Protective and Curative Effects of Trichoderma asperelloides Ta41 on Tomato Root Rot Caused by Rhizoctonia solani Rs33

Two molecularly identified tomato isolates, Trichoderma asperelloides Ta41 and Rhizoctonia solani Rs33, were characterized and antagonistically evaluated. The dual culture technique showed that Ta41 had a high antagonistic activity of 83.33%, while a light microscope bioassay demonstrated that the Ta41 isolate over-parasitized the pathogen completely. Under greenhouse conditions, the application of Ta41 was able to promote tomato plant growth and had a significant increase in plant height, root length, and shoot fresh, shoot dry, root fresh, and root dry weight. It also improved chlorophyll content and total phenol content significantly, both in protective and in curative treatments. The protective treatment assay exhibited the lowest disease index (16.00%), while the curative treatment showed a disease index of 33.33%. At 20 days post-inoculation, significant increases in the relative expression levels of four defense-related genes (PR-1, PR-2, PR-3, and CHS) were observed in all Ta41-treated plants when compared with the non-treated plants. Interestingly, the plants treated with Ta41 alone showed the highest expression, with relative transcriptional levels of CHS, PR-3, PR-1, and PR-2 that were, compared with the control, 3.91-, 3.13-, 2.94-, and 2.69-fold higher, respectively, and the protective treatment showed relative transcriptional levels that were 3.50-, 3.63-, 2.39-, and 2.27-fold higher, respectively. Consequently, the ability of Ta41 to promote tomato growth, suppress Rs33 growth, and induce systemic resistance supports the incorporation of Ta41 as a potential bioagent for controlling root rot disease and increasing the productivity of crops,


Introduction
The tomato (Lycopersicon esculentum L.) is the second-most commonly consumed vegetable crop after the potato worldwide [1]. Several pathogens can infect tomato plants and cause diseases. Many common diseases that attack tomatoes are caused by fungi, bacteria, nematodes, and viruses [2,3]. Among the fungal pathogens causing several diseases in tomatoes, Rhizoctonia solani is the worst fungus that could damage tomato plants and reduce the yield [4][5][6].
R. solani is a destructive soil-borne pathogen that causes severe losses in many crops worldwide. R. solani does not form asexual spores (conidia) but reproduces a survival form called sclerotia [7], which considers a major cause of R. solani infection. The excessive use of chemical fungicides, the most common strategy used by farmers to control R. solani, poses severe risks to human health and the environment and leads to pathogen-resistant strains. Therefore, biological control has been used more frequently as an alternative for controlling plant diseases. Biological control is environment-friendly and effective in managing most plant fungal pathogens. Most research on disease control was done using different Trichoderma fungus strains [8][9][10]. Internal transcribed spacer (ITS), RNA polymerase II subunit 2 (Rpb2), and translation elongation factor 1 alpha (Tef-1) genes are the most used molecular markers in phylogenetic analysis for the high throughput sensitive identification and characterization of Trichoderma spp. for the early screening of potential antagonists against soil-borne pathogens [11].
Trichoderma species use different mechanisms for pathogen inhibition, such as mycoparasitism via hydrolytic enzyme secretion, antibiosis via secondary metabolite production, competition for space and nutrients, promoting plant growth, and inducing plant systemic resistance mechanisms [12]. Trichoderma spp. are effective biocontrol agents due to the rapid multiplication or the tolerance of harsh conditions [13]. Trichoderma spp. have potent antagonism and mycoparasitic actions on plant pathogens, allowing them to reduce the incidence of plant diseases, and the main mechanism for Trichoderma species is hyperparasitism [14,15]. Many genes in Trichoderma spp. encoding extracellular proteases and oligopeptide transporters are expressed when contacts occur between Trichoderma spp. and the host-pathogen [16,17]. During the hyperparasitic process, cell wall degrading enzymes (CWDEs), i.e., glucanases, chitinase, and proteinases, can be secreted by Trichoderma spp. [18]. The secreted CWDEs can degrade the plant pathogen's cell wall [19]. The Trichoderma colonization of roots causes root hair growth and triggers defense activities, such as significant changes in a variety of metabolic pathways and the activation of genes involved in plant host defense, primarily through signaling pathways involving jasmonic acid and ethylene [20,21]. In Arabidopsis, colonization by Trichoderma fungus before infection by biotrophic or necrotrophic plant pathogens triggered an oxidizing status that enhanced resistance systemically [22].
Microbial communities have attracted much attention as an eco-acceptable and costeffective disease resistance enhancement via induced systemic resistance (ISR) by releasing proteins, secondary metabolites, and plant growth stimulation for long-term crop production [23][24][25]. Secondary metabolites produced by Trichoderma spp. offer selective advantages in mechanisms such as competitiveness, symbiotic relationships, mineral transportation, growth production, sensing, and mycoparasitic behavior [26,27]. Recognizing the importance of screening new Trichoderma species with more potent antifungal activity for agricultural use, the current study aimed to evaluate the protective and curative activities of Trichoderma asperelloides Ta41 on tomato root rot caused by Rhizoctonia solani Rs33 under controlled greenhouse conditions. Moreover, the effects of Ta41 on the plant growth parameters, chlorophyll content, total phenol content, and expression levels of defense-related genes with or without Rs33 were estimated.

