**2. Materials and Methods**

#### *2.1. Fruit Rot Pathogens*

Fruit rot fungal pathogens *viz*., *Colletotrichum scovillei*, *Colletotrichum truncatum* and *Fusarium oxysporum* were isolated from infected chilli fruits collected from various locations of Tamil Nadu, India. The infected portion of the fruits were cut into small pieces (5 mm) using a sterile blade and surface sterilized with 1% NaOCl4 for 1–2 min followed by 70% ethanol for 30 s and rinsed thrice with sterile distilled water [36]. The surface

disinfected fruit pieces were placed onto sterile Potato Dextrose Agar (PDA) medium amended with streptomycin sulphate (0.03 g L−1) and incubated at 28 ± <sup>2</sup> ◦C for 7 days. Pure cultures of the pathogens were obtained by single hyphal tip method. The stock cultures of the pathogens were maintained as pure cultures on PDA slants at 4 ◦C.

#### *2.2. Antagonistic Actinobacteria*

Rhizosphere actinobacteria were isolated from the rhizosphere soil of healthy chilli plants as described by Anwar et al. [37]. The soil samples were taken from a depth of 10–20 cm and subjected to dry heat pre-treatment for 4 h at 45 ◦C [38] to diminish the fast growing and abundant soil bacteria that would hinder slow growing actinobacteria [39]. Ten grams (10 g) of pre-treated soil was suspended in 90 mL of sterile distilled water, shaken thoroughly for 1 h at 100 rpm in an orbital shaker and allowed to settle for an hour. Subsequently, samples were serially diluted up to 10−<sup>5</sup> dilutions and 1 mL aliquot from 10−3–10−<sup>5</sup> dilutions were plated on sterile Starch Casein Agar (SCA) supplemented with 25 μg/mL nalidixic acid and 50 μg/mL nystatin as antibacterial and antifungal agents [40].

Phyllospheric actinobacteria were isolated from the leaf, stem, flower and fruits of healthy chilli plants [41]. Ten grams (10 g) of samples were preheated at 70 ◦C for 15 min and transferred to 90 mL of 0.85% saline buffer (NaCl) and kept in an orbital shaker at 250 rpm for 30 min at 28 ± 2 ◦C. The solution thus obtained was subjected to the standard serial dilution pour plate technique on Starch Casein Agar (SCA) supplemented with nalidixic acid (25 μg/mL) and nystatin (50 μg/mL).

The actinobacteria from the surface sterilized plant tissues were isolated as per the procedure described by Li et al. [42]. A five-step procedure was employed for the sterilization of the plant tissues: (i) the tissue segments were surface-sterilized in 0.1% sterile Tween 20 for 1 min, (ii) the samples were sterilized with 5% sodium hypochlorite for 4 min (leaf samples) or 6 min (stem and root samples), (iii) the samples were then rinsed in 2.5% (*w*/*v*) sodium thiosulfate for 10 min and washed three times with sterile distilled H2O, followed by (iv) immersing in 70% (*v*/*v*) ethanol for 4 min (leaf samples) or 6 min (stem and root samples), and finally (v) the samples were washed with sterile distilled water for a minimum of three times. To validate the successful surface disinfection process, 0.2 mL of water from the final wash was spread onto the isolation medium and incubated at 28 ± 2 ◦C. One gram of surface-sterilized plant tissues was homogenized in a mortar and pestle with 1 mL of 0.9% saline buffer (*w*/*v*). One millilitre of the tissue suspension was serially diluted and 10−3–10−<sup>5</sup> dilutions were plated on Starch Casein Agar plates. The plates were incubated at 28 ± 2 ◦C for 7–10 days. Powdery, bright actinobacterial colonies were purified, suspended in 20% glycerol and stored at −80 ◦C as stock culture [43].

