The antagonistic activity of Trichoderma (T27, MH908510) was evaluated in vitro against H. maydis using the dual culture technique at 28°C ± 2°C (Pan and Bhagat, 2008). The pathogen inhibition percentage (IP) against mycelial growth was calculated according to the following formula (Hajieghrari et al., 2008):

where, C = radial growth in control (pathogen only), T = radial growth in treatment.

Next, the hyphal interaction in the contact area was examined and photographed using a light microscope equipped with a USB camera at a magnification power of 800× (OPTIKA C-B5 5.1 Megapixel CMOS USB 2.0 Camera, Microscope Company, USA).

The antifungal activity of the culture filtrate of T27 against H. maydis was tested in vitro as described by (Elshahawy and El-Sayed, 2018). Trichoderma T27 was grown for ten days at 28°C ± 2°C with agitation (150 rpm) in 100 mL Erlenmeyer flasks containing 50 mL of sterilized PDB. Next, the mycelial growth was removed by filtration using filter paper (Whatman filter paper #1, WHA1001090) and centrifuged at 5000 rpm with slight modifications. Subsequently, 5 mL of Trichoderma filtrate was added to 45 mL of PDB medium to make a final concentration of filtrate (10% v/v). This was performed in triplicate, and a negative control was prepared using 5 mL sterile distilled water instead of fungal filtrate (Rahman et al., 2023).

The amended flasks were then inoculated with a five-day-old (5 mm) disk of H. maydis and incubated at 28°C with shaking at 150 rpm for nine days. All flasks were weighed at zero time and after 3, 6, and 9 days of inoculation. The reduction of H. maydis growth was measured according to the following equation:

where, Wc = weight of control flask, Wt = weight of treatment flask at the same time as control.

The synergistic effect of culture filtrates of T27 and A. brasilense was also studied. Briefly, 5 mL of bacterial culture (A. brasilense) was transferred to 100 mL Erlenmeyer flasks containing 50 mL of Dobereiner’s broth (DB) medium and incubated at 28°C ± 2°C for four days. The microbial cells were then removed from each culture by filtration through filter paper (Whatman no. 1) and then centrifugation (centrifuge Tube GKF, China) at 5,000 rpm for 10 min to obtain cell-free filtrate for further experiments (Abdulkareem et al., 2014).

Subsequently, 5 mL of bacterial filtrate was mixed separately with 45 mL of PDB. In comparison, 2.5 mL of bacterial filtrate, 2.5 mL of the T27 filtrate, and 45 mL of PDB were prepared for a final concentration of 10% (v/v). Sterilized DB medium and distilled water served as the negative control. Next, control and treatment flasks were inoculated separately with 5 mm diameter mycelial discs of H. maydis and incubated at 28°C ± 2°C with shaking at 150 rpm. The weight of each flask was measured at zero time and after 3, 6, and 9 days of inoculation. The reduction in H. maydis growth was calculated as described in the equation above. All treatments and controls were performed in triplicate.

Mono- and dual cultures of T. viride T27 alone and Trichoderma in the presence of H. maydis C5 were tested to produce bioactive secondary metabolites. T. viride (T27) was grown separately or in combination on a PDB medium at optimum temperature for 20 days. Cultures were then filtered through filter papers (Whatman no. 1.), and equal volumes of filtrate and hexane (1:1 v/v) were mixed gently to extract the metabolites. Subsequently, hexane was evaporated using a rotary evaporator with a rotor speed of 120 rpm at 400 °C until the precipitate was formed. The precipitate was re-suspended in acetone for further characterization by GC/MS (GCMS-QP2010 Plus ultra), as previously described by (Bhardwaj and Kumar, 2017).

