Application of Bio-Friendly Formulations of Chitinase-Producing Streptomyces cellulosae Actino 48 for Controlling Peanut Soil-Borne Diseases Caused by Sclerotium rolfsii

Of ten actinobacterial isolates, Streptomyces cellulosae Actino 48 exhibited the strongest suppression of Sclerotium rolfsii mycelium growth and the highest chitinase enzyme production (49.2 U L−1 min−1). The interaction between Actino 48 and S. rolfsii was studied by scanning electron microscope (SEM), which revealed many abnormalities, malformations, and injuries of the hypha, with large loss of S. rolfsii mycelia density and mass. Three talc-based formulations with culture broth, cell-free supernatant, and cell pellet suspension of chitinase-producing Actino 48 were characterized using SEM, Fourier transform infrared spectroscopy (FTIR), and a particle size analyzer. All formulations were evaluated as biocontrol agents for reducing damping-off, root rot, and pods rot diseases of peanut caused by S. rolfsii under greenhouse and open-field conditions. The talc-based culture broth formulation was the most effective soil treatment, which decreased the percentage of peanut diseases under greenhouse and open-field conditions during two successive seasons. The culture broth formulation showed the highest increase in the dry weight of peanut shoots, root systems, and yielded pods. The transcriptional levels of three defense-related genes (PR-1, PR-3, and POD) were elevated in the culture broth formulation treatment compared with other formulations. Subsequently, the bio-friendly talc-based culture broth formulation of chitinase-producing Actino 48 could potentially be used as a biocontrol agent for controlling peanut soil-borne diseases caused by S. rolfsii.


Introduction
Peanut, or groundnut (Arachis hypogaea L.), is one of the most important oilseed crops in the world, including Egypt. Peanut is susceptible to diseases caused by abundant soilborne pathogens. One of the most important soil-borne fungal diseases of peanut is stem, root, and pods rot caused by Sclerotium rolfsii (teleomorph Athelia rolfsii (Curzi) C.C. Tu & Kimbr.), which has the ability to infect more than 500 plant species [1]. Circular and light-tan to brown clusters of seed-like bodies less than 1/10th of an inch in diameter called sclerotia form on the mat of fungal growth on the soil surface, decaying stems and pods

Antagonistic Effect of Actinobacterial Isolates against S. rolfsii
Estimation of S. rolfsii biomass development in the presence of actinobacterial isolates, to determine their antagonistic effect, was performed according to Trivedi et al. [26] with slight modifications. We added 1 mL of the 5-day-old pre-culture of actinobacterial isolates into 50 mL potato dextrose broth (PDB) containing S. rolfsii plug (6 mm diameter) from freshly grown culture on potato dextrose agar (PDA). The media were incubated at 30 • C for seven days. Another flask containing PDB with only the fungal plug was used as a control. The contents of the flasks were filtered through pre-weighed Whatman No. 1 filter paper and allowed to dry at 50 • C. The percentage of weight reduction of the tested fungus was calculated using the formula: (W1 − W2)/W1 × 100, where W1 represents the weight (g) of the tested fungus in a control flask and W2 is the weight of the fungus in the presence of antagonistic bacteria (g).

Qualitative and Quantitative Evaluation of Chitinase Production from S. cellulosae Actino 48 2.3.1. Detection of Chitinase Production
The promising actinobacterial isolate Actino 48 identified as S. cellulosae, which produced a higher inhibition percentage against S. rolfsii than other isolates, was streaked onto a colloidal chitin agar plate and incubated at 30 • C for 10 days. The formation of a halo zone surrounding the colony indicated a positive result for chitinase production.

Chitinase Assay
For the chitinase assay, a fresh culture of S. cellulosae isolate Actino 48 (10 7 colony forming units (CFU) mL −1 ) was grown in minimal liquid medium (MLM, containing (g/L) MgSO 4 .7H 2 O, 0.2; K 2 HPO 4 , 0.9; KCl, 0.2; NH 4 NO 3 , 1.0; FeSO 4 .7H 2 O, 0.002; MnSO 4 , 0.002; ZnSO 4 , 0.002; pH 6.8), supplemented with colloidal chitin (1% w:v) (LOBA Chemie PVT. LTD., Maharashtra, India) and incubated for 8 days at 30 • C in flasks. Samples were used for the colorimetric estimation of chitinase every day using the method of Boller and Mauch [27]. We incubated 1 mL of cell-free supernatant with 1 mL of colloidal chitin (1% v:v) in a citrate phosphate buffer (0.1 M pH 6.5) at 40 • C for 2 h in a shaking water bath. The reaction was stopped by adding 2 mL 3,5-dinitrosalicylic acid (DNS) reagent and kept in a boiling water bath for 5 min to develop the color. The tubes were cooled, centrifuged at 5000× g for 10 min, and absorbance was measured at optical density (OD) 575 nm against the blank prepared with 0.1 M citrate phosphate buffer and 0.45% colloidal chitin without enzyme. One unit of chitinase is defined as the amount of enzyme that releases 1 µmol of N-acetylglucosamine per minute under the reaction condition.

