum PP002272 CFs led to a gradual increase in PAL activity, reaching its highest peak at 72 hpt. However, it quickly decreased at 96 hpt (Figure 6d).

3.7. Non-Enzymatic Antioxidants: Total Soluble Phenolics and Flavonoids

Treatment with T. longibrachiatum PP002254 and T. harzianum PP002272 CFs also led to a significant increase in total soluble phenolics and flavonoids compared to untreated controls. At 72 hpt, total phenolic content in oranges treated with T. longibrachiatum PP002254 reached 9.60 ± 0.072 mg GAE g⁻¹ FW, significantly higher than in controls (p < 0.05), while T. harzianum PP002272 treatment resulted in 6.96 ± 0.077 mg GAE g⁻¹ FW (

Table 3. Effect of T. longibrachiatum PP002254 and T. harzianum PP002272 culture filtrate on total soluble phenolics (mg GAE g⁻¹ FW) in orange fruit infected with P. italicum.

Treatment	Hours Post-Treatment (hpt)
	24	48	72	96
Control	2.15 ± 0.036 b	2.00 ± 0.026 b	1.82 ± 0.036 c	2.05 ± 0.039 b
T. longibrachiatum PP002254	0.98 ± 0.039 c	1.32 ± 0.068 c	9.60 ± 0.072 a	5.72 ± 0.380 a
T. harzianum PP002272	3.17 ± 0.029 a	3.77 ± 0.063 a	6.96 ± 0.077 b	5.26 ± 0.042 a

Values are mean ± standard error (SE). This means values within each column followed by different letters are significantly different according to Tukey’s HSD test at p < 0.05.

). Similarly, total flavonoid content peaked at 72 hpt, with T. longibrachiatum PP002254 CF-treated fruits reaching 2.69 ± 0.072 mg RE g⁻¹ FW, compared to 2.36 ± 0.047 mg RE g⁻¹ FW in T. harzianum PP002272-treated fruits (

Table 4. Effect of T. longibrachiatum PP002254 and T. harzianum PP002272 culture filtrate on total soluble flavonoids (mg RE g⁻¹ FW) in orange fruit infected with P. italicum.

Treatment	Hours Post-Treatment (hpt)
	24	48	72	96
Control	2.11 ± 0.081 a	1.45 ± 0.087 c	1.35 ± 0.049 c	1.12 ± 0.011 b
T. longibrachiatum PP002254	1.64 ± 0.085 b	1.85 ± 0.081 a	2.69 ± 0.072 a	1.51 ± 0.013 ab
T. harzianum PP002272	1.62 ± 0.090 b	1.71 ± 0.0421 b	2.36 ± 0.047 b	1.68 ± 0.197 a

Values are mean ± standard error (SE). This means values within each column followed by different letters are significantly different according to Tukey’s HSD test at p < 0.05.

). These increases in phenolic and flavonoid content highlight the non-enzymatic antioxidant response induced by Trichoderma CFs.

3.8. Reduction of Lipid Peroxidation in Orange Fruits

Although the infection with P. italicum markedly elevated the lipid peroxidation in infected orange fruit as expressed by the level of malondialdehyde, both Trichoderma filtrates (T. longibrachiatum PP002254 and T. harzianum PP002272) significantly reduced the concentration of MDA. The results indicated that treatment of orange fruit significantly reduced the MDA content in P. italicum-infected orange fruit during the whole time compared with the untreated fruit (

Table 5. Effect of T. longibrachiatum PP002254 and T. harzianum PP002272 culture filtrate on lipid peroxidation (Malondialdehyde (MDA; nmol g⁻¹ FW) in orange fruit infected with P. italicum).

Treatment	Hours Post-Treatment (hpt)
	24	48	72	96
Control	110.04 ± 7.11 a	124.71 ± 2.85 a	135.71 ± 4.95 a	137.56 ± 3.65 a
T. longibrachiatum PP002254	52.94 ± 4.54 b	62.61 ± 1.60 c	79.67 ± 8.53 b	100.77 ± 6.43 c
T. harzianum PP002272	70.46 ± 11.83 b	98.63 ± 3.61 b	110.62 ± 8.40 a	120.43 ± 1.88 b

Values are mean ± standard error (SE). This means values within each column followed by different letters are significantly different according to Tukey’s HSD test at p < 0.05.

). The maximum value of MDA content was 137.56 ± 3.65 nmol g⁻¹ FW of P. italicum-infected orange fruit on the fourth day compared to the treatment with culture filtrate of T. longibrachiatum PP002254 and T. harzianum PP002272 (100.77 ± 6.43 and 120.43 ± 1.88 nmol g⁻¹ FW, respectively) on the same day (Table 5).