Sample Collection, Isolation, and Identification
Ten tomato plants (cv. Peto 86) showing root rot symptoms were collected from El-Behira governorate, Egypt. The phytopathogen was isolated from the 10 symptomatic samples and identified by cultural, morphological characteristics, and sequencing of the ITS, as described previously [6,28].
The Trichoderma isolate was isolated from soil rhizosphere samples collected from tomato cultivated areas, El-Behira governorate, Egypt. The serial dilution plate technique was used to isolate the antagonistic Trichoderma spp. using the Trichoderma specific medium (TSM). One milliliter of 1 × 10 −3 dilution was poured onto a selective medium and the receipt described by Elad et al. [29]. The obtained culture was purified by the hyphal tip isolation technique and maintained on PDA slants for further identification processes. The identification was performed based on their morphological characteristics and molecular typing using ITS, Rpb2, and Tef-1 genes [30][31][32][33]. Primer sequences are given in Table 1. PCR reactions containing 0.5 µL of each primer pair (forward and reverse), 10 µL of 2x Taq Ready Mix (Enzynomics Inc., Daejeon, Korea), and 1 µL of template DNA, and the Milli-Q water was added up to a volume of 25 µL. Cycling was done using a Techne Prime Thermal Cycler (Cole-Parmer, Staffordshire, UK) as follows: an initial denaturation of 95 • C for 4 min, followed by 35 cycles of 94 • C for 45 s, 55 • C for 45 s, and 72 • C for 1 min, and a final extension step at 72 • C for 5 min. The PCR amplifications were sequenced, and the nucleotide sequences were aligned using MEGA 6 software. GenBank BLAST tool was used to compare the obtained sequences with those in the GenBank database.

Dual Culture Technique
The Trichoderma asperelloides and Rhizoctonia solani isolates were grown on PDA individually for a week. After that, 5 mm mycelial plugs of the two fungal isolates T. asperelloides and R. solani were placed opposite each other on a PDA plate [34]. The antagonistic tests were conducted in triplicate, and hyphal interactions between the two isolates were studied. The radial growth of R. solani was measured (mm), and inhibition percentage was calculated as follows: Inhibition % = C−T C × 100, where C is the R. solani radial growth in the control, and T is the radial growth of R. solani in the presence of T. asperelloides. The mycoparasitic interacted regions on PDA were observed after 4 days under the 10x light microscope.

Greenhouse Assessments of R. solani, Samples Collection, and Plant Growth-Promoting Abilities of T. asperelloides
The effect of T. asperelloides isolates on the activity of R. solani and plant growth was evaluated based on a pot experiment under controlled greenhouse conditions (temperature: 28 ± 2 • C; humidity: 75 ± 5%; photoperiod: 14 light/10 dark h) The 4-week-old tomato seedlings (cv. Peto 86) were transplanted to plastic pots (20 cm in diameter) filled with sterile soil. After 5 days of seedlings transplanting, 10 mL of T. asperelloides inoculum with a concentration of 1 × 10 8 spores/mL was added to each pot as described by Fahmi et al. [35]. An R. solani inoculum was added to the pots at 1% (W:W). Treatments were distributed in the greenhouse with 5 replicates as follows: T1: tomato plant control inoculated with media-free microorganisms; T2: tomato plants inoculated with R. solani only; T3: tomato plants inoculated with T. asperelloides; T4: tomato plants inoculated with T. asperelloides 48 h before inoculation with R. solani (protective application); T5: tomato plants inoculated with T. asperelloides 48 h after inoculation with R. solani (curative application). Twenty days after inoculation with R. solani, tomato leaf samples from all treatments were collected to test the total phenolic compounds and defense-related genes as a response to different treatments under greenhouse conditions. One month after transplanting, the plants screened for disease severity according to a 0-5 scale according to root browning appears on the root system [36], where 0 = No symptom, 1 = 0-25% root browning, 2 = 26-50% root browning, 3 = 51-75% root browning, 4 = 76-100% root browning, and 5 = plant death. Tomato plants were observed, and the disease index (DI) was calculated using the following formula: DI % = ∑ of observed numerical rating Max. disease rating × Total number of observed plants × 100 The plants were also used to record the effect of T. asperelloides on the following growth parameters, plant height (cm), root length (cm), shoot and root fresh weight (g), shoot and root dry weight (g), and total chlorophyll content (SPAD-502 Plus, Konica Minolta, Inc., Tokyo, Japan).