Detection of Interaction between S. cellulosae Actino 48 and S. rolfsii
Scanning electron microscope (SEM, JEOL JSM-6360LA, Tokyo, Japan) was used to detect and analyze the inhibition interaction between S. rolfsii and S. cellulosae Actino 48. A dual-culture agar plate assay was used to detect the previous interaction.

Formulation of Culture Broth, Cell-Free Supernatant, and Cell Pellet Suspension of Chitinase-Producing S. cellulosae Actino 48
Culture broth, cell-free supernatant, and cell pellet suspension of the antagonistic chitinase-producing S. cellulosae isolate Actino 48, which showed a higher inhibition percentage against S. rolfsii than other actinobacterial isolates, were used for the preparation of a bioformulation to reduce peanut soil-borne diseases. Talc powder (TP) was used as a carrier for the preparation of biofriendly formulations. We added 10 g of colloidal chitin as a carbon source and an adhesive agent to 400 mL of culture broth (including 10 7 CFU mL −1 of S. cellulosae Actino 48), cell-free supernatant, and cell pellet suspension. The broth, supernatant, and pellet including additives were mixed homogeneously in a vortex mixer. We adjusted the pH of the formulations to 7.0 by adding 15 g of calcium carbonate to 1 kg of sterilized talc powder (TP) and combined well. We mixed 400 mL of culture broth, cell-free supernatant, and cell pellet suspension with additives with 1 kg of talc powder. The humidity content of bioformulations was decreased to less than 20% by drying, and the bioformulations were stored at 4 • C until use [28]. The morphological features and microstructure of all talc-based formulations of S. cellulosae isolate Actino 48 were examined using SEM (JEOL JSM-6360LA, Tokyo, Japan). The sample was operated at an acceleration voltage of 10 KV. Magnification power varied from 300 to 5000×.

Fourier Transform Infrared (FTIR) Spectroscopy
The surface functional groups with binding sites and the structure of the materials used in talc formulations were studied by Fourier transform infrared spectroscopy (FTIR) (Shimadzu FTIR-8400 S, Kyoto, Japan).

Particle Size Analysis
A particle size analyzer (PSA; Mod.: N5, Beckman Coulter, Brea, CA, USA) was used to detect the size of particles of talc-based formulations of S. cellulosae isolate Actino 48. Sorghum, coarse sand, and water (2:1:2 v/v) medium was prepared for inoculation of S. rolfsii. After sterilization, the medium was inoculated using agar discs, obtained from the margin of a 4-day-old colony of the tested fungus. The inoculated media were incubated at 28 • C for 2 weeks and then used for soil infestation [29].

Soil Infestation
Inoculum of S. rolfsii was added to the soil surface of each pot at the rate of 2% w/w and was covered with a thin layer of sterilized soil. The infested pots were irrigated and kept for 14 days before sowing.

Application Dose of Bio-Friendly Formulations and Recommended Fungicide
Rizolex-T 50% Wettable Powder (WP) Seeds of peanut (Giza 6 cv.) were treated with talc-based culture, supernatant, and pellet formulations of chitinase-producing S. cellulosae Actino 48 as a seed dressing at a rate of 10 g kg −1 of seeds or Rizolex-T 50% WP at a ratio of 3 g kg −1 of seeds. Formulations were applied again at a rate of 3 kg acre −1 two times, 30 and 50 days after seed sowing, as a soil drench.

Open-Field Experiment
During the 2018 and 2019 growing seasons, the field experiments were conducted at El-Nobaria, El-Behaira Governorate, Egypt, to study the effect of the talc-based bioformulations of S. cellulosae Actino 48 in controlling damping-off, root, and pod rot diseases. The fields had a heavy natural infestation with phytopathogenic fungus S. rolfsii. Peanut seeds (Giza 6 cv.) were sown in the first week of May through the two evaluated seasons with 10 cm spacing between rows. Talc-based formulations of culture broth, cell-free supernatant, and cell pellet suspension of S. cellulosae Actino 48 and the fungicide Rhizolex-T 50% WP were applied as previously mentioned. Cultural practices and fertilization for the peanut crop were performed as recommended. The experimental unit area was 10.5 m 2 (1/400 acre). The treatments were applied using a randomized block design with four replicates. Diseases assessments, peanut yield, and dry weight of shoot and root systems were recorded as mentioned before.