Ammonia (NH₃) and hydrogen cyanide (HCN) produced from T27 culture filtrate were estimated spectroscopically according to previously described methods. Briefly, a fresh culture of T27 was prepared in a test tube containing 10 mL of peptone water and incubated for five days at 28°C ± 2°C. After incubation, 1 mL of the culture was transferred to Eppendorf and combined with 50 μL of Nessler’s reagent, which was prepared by mixing 2 g KI in 5 mL of H₂O. Next, 3 g of HgI₂ was added, and the resulting solution was made up to 20 mL. Finally, 40 g of KOH (30%) was added to provide the alkaline base. A color change from a clear solution to a faint yellow indicated the presence of a small amount of ammonia, while a deep yellow or brown color was indicative of higher ammonia content. The color change was measured using a spectrophotometer (Sco. Tech, SP UV-19) at 450 nm. A standardized curve was generated by titrating ammonium sulfate from 0.1 – 5 μmol/mL (Cappuccino and Sherman, 1992; Reetha et al., 2014; Abdenaceur et al., 2022).

Next, 10 mL of T27 was inoculated in a 100 mL Erlenmeyer flask containing King’s B broth medium amended with 4.4 g/L glycine to detect HCN as previously described (El-Rahman et al., 2019). Non-inoculated flasks were used as negative controls, and all treatments and controls were performed in triplicate. Sterilized filter paper strips dipped in picrate solution (0.5% picric acid in 2% sodium carbonate) were attached to the neck of the flasks. Each flask was then plugged and sealed off with Parafilm and incubated with shaking at 140 rpm for four days at 28 °C ± 2°C. A change in the color of the filter paper strips from yellow to light brown, brown, or brick red was recorded as a weak (+), moderate (++), or strong (+++) reaction, respectively. A lack of color change was recorded as a negative (−) reaction. Moreover, the color intensity was detected and measured by spectrophotometry at 625 nm by dipping the filter paper strips into 10 mL of distilled water to elute the produced color. Likewise, qualitative and quantitative assessment of siderophores using chrome azurol S (CAS) reagent was also performed (Payne, 1994; Dutta et al., 2015).

For the qualitative evaluation, T27 was grown on CAS agar plates at 28°C for five days. Siderophore production was detected when the color of the medium changed to orange color. The CAS-shuttle assay was used for quantitative detection of siderophores. A total of 0.5 mL of supernatant was obtained from the filtration of broth cultures of the tested T27, which was mixed with an equal volume of CAS reagent. Siderophores were analyzed by spectrophotometry at 630 nm, where uninoculated broth medium was used as control, and the proportion of siderophore units was calculated as a percentage according to the following formula:

where, Ac = absorbance of the control, As = absorbance of the sample.

Chitinase (3.2.1.14) activity was qualitatively estimated using the Lukewarm agar medium, amended with bromocresol purple for colored zone formation. The constituents of the medium were as follows: MgSO₄.7H₂O, 0.3 g/L; (NH₄)₂SO₄, 3; KH₂PO₄, 2 g/L; citric acid monohydrate,1 g/L; agar, 15 g/L; colloidal chitin, 4.5 g/L; bromocresol purple, 0.15 g/L; and 200 μL Tween-80, pH 4.7 (Sigma-Aldrich, USA). Solidified medium plates were inoculated with T27, incubated at 28°C ± 2°C for seven days, and observed for colored zone formation (Agrawal and Kotasthane, 2012). Moreover, for quantitative assessment of chitinase, T27 was inoculated in Lukewarm broth medium without bromocresol purple and incubated in a shaker at 28°C with shaking at 150 rpm for five days. After the incubation period, the culture was centrifuged at 5000 rpm for 15 min, and chitinolytic activity was quantitatively assayed in culture filtrate by measuring the released reducing sugars from colloidal chitin. Briefly, 0.3 mL of 1 M sodium acetate buffer (pH 4.6) and 0.2 mL of colloidal chitin were transferred to a test tube containing 1 mL of culture filtrate, then incubated at 40°C for 20 h. After incubation, the mixture was centrifuged at 10,000 rpm for 5 min. Next, 0.75 mL of the tested mixture was combined with 0.25 mL of DNS solution (i.e., 1.0 g of 3,5 dinitro salicylic acid in 20 mL 2 M NaOH, to which 30 g of sodium potassium tartrate was slowly added before dilution to a final volume of 100 mL using distilled water) were mixed in test tubes and heated at 100°C for 5 min. After cooling, the absorbance was detected at 582 nm using a spectrophotometer (Sco. Tech, SP UV-19) (Miller, 1959). A standard N-acetyl-glucosamine (NAGA) curve was used to calculate chitinolytic activity using the concentration of released NAGA as the readout. Both amylase and cellulase activities were estimated in crude culture filtrate of T27. Starch broth and carboxy methyl cellulose media were used to estimate amylase and cellulase. The dinitrosalicylic acid method was applied for both enzymes to measure the released amounts of glucose by spectrophotometry at 575 nm using a glucose standard curve as previously described (Miller, 1959).