Total Phenolic Compounds Content in Tomato Plants
To measure the Total Phenolic Compounds (TPCs) of tomato leaf samples collected from all treatments, the Folin-Ciocalteu (FC) method was chosen [37]. Ten milligrams of leaves extract were liquefied in 10 mL of ethanol to obtain 1 mg/mL as a final concentration. One hundred microliters of the liquefied extract were blended with 750 µL of the FC reagent. The liquid composite was allowed to remain at 25 • C for 5 min. After that, 750 µL of Na 2 CO 3 was applied to the mixture, and the tube was gently shaken to combine it. The mix was measured after incubation for 1 h at 725 nm using an OPTIMA SP-300 spectrophotometer (Optima Inc., Tokyo, Japan). Gallic acid was used to establish a calibration curve. TPC was calculated as gallic acid equivalents (GAE, mg g-1 of extract). One hundred milligrams of tomato leaves collected at 20 days post-inoculation (dpi) were used as starting materials for total RNA extraction, using the guanidium isothiocyanate method [38]. The concentration and purity of the extracted RNA were estimated by using a Nano SPECTROstar (BMG Labtech, Ortenberg, Germany), while the RNA integrity was checked by agarose gel electrophoresis. For cDNA synthesis, two micrograms of DNase-treated total RNA were used as a template in a reverse transcription reaction, as described previously [39]. The cDNA samples were stored at −80 • C until use as a template in qRT-PCR.

qRT-PCR and Data Analysis
The transcriptional levels of 4 tomato defense-related genes, PR-1, PR-2, PR-3, and CHS (Table 1) were evaluated at 20 dpi using the qRT-PCR technique. The expression detected from the β-actin gene was used as an internal reference ( Table 1). The experiments were run in triplicate reactions for each sample. The qRT-PCR was performed on a Rotor-Gene 6000 using SYBR Green PCR Master Mix [40,41]. The relative changes of the transcript level of each tested gene were measured according to the 2 −∆∆CT method [42].

Statistical Analysis
The obtained data were statistically analyzed by one-way ANOVA using the CoStat software. At the same time, the statistical differences in the mean were determined by Tukey's honest significant differences method (H.S.D.) at a p ≤ 0.05 level of probability, and standard deviation (± SD) was shown as a column bar. Columns with the same letter do not differ significantly. Compared to the control, relative transcript levels greater than 1 demonstrate an increase in gene transcription (upregulation), while values lower than 1 indicate a decrease in transcription levels (downregulation).

Isolation and Identification of R. solani and T. asperelloides
Out of 10 R. solani isolates isolated from root rot diseased tomato plants, the most aggressive isolate of R. solani was chosen based on a pre-pathogenicity test (data not shown). The vegetative characteristics of R. solani showed that hyphae septate multinucleate and clamp connection, conidia, and rhizomorphs were never observed. Several Trichoderma isolates were obtained, and the best effective antifungal Trichoderma isolate (data not shown) was chosen to complete the study.
Molecular results confirmed the initial identification of the two confronting isolates used in this study. The sequence of amplified ITS regions (approximately 650 bp) R. solani Rs33 isolates were deposited in GenBank under accession no. MW664424. Comparing R. solani Rs33 nucleotide ITS sequence with those isolates of R. solani in the GenBank database ( Figure 1) clarified that the highest homogeneity was 100% with the isolated R. solani from tomatoes in Egypt (MH687913). Minimum nucleotide sequence similarity (99.69%) with R. solani isolates from China (MH172598, MH172624, MH483966, and MH483967), Turkey (MT380171 and MT408040), Azerbaijan (MG654491), and Canada (MN313269) was found.
Agronomy 2021, 11, x FOR PEER REVIEW 5 of 16 1 demonstrate an increase in gene transcription (upregulation), while values lower than 1 indicate a decrease in transcription levels (downregulation).