Disease Evaluation
Disease was evaluated according to Hussien et al. [29]. The percentage of damping-off (pre-and post-emergence) was estimated 15  Total RNA was extracted from peanut leaves (0.1 g, fresh weight) collected at 72 and 96 h post-inoculation (hpi) with the talc-based culture, supernatant, and pellet formulations of S. cellulosae Actino 48 using the guanidium isothiocyanate (GITC) extraction method with some modifications [30]. The purity and concentration of extracted RNA were determined using SPECTROstar Nano (BMG Labtech, Ortenberg, Germany), while the integrity was assessed using agarose gel electrophoresis. One microgram of DNase-treated total RNA was used to synthesize cDNA in a reverse-transcription reaction as described previously [31]. The RT-PCR reaction mixture was stored at -20 • C until use.

qRT-PCR Assay and Data Analysis
The transcriptional levels of three peanut defense-related genes (peroxidase (POD), pathogenesis-related protein-1 (PR-1), and chitinase (PR-3)) in all treatments were evaluated using qRT-PCR at 72 and 96 h post-inoculation with the talc-based culture, supernatant, and pellet formulations of S. cellulosae Actino 48 ( Table 1). The β-actin gene ( Table 1) was used as a reference gene to normalize the transcript expression levels. Each biological sample was run in triplicate reactions on a Rotor-Gene 6000 (QIAGEN, ABI System, Valencia, CA, USA) using SYBR Green PCR Master Mix (Thermo, Waltham, MA, USA) as previously described [32]. The relative expression level of each tested gene was truthfully calculated according to Reference [33].

Statistical Analysis
The relative expression levels were analyzed by one-way analysis of variance (ANOVA) using the CoStat software, and the significant differences were determined according to the least significant difference (LSD). p ≤ 0.05 level of probability and standard deviation (±SD) are shown as a column bar. Compared to the healthy control, the relative expression levels higher than 1 demonstrated an increase in gene expression (upregulation), whereas values lower than 1 indicated a decrease in expression levels (downregulation).

Antagonistic Effect of Actinobacterial Isolates against S. rolfsii
Ten actinobacterial isolates were tested as potential biological control agents for their antagonistic effect on the in vitro growth of S. rolfsii. The data obtained in the current study revealed significant differences between actinobacterial isolates. Isolate Actino 48 was more effective in inhibiting the fungal mycelia growth of S. rolfsii than other isolates and had the highest inhibition percentage against the pathogen, which reached 98.7%, followed by actinobacterial isolate Actino 32, which reached 95% (Figure 1).

Qualitative and Quantitative of Chitinase Production from S. cellulosae Actino 48
The ability of the actinobacterial isolate Actino 48, identified as S. cellulosae, to produce the chitinase enzyme was tested qualitatively on a chitin agar plate. S. cellulosae Actino 48 formed a large halo zone surrounding the colony, which indicated the chitinase production and chitin degradation abilities. The Actino 48 isolate that showed the highest inhibition percentage against S. rolfsii was cultured for chitinase production. The maximum chitinase activity was observed at seven days of cultivation (49.2 U L −1 min −1 ). After that, enzyme activity decreased to reach 47 U L −1 min −1 at eight days ( Figure 2).

Antagonistic Effect of Actinobacterial Isolates against S. rolfsii
Ten actinobacterial isolates were tested as potential biological control agents for their antagonistic effect on the in vitro growth of S. rolfsii. The data obtained in the current study revealed significant differences between actinobacterial isolates. Isolate Actino 48 was more effective in inhibiting the fungal mycelia growth of S. rolfsii than other isolates and had the highest inhibition percentage against the pathogen, which reached 98.7%, followed by actinobacterial isolate Actino 32, which reached 95% ( Figure 1).

Qualitative and Quantitative of Chitinase Production from S. cellulosae Actino 48
The ability of the actinobacterial isolate Actino 48, identified as S. cellulosae, to produce the chitinase enzyme was tested qualitatively on a chitin agar plate. S. cellulosae Actino 48 formed a large halo zone surrounding the colony, which indicated the chitinase production and chitin degradation abilities. The Actino 48 isolate that showed the highest inhibition percentage against S. rolfsii was cultured for chitinase production. The maximum chitinase activity was observed at seven days of cultivation (49.2 U L −1 min −1 ). After that, enzyme activity decreased to reach 47 U L −1 min −1 at eight days ( Figure 2).

Detection of Interaction between Actinobacteria and S. rolfsii
Scanning electron microscope (SEM) micrographs of the interaction between S. rolfsii and chitinase-producing S. cellulosae Actino 48, which showed a higher inhibition percentage against S. rolfsii than other isolates, showed abnormal, malformed, and injured fungal hypha of S. rolfsii, and large losses in the density and mass of the mycelia ( Figure 3).

Detection of Interaction between Actinobacteria and S. rolfsii
Scanning electron microscope (SEM) micrographs of the interaction between S. rolfsii and chitinase-producing S. cellulosae Actino 48, which showed a higher inhibition percentage against S. rolfsii than other isolates, showed abnormal, malformed, and injured fungal hypha of S. rolfsii, and large losses in the density and mass of the mycelia ( Figure 3).