Indeed, most endophytic microbes, either fungi or bacteria, live in association with the roots of many plants. The ability of Trichoderma T27 and A. brasilense to produce indole acetic acid (IAA) and gibberellic acid (GA₃) in vitro was assessed using Salkowski’s and Folin–Ciocalteu (FC) reagents as previously described (Crozier et al., 1988; Pastrana et al., 1995; Perrig et al., 2007; Spaepen et al., 2008; Zhang et al., 2013; Nieto-Jacobo et al., 2017; Abdenaceur et al., 2022). T27 and A. brasilense cultures were grown for seven days on Czapek-dox broth and nutrient broth media supplemented with L-tryptophan (1 mg/L), respectively. After the incubation period, the microbial growth was removed by filtration. Next, 20 mL of each culture filtrate was centrifuged at 3000 rpm for 5 minutes. IAA production was then tested by adding 2 mL of the filtrate to 2 mL of Salkowski reagent (0.5 M ferric chloride (FeCl₃) and 35% perchloric acid (HClO₄)) and allowed to stand for 15 minutes. A color change to pink (measured at 535 nm) was indicative of a positive result. Additionally, for the GA₃ assay, 1 mL of microbial supernatant was combined and boiled with 1 mL of the reagent, 1 mL of concentrated HCl, and 3 mL dH₂O for 5 min in a water bath. Finally, after cooling to room temperature, the produced color change from green to blue was measured at 750 nm using a spectrophotometer (Sco. Tech, SP UV-19).

Mycoparasitism activity was studied as previously described (Bhat, 2017; Mukherjee et al., 2022) with minor modifications. Briefly, 15 mL of PDA was poured into a 90 mm petri dish and allowed to solidify. Then, 5 mm discs of T. viride (T27) and H. maydis C5 were inoculated at opposite points on the edges of PDA agar and then incubated for 5–7 days at 28°C, allowing the two fungi to grow toward each other. Upon interaction, a small portion was carefully separated without destroying the interacting mycelia and transferred to a clean glass slide. The interfering mycelia were then examined under a light microscope (800×) with a USB camera (OPTIKA C-B5 5.1 Megapixel CMOS USB 2.0 Camera, Microscope Company, USA).

The results of the in vitro studies on maize plants (Zea mays L.) were applied in the field during the growing season (Summer 2020). This experiment was carried out at the Faculty of Agriculture, Benha University, Egypt. The current study applied a randomized complete block design in triplicate. Each experimental plot (21 m²) was split into six rows. The in planta and field treatments were designed as follows: T1 as the uninfected control; T2 as the infected control by the pathogen H. maydis; T3: bioagent (T. viride T27 only) inoculated plants; T4: plants inoculated with a combination of (T27 + A. brasilense); T5: plants infected with a combination of (T27 + H. maydis); and T6: the last treatment which consisted of the inoculated plants with a combination of (T27 + A. brasilense + H. maydis). The grains of two yellow solitary hybrid cultivars of maize varied in their susceptibility to LWD and were purchased from Pioneer Company, Egypt (https://www.pioneer.com/landing), ( Supplementary Figure S1 ). The first hybrid (11N30) was registered as a tolerant cultivar, while the second (3062) was considered susceptible. This experiment evaluated the influence of T. viride T27 and A. brasilense on the plant growth criteria and incidence of LWD caused by H. maydis. The soil was clay loam comprised of 1.52% organic matter (pH 8.2).