Isolation and Identification of R. solani and T. asperelloides
Out of 10 R. solani isolates isolated from root rot diseased tomato plants, the most aggressive isolate of R. solani was chosen based on a pre-pathogenicity test (data not shown). The vegetative characteristics of R. solani showed that hyphae septate multinucleate and clamp connection, conidia, and rhizomorphs were never observed. Several Trichoderma isolates were obtained, and the best effective antifungal Trichoderma isolate (data not shown) was chosen to complete the study.
Molecular results confirmed the initial identification of the two confronting isolates used in this study. The sequence of amplified ITS regions (approximately 650 bp) R. solani Rs33 isolates were deposited in GenBank under accession no. MW664424. Comparing R. solani Rs33 nucleotide ITS sequence with those isolates of R. solani in the GenBank database ( Figure 1) clarified that the highest homogeneity was 100% with the isolated R. solani from tomatoes in Egypt (MH687913). Minimum nucleotide sequence similarity (99.69%) with R. solani isolates from China (MH172598, MH172624, MH483966, and MH483967), Turkey (MT380171 and MT408040), Azerbaijan (MG654491), and Canada (MN313269) was found. Regarding the Trichoderma isolate, the three specific primers ITS1/ITS4, EF1-728F/TEF1LLErevR, and RPB2-5F/RPB2-7cR successfully amplified approximately 650 bp, 500 bp, and 1050 bp, respectively. The NCBI-Blast alignment revealed that the Trichoderma isolate was highly similar to T. asperelloides. To describe species limits, phylogenetic analysis was carried out using the three-locus combined ITS, Tef-1, and Rpb2 datasets. The significance of each branch in alignment was indicated by bootstrap 2000 subsets (only values higher than 40% are indicated). A multiple sequence alignment in the neighborjoining method using Mega 6 revealed that the relationship of almost all Trichoderma reference isolates could be clearly distinguished on the level of species and separated into different clusters and clades ( Figure 2). The isolates T. asperelloides strain CEN1427 and T. asperelloides strain CEN1431 clustered with our isolate Ta41 (>98% similarity in three genes), so they should be classified within this species. Sequences of T. asperelloides Ta41 were analyzed and deposited in the GenBank database (accession no. MW797033, MZ269255, and MZ269254) for the three genes, respectively. Regarding the Trichoderma isolate, the three specific primers ITS1/ITS4, EF1-728F/TEF-1LLErevR, and RPB2-5F/RPB2-7cR successfully amplified approximately 650 bp, 500 bp, and 1050 bp, respectively. The NCBI-Blast alignment revealed that the Trichoderma isolate was highly similar to T. asperelloides. To describe species limits, phylogenetic analysis was carried out using the three-locus combined ITS, Tef-1, and Rpb2 datasets. The significance of each branch in alignment was indicated by bootstrap 2000 subsets (only values higher than 40% are indicated). A multiple sequence alignment in the neighbor-joining method using Mega 6 revealed that the relationship of almost all Trichoderma reference isolates could be clearly distinguished on the level of species and separated into different clusters and clades ( Figure 2). The isolates T. asperelloides strain CEN1427 and T. asperelloides strain CEN1431 clustered with our isolate Ta41 (>98% similarity in three genes), so they should be classified within this species. Sequences of T. asperelloides Ta41 were analyzed and deposited in the GenBank database (accession no. MW797033, MZ269255, and MZ269254) for the three genes, respectively.

Effect of T. asperelloides Ta41 on the Mycelial Growth of R. solani Rs33 In Vitro
The reduction of R. solani Rs33 growth in response to the T. asperelloides Ta41 isolate is presented in Figure 3. The growth of the Rs33 isolates inhibited significantly with Ta41 isolate in the dual culture technique with a value of 15 mm compared with the control. The percentage of growth inhibition in the confrontation assays raised values to 83.3%. 10X light microscope graphs of the Ta41 hyperparasite on the Rs33 cell wall were observed and describe the spore germination and penetration peg formation ( Figure 3). The advanced stages of mycoparasitism of Ta41 started coiling around the Rs33 cell wall and tightly encircled the hyphae. The Ta41 isolate penetrated the hyphae of the Rs33 isolate and entered the mycelium. (Figure 3).

Effect of T. asperelloides Ta41 on the Mycelial Growth of R. solani Rs33 In Vitro
The reduction of R. solani Rs33 growth in response to the T. asperelloides Ta41 isolate is presented in Figure 3. The growth of the Rs33 isolates inhibited significantly with Ta41 isolate in the dual culture technique with a value of 15 mm compared with the control. The percentage of growth inhibition in the confrontation assays raised values to 83.3%. 10× light microscope graphs of the Ta41 hyperparasite on the Rs33 cell wall were observed and describe the spore germination and penetration peg formation ( Figure 3). The advanced stages of mycoparasitism of Ta41 started coiling around the Rs33 cell wall and tightly encircled the hyphae. The Ta41 isolate penetrated the hyphae of the Rs33 isolate and entered the mycelium. (Figure 3).