SEM
We used SEM for the morphological analysis of talc-based culture broth, cell-free supernatant, and pellet formulations of S. cellulosae Actino 48 under different magnifications. Figure 4 illustrates talc formulation at 5000×: it has both small and big particle aggregates with sharp edges. The spores of S. cellulosae Actino 48 are shown on talc particles in the culture broth and pellet formulations.  Figure 4 illustrates talc formulation at 5000×: it has both small and big particle aggregates with sharp edges. The spores of S. cellulosae Actino 48 are shown on talc particles in the culture broth and pellet formulations.

FTIR Spectroscopy
The vibrations in the bands of the FTIR spectrum for talc powder and talc-based culture, supernatant, and pellet formulations of S. cellulosae Actino 48 are shown in Figure

Particle Size Analysis
The particles size of talc powder was 958 nm, whereas those of talc-based culture, supernatant, and pellet formulations of S. cellulosae Actino 48 were 1070, 796, and 754 nm, respectively ( Figure 6). The data demonstrated in Figure 7 and confirmed in Table 2 show that treatments using talc-based culture and cell-free supernatant formulations of Actino 48 in a soil infected with S. rolfsii more significantly reduced damping-off percentage, which decreased to 15% and 17.5% respectively, and Rizolex-T reduced damping-off percentage to 12.5% with no significant differences between treatments. Damping-off percentage caused by S. rolfsii (infected control) was 27.5%. Root rot percentage caused by S. rolfsii was 32.5%, whereas treatments using talc-based culture and cell-free supernatant formulations effectively reduced root rot percentage, to 12.5% and 20% respectively, with no significant differences between them. Rizolex-T reduced root rot percentage to 10%. The healthy survival percentage of peanut plants for treatments using the talc-based culture formulation of S. cellulosae Actino 48 and Rizolex-T (standard) in a soil infected with S. rolfsii increased to 72.5% and 77.5% respectively, and 40% with S. rolfsii alone. The pellet formulation was less effective than culture and cell-free supernatant formulations in its ability to reduce the incidence of peanut damping-off and root rot diseases.
Mostly all the formulations stimulated the growth of peanut plants whether in uninfected soil or soil infected with S. rolfsii (Figure 7). Dry weights of shoot and root systems increased significantly in treatments with bioformulations of Actino 48. The talc-based culture formulation more significantly increased the dry weight of shoot and root systems of the peanut in uninfected soil or soil infected with S. rolfsii than other treatments. The dry weights of the shoot system of peanut plants were 41.56 and 29.70 g, and 4.59 and 3.01 g for the root system of culture formulation in uninfected soil or soil infected with S. rolfsii, respectively. Treatment with S. rolfsii alone decreased the dry weights of the shoot and root system to 8.31 and 1.48 g, respectively (Table 3). We found no difference between treatment with the talc-based culture formulation of Actino 48 in soil infected with S. rolfsii and Rizolex-T treatment on increasing the dry weight of the shoot system of peanut plants, but variations were observed in the root systems (Table 3).    * All data are averages of four measurements (replicates) ± standard deviation (SD). ** Means in each column followed by the same letter do not differ significantly (p ≤ 0.05). *** Significant letters.   Treatments with talc-based culture, cell-free supernatant, and pellet formulations of Actino 48 in soil infested with S. rolfsii effectively decreased the percentage of infected peanut pods, which showed no significant differences between them (14.08%, 16.97%, and 16.50%, respectively). The percentage of infected peanut pods in the treatment with S. rolfsii alone was 56.35% ( Figure 8). The same treatments resulted in a high percentage of apparently healthy pods (85.92%, 83.03%, and 83.50%, respectively) compared to the infected treatment, for which we recorded 43.65% healthy pods (Table 3). Treatment with Rizolex-T was highly effective in decreasing the percentage of infected peanut pods, which was 9.13%, for a high percentage of apparently healthy pods of 90.87%.  * All data are averages of four measurements (replicates) ± standard deviation (SD). ** Means in each column followed by the same letter do not differ significantly (p ≤ 0.05). *** Significant letters.  Table 4 shows the effect of treatment by talc-based culture, cell-free supernatant, and pellet formulations of Actino 48 on peanut yield under greenhouse conditions. Treatments with talc-based culture formulation and Rizolex-T in a soil infested with S. rolfsii gave high dry weight of healthy pods (11.62 and 10.63 g pot −1 , respectively) with yield increases  Table 4 shows the effect of treatment by talc-based culture, cell-free supernatant, and pellet formulations of Actino 48 on peanut yield under greenhouse conditions. Treatments with talc-based culture formulation and Rizolex-T in a soil infested with S. rolfsii gave high dry weight of healthy pods (11.62 and 10.63 g pot −1 , respectively) with yield increases of 22.57% and 12.13%, respectively. On the other hand, treatment with S. rolfsii alone decreased the dry weight of healthy pods to reach 4.59 g pot −1 (Figure 8). * All data are averages of four measurements (replicates) ± standard deviation (SD). ** Means in each column followed by the same letter do not differ significantly (p ≤ 0.05). *** Significant letters.