Next, we tested antagonistic microorganisms following inoculation. Briefly, T. viride (27) was prepared on PDB medium at 28 °C ± 2°C with shaking (150 rpm) for seven days before it was homogenized and mixed with soil one week before cultivation (excluding the treatment of the control rows). Then, T. virdie homogenized culture containing 10⁶ Spore/ml was added at the rate of 100 ml/Jura (10 cm and depth 10 cm). Extra doses of T. viride suspension were added to the plants three times during the growing season at a rate of 50 mL/plant. Further, cell suspensions of A. brasilense MC12 were grown on DB medium and incubated at 28°C ± 2°C for four days. Then, maize seeds were soaked for 30 min in a mixture of the cell suspension (10⁸ colony forming unit (cfu) mL⁻¹) and 10% Arabic gum as an adhesive agent before cultivation. An excessive dose of A. brasilense inoculum was added to the soil rhizosphere near each plant three times during the growing season at a rate of 50 mL/plant.

Maize seeds were sown at a 20 cm distance between plants. After 21 days of emergence, plants were manually thinned to one plant/Jura. Plants were irrigated and chemically fertilized with a nitrogen (N; 140 kg N₂ as ammonium sulfate), phosphate (P; 200 kg P₂O₅ as calcium superphosphate), and potassium (K; 50 kg K₂O as potassium sulfate) mix as recommended by the Ministry of Agriculture and Land Reclamation of Egypt in two equal doses at vegetative and flowering stages. The dose of N was reduced to half in treatments with A. brasilense.

H. maydis was cultured on PDB at 28 °C ± 2°C with shaking (150 rpm) for seven days, then used to infect maize plants as previously described (Shekhar and Kumar, 2012) with some modifications. A sterilized thin syringe was filled with homogenized H. maydis inoculum under sterilized conditions. Subsequently, the outer surface of the plants (40 days old) was sterilized with 70% (v/v) ethyl alcohol, and the pathogen was injected into the second lower internodes above the soil level. In addition, the infected plants were apparently observed with late wilt symptoms at 20–25 days after infection. Each experimental plot (21 m²) was split into six rows; Maize seeds were sown at a 20 cm distance between plants. After 21 days of emergence, plants were manually thinned to one plant/Jura.

Disease assessment was performed periodically as disease incidence and severity after 60, 80, and 120 days of sowing (or after 20, 40, and 80 days of infestation). Disease incidence and severity were periodically recorded by examining the stems and leaves of 10 randomly selected infected plants after 60, 80, and 120 days of sowing (or after 20, 40, and 80 days of infection).

Ten plants of each replicate were randomly selected and labeled with all treatments to determine disease severity. A previously published scoring scale, which is divided into six grades as follows, was used for estimating LWD severity (El-Naggarr et al., 2015):

0: No symptoms on stalk and leaves.

1: Dark green longitudinal streaks appear with healthy-appearing leaves on the first basal internode.

2: Shrinking appears on the first internode; dark green longitudinal streaks extend to the second internode, and a slight yellowing occurs on the lower leaves.

3: Shrinking extends to the second and third internodes while a few lower leaves appear slightly dry.

4: Shrinking overcomes most internodes, twists the first internode, and dries most leaves.

5: All stalk internodes and leaves are dried, and the plant has died.

Mature maize plants were used to determine all growth parameters such as plant height (cm), number of leaves, fresh weight/plant (g), dry weight/plant (g), cone length (cm), cone diameter (cm), cone weight (g), number of rows/cones, number of grains/rows, weight of seeds/cone, weight of cones/15 plant (g) and weight of seeds/15 plant (g).

