**1. Introduction**

*Lycopersicon esculentum* Mill. (Tomato) is one of the widely used vegetables cultivated all over the world. It is the important source of vitamin C and vitamin A [1], lycopene (carotenoids), pro-vitamin A, β-carotene and flavonoids [2]. In the recent years, its yield is significantly reduced by the infestation of leaf caterpillars.

Leaf caterpillar *S. litura* (Fabricius) (Lepidoptera: Noctuidae), also known as tropical armyworm, is among the main pests of cultivated crops that can cause significant damage to tomato crop. To this date, *S. litura* has infected about 290 plant species, belonging to 99 families [3,4]. It grows throughout

the year, and mounts nearly 7 to 8 generations per year. The larvae of *S. litura* feed initially on plant leaves and latterly feed on almost every part of the plant. The larvae can cause 12 to 23% damage to tomatoes in the monsoon and 9.4 to 27.4% in winter [5]. This insect had shown strong resistance to all conventional and some new chemically synthesized insecticides [6,7]. To combat this notorious insect attack, one can develop new insect resistant cultivars. The main drawbacks of the new cultivars' development are time and expenses. Alternatively, the use of plant growth promoting rhizobacteria having biocontrol properties is a sustainable and eco-friendly approach.

Rhizosphere bacteria form a close association with the roots of plants, they nourish on the soil nutrients and root-exudates of plants; in return they protect the host against the biotic and abiotic stresses and help in host growth [8,9]. Plant growth promoting rhizobacteria (PGPR) boost plant growth directly through the production of phytohormones and indirectly as biocontrol agents [10]. PGPR employs different mechanisms to promote plant growth and control phyto-pathogens. One of the widely recognized mechanisms is the production of inhibitory allelo-chemicals, the production of antibiotics, siderophore, lytic enzymes and the induction of systemic resistance (ISR) in host plants against a broad spectrum of pathogens [11]. Induced systemic resistance (ISR) protects the plant against a broad range of diseases [12,13], triggered by a wide variety of beneficial microbes [14].

PGPR consortium of *S. marcescens, B. amyloliquefaciens*, *P. putida*, *P. fluorescens* and *B. cereus* significantly increased the number of fruit/plant [15]. The three bacterial species viz. *B. amyloliquefaciens, B. subtilis* and *B. brevis* have significantly improved the activity of defense related enzymes in tomato plants infected with bacterial canker [16]. Several bacterial species *(Pseudomonas, Azotobacter, Azospirillum, Pseudomonas* + *Azotobacter, Pseudomonas* + *Azospirillum, Azotobacter* + *Azospirillum and Pseudomonas* + *Azotobacter* + *Azospirillum*) played a key role in nutrient uptake by tomato plants. Also, the rhizospheric bacteria significantly improved shoot and root dry weights, enhanced and modulated production of secondary metabolites [17] and induced resistance to various diseases [18]. *Pseudomonas aeruginosa* is an aerobic, gram-negative rod-shaped bacterium of *Pseudomonadaceae* [19] that was reported to have antifungal activity against *Fusarium moniliforme* [20]. Both *Pseudomonas aeruginosa* and *Bacillus endophyticus* were catalase and oxidase positive, solubilize phosphorus and produce bacteriocin. These bacterial strains showed significant (*p* < 0.05) increase in dry matter production, plant height and root length of maize [21]. They were found positive for the production of antibiotics [22] and had a protruding impact on plant metabolism and plant defense against environmental stresses [23,24].

The present investigation was based on the hypothesis that rhizobacteria isolated from stressed habitats can induce tolerance to plants against environmental stresses in a much better way than those from normal conditions [25]. The rhizobacteria *Bacillus endophyticus* strainY5 (Accession no. JQ792035) and *Pseudomonas aeruginosa* JYR (Accession no JQ792038) were isolated from the semiarid areas of Yousaf wala Sahiwal (15% soil moisture) and arid areas of Jhang (9% soil moisture), where maize is grown as a main crop. Soil sampling was done at the tasseling stage of maize. The role of those two PGPRs used as bioinoculants was studied on growth and yield of tomato (*Solanum lycopersicum* L.) infested with *S. litura*.

### **2. Materials and Methods**

#### *2.1. Plant Material*

The experiment was conducted in the green house of Quaid-i-Azam University, Islamabad. Seeds of *Solanum lycopersicum L*. cv. Rio Grande was obtained from the National Agricultural Research Centre (NARC) Islamabad. Prior to sowing the seeds were surface sterilized with 70% ethanol for 2–3 min, followed by shaking in 10% clorox for 2–3 min. The seeds were finally washed with autoclaved distilled water to remove the traces of treated chemicals [13].