Open-Field Experiment
As attained in greenhouse experiments, talc-based culture broth, cell-free supernatant, and pellet bioformulations of chitinase-producing S. cellulosae Actino 48 were estimated to reduce peanut soil-borne diseases caused by S. rolfsii compared to the standard fungicide (Rizolex-T) under open-field conditions during two successive seasons, 2018 and 2019. Data presented in Table 5 showed that there were significant effects of all treatments in reducing peanut soil-borne diseases caused by S. rolfsii during the two tested seasons compared to untreated infected control. In general, Rizolex-T was a more effective treatment in reducing damping-off and root rot diseases caused by S. rolfsii in the two tested seasons, which reached 8.04% and 7.4% in the first season and 8.26% and 8.7% in the second one respectively, compared to the control (20.22% and 21.52%, and 22.18% and 21.31%). Treatment with talc-based culture broth formulation of chitinase-producing Actino 48 was the most effective one from all bioformulations to reduce the percentage incidence of the two tested diseases, which reached 12.61% and 11.74% in the first season and 13.48% and 10.87% in the second one, respectively. No significant differences, in reducing the percentage incidence of the two tested diseases, showed between treatments of talc-based culture broth and cell-free supernatant in the first season but they showed in the second one. On the other hand, a significant difference was demonstrated between treatments, talc-based culture broth and pellet bioformulations, in the first season but they did not demonstrate to reduce the percentage incidence of the two tested diseases in the second season.
Under open-field conditions and through the first season, 2018, talc-based culture formulation was more effective in increasing the dry weight of shoot and root systems of peanut plants grown in infected soil with S. rolfsii than other treatments, with dry weight of shoot system reaching 385.01 g and root system reaching 41.65 g for experimental unit followed by Rizolex-T (380.20 g and 40.92 g), without any significant difference compared to untreated and infected control (095.81 g and 19.27 g), as mentioned in Table 6. Moreover, no significant differences were found between treatments, talc-based culture broth biofor-mulation and Rizolex-T, in the second season, 2019, on dry weight of peanut shoot and root systems grown in infested soil with S. rolfsii. In addition, no significant differences were found between treatments, cell-free supernatant and pellet bioformulations, in the same season, 2019, but they were less effective than talc-based culture broth bioformulation and Rizolex-T on dry weight of peanut shoot and root systems ( Table 6). * All data are averages of four measurements (replicates) ± standard deviation (SD). ** Means in each column followed by the same letter do not differ significantly (p ≤ 0.05). *** Significant letters. As demonstrated in Table 7, competence of talc-based culture broth, cell-free supernatant, and pellet bioformulations of chitinase-producing Actino 48 and Rizolex-T were verified to reduce peanut pods rot incidence (%) under open-field conditions during the two seasons, 2018 and 2019. Rizolex-T and talc-based culture broth gave high percentages of healthy pods (94.26%, 90.93%, and 94.69%, 92.76%) and low percentages of infected pods (5.74%, 9.07%, and 5.31%, 7.24%) in the two seasons respectively, compared to the untreated and infected control. Moreover, no significant differences were found between treatments, cell-free supernatant, and pellet bioformulations in the two tested seasons, 2018 and 2019, but they were less effective than talc-based culture broth bioformulation and Rizolex-T on percentage of infected peanut pods. * All data are averages of four measurements (replicates) ± standard deviation (SD). ** Means in each column followed by the same letter do not differ significantly (p ≤ 0.05). *** Significant letters. Table 8   Compared to the control, the relative expression levels of POD were differentially expressed at 72 and 92 h in different treatments (Figure 9). The POD transcripts of plants infected with S. rolfsii were significantly downregulated with relative expression levels 0.356-and 0.473-fold change lower than the control at 72 and 96 h, respectively (Figure 9). Like S. rolfsii treatment, the Rizolex-T + S. rolfsii treatment exhibited relative expression levels 0.314-and 0.460-fold change lower than control at 72 hand 96 h, respectively.