Disease Index of R. solani Rs33 on Tomato Plants In Vivo
Disease index was recorded 30 days after transplanting and was estimated according to the browning on the roots, regardless of its extent (using a 0-5 scale) depending on treatment (Figures 4 and 5). The Ta41 isolate (T3) application significantly reduced the disease index compared to the infested control plants (T2). The application of the Ta41 isolate as a pre-inoculation of the R. solani Rs33 isolate (T4) showed a low disease index (16.00%), followed by T5, the Ta41 isolate applied after inoculation with the Rs33 isolate (33.33%). The disease index was 81.00% with the R. solani Rs33 isolate alone (T2) compared to treatments that showed no symptoms-T1 and T3 ( Figure 5). isolate in the dual culture technique with a value of 15 mm compared with the control. The percentage of growth inhibition in the confrontation assays raised values to 83.3%. 10X light microscope graphs of the Ta41 hyperparasite on the Rs33 cell wall were observed and describe the spore germination and penetration peg formation (Figure 3). The advanced stages of mycoparasitism of Ta41 started coiling around the Rs33 cell wall and tightly encircled the hyphae. The Ta41 isolate penetrated the hyphae of the Rs33 isolate and entered the mycelium. (Figure 3).

Disease Index of R. solani Rs33 on Tomato Plants in Vivo
Disease index was recorded 30 days after transplanting and was estimated according to the browning on the roots, regardless of its extent (using a 0-5 scale) depending on treatment (Figures 4 and 5). The Ta41 isolate (T3) application significantly reduced the disease index compared to the infested control plants (T2). The application of the Ta41 isolate as a pre-inoculation of the R. solani Rs33 isolate (T4) showed a low disease index (16.00%), followed by T5, the Ta41 isolate applied after inoculation with the Rs33 isolate (33.33%). The disease index was 81.00% with the R. solani Rs33 isolate alone (T2) compared to treatments that showed no symptoms-T1 and T3 ( Figure 5).

Effect of T. asperelloides Ta41 on Tomato Growth under Greenhouse Conditions
The efficacy of T. asperelloides Ta41 isolate against R. solani Rs33 on tomato plants was tested under controlled greenhouse conditions. Both the pre-and post-inoculation treatments of the Ta41 isolate significantly improved the growth of tomato plants. Application of the T3 treatment was found to be more effective in increasing chlorophyll content (39.00 SPAD unit), followed by the T4 application, which was found to be more effective than T5 (37.90 and 33.90 SPAD unit, respectively) without significant differences ( Figure 6). On the other hand, the effect on plant height was significant due to the application of Ta41, which recorded 39.80 cm in T3, followed by T4 (34.20 cm). The root length increased with T3, T4, and T5 (18.4, 18.4, and 17.70 cm, respectively). These treatments displayed an increase in shoot fresh weight (16.4, 13.5, and 9.5 g, respectively) and root fresh weight (5.8, 5.6, and 4.5 g, respectively). Moreover, positive effects on the dry weight of the shoot were observed with T3 followed by T4 and T1 (3.50, 3.20, and 3.20 g, respectively) but without a Agronomy 2021, 11, 1162 8 of 17 significant effect. The root dry weight of the tomato plants was affected by all treatments that contained Ta41 (T3, T4, and T5) significantly more than the other two treatments (T1 and T2) ( Table 2).

Effect of T. asperelloides Ta41 on Tomato Growth Under Greenhouse Conditions
The efficacy of T. asperelloides Ta41 isolate against R. solani Rs33 on tomato plants was tested under controlled greenhouse conditions. Both the pre-and post-inoculation treatments of the Ta41 isolate significantly improved the growth of tomato plants. Application of the T3 treatment was found to be more effective in increasing chlorophyll content (39.00 SPAD unit), followed by the T4 application, which was found to be more effective than T5 (37.90 and 33.90 SPAD unit, respectively) without significant differences ( Figure 6). On the other hand, the effect on plant height was significant due to the application of Ta41, which recorded 39.80 cm in T3, followed by T4 (34.20 cm). The root length increased with T3, T4, and T5 (18.4, 18.4, and 17.70 cm, respectively). These treatments displayed an increase in shoot fresh weight (16.4, 13.5, and 9.5 g, respectively) and root fresh weight (5.8, 5.6, and 4.5 g, respectively). Moreover, positive effects on the dry weight of the shoot were observed with T3 followed by T4 and T1 (3.50, 3.20, and 3.20 g, respectively) but without a significant effect. The root dry weight of the tomato plants was affected by all treatments that contained Ta41 (T3, T4, and T5) significantly more than the other two treatments (T1 and T2) ( Table 2).