4. Discussion

Penicillium digitatum and Penicillium italicum are the primary agents responsible for citrus fruit decay during postharvest handling, leading to substantial economic losses [47]. Blue mold, caused by P. italicum, is a particularly severe postharvest disease affecting citrus fruits, including oranges. It can result in economic losses ranging from 10% to 30% and in extreme cases, up to 50% [48,49]. Accordingly, managing postharvest diseases, especially blue mold, is a critical challenge for orange production worldwide. Chemical control has traditionally been the most effective method for managing blue mold [50]. However, its efficacy is waning due to the widespread and repeated use of various fungicides, leading to serious concerns. Therefore, it is essential to search for efficient and eco-friendly management alternatives. Biological control offers a promising solution for managing blue mold while preserving the quality of oranges [9,51].

Trichoderma species are among the most significant fungal genera for controlling postharvest diseases due to their diverse mechanisms of action, including nutrient competition, mycoparasitism, and the production of antibiotic and hydrolytic enzymes [11,52]. Furthermore, isolating and identifying new Trichoderma strains are essential to improve their application as biological control agents [53]. Despite their potential, there is still limited information about the use of Trichoderma in controlling blue mold caused by P. italicum in oranges and its impact on the physiological changes in fruits. Therefore, this study aimed to isolate, identify, and evaluate the antagonistic activities of new Trichoderma isolates against P. italicum. Additionally, this study examined the role of Trichoderma CFs in enhancing the defense mechanisms in orange fruits.

In the current study, the results of the dual culture assay showed that the rapid growth rates of T. longibrachiatum PP002254 and T. harzianum PP002272, in comparison to P. italicum, suggest a strong potential for these strains to act as biocontrol agents against P. italicum, mainly through competition for space and resources. Several studies have shown that these mechanisms, along with mycoparasitism, are key strategies employed by various Trichoderma species to control fungal phytopathogens [54]. T. longibrachiatum is recognized for its role as a significant biocontrol agent. It engages in parasitism, stimulates plant defense mechanisms, and produces various secondary metabolites. For instance, T. longibrachiatum EF5 demonstrated mycoparasitic activity against M. phaseolina, as evidenced by hyphal entanglement between the two fungi. Additionally, it exhibited antagonistic activity, leading to mycelial modifications of both M. phaseolina and S. rolfsii in dual confrontation assays [55,56].

Furthermore, in a dual culture assay, T. harzianum parasitized Fusarium sudanense, breaking down its hyphae and restricting its growth by competing for space and nutrients, thus preventing seed rot in wheat [54]. T. harzianum has shown antagonistic abilities in vitro against the pathogen Alternaria cerealis, effectively inhibiting its growth [57]. In a different study, two strains of T. harzianum exhibited significant antagonistic activity against a range of sweet potato postharvest pathogens, such as Fusarium ipomeae, F. solani, F. oxysporum, Penicillium rotoruae, P. citrinum, Aspergillus wentii, Mucor variicolumellatus, and M. phaseolina, in dual confrontation assays while competing for space [58]. Other species of Trichoderma suppressed the growth of many postharvest pathogenic fungi through mycoparasitism; for example, T. asperellum parasitized P. chrysogenum, significantly reducing its growth through direct mycoparasitism and enzymatic degradation [59].

T. atroviride has been reported to parasitize P. italicum by wrapping around its fungal hyphae and secreting enzymes that break down the pathogen’s cell walls [30]. These examples illustrate the diverse mechanisms by which Trichoderma species can parasitize and control pathogenic fungi, highlighting their potential as effective biocontrol agents. Thus, the present study suggests that T. longibrachiatum PP002254 and T. harzianum PP002272 suppressed P. italicum and inhibited its growth by competing for space and resources. However, further research is needed to investigate additional mechanisms underlying their mycoparasitic activities and to evaluate their efficacy more comprehensively.

Furthermore, the culture filtrates of T. longibrachiatum PP002254 and T. harzianum PP0022272 significantly inhibited the fungal growth of P. italicum. T. longibrachiatum PP002254 showed higher efficacy with 77.22% inhibition. This is consistent with prior research that has investigated the use of culture filtrates from various Trichoderma species to control phytopathogenic fungi for example, the culture filtrate of T. longibrachiatum demonstrated inhibitory effects on the growth of various pathogens, including Alternaria brassicicola, B. cinerea, Colletotrichum coccodes, Cladosporium cucumerinum, Cylindrocarpon destructans, Magnaporthe oryzae, and Phytophthora infestans [60]. Additionally, the culture filtrate of T. harzianum strongly inhibited the growth of B. cinerea, F. oxysporum [61], Mucor circinelloides, A. flavus, A. fumigatus, and Rhizoctonia solani [62].