Effects on the Transcriptional Level of Peroxidase (POD)
Compared to the control, the relative expression levels of POD were differentially expressed at 72 and 92 h in different treatments (Figure 9). The POD transcripts of plants infected with S. rolfsii were significantly downregulated with relative expression levels 0.356-and 0.473-fold change lower than the control at 72 and 96 h, respectively (Figure 9). Like S. rolfsii treatment, the Rizolex-T + S. rolfsii treatment exhibited relative expression levels 0.314-and 0.460-fold change lower than control at 72 hand 96 h, respectively. Compared to the healthy control, the relative expression levels higher than 1 demonstrate an increase in gene expression (upregulation), while values lower than 1 indicate a decrease in expression levels (downregulation).
Significant upregulation of the POD transcript was observed in plants treated with culture formulation, pellet formulation, and CU-F + S. rolfsii at the two-time intervals (Figure 9). The highest expression level was observed in the pellet and culture formulation treatments, with no significant differences, followed by CU-F + S. rolfsii with relative expression levels 4.119-, 3.784-, and 2.969-fold change increased compared to the control at Compared to the healthy control, the relative expression levels higher than 1 demonstrate an increase in gene expression (upregulation), while values lower than 1 indicate a decrease in expression levels (downregulation).
Significant upregulation of the POD transcript was observed in plants treated with culture formulation, pellet formulation, and CU-F + S. rolfsii at the two-time intervals (Figure 9). The highest expression level was observed in the pellet and culture formulation treatments, with no significant differences, followed by CU-F + S. rolfsii with relative expression levels 4.119-, 3.784-, and 2.969-fold change increased compared to the control at 72 h, respectively. At 96 h, the highest level (5.329-fold) was demonstrated by culture formulation treatment, followed by the CU-F + S. rolfsii treatment with a relative expression level 2.329-fold higher than the control (Figure 9).

Effects on Transcriptional Level of Pathogenesis-Related Protein 1 (PR-1)
Compared with the control at 72 h, the relative expression level of PR-1 was induced in different treatments (Figure 10). The highest transcriptional level (71.671-fold) was shown in the CU-F + S. rolfsii treatment, followed by PE-F + S. rolfsii, which achieved a 17.959-fold higher change than S. rolfsii treatment, which showed 9.713-fold ( Figure 10). Although culture and supernatant formulation treatments showed slight increases of 1.794-and 1.155-fold change respectively, no significant changes were found compared with the control. At 96 h, the transcriptional levels of PR-1 dramatically decreased in all treatments except for the PE-F + S. rolfsii treatment, in which levels increased and exhibited the highest expression level (37.014-fold) compared with the control (Figure 10). The transcript of PR-1 of S. rolfsii-treated plants was significantly downregulated with a relative expression level of 0.567-fold lower than the control ( Figure 10). PR-1 transcripts with relative expression levels were 18.895-, 12.125-, and 5.464-fold higher than the control in CU-F + S. rolfsii, pellet formulation, and Rizolex-T + S. rolfsii at 96 h, respectively ( Figure 10).

Effects on Transcriptional Level of Chitinase (PR-3)
Similar to POD, peanut plants in both S. rolfsii and Rizolex-T + S. rolfsii treatments showed downregulation of PR-3 with relative expression levels of 0.414-and 0.406-fold change lower at 72 h respectively, and 0.229-and 0.376-fold change lower than the control at 96 h, respectively (Figure 11). At 72 h, the highest transcriptional level of PR-3 (5.187fold) was exhibited in CU-F + S. rolfsii-treated plants, whereas at 96 h, the supernatant formulation treatment showed the higher expression level, 11.236-fold change higher than healthy control, followed by the CU-F + S. rolfsii treatment, which was 6.727-fold higher ( Figure 11). The moderate induction of PR-3 was observed in the culture formulation, SU-F + S. rolfsii, and PE-F + S. rolfsii at 72 and 96 h with relative expression levels of 1.390-, 2.211-, and 3.149-fold, and 1.263-, 1.357-, and 1.636-fold, respectively ( Figure 11).

Effects on Transcriptional Level of Chitinase (PR-3)
Similar to POD, peanut plants in both S. rolfsii and Rizolex-T + S. rolfsii treatments showed downregulation of PR-3 with relative expression levels of 0.414-and 0.406-fold change lower at 72 h respectively, and 0.229-and 0.376-fold change lower than the control at 96 h, respectively (Figure 11). At 72 h, the highest transcriptional level of PR-3 (5.187-fold) was exhibited in CU-F + S. rolfsii-treated plants, whereas at 96 h, the supernatant formulation treatment showed the higher expression level, 11.236-fold change higher than healthy control, followed by the CU-F + S. rolfsii treatment, which was 6.727-fold higher ( Figure 11). The moderate induction of PR-3 was observed in the culture formulation, SU-F + S. rolfsii, and PE-F + S. rolfsii at 72 and 96 h with relative expression levels of 1.390-, 2.211-, and 3.149-fold, and 1.263-, 1.357-, and 1.636-fold, respectively ( Figure 11).