Total Phenolic Compound Content
Total phenol content (TPC) was accumulated in high concentrations in plants in the T4 (51.4 GAE mg/g dry extract) and T5 (33.6 GAE mg/g dry extract) treatments, as well as in T2 (36.8 GAE mg/g dry extract), compared to their corresponding control, T1 (13.2 GAE mg/g dry extract). All plants treated with T3 showed a low accumulation of total phenol (26.8 GAE mg/g dry extract) compared to the control and the other treatments.

Total Phenolic Compound Content
Total phenol content (TPC) was accumulated in high concentrations in plants in the T4 (51.4 GAE mg/g dry extract) and T5 (33.6 GAE mg/g dry extract) treatments, as well as in T2 (36.8 GAE mg/g dry extract), compared to their corresponding control, T1 (13.2 GAE mg/g dry extract). All plants treated with T3 showed a low accumulation of total phenol (26.8 GAE mg/g dry extract) compared to the control and the other treatments.

Transcriptional Levels of Defense-Related Genes
At 20 dpi, significant increases in the relative expression levels of four defense-related genes (PR-1, PR-2, PR-3, and CHS) were observed in the treated plants when compared with the non-treated plants (p ≤ 0.05). Compared to the control (T1), a significant upregulation of PR-1 was observed in all treatments, while the infested plants in T2 showed downregulation (Figure 7). The highest relative expression level (2.94-fold higher than the control) was reported in T3, followed by the protective (T4) and curative (T5) treatments with expression levels that were 2.39-and 1.46-fold higher, respectively ( Figure 6). Like PR-1, the transcriptional level of PR-2 exhibited upregulation in all plants treated with T. asperelloides Ta41 (Figure 7). The greatest expression level (2.69-fold higher than the control) was reported in T3, followed by T5 (2.42-fold higher) and T4 (2.12-fold higher). On the other hand, the inoculated plants with T2 showed a downregulation, with a relative expression level that was 0.94-fold lower than the control (Figure 7). Regarding PR-3 expression, significant upregulation was found in all treatments compared to the control (Figure 7). The highest transcriptional level (3.13-fold higher than the control) was observed with T3, followed by T4, T5, and T2 with relative expression levels that were 2.63-, 2.32-, and 1.43-fold higher, respectively, higher than the control (Figure 7). Similar to PR-3, the expression level of CHS exhibited upregulation in all treatments compared to the control. The expression level of T3 was the most extraordinary (3.91-fold higher than the control), while the levels for the T4, T5, and T2 treatments were 3.50-, 2.81-, and 1.52-fold higher, respectively ( Figure 7). pression, significant upregulation was found in all treatments compared to the control (Figure 7). The highest transcriptional level (3.13-fold higher than the control) was observed with T3, followed by T4, T5, and T2 with relative expression levels that were 2.63-, 2.32-, and 1.43-fold higher, respectively, higher than the control (Figure 7). Similar to PR-3, the expression level of CHS exhibited upregulation in all treatments compared to the control. The expression level of T3 was the most extraordinary (3.91-fold higher than the control), while the levels for the T4, T5, and T2 treatments were 3.50-, 2.81-, and 1.52-fold higher, respectively (Figure 7).

