*2.6. Thin Layer Chromatography of Methanolic Extract of Tomato Leaves*

Leaves of tomato plant were harvested 24 h after infestation (80 DAS); shade dried at room temperature and grinded to fine powder. Powdered leaves (20 g) was extracted in 400 mL methanol for 72 h.The methanolic extract was dried using rotary evaporator (RFE), the residue (3 mg) was dissolved in 500 μL methanol and collected in eppendorf tube and stored at −4 ◦C.

Extract was spotted on a TLC plate (20 × 20 cm) coated with silica gel HF (250-350 nm). The mobile phase used was chloroform: methanol (95:5 *v*/*v*). The bands, representing various compounds were visualized under UV (254 nm and 380 nm) [39]. The Rf value of each band was calculated and identification of the compound from each band at specific Rf was made from the literature documented.

#### *2.7. FTIR Spectroscopy*

All spectra were obtained with the help of an OMNI-sampler attenuated total reflectance (ATR) accessory on a Nicolet FTIR spectrophotometer followed by the method of Lu et al. [40] and Liu et al. [41] with some modifications. Small amount of TLC eluent corresponding to the Rf value of major bands were placed directly on the germanium piece of the infrared spectrometer with constant pressure applied and data of infrared absorbance, collected over the wave number ranged from 4000 cm–1 to 675 cm–1 and computerized for analyses by using the Omnic software [42].

#### *2.8. Statistical Analysis of Data*

The data was subjected to analysis of variance using Statistix 8.1 software. The differences among various treatment means were compared using the least significant differences test (LSD) at *p* ≤ 0.05 probability level (Table S1).

#### **3. Results**

#### *3.1. Plant Growth Attributes*

The plant spread, which is a measurement of plant width, was significantly (31%) higher in PGPR treated plants under unstressed condition over control (Figure 1). Insect infestation decreased the plant spread by 41%, the decrease was ameliorated by the PGPRs and the value was even greater than the control. The plant height was significantly increased in PGPR inoculated plants (Figure 1). The insect infestation significantly reduced (*p* ≤*0.05*) the height of the plant by 40%, and root area by 50% of the control (Figure 1). The PGPR inoculated plants alleviated the inhibitory effects of insect infestation on plant height and root area such that the root area was significantly higher than the control. Both the shoot and root fresh weights were significantly (44% and 34%) increased in PGPR inoculated plants (Figures 2 and 3). Infestation with the insect decreased the fresh weights of both the root and shoot, the shoot fresh weight was more adversely affected. The PGPR inoculation had ameliorated the insectinduced decrease in the root and shoot fresh weight.

**Figure 1.** Mean plant height and plant spread (cm) of tomato under control and infested conditions. Data are means of four replicates along with standard error bars. Different letters on the bar represent significant differences (*p* < 0.05) among treatments.

**Figure 2.** Root area (cm2) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 3.** Fresh weight of shoot and root (g) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

The dry weight of root and shoot was also higher (*p* ≤*0.05*) in PGPR inoculated plants (Figure 4). The root was more responsive and the % increase in root weight was greater. The leaves were almost eaten by the insect and the shoot weight was significantly decreased to 81% whereas root weight was decreased by 38% over the control.

**Figure 4.** Dry weight of leaf, shoot and root (g) of tomato plant infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

C-uninoculated uninfested control, T1-Seeds inoculated with *Bacillus endophyticus*, T2-Seeds inoculated with *Pseudomonas aeruginosa*, T3-Plants infested with *S. litura*, T4-Seeds inoculated with *Bacillus endophyticus* and plants infested with *S. litura*, T5-Seeds inoculated with *Pseudomonas aeruginosa* and plants infested with *S. litura.*

#### *3.2. Physiological Parameters*

The proline production was lower (*p* ≤ *0.05*) in the untreated control plants (Figure 5). Under unstressed conditions the PGPR treatments stimulated proline content of leaves by 18% over control. Similar percent of increase was recorded in plants infested with *S. litura*. Both the PGPR inoculated plants infested with *S. litura* exhibited marked increase in proline content of leaves over infested plants. The maximum (59%) increase was recorded in the *Bacillus endophyticus* inoculated plants infested with *S. litura.* Chlorophyll a, b and carotenoids followed the similar pattern of response to PGPR and *S. litura* infestation (Figure 6). The response of PGPR was higher (*p* ≤ *0.05*) particularly for carotenoids content. Both the protein and the sugar contents were higher (*p* ≤ *0.05*) in PGPR inoculated plants (Figure 7) under unstressed conditions. *Pseudomonas aeruginosa* showed maximum (1.4 fold) increase in sugar content over infested plants. The infestation with *S. litura* had increased sugar and protein contents significantly higher than the control. The inoculated plants receiving insect infestation exhibited up to 2.25 fold increase in sugar content as compared to that of infested plants.