#### *2.3. In Vitro Antifungal Bioassay*

2.3.1. Primary Screening of Actinobacterial Isolates for the Antifungal Activity against Chilli Fruit Rot Pathogens

Fifty-two actinobacterial isolates were screened for their antifungal activity against chilli fruit rot pathogens by dual-culture assay [44]. The test isolates were streaked at one corner of the PDA plates (10 mm from the periphery of a 90 mm diameter Petri dish) and incubated at 28 ± 2 ◦C for 4 days. After incubation, the 5-day-old pathogen fungal disc was placed opposite to actinobacterial streak (10 mm away from the periphery). Petri dishes without actinobacterial isolates served as the control. All plates were incubated at 28 ± 2 ◦C for 7 days. All the isolates were tested in triplicate. After incubation, the zone of inhibition was measured and the per cent inhibition of mycelial growth was calculated. The zone of inhibition (ZI) was measured as the diameter of the halo zone (in cm) between the actinobacteria and pathogen colony as and when the pathogen in the control plate covered the entire plate. Per cent inhibition of mycelial growth (PIMG) was determined according to the formula: PIMG = (C − T)/C 100, where C and T are the mycelial growth of pathogenic fungus in the control plate and dual culture plate, respectively. The degree of antifungal activity of various actinobacterial isolates against the

tested pathogens were evaluated based on the zone of inhibition (ZI) (in cm) and per cent inhibition of mycelial growth (PIMG) [45]. Based on the zone of inhibition, the antagonistic activity of actinobacterial isolates were grouped into four categories according to Lee and Hwang [46] as: − no inhibition (ZI ≤ 0); + weak inhibition (ZI = 0.1–1.0 cm); ++ moderate inhibition (ZI = 1.01–2 cm); and +++ strong inhibition (ZI ≥ 2 cm).

#### 2.3.2. Secondary Screening for the Antifungal Activity of Actinobacterial Isolates

The antifungal activity of six actinobacterial isolates which exhibited the strongest inhibition against the tested pathogens by dual culture assay was further confirmed by paired culture antibiosis assay as per the protocol of Liotti et al. [47] with slight modification. An 8 mm mycelial disc of the pathogen was placed at the centre of a Petri dish containing PDA medium and the actinobacterial isolate was streaked at equidistance on both sides of the pathogen, about 10 mm from the periphery of Petri dish. A control plate was maintained without actinobacteria. The experiment was replicated thrice. After 7 days of incubation at 28 ± 2 ◦C, the percentage inhibition of mycelial growth (PIMG) of the pathogen was calculated as per the formula described above.

2.3.3. In Vitro Screening of Actinobacterial Isolates for Production of Extracellular Lytic Enzymes and Siderophore

The actinobacterial isolates were assayed for their biocontrol traits *viz*., amylase, cellulase, chitinase and protease production by spot inoculating 10 μL of culture in starch agar medium [48], Carboxy Methyl Cellulose (CMC) agar medium [49], colloidal chitin agar medium [50] and skim milk agar medium [51], containing starch, cellulose, colloidal chitin and casein as the respective substrates. Siderophore production was assayed on Chrome Azurol Sulphonate (CAS) agar medium according to the methodology of Sadeghi et al. [52]. The plates were incubated for 5–7 days at 28 ± 2 ◦C. Three replications were maintained for each actinobacterial isolate.

The amylase activity of the actinobacteria was evaluated by flooding the plate with Lugol's iodine solution for 30 s. A clear hydrolysis zone around the colonies against the blue background indicated the hydrolysis of starch by the amylase enzyme [53]. Cellulase activity was determined by flooding the plates with 0.1% Congo red solution and counter staining with 1 M NaCl for 15–20 min. The formation of a clear zone around the colony due to the hydrolysis of cellulose indicated a positive result for the production of the cellulase enzyme by the actinobacterial isolates. The isolates positive for chitinolytic and proteolytic activity produced a clear halo zone around the colonies due to the hydrolysis of chitin and casein in the respective media. The formation of a yellow to orange halo around the actinobacterial colonies due to the removal of iron from CAS represented a positive result for the production of siderophore.