Discussion
The application of plant-growth-promoting fungi (PGPFs) as biocontrol agents is a safe substitution for undesirable chemical fungicide use and is considered a sustainable and environmentally friendly alternative [43,44]. Among PGPFs, Trichoderma spp. are highly efficient antagonist fungi and possess biocontrol activity on many phytopathogenic fungi such as R. solani, S. rolfsii, and V. dahliae, among others [45,46]. The results of ITS molecular characterization of the isolated pathogen from root rot tomato plants revealed that the pathogen was Rhizoctonia solani Rs33. For Trichoderma isolate identification, we performed a full multigenic analysis with three markers: ITS, Tef-1, and Rpb2. Among the sequenced markers, the most informative gene was Tef-1, followed by Rpb2 and ITS. The phylogram based on the concatenation of three gene sequences confirmed the identification of Trichoderma asperelloides Ta41 among reference sequences, based on their clustering with reference taxa [47,48].
Our results indicate an ability of the bioagent Ta41 to stop the progress of the pathogen Rs33 by up to 83.33% as Ta41 grew three times faster than Rs33 in the early in vitro experiments conducted in this study. Ramírez-Cariño et al. [49] reported that T. asperelloides has a major advantage over the pathogens A. alternata and F. oxysporum in the competition for space and nutrients due to its faster growth rate. T. asperelloides Ta41 prevented pathogen development and triggered hyperparasitism, mostly on phytopathogens, using a dual culture technique. 10× microscopic photographs of the interaction assays were used to confirm this mechanism. The images indicate that, when Ta41 and Rs33 interact, the Ta41 hyphae wrap around the Rs33 hyphae. Mycoparasitism is a complicated process involving recognition, invasion, and eventual penetration, and pathogen destruction [50]. The findings of this study are aligned with those of Alamri et al. [51], who found that T. harzianum parasitized R. solani hyphae by winding around them and then secreting secondary metabolites such as lytic enzymes to weaken and destroy the target cell wall and promote the feed intake of nutrients.
Similarly, several authors mentioned the role of secondary metabolites of several strains from Trichoderma spp. to control several plant pathogens, especially in the rhizosphere. Trichoderma spp. and R. solani interacted with many mechanisms to destroy the hyphae cell wall and the membrane permeability. T. asperelloides has been shown to suppress the incidence of R. solani, which causes damping-off disease in beans and induces defensin genes in cucumber seedlings against Pseudomonas syringae pv. lachrimans [52]. Furthermore, Trichoderma spp. has been confirmed to treat Sclerotinia sclerotiorum in soybean crops [45] effectively. Doley et al. [53] found that T. asperelloides decreased the prevalence of Sclerotium rolfsii pathogen in peanut plants. There are no other studies in the literature about using T. asperelloides to control R. solani in tomato plants besides this study.
Under controlled greenhouse conditions, the antifungal protective (T4) and curative (T5) treatments of Ta41 against Rs33 were evaluated in this study. Ta41 application significantly improved the tomato growth parameters, including shoot and root length, the fresh and dry weight of shoots and roots, and chlorophyll content, with a significant difference compared to the control (T1) and the infected tomato plants (T2). Our results showed that Rs33 could be inhibited by treating tomato plants with Ta41, either before or after the challenge with Rs33. These results agree with the enhancement of shoot and root growth, as reported previously [54,55]. The obtained data are also in agreement with those found by Yedidia and coworkers, who reported a stronger effect with treatment by T. harzianum on cucumber plants, which increased the root length by 75%, the shoot length by 95%, and the dry weight by 80% compared to the control plants [56]. The application of T. harzianum and T. asperellum, increased the chlorophyll content in melon-treated plants [57,58].
As an organic fertilizer, Trichoderma can degrade soil nutrients and enhance plant photosynthesis, resulting in improving plant growth; plants and several microorganisms can synthesize indole acetic acid (IAA) [59,60]. The indole acetic acid hormone is essential for root and shoot growth in plants and is considered a key regulator for root hair and lateral root development [46,61]. Studies have shown that Trichoderma spp. from various geographical areas can produce IAA and encourage plant growth such as cucumber, tomato, and bitter gourd [46,61]. Contreras-Cornejo et al. [62] revealed that the secretion of IAA by Trichoderma spp. can significantly improve plant and lateral root growth. Trichoderma spp. can produce volatile and non-volatile secondary metabolites including 6-n-pentyl-6H-pyran-2-one (6PP), viridin, gliotoxin, harziandione, harzianopyridone, and peptaibols [27,63], which, as plant-growth promoters, have a significant effect [8,55]. Secondary metabolites of the T22 and T39 commercial strains of T. harzianum and T. atroviride P1, as well as T. harzianum A6, also significantly affected plant-growth promoters [64].
Through invading the epidermis, Trichoderma spp. colonize plant roots that are usually associated with and triggering plant metabolism by changing gene expression [62,65]. In the current study, the impact of Ta41 on the relative expression levels of four defenserelated genes (PR-1, PR-2, PR-3, and CHS) at 20 dpi were evaluated. It was reported that, when the plant contacts with a pathogen, a mechanism of SAR is activated, while when it interacts with a non-pathogen organism, a mechanism for ISR was activated [66,67]. During the Trichoderma-plant interaction, various secondary metabolites induce PR proteins' expression that triggers plant defense mechanisms against the pathogen [68,69].