Eighty-day-old leaves were collected to extract and measure photosynthetic pigments (Nornal, 1982; Zhao et al., 2003). Different photosynthetic pigments (i.e., chlorophyll A, chlorophyll B, and carotenoids) in the leaf tissues were detected by extracting 1 g of leaf sample in 10 mL 80% (v/v) acetone for 2 mins before filtration through Whatman no. 1 filter paper, and the volume was then made up to 100 mL. Next, the optical density (OD) was measured at 660, 644, and 440 nm, and the quantity of each pigment was calculated as mg/L according to the equations:

After 80 days of cultivation, 2.0 g of fresh maize leaves were collected and grinded using a mortar and pestle in washed and dried sand with 4 mL of 0.1 M sodium phosphate buffer (pH 7) under sterilized conditions. The samples were then filtered and centrifuged at 3000 rpm for 20 mins, and the supernatant was taken to measure oxidative enzyme activity. All measurements and assays were performed in triplicate. Polyphenol oxidase (PPO, 1.10.3.1) activity was estimated (Lippolis et al., 2008). In a clean test tube, 0.2 mL of crude enzyme extract was mixed with 1 mL of 0.2 M sodium phosphate buffer (pH 7) and 1 mL of 1 mM catechol. Then, the final volume was adjusted to 6 mL with distilled water. The reaction mixture was incubated at 30°C for 30 min, and the absorbance was detected at 495 nm (Matta and Dimond, 1963). Catalase (CAT, 1.11.1.6) activity was evaluated according to published methods (Shim et al., 2003). In a reaction mixture of 0.5 mL 0.2 M sodium phosphate buffer (pH 7.6) and 0.3 mL of 0.5% (v/v) hydrogen peroxide (H₂O₂), the enzyme extract (0.4 mL) was added, and the final volume was completed to 3 mL with distilled water. The decrease in H₂O₂ absorbance at 240 nm was monitored, and CAT activity was calculated as µmol/min/g of fresh weight. Peroxidase (PO, 1.11.1.7) activity was measured by mixing 0.3 mL enzyme extract with a reaction mixture containing 0.5 mL 0.1 M potassium phosphate buffer (pH 7), 0.3 mL 0.05 M pyrogallol, 0.1 mL 1% (v/v) H₂O₂ and completed to 3 mL with distilled water. H₂SO₄ (5% (v/v)) was used to terminate the reaction after incubation for 15 min at 25°C. Subsequently, the change in absorbance at 470 nm was monitored, and PO activity was calculated as µmol/min/g of fresh weight (Allam and Hollis, 1972). Next, phenylalanine ammonia-lyase (PAL) activity was measured by combining 3.8 mL of sodium borate buffer (pH 8.8) with 1 mL of 0.33% (w/v) L-phenyl alanine and 0.2 mL of crude enzyme. After incubation at 40°C for 15 mins, the mixture was left to cool at room temperature, and the absorbance was measured at 290 nm (Cheng and Breen, 1991). Chitinase (Chit, 3.2.1.14) activity was measured as previously described (Mauch et al., 1988) by measuring absorbance at 540 nm and reported as mM N-acetyl glucose amine released/g of fresh weight/60 mins.

Comparative anatomical characteristics of the maize roots between treated and non-treated plants (control) were examined approximately 150 days after sowing (Johanson, 1940). Briefly, root cross-sections were taken from the primary roots and examined for histological changes. Maize primary roots were collected from each plant during the flowering stage and fixed in FAA (5 mL formalin, 5 mL glacial acetic acid, and 90 mL 70% (v/v) ethyl alcohol). Next, the samples were prepared for analysis with some modifications according to (Sass, 1951). Briefly, the samples were washed in 50% (v/v) ethyl alcohol, dehydrated in serial dilutions of ethyl alcohol (70, 90, 95, and 100% (v/v)), incubated in xylene, embedded in paraffin wax with a melting point of 60°C–63°C, sectioned to 12 μm sections, double-stained with fast green and safranin, cleared in xylene, and finally, mounted in Canada balsam. Next, root cross-sections were examined for histological changes. The prepared sections were examined, counted, and measured under a light microscope with an optical camera (magnification power is 100×).