**Figure 5.** Proline content (μg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 6.** Chlorophylls and carotenoids content (mg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

**Figure 7.** Sugar and protein content (mg/g) of tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

The weight of tomato fruit was about 35% greater in plants inoculated with *Bacillus endophyticus* while *Pseudomonas aeruginosa* inoculated plants exhibited 44% increase over control. There was 26% decrease in the weight of tomato fruit in infested plants (Figure 8). The PGPR inoculated plants ameliorated the inhibitory effect of the insect and showed up to 78% increase in the fruit weight over infested plants.

**Figure 8.** Weight of tomato fruits/plant (g) infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

The infestation with insects enhanced the SOD activity. The SOD activity was three fold higher in leaves of plants inoculated with *Bacillus endophyticus* (T1). Plants inoculated with *Pseudomonas aeruginosa* (T2) on infestation further augmented SOD (Figure 9).

**Figure 9.** Superoxide dismutase (SOD) activity in tomato leaves infested with *S. litura* and under control condition. Data are means of four replicates along with standard error bars. Different letters are indicating significant differences (*p* < 0.05) among treatments.

#### *3.3. Phytohormones Contents of Leaves*

The data in Figure 10 revealed that uninoculated uninfested control leaves of tomato had traceable amounts of Salicylic acid. Insect infestation produced very little amounts of SA. Both the PGPR produced significantly higher amounts of SA in plants, *Pseudomonas* sp. being more efficient. The SA was 1.8 folds greater than infested plant leaves. In *Pseudomonas* inoculated plants, this was further augmented and significantly higher (3.6 fold) SA was recorded in infested plant leaves pretreated with *Pseudomonas aeruginosa*. IAA was not detected in the control and insect infested plants but both the PGPR produced significant amount of IAA in the leaves of inoculated plant which was further augmented and up to 449 μg IAA/g leaves was detected in the leaves of plants infested with *S. litura* and pretreated with *Pseudomonas aeruginosa* (Figure 10). Insect infestation increased the GA content of leaves significantly over control. Several fold increases in GA production were recorded in both the PGPR inoculated plants: *Pseudomonas aeruginosa* being most efficient. Both the PGPR inoculated plants overcame the insect infestation induced decrease in GA content (Figure 10). The ABA content was significantly lower in the infested plant leaves as compared to control. *Bacillus endophyticus* inoculation showed significantly higher ABA production under controlled conditions and the value was several times greater than control in the inoculated plant infested with *S. litura*.

**Figure 10.** Phytohormone content in the leaves of tomato plants infested with *S. litura* and under control condition. (**A**): Salicylic acid; (**B**): Indole acetic acid; (**C**): Gibberellic acid; (**D**): Abscisic acid.

#### *3.4. Detection of Secondary Metabolites from Extract of Tomato Leaves*

Thin layer chromatography of tomato leaf extract showed 29 bands of different colors under UV light (Table 1). Calculated Rf values of leaf extract were compared with Rf values of standard compounds ferulic acid (0.72), salicylic acid (0.60), o-coumeric acid (0.74), trans-cinnamic acid (0.74), caffeic acid (0.85), p-coumaric acid (0.77).

The un-inoculated non infested control plant leaves extract contained caffeic acid (Rf 0.85) and quercetin (Rf 0.88). This was in contrast to *Bacillus endophyticus* inoculated plant leaves which exhibited some unidentified compounds at Rf 0.50 in addition to myricitin (Rf 0.73) o-coumaric (Rf 0.74) whereas, *Pseudomonas aeruginosa* inoculated plants showed the presence of flavonoids, ferulic acid, o-coumaric, kaempferol-7-neoheps-eridiside-glycosides in addition to some unidentified compounds of low polarity. Infestation with *S. litura* resulted in the production of caffeic acid and o-coumaric acid in addition to low and high polarity unidentified compounds. PGPR inoculated plants on infestation produced salicylic acid, rutin and kaempferol in addition to p-Coumaric acid and some unidentified compounds.


**Table 1.** Putative secondary metabolites identified on the basis of the Rf values in the extract of tomato leaves of different treatments.