Discussion
Given the increasing demand for peanut as food and an oilseeds crop, peanut production must be substantially increased. The national production of comestible oils must be raised to reduce the need to import oils. The huge number of pathogenic fungi affecting the peanut crop is a task for phytopathologists concerned with enhancing peanut yield. Incidences of major fungal diseases can decrease the productivity as much as 50%. Given the changing climatic conditions and reports of incidences of minor diseases becoming virulent, many diseases pose threats to peanut production. Given the health hazards and environmental concerns due to the indiscriminate use of pesticides, biological control agents have been developed. Native bioagents and plant growth-promoting potential are being investigated to control fungal phytopathogens of peanut. Bacteria isolated from the rhizosphere and belonging to a wide variety of genera have the potential to suppress diseases caused by soil-borne phytopathogens [34].
Actinobacteria are considered potential biocontrol agents of plant diseases. Martinez-Alvarez et al. [35] reported that spores-producing bacteria can be used as an alternative to chemical pesticides for controlling plant diseases. Several modes of action of actinobacteria have been suggested as involved in the biocontrol of plant pathogens such as the production of antibiotic compounds, siderophores, hydrogen cyanide (HCN), and hydrolytic enzymes, such as chitinases and glucanases [36,37]. Induced resistance may be implicated in the management of root and pods rot of peanut by actinobacteria. SAR and JA/ET gene expression in Arabidopsis thaliana were induced by inoculation with endophytic actinobacteria [38].
In our study, ten actinobacterial isolates were tested in vitro as biocontrol agents for their ability to suppress the mycelium growth of S. rolfsii. Actinobacterial isolate Actino 48 more effectively inhibited mycelia growth of S. rolfssii than other actinobacterial isolates. These results agree with those obtained by Adhilakshmi et al. [39], who reported that Streptomyces sp. MDU most effectively inhibited the growth of S. rolfsii. The B. subtilis isolate B4 showed the strongest antagonistic effect and produced a higher inhibition zone diameter against S. rolfsii compared to other isolates [34].