#### *2.2. Preparation of Inocula and Method of Inoculation*

Fresh cultures (24 h old) of *Bacillus endophyticus* and *Pseudomonas aeruginosa* were used to inoculate Luria-Bertani (LB) broth, incubated on a rotary shaker for 48 h at 28 ◦C. The cultures were centrifuged at 3000 rpm for 10 min. Supernatant was discarded, and the pellet containing the bacterial cells was suspended in the autoclaved distilled water to adjust the optical density (λ = 1) at 660 nm. The inoculum prepared was found to have 10<sup>6</sup> cells/mL. Sterilized seeds were soaked in the bacterial inoculum for 3 to 4 h. The seeds soaked in autoclaved distilled water for the same period were treated as a control [5].

#### *2.3. Growing Conditions and the Treatments*

Seeds were sown in pots containing autoclaved sand and soil mixed in 1:3 ratio [26]. Pots were kept in the greenhouse of Quaid-i-Azam University using randomized complete block design with four replicates per treatment. The growing conditions were: photoperiod 16 h, temp 22–28 ◦C and humidity 60–80%.

The treatments included: Tomato seeds uninoculated uninfested control (C); Tomato seeds inoculated with *Bacillus endophyticus* (T1); Tomato seeds inoculated with *Pseudomonas aeruginosa* (T2); plants infested with *S. litura* (T3); Tomato seeds inoculated with *Bacillus endophyticus* and latterly infested the leaves at 6 to 7 branching stage with *S. litura* (T4); Tomato seeds inoculated with *Pseudomonas aeruginosa* and infested the leaves at 6 to 7 branching stage with *S. litura* (T5).

The tropical armyworm was obtained from the Insectary department, National Agricultural Research Centre (NARC), Islamabad. The leaves of tomato seedlings at 6 to 7 branching stage were infested with larvae of *S. litura* at larval stage of 2nd instar. The larvae were starved for 2 h prior to infestation.

#### *2.4. Height and Weight of Plants and Weight of Tomato Fruit*

At the time of harvesting, four plants were marked from each treatment to measure the average height (cm) of the plant and their fresh and dry weights were recorded. After 180 days of sowing, the red ripened fruits were harvested and their fresh weight was measured [27].

### *2.5. Physiological and Biochemical Attributes of Plants*

The physiological and biochemical parameters of leaves were measured after insect infestation.

#### 2.5.1. Leaf Protein Content

Protein content of fresh leaves of tomato plant was estimated following the method of Lowry et al. [28], using Bovine Serum Albumin (BSA) as a standard. Fresh leaves (0.1 g) were grinded in 1 mL of phosphate buffer (pH 6.8) and centrifuged for 10 min at 3000 rpm. The supernatant (0.1 mL) was poured into the test tube and a total volume of 1 mL was made with distilled water. A mixture of 50 mL of Na2CO3, NaOH and Na-K tartrate and 1mL of CuSO4.5H2O was added. After shaking for 10 min, 0.1 mL of Folin phenol reagent was added. The absorbance of each sample was recorded at 650 nm after 30 min incubation. The concentration of protein was determined using the following formula:

$$Protzin \left(\frac{mg}{g}\right) = \frac{K - value \times diffusion\ factor \times absorber}{weight\ of\ sample}$$

K value = 19.6 Dilution factor = 2 Weight of leaf sample = 100 mg

#### 2.5.2. Chlorophyll and Carotenoids Content

Estimation of chlorophyll contents was made according to the method of Arnon [29]. The tomato leaves (0.05 g) were grinded in 10 mL dimethyl sulfoxide (DMSO). The tubes were incubated at 65 ◦C for 4 h and then the optical density of the sample was recorded at 665 nm and 645 nm. The carotenoids content was determined following the method of Lichtenthaler and Welburn [30].

$$Chlorophyll \, a \left(\frac{mg}{g}\right) = 1.07(OD\_{663}) - 0.09(OD\_{645})$$

$$Chlorophyll \, b \left(\frac{mg}{g}\right) = 1.77(OD\_{645}) - 0.28(OD\_{663})$$

$$Carotonoids \left(\frac{mg}{g}\right) = Absorbance \, (OD\_{663}) \times 4$$

2.5.3. Proline Content of Leaves (μg/g)

Free proline content in tomato plant leaves was estimated following the method of Bates et al. [31]. Fresh plant leaf (0.5 g) was grounded in 3% sulfosalicylic acids and kept overnight at 4 ◦C. The extract was centrifuged at 3,000 rpm for 10 min. The supernatant was mixed with acidic ninhydrin and boiled for 1 h. The solution was then cooled and toluene was added. The absorbance of the toluene layer was recorded at 520 nm against toluene blank. The content of free proline was estimated on fresh weight basis following the formula:

$$Proline\left(\frac{\mu g}{g}\right) = \frac{K - value \times diffusion\ factor \times absorber}{leaf\ weight}$$

Value of K= 17.52 Dilution factor= 2 Weight of leaf sample= 100 mg

#### 2.5.4. Sugar