#### 2.3.4. Antifungal Activity of Volatile Organic Compounds

The four most active actinobacterial isolates that showed significant positive results for the production of cell wall-degrading enzymes and siderophore were subjected to additional assays on the production of volatile, non-volatile and thermostable compounds. The antifungal activity of volatile organic compounds (VOCs) produced by the actinobacterial isolates was tested against the fruit rot pathogens by the double-sealed plate method [54]. A 90 mm diameter Petri dish bottom containing 15 mL of ISP4 medium was streaked with a loopful of actinobacterial culture. An 8 mm diameter mycelial plug of the pathogen was inoculated in the centre of another Petri dish bottom containing 15 mL of potato dextrose agar medium. A Petri dish "sandwich" was made with the antagonist Petri dish placed over the pathogen plate in such a way that the pathogen plate was at the bottom and antagonist plate was on the top. The sandwiched Petri dish was sealed together with a parafilm without any gaps and incubated at 28 ± 2 ◦C for 7–10 days. A Petri dish containing ISP4 medium without the antagonist placed over the pathogen plate served as the control. The parafilm-sealed plates ensured no physical contact between the pathogen and

antagonist. The experiment was conducted with three replications for each isolate. The rate of inhibition (%) of mycelial growth was calculated as described previously.

#### 2.3.5. Antifungal Activity of Non-Volatile Metabolites

The antifungal activity of non-volatile metabolites in the cell-free culture filtrate of actinobacterial isolates was determined using the seeded agar method [55,56]. Actinobacterial isolates were cultured in a 250 mL conical flask containing 100 mL ISP4 broth and incubated in an orbital shaker at 150 rpm for 7 days at 28 ± 2 ◦C. Then the culture broth was centrifuged at 10,000 rpm for 15 min at 4 ◦C. The supernatant obtained was filtered through a 0.22 μm nitrocellulose membrane filter to obtain cell-free culture filtrate and subjected to an antifungal assay. The filtrate was mixed with warm PDA (25%) and plated in a sterile Petri dish. Finally, an 8 mm mycelial disc of the pathogen was placed at the centre of the seeded PDA medium in the Petri dish. The pathogen growth on the Petri dish without the cell-free culture filtrate served as the control. The plates were incubated at 28 ± 2 ◦C until the mycelial disc in the control plate completely covers the plate. Three replicates were maintained for each isolate. Per cent inhibition (PI) of mycelial growth was calculated as described previously.

#### 2.3.6. Antifungal Activity of Thermostable Compounds

The actinobacterial isolates were cultured in 100 mL ISP4 broth in a 250 mL conical flask with constant agitation in an orbital shaker (150 rpm) for 7 days at 28 ± 2 ◦C. The actinobacterial cells were harvested by centrifugation at 10,000 rpm for 15 min. Twenty-five millilitres of the supernatant were transferred to a conical flask containing 75 mL PDA medium and sterilized at 121 ◦C for 20 min. The actinobacterial metabolite-amended sterile medium was plated into Petri dish and a 9 mm mycelial disc of the tested pathogen was placed at the centre of solidified medium. The pathogen growth on PDA medium without actinobacterial metabolite served as the control. The Petri dishes were incubated at room temperature for 7 days and the Per cent Inhibition (PI) of mycelial growth of the pathogen was assessed as per the formula described above.

#### 2.3.7. Assessment of In Vitro Antifungal Traits

Among the six isolates, the best isolate with the highest antagonistic potential was selected based on a bonitur scale as described by Passari et al. [57] and El-Sayed et al. [58]. In this scale, points were given for each in vitro antifungal trait and the maximum bonitur score is 24 points. The per cent inhibition of mycelial growth (PIMG) was evaluated as follows: if PIMG is 30–54.9% = 1 point; 55–74.9% = 2 points; 75–95% = 3 points. Lytic enzyme production was evaluated with 1 point and siderophore with 2 points each.