Discussion
Given the increasing demand for peanut as food and an oilseeds crop, peanut production must be substantially increased. The national production of comestible oils must be raised to reduce the need to import oils. The huge number of pathogenic fungi affecting the peanut crop is a task for phytopathologists concerned with enhancing peanut yield. Incidences of major fungal diseases can decrease the productivity as much as 50%. Given the changing climatic conditions and reports of incidences of minor diseases becoming virulent, many diseases pose threats to peanut production. Given the health hazards and environmental concerns due to the indiscriminate use of pesticides, biological control agents have been developed. Native bioagents and plant growth-promoting potential are being investigated to control fungal phytopathogens of peanut. Bacteria isolated from the rhizosphere and belonging to a wide variety of genera have the potential to suppress diseases caused by soil-borne phytopathogens [34].
Actinobacteria are considered potential biocontrol agents of plant diseases. Martinez-Alvarez et al. [35] reported that spores-producing bacteria can be used as an alternative to chemical pesticides for controlling plant diseases. Several modes of action of actinobacteria have been suggested as involved in the biocontrol of plant pathogens such as the production of antibiotic compounds, siderophores, hydrogen cyanide (HCN), and hydrolytic enzymes, such as chitinases and glucanases [36,37]. Induced resistance may be implicated in the management of root and pods rot of peanut by actinobacteria. SAR and JA/ET gene expression in Arabidopsis thaliana were induced by inoculation with endophytic actinobacteria [38].
In our study, ten actinobacterial isolates were tested in vitro as biocontrol agents for their ability to suppress the mycelium growth of S. rolfsii. Actinobacterial isolate Actino 48 more effectively inhibited mycelia growth of S. rolfssii than other actinobacterial isolates. These results agree with those obtained by Adhilakshmi et al. [39], who reported that Streptomyces sp. MDU most effectively inhibited the growth of S. rolfsii. The B. subtilis isolate B4 showed the strongest antagonistic effect and produced a higher inhibition zone diameter against S. rolfsii compared to other isolates [34].
Several antibiotics with different chemical structures produced by actinobacteria, such as polyketides, β-lactams, and peptides, show antagonistic effects against bacteria, fungi, and protozoa [40,41]. In addition, various species of Streptomyces have the ability to produce chitinase as a lytic enzyme, which works on the chitin of the fungal cell wall, resulting in the suppression of fungal growth [42]. Various chitinolytic Streptomyces spp. showed antagonistic activity against S. rolfsii of chickpea [43]. Ningthoujam et al. [44] reported that chitinase-producing S. vinaceusdrappus effectively inhibited the mycelial growth of rice fungal pathogens Curvularia oryzae, Pyricularia oryzae, Bipolaris oryzae, and F. oxysporum. Inhibition of the mycelia growth of S. rolfsii may be related to the ability of actinobacteria to produce antifungal compounds and lytic enzymes such as chitinase.
Three talc-based bioformulations were prepared using culture broth, cell-free supernatant, and pellet suspension of chitinase-producing S. cellulosae Actino 48. The bioformulations effectively reduced soil-borne diseases incidence on peanut plants when applied as biocontrol agents in soil infested with S. rolfsii. Damping-off, root rot, and pod rot and healthy survival percentages were evaluated. In addition, the dry weights of peanut shoot and root systems and the dry weights of infected, healthy, and total pods were determined.
The obtained data revealed that the talc-based culture broth formulation compared to the talc-based supernatant formulation and talc-based pellet formulation effectively reduced peanut damping-off and root rot diseases caused by S. rolfsii under greenhouse and open-field conditions during two successive seasons, 2018 and 2019. Their effects were close to those of Rizolex-T, which is the recommended fungicide. The talc-based culture broth formulation efficiency may refer to this formulation containing S. cellulosae Actino 48 spores and chitinase enzyme activity. These results agree with the outcomes reported by Errakhi et al. [45] and Abdel-Gayed et al. [34], who found that Streptomyces isolate J-2 and B. subtilis isolate B4 significantly reduced the disease severity of sugar beet damping-off and root rot of peanut caused by S. rolfsii, respectively. Zacky and Ting [46] reported that chitinase-producing Streptomyces spp. are usually involved in the biocontrol of several plant fungal pathogens and formulated as active biofungicides. A wettable talc powder formulation of S. philanthi RL-1-178 was more effective in controlling root and stem rot of chili pepper (Capsicum annuum L.) caused by S. rolfsii than granules and encapsulated granules formulations [47].
In this study, a talc-based culture broth formulation of chitinase-producing S. cellulosae Actino 48 (closed to Rizolex-T) decreased peanut pods rot caused by S. rolfsii and increased the dry weight of healthy and total pods compared to the control under greenhouse and open-field conditions during two successive seasons, 2018 and 2019. These conclusions agree with those obtained by Abdel-Gayed et al. [34], who reported that a talc-based formulation of B. subtilis isolate B4 resulted in a higher number of healthy pods and a low percentage of pods infected with S. rolfsii compared to other treatments under greenhouse and open-field conditions. It also increased the dry weight of healthy and total pods.
In the current study, we employed qRT-PCR to analyze the expression patterns of some defense genes (POD, PR-1, and PR-3) that are regulated in peanut plants in response to S. rolfsii infection. Talc-based culture, supernatant, and pellet bioformulations of S. cellulosae Actino 48 were tested against S. rolfsii under greenhouse conditions. Their effects on the relative expression levels of three defense-related genes (POD, PR-1, and PR-3) at 72 and 96 h post-inoculation were evaluated. The results of our study indicated that treatment with the talc-based culture formulation of S. cellulosae Actino 48 in soil infected with S. rolfsii induced higher expression levels of POD, PR-1, and PR-3, which activated the defense mechanism of peanut against S. rolfsii infection. Our results agree with those recorded by several investigators who have demonstrated the induction of PR genes and their role in plant disease resistance mechanisms using real-time qPCR. It was reported that the induction of pathogenesis related-protein (PR) genes and increasing POD activity were correlated with activation of the defense system [48,49]. PR-1, a salicylic acid (SA) marker gene, is a principal regulator of systemic acquired resistance (SAR) and could be an indicator of the plant defense response and increasing resistance [50][51][52][53]. Increased POD activity has been associated with improvement in plant defense against pathogens [54,55]. PR-3 (chitinase) is known to inhibit fungal growth and plays an important role in protecting plants from fungal infestations [56]. In the present study, the peanut plants infected with S. rolfsii were only associated with downregulation of POD, PR-1, and PR-3 at the two time-intervals, except PR-1 at 72 h showed upregulation with relative expression levels 9.713-fold higher than the control. Compared to plants infected with S. rolfsii, the CU-F + S. rolfsii plants exhibited the highest transcripts of POD, PR-1, and PR-3 genes with relative 2.969-, 71.671-, and 5.187-fold changes, and 2.329-, 18.895-, and 6.727-fold changes higher than the control at 72 and 96 h, respectively. The changes in the relative expression of studied genes at 72 and 96 hpi may depend on the time of pathogen and/or biocontrol agent treatment. It was reported that the transcriptional levels of many defense-related genes during the plant-fungal interaction depend on the time of the infection process and biocontrol agents treatment [57,58]. As a result, the application of the talc-based culture formulation of S. cellulosae Actino 48 induced the peanut immune defense system, resulting in the development of SAR activation against S. rolfsii infection. These results indicated that S. cellulosae Actino 48, and specifically the talc-based culture formulation, produces strong biocontrol effects on S. rolfsii in peanut and could be used as a biocontrol agent against plant fungal infection. Finally, the physico-chemical structure investigation of a talc-based culture broth formulation of chitinase-producing S. cellulosae Actino 48 using different techniques such as high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) could provide an investigation lead for further development of a novel biofungicide. Consequently, we will address these investigations in more details in future studies.

Conclusions
We can decrease the amounts of chemical fungicides that are broadly applied to control numerous fungal plant diseases using biocontrol agents as an alternative management method. Our study showed that bioformulations of S. cellulosae Actino 48 can be employed as a biofungicide to control peanut soil-borne diseases caused by S. rolfsii as a substitute for chemical fungicides.