The fungus, Trichoderma viride is one such biocontrol agent, mainly used for the control of various fungal pathogens. T. viride appears to be a bit granular on PDA, with green conidia distributed throughout. The cultures are typically fast growing at 28–30°C on PDA media ( Figures 1A, B ). A molecular phylogenetic tree based on rDNA internal transcribed spacers (ITS) identifies T. viride T27 (MH908510) by the maximum Likelihood Model of MEGA11.0 ( Figure 1C ). T. viride T27 (MH908510) was tested using a dual culture assay for its antagonistic activity against H. maydis in vitro ( Figure 2A ). The results of this assay indicated that T. viride grew faster than H. maydis and occupied the whole plate. ( Figure 2A ) showed the overgrowth of T27 upon the mycelia of H. maydis, where the inhibition percentage (IP) was 68.33%. Using microscopy, we also assessed the interaction area between two fungi for mycoparasitism activity. Results showed that T. viride approached H. maydis, began attaching to it, and ultimately penetrated its cell wall ( Figure 2B ). Subsequently, T. viride formed structures such as haustoria and appressorium that absorb nutrients from cells of the pathogenic fungus, which finally led to cell denaturation and lysis. Specifically, cell wall degrading enzymes accumulate inside the pathogenic fungal cell and cause progressive degradation of its cell wall until the fungal cell walls are entirely decomposed ( Figures 2C-G ). This may be due to the action of lytic enzymes such as chitinase and other secreted secondary metabolites by T27( Figure 3A ).

The inhibitory nature of secreted T. viride metabolites was tested, and cell-free filtrate of 7-day-old T. viride was examined to verify its activity against the innate growth of the phytopathogenic fungus, H. maydis, during three incubation periods (3, 6, and 9 days). Furthermore, the combination of T. viride and A. brasilense cell filtrates on H. maydis growth was also compared. Here, we found that the cell-free filtrate of T. viride T27 was more effective than A. brasilense in suppressing H. maydis growth during all incubation periods, with the exception of day 6. The reduction rate gradually increased from three days until it reached its maximum after nine days of incubation. In addition, the combination of the cell-free filtrates of A. brasilense and T. viride significantly suppressed the growth of H. maydis compared to the separate mono-treatments ( Figure 2H ).

The production of secondary antifungal metabolites was evaluated in a single culture of T. viride and its co-culture with H. maydis using GC/MS. The similarity of the metabolic profiles in both single and mixed cultures is shown in ( Table 1 ). Results indicated that 17 bioactive compounds were detected in the single culture of T27. Conversely, the interaction of H. maydis and T. viride stimulates the production of 10 new bioactive compounds compared to the individual culture of T27. These include cis-1,4-Cyclohexanediamine, N-methyl; 10-Undecen-1-al,2-methyl-; 7-Hexadecenal, (Z)-; 9,12-Octadecadienoyl chloride, (Z, Z)-; 2,5-Octadecadiynoic acid, methyl ester; Cyclobarbital (1); Cyclobarbital (5); Cyclobarbital (6), and Cyclobarbital (7). We also found that five compounds were inhibited in the mixed culture compared to the single culture, namely 1,2-15,16-Diepoxyhexadecane; Cyclobarbital (2); Cyclobarbital (3); Cyclobarbital (4), and Cyclobarbital (8). Otherwise, 12 compounds were detected in various proportions in single and mixed cultures. The analyzed metabolites included organic acids, aromatics, fatty acids, alcohols, esters, and hydrocarbons.

Various mechanisms were studied to understand the biological antifungal activity of T. viride strain T27 against H. maydis by producing certain volatile compounds such as hydrogen cyanide, ammonia, and siderophores. A color change to orange at the bottom of the T27 culture plates on CAS agar medium was considered an indicator of siderophores formation, where the concentration was 0.765% ( Supplementary Table S1 , Figure 3B ). Moreover, Ammonia was detected using a colorimetric assay (i.e., color change to yellow) and was quantified at 3.12 mg/L ( Supplementary Table S1 , Figure 3C ). Further, a color change to light brown in filter paper strips indicated moderate (++) HCN production, which was then quantified by measuring the OD (0.208; Supplementary Table S1 , Figure 3D ).