Several mechanisms have been proposed to elucidate the antifungal properties of Trichoderma species. These fungi are known to produce a variety of antibiotics and bioactive compounds that can inhibit the growth of phytopathogens [63]. Trichoderma species produce numerous antimicrobial secondary metabolites, such as trichomycin, gelatinomycin, chlorotrichomycin, and antibacterial peptides [64]. Secondary metabolites from T. longibrachiatum play important roles in its biocontrol capacity. In addition, T. harzianum is known for producing secondary metabolites with significant biocontrol properties [65]. For example, T. longibrachiatum produces peptaibols as secondary metabolites, which have been shown to inhibit the growth of various plant pathogens, particularly R. solani and A. solani [66].

Additionally, some Trichoderma species secrete enzymes such as chitinase and β-1,3-glucanase, which are essential for degrading the cell walls of fungal pathogens [67]. The antifungal activities of the culture filtrates of T. longibrachiatum PP002254 and T. harzianum PP0022272 might be attributed to their destructive effects on the structure of P. italicum. SEM analysis revealed that the CFs of T. longibrachiatum PP002254 and T. harzianum PP0022272 caused deformation, collapse, and erosion of the hyphae of P. italicum. These observations suggest that the in vitro antifungal mechanism of Trichoderma CFs against P. italicum might involve disrupting the fungal cell membrane and inhibiting cell growth [68]. The destructive effects of T. longibrachiatum PP002254 and T. harzianum PP0022272 CFs on the hyphae of P. italicum highlight their potential as effective biocontrol agents. However, further research is needed to identify the specific enzymes and metabolites responsible for these effects and to evaluate their efficacy.

Moreover, in the present study, the CFs of T. longibrachiatum PP002254 and T. harzianum PP0022272 significantly reduced the development of blue mold disease in treated oranges compared to untreated ones. This reduction is probably due to the stimulation of the antioxidant defense system in treated orange fruits. The antioxidant defense system in plants is an intricate network that safeguards cells from oxidative harm induced by reactive oxygen species (ROS). This system encompasses a range of enzymatic components, which serve as the first line of defense against reactive oxygen species, and non-enzymatic components, which form the second line of defense, working together to neutralize reactive oxygen species and mend oxidative damage [69]. Superoxide dismutase, catalase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase, and peroxidase are key antioxidant enzymes detoxifying reactive oxygen species [70].

Catalase is a principal enzyme that either directly or indirectly aids in eliminating ROS, particularly hydrogen peroxide (H₂O₂), thereby averting oxidative damage by converting it into water (H₂O) and oxygen (O₂) [71,72]. Additionally, it is important to note that peroxidase also neutralizes H₂O₂, helping to preserve the redox balance [70]. Furthermore, polyphenol oxidase is a crucial enzyme in plant defense, particularly in response to wounding and pathogen attacks. It catalyzes the oxidation of phenolic compounds into quinones, which are more toxic than phenolic compounds and help repel pathogens [73]. Although polyphenol oxidase is not a primary component of the antioxidant defense system that directly neutralizes reactive oxygen species, it plays a vital role in the overall defense mechanism of plants. Moreover, phenylalanine ammonia-lyase (PAL) is a significant enzyme in the phenylpropanoid and shikimate pathways and is responsible for the biosynthesis of several compounds, such as phytoalexins, lignins, tannins, phenolic compounds, and salicylic acid, which enhance plant resistance to disease [74].

Our findings revealed a significant increase in the enzymatic activities of catalase (CAT), peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) in oranges treated with CFs of T. longibrachiatum PP002254 and T. harzianum PP0022272. Numerous studies have demonstrated that the application of Trichoderma species can lead to significant increases in the activities of defense-related enzymes, which are crucial for plant resistance mechanisms. For instance, [17] found that apple fruits treated with the CFs of T. harzianum (Tha739) displayed a significant increase in the activities of POD and PPO enzymes compared with untreated fruit. Another study conducted by [75] reported that tomato plants under early blight stress and treated with culture filtrate of three isolates of Trichoderma such as T. harzianum, T. atroviride, and T. longibrachiatum showed a significant increase in POD, PPO, and PAL activities. Furthermore, the CAT activity was increased with the application of T. harzianum on pak choi infected with Clubroot disease and reduced H₂O₂ levels and MDA concentration [76]. Trichoderma spp. stimulate plants to produce defense-related enzymes like polyphenol oxidase and peroxidase, enhancing the plant’s defense mechanisms against fungal, viral, and bacterial pathogens [77,78].