PR-1, the salicylic acid (SA) marker gene, is a crucial regulator of SAR and could be an indicator for early defense response in plants [70,71]. Meanwhile, the increasing resistance of plants is often associated with PR-1 induction and SA content accumulation [72,73]. In the present study, the tomato plants challenged with Rs33 only (T2) showed downregulation of PR-1, with a relative expression level 0.63-fold lower than the control (T1). Interestingly, the tomato plants inoculated with Ta41 only (T3) showed the highest expression level (2.94-fold higher), followed by those of the protective (T4) and curative (T5) treatments with relative transcriptional levels 2.39-and 1.46-fold higher than the control (T1). Consequently, we suggest that Ta41 may produce an elicitor metabolite molecule that induces the immune defense system resulting in SAR activation. The upregulation of PR1 could be related to SAR activation and the ISR status [74]. Thus, it could be deduced that the T. asperelloides Ta41 can modulate the response of the plant, increase resistance, and prevent the suppression of defense genes caused by R. solani Rs33.
PR-2 proteins have β-1,3-glucanase activity, are involved in pathogenic defense and different physiological plant functions, and are induced mainly by SAR and SAR inducers, such as SA [75][76][77]. In the current study, all Ta41-treated tomato plants (T3, T4, and T5) showed an upregulation of PR-2 compared with the control (T1). On the other hand, the decrease in the PR-2 expression of T2 plants (0.94-fold lower than the control) does not show a significant difference with respect to the control. The activity of PR-2 increased in plants treated with Ta41, which could be due to the elicitors (the oligosaccharides released) of the plant response and the fungal secondary metabolism [9]. This finding is consistent with other studies indicating that Trichoderma strain colonization causes an increase in PR-2 transcript in plants [22,78,79]. It has been documented that increasing 1,3-glucanase activity in the cell wall increased the amount of oligosaccharides released, acting as elicitors of plant defense responses and/or a secondary metabolism of the bioagents [9,78].
PR-3, which encodes a chitinase enzyme that catalyzes the hydrolysis of chitin, is a fungicide that protects plants from fungal infestations by inhibiting fungal growth [79,80]. The ability of each Trichoderma strain to inhibit R. solani growth may be due to differences in mycoparasitism activity through the secretion of enzymes, e.g., chitinase, that degrade the fungal cell wall [79]. In mycoparasitic action, chitinase is one of the most critical extracellular lytic enzymes [81]. In the present study, the tomato plants challenged with RS33 only or treated with Ta41 exhibited upregulation of the PR-3 gene. The highest relative expression (3.13-fold higher than the control) was shown in T3 plants, followed by that of the T4, T5, and T2 plants (2.63-, 2.32-and 1.43-fold higher, respectively) (T1). The obtained results show the role of PR-3 in increasing plant resistance against fungal infection. The colonization of roots with Trichoderma species promotes leaf tissue for the enhanced activation of many defensin genes, including PR-3, which results in higher resistance to pathogens [68,82].
The accumulation of phenolic compounds in exposure to Trichoderma species has been correlated with oxidative biochemical defense against pathogenic fungi. Yedidia et al. [83] reported that the roots invaded and conquered by T. harzianum showed a high defense against harmful organisms, and this was correlated with changes in the accumulation of phenolics. In the T4 treatment, the phenolic content was nearly four-fold higher than the content in the control, while in T3 or T5, it was 2.78 and 2.54-fold higher. Additionally, in the pathogen attack treatment (T2), the phenolic concentrations were lower than those found in all other treatments but still higher, by about two-fold, than those of the untreated plants (T1). Therefore, phenolics accumulating in Trichoderma-treated plants can serve as electron and hydrogen donors, preventing root tissue from oxidative damage throughout pathogen attacks [84]. Our TPC results are consistent with protection from R. solani phytopathogen in tomatoes. This was also found by Ortega-García et al. [85] in their studies on onions.
CHS, the initial enzyme of the pathway of flavonoids, converts the p-coumaroyl CoA to naringenin chalcones and is considered a strict precursor required for plant flavonoids [41,86]. Among the four tested genes, CHS exhibited the highest expression, with relative transcriptional levels in T3, T4, T5, and T2 that were 3.91-, 3.50-, 2.81-, and 1.52-fold higher than those of the control. In earlier studies, the overexpression of CHS was found to result in a high accumulation of flavonoid and isoflavonoid compounds that showed broad antifungal activity against a variety of fungal phytopathogens [87][88][89][90]. Consequently, the treatment of tomato plants with Ta41, in protective or in curative treatments, may increase the number of many flavonoid compounds. Thus, Ta41 could be useful as a biocontrol agent against R. solani infections. However, for future field applications, more research is required.

Conclusions
The current study aims to assess the in vitro biological control activities and in vivo activation of defense response of tomato by T. asperelloides either with or without R. solani, given the significance of implementing new Trichoderma species with much more efficient antimicrobial activities for agriculture applications. The application of T. asperelloides Ta41 was able to promote tomato plant growth and caused a significant increase in plant height, root length, and shoot fresh, shoot dry, root fresh, and root dry weight. It also improved chlorophyll content and total phenol content significantly, both in protective or in curative treatments. The protective treatment assay exhibited the lowest disease index. At 20 dpi, significant increases in the relative expression levels of four defense-related genes (PR-1, PR-2, PR-3, and CHS) were observed in all Ta41-treated plants compared to untreated plants.