Besides the production of the bioactive compounds, three cell wall degrading enzymes (cellulase, chitinase, and amylase) were considered in this study. Qualitative analysis of chitinase activity by T. viride strain T27 was observed by conversion of Lukewarm agar medium to a violet color ( Figure 3A ). We observed superior production of chitinase rather than cellulase and amylase in T27 (2.206, 0.121, and 0.493 mg/mL, respectively; Supplementary Table S1 ). Conversely, the plant growth promotion activity of T27 was compared to that of A. brasilense through their ability to produce the plant growth hormones IAA and GA₃. Here, A. brasilense produced considerable amounts of GA₃ compared to T27, where concentrations were 46.9 and 30.8 mg/L, respectively ( Figures 3E, F ; Supplementary Table S1 ). In contrast, we found that T27 produced IAA in higher quantities than A. brasilense, where concentrations were 33.2 and 15.3 mg/L, respectively ( Figures 3G, H ; Supplementary Table S1 ). Taken together, we confirmed that T. viride produces all estimated antifungal compounds besides two growth promotors.

Both microbial application and LWD progression were evaluated via a field trial, which designed to assess the efficacy of the biocontrol agent, T. viride, and the bacterial strain, A. brasilense, in controlling the maize LWD. Generally, no disease symptoms were recorded in uninfected plants (T1, T3, T4).

However, the infection of maize plants with H. maydis resulted in disease symptoms at varying severities with or without any treatment. In contrast, uninfected control plants (+ve; T1) appeared healthy without any disease symptoms ( Figure 4A ), compared with the infected control plants (–ve; T2), which showed rapid wilting of the near ground leaves and gradually lost their color and appearance of yellow streaks on the leaves ( Figure 4B ) and the lower internodes ( Figures 4C–E ).

Characteristically, the disease symptoms first appeared in the lower part of the plant and then spread to the upper parts over time. Data in ( Figures 5A–C ) shows two types of disease progression in the form of disease severity (DS) and disease incidence (DI) in the treated hybrid maize plants. Plants treated with Trichoderma T27 and A. brasilense separately in T3 and T4 treatments appeared healthy and vigorous ( Figures 5A–C ). Additionally, promising results were observed in the T6 treatment, which showed a high suppressing effect on DS and DI, indicating the synergistic activity between Trichoderma T27 and A. brasilense under pathogen stress. Meanwhile, treatment of infected plants with only T27 in T4 treatment showed moderate suppression of DS and DI. DS and DI data were higher in the susceptible hybrid than in the resistant plants under the same treatments. The DS and DI increased throughout the tested time intervals till they reached their maximum after 120 days of infection in the susceptible hybrid ( Figures 5A–F ).

In conclusion, during the experimental trial, no symptoms were recorded in plants infected with Azospirillum and Trichoderma. Results in ( Figure 6 ) were obtained from the two-way ANOVA analysis for all maize plants, which assessed parameters following inoculation with H. maydis, treatment with T. viride and/or A. brasilense, and their combined interactions. These data indicated that H. maydis significantly influences the measured parameters of both hybrids of maize plants ( Figure 6 ). In addition, the weight of seeds per plant decreased by 42.8% and 32.5% for susceptible and resistant cultivars, respectively. Conversely, grain yield/plant in the treatments (T5 & T6) for the susceptible hybrid increased by 44.3% and 67%, respectively, compared to the control plants in the T2 treatment. In addition, the increment in grain yield/plant for the tolerant cultivar increased by 16.9% and 55.9% for the same treatments ( Figure 6D ). Moreover, this affects the cone criteria (i.e., length, weight, diameter, number of rows/cones, and number of grains/rows), of which the lowest values were recorded in the combined treatments ( Figure 6E , Figure 7 ). Notably, vegetative and cone parameters were highest in T4 plants (i.e., plants treated with T27 and A. brasilense without H. maydis).