#### *2.4. Scanning Electron Microscopy (SEM)*

The interaction of the actinobacterial isolate AR26 which exhibited strong antifungal activity against the pathogens in the dual culture plate was documented by Scanning Electron Microscope (SEM) (Model: FAI QUANTA 250, Czech Republic) at 15 KV [59]. Mycelial discs (5 mm) of the pathogen from the periphery of inhibition zone in the dual culture plate as well as in the control plate were cut with a sterile scalpel and transferred to perforated capsules and fixed in 1.5% glutaraldehyde in phosphate buffer for 4 h [60]. Then, the specimens were washed with 0.2 M sodium cacodylate buffer (pH 6.2) and dehydrated with an increasing concentration of ethanol washes from 0–100% at 10 min intervals (0%, 30%, 50%, 70%, 80%, 90% and 100%). Later the specimens were mounted on aluminium stubs using conductive double-sided carbon tape. The stubs were then lyophilized, and sputter coated with gold (5 nm thickness). Finally, any morphological changes of the pathogen mycelium in the dual culture plate as well as in the control plate were examined under scanning electron microscope.

#### *2.5. Molecular Characterization of Actinobacterial Isolates*

The genomic DNA of the actinobacterial isolate was extracted from the spore masses using the Cetyl Trimethyl Ammonium Bromide (CTAB) method [61]. The 1.5 kb full length 16S rRNA gene of actinobacteria was amplified by Polymerase Chain reaction (PCR) with a forward primer 27F (5- AGAGTTTGATCCTGGCTCAG-3- ) and reverse primer 1492R (5- -GGTTACCTTGTTACGACTT-3- ) [62]. The PCR amplification was performed with a 25 μL reaction mixture which contained 10 μL of master mix, 1 μL of bacterial genomic DNA at a concentration of 20 ng, 1 μL of each primer at a concentration of 10 pM and 12 μL of sterilized deionized water. The PCR amplification conditions included an initial denaturation at 94 ◦C for 5 min, 35 cycles of denaturation at 94 ◦C for 1 min, annealing at 55 ◦C for 1 min, extension at 72 ◦C for 40 s and final extension at 72 ◦C for 10 min. The PCR amplified products were visualized on 1% agarose gel with a UV transilluminator and photographed using the gel documentation system and sequenced at Biokart India Pvt. Ltd., Bangalore, India. The sequence similarities were determined by BLAST analysis (Basic Local Alignment Search Tool) (BLAST, (https://www.ncbi.nlm.nih.gov) (accessed on 27 November 2022) and submitted in GenBank. The most homologous sequences showing the highest similarity were retrieved from the NCBI GenBank database and multiple sequences were aligned using the ClustalW algorithm. A phylogenetic tree was constructed with closely related nucleotide sequences using the Neighbour-Joining (NJ) method [63] using MEGA (Molecular Evolutionary Genetics Analysis) 11 software [64] with bootstrap values of 1000. Evolutionary distances were calculated using the maximum combined likelihood method and are given in units of the number of base substitutions per site.

#### *2.6. Antifungal Bioassay of Liquid Formulation of Actinobacterial Isolate on Chilli Fruits*

The antifungal activity of actinobacterial isolate AR26 on green chilli fruits was determined as per the antifungal bioassay of Liottia et al. [47]. Fresh fruits of the chilli hybrid "Ganga" of uniform size and maturity without wounds, scars and rots on their surface were surface sterilized as described previously. The surface sterilized green chilli fruits were wounded to the depth of 1 mm with a sterile needle and subjected to following treatments. (i) healthy control: chilli fruits were inoculated with 20 μL of sterile distilled water, (ii) pathogen-inoculated control: chilli fruits were inoculated with 6 mm mycelial disc of pathogen culture, (iii) antagonist inoculated control: chilli fruits were inoculated with 20 <sup>μ</sup>L of liquid formulation of *S. tuirus* AR26 at10 mL/L containing 9 × <sup>10</sup><sup>8</sup> CFU/mL, (iv) chilli fruits were first inoculated with 20 μL of liquid formulation of *S. tuirus* at 5 mL/L and after an hour of incubation, 6 mm mycelial disc of respective pathogens were placed over it, (v) chilli fruits were first inoculated with 20 μL of liquid formulation of *S. tuirus* at 10 mL/L and 6 mm mycelial disc of respective pathogens were placed over it.