Currently, over 20 elicitors are produced by Trichoderma to induce plant resistance. These include antitoxins, polypeptides, lipopeptides, cellulases, hydrophobic proteins, non-toxic gene proteins, terpenoids, phenol derivatives, glycosidic ligands, and flavonoids [79]. These secondary metabolites trigger plant defense mechanisms and enhance growth. The interaction between Trichoderma and plants boosts the production of enzymes and substances related to defense [80]. The marked increase in the activities of CAT, POD, PPO, and PAL in oranges treated with the culture filtrates of T. longibrachiatum PP002254 and T. harzianum PP0022272 highlighted the potential of these Trichoderma strains as effective biocontrol agents. These strains enhance the inherent plant defense mechanisms, providing strong protection against various pathogens. Nonetheless, further research is necessary to pinpoint the specific elicitors and metabolites responsible for these effects and to assess their effectiveness.

Additionally, the current study noted increased levels of total soluble phenolics and flavonoids in oranges after treatment. These non-enzymatic antioxidants, phenolics, and flavonoids are crucial for plant disease resistance. They aid in reducing oxidative stress and maintaining cellular balance [69]. Phenolic compounds are known to hinder the growth of various pathogens by interfering with their cell walls and membranes. Flavonoids are important in plants serving as detoxifying agents, phytoalexins, signaling molecules, and allelochemicals [81]. The rise in phenolics and flavonoids suggests that Trichoderma species effectively stimulate systemic resistance releasing not just proteins but also secondary metabolites [82]. Our findings are consistent with previous reports and confirm that the CFs of Trichoderma species accumulate phenolic compounds, which impede the invasion of pathogens in plants [75]. The elevated presence of these compounds in treated oranges may have played a role in suppressing the growth of P. italicum.

Malondialdehyde (MDA) is a marker of oxidative stress and lipid peroxidation in plant tissues [83]. Our current findings revealed that the culture filtrates of T. longibrachiatum PP002254 and T. harzianum PP002272 significantly reduced MDA concentrations in treated orange fruits compared to untreated controls. This reduction in MDA levels can be attributed to several mechanisms associated with Trichoderma spp., particularly their ability to enhance antioxidant enzyme activity. Trichoderma species are known to boost the activity of crucial antioxidant enzymes such as catalase, superoxide dismutase, and peroxidase in plants. These enzymes play vital roles in scavenging reactive oxygen species thereby mitigating oxidative stress and lipid peroxidation [80].

It is worth noting that treatment with T. longibrachiatum PP002254 and T. harzianum PP0022272 culture filtrate significantly increased the activity of antioxidant enzymes such as catalase and peroxidase as well as non-enzymatic antioxidants (phenolics and flavonoids) which reduced the damage caused by P. italicum infection and hence could be attributed to the decreased levels of MDA. Moreover, Trichoderma spp. produce various secondary metabolites with potent antioxidant properties. These metabolites can directly neutralize the levels of reactive oxygen species, further contributing to the reduction of MDA levels [65]. Overall the application of T. longibrachiatum PP002254 and T. harzianum PP002272 filtrates appears to be a promising strategy for enhancing oxidative stress tolerance and improving the quality of orange fruits.

5. Conclusions

Blue mold caused by Penicillium italicum represents a significant postharvest disease of citrus fruits, leading to substantial production losses. Traditionally, control of P. italicum has relied on synthetic fungicides. However, the associated risks to consumers and the environment necessitate the exploration of safe and effective alternatives. The present study demonstrated that Trichoderma longibrachiatum PP002254 and Trichoderma harzianum PP002272 or their culture filtrates significantly suppressed the growth of P. italicum and delayed the development of blue mold in orange fruits. The antifungal properties of these Trichoderma isolates are likely attributed to the enhanced activities of defense-related enzymes such as catalase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase and non-enzymatic antioxidants (total soluble phenolics and total soluble flavonoids), which mitigate disease impact and reduce malondialdehyde (MDA) levels. These findings suggest that T. longibrachiatum PP002254 and T. harzianum PP002272 hold considerable potential as environmentally friendly biocontrol agents for controlling citrus blue mold.