In conclusion, inoculated maize plants with Trichoderma alone reduced both DI and DS compared to plants without inoculation.

Regarding the indices of plant development, plant height, the number of leaves, shoot fresh weight, and shoot dry weight were recorded. Interestingly, all treatments recorded higher values than plants infected with H. maydis only ( Figure 6 ). This trend was confirmed in both susceptible and resistant hybrids. As a result of maize plant infestation with H. maydis, all cone traits (length, weight, diameter, number of rows/cones, number of grains/row, and seeds weight/cone (g)) were significantly affected and gave lower values in both susceptible and resistant hybrids compared to uninfected plants ( Figures 7A–D ). In addition, the weight of seeds per plant decreased by 42.8% and 32.5% for susceptible and resistant cultivars, respectively. On the contrary, the inoculation of the LWD infected-susceptible hybrid with T. viride only (T5) or with A. brasilense (T6) caused an increase in weight of seeds per cone compared to the LWD-infected plants (T2) by 44.3% and 67%, respectively. Moreover, the weight of seeds per cone of tolerant cultivar increased by 16.9% and 55.9% in inoculated plants with T. viride only (T5) or with A. brasilense (T6), respectively, compared to plants infected with H. maydis only (T2) ( Figure 6D ).

A significant difference was observed among hybrids regarding photosynthetic pigments for the foliar pigments index. The photosynthetic pigment content in maize leaves was detected in all treatments, where chlorophyll values were higher in the resistant hybrid, while chlorophyll b and carotenoid contents were higher in the susceptible hybrid ( Figures 8A–C ). Moreover, a significant decrease in chlorophyll b and carotenoids was recorded in the infected plant with H. maydis (T2). This trend was the opposite for chlorophyll a, which was higher in infected fungal plants compared to the control. The chlorophyll content was recorded at higher values in infected control plants than in uninfected control plants. However, we observed a significant increase in chlorophyll b and carotenoid content in T4 plants for both hybrids. These data suggest that the inoculation of maize with a mixture of A. brasilense and T. viride in the absence of H. maydis caused a significant increase in chlorophyll b and carotenoid content compared to control plants in both hybrids ( Figure 8D ). Additionally, results showed that carotenoids increased in maize leaves inoculated with Trichoderma and Azospirillum in the presence or absence of H. maydis but decreased in maize plants infected with H. maydis only ( Figure 8C ).

Regarding oxidative enzymes, we showed that chitinase and CAT activities were significantly higher in control plants infected with H. maydis ( Supplementary Table S2 ). However, no significant differences were observed in PO activity among various treatments. PPO activity was higher in T4 and T6 plants than in T5 plants. These results showed the impact of Azospirillum treatment on pathogen suppression compared with the Trichoderma alone and control with or without the pathogen. Furthermore, PPO and POD activity was higher in resistant hybrids than in susceptible plants.

Infection of maize roots with H. mayids decreased whole root thickness by 40.9% and 34.5% compared to control plants in susceptible and resistant hybrids, respectively. Notably, the combination of T. viride and A. brasilense enhanced the root thickness in the presence or absence of H. mayidism. We also noted that the diameter of the phloem and pith and the endodermis thickness was thinner in H. mayids-infected roots than in other treatments, and the same trend was observed in both hybrids. Moreover, we also found that susceptible hybrids increased the number and thickness of their xylem vessels compared to resistant hybrids. The highest degradation area of the root cortex was observed in H. maydis-infected plants in both hybrids. We also noted changes in the cortex area with plant response to fungal infection. Lateral root (LR) development was also observed in plants infected with T. viride only or H. maydis in a suspectable hybrid. Conversely, the recorded number and thickness of aerenchyma (i.e., air pockets forming in the cortex cell layers that may aid in gas exchange) was lower in H. maydis-infected plants ( Supplementary Table S3 ; Figure 9 ). Taken together, we consider T. viride to be a potentially promising approach as a safe and effective natural fungicide compared to other chemical fungicides due to its potent suppression of LWD in maize.