Inoculated fruits of each treatment were placed in separate glass Petri dishes, sealed with parafilm and incubated at 28 ± 2 ◦C for 7 days. The experiment was conducted statistically as a completely randomized design (CRD) in three replicates of five fruits each. The progress of the symptom on the fruits was measured as the lesion diameter after seven days of incubation. The percentage of inhibition of fruit rot symptom and disease incidence was calculated as per the formula given below. Per cent disease reduction = [(D − d) × 100]/D, where D is the lesion diameter in pathogen-inoculated control fruits, and d is the lesion diameter in actinobacteria and pathogen co-inoculated fruits.

Disease incidence = (Number of diseased chili fruits/Total number of chili fruits) × 100.

### *2.7. Antifungal Bioassay of Soluble Metabolites of Actinobacterial Isolate on Chilli Fruits*

This assay was conducted to differentiate whether the antifungal activity was mediated by the presence of actinobacterial culture or by its metabolites. The soluble metabolites produced by the isolate AR26 in the dual culture plate in PDA medium were extracted from the zone of inhibition by excising the PDA medium from the inhibition zone using a sterile scalpel. Excised PDA medium was blended with HPLC-grade acetonitrile in a 1:4 ratio (5 g agar in 20 mL of HPLC grade acetonitrile). The mixture was incubated overnight at 28 ± 2 ◦C in an orbital shaker at 150 rpm. The homogenised samples were subjected to 10 min centrifugation at 10,000 rpm, and then filtered through Whatman No.1 filter paper to separate the agar particles and supernatant. The supernatant was dried in a vacuum flash evaporator (Roteva Equitron Make). After discarding the eluent, the final product was diluted in 1 mL of HPLC-grade methanol [65]. The extract obtained was tested for its ability to control chilli fruit rot pathogens. The assay was performed as described above with four treatments, using 20 μL of methanol extract of *S. tuirus* AR26 for treatments and 20 μL of methanol alone for control.

#### *2.8. Statistical Analysis*

The data was subjected to a single factor test of significance (ANOVA) using the analytical software SPSS version 16.0. Significant differences between the average values of each treatment (*p* ≤ 0.05) were determined using critical difference.

#### **3. Results**

#### *3.1. Primary Screening for Antifungal Activity of Actinobacterial Isolates*

In this study, 52 actinobacterial isolates obtained from rhizospheric (26), phyllospheric (16) and surface sterilized plant tissues (10) of chilli plants were screened for their antagonistic potential against chilli fruit rot pathogens *viz*., *C. scovillei*, *C. truncatum* and *F. oxysporum* by dual culture technique. About 19.2% of the rhizospheric isolates, 12.5% of phyllospheric isolates and 10.0% of the endophytic isolates exhibited strong antifungal activity against *C. scovillei*, whereas 15.4% of the rhizospheric isolates and 12.5% of the phyllospheric isolates and again 10.0 % of endophytic isolates showed antifungal activity against *C. truncatum*. With regard to *F. oxysporum*, 23.1% of rhizospheric, 12.5% of phyllospheric isolates and 10.0% of endophytic isolates showed strong antagonistic activity. Thirty-eight (73.07%) out of 52 isolates inhibited the mycelial growth of at least one out of the three pathogens with varying degrees of inhibitory action, ranging from 4.82% to 67.90% (weak to strong inhibition) (Supplementary Table S1 and Figure 1). Six isolates designated as AR1, AR10, AR26, AL5, AL7, and AFE2 strongly inhibited the growth of all three pathogens (Figure 2) with an inhibition zone (ZI) greater than 2 cm. Isolate AR26 was found to be significantly superior to other isolates, with the highest mycelial growth inhibition of 67.90%, 63.21%, and 60.37% and inhibition zones of 3.2 cm, 2.8 cm, and 2.7 cm respectively for *C. scovillei*, *C. truncatum*, and *F. oxysporum*, followed by the isolate AR10 (Figure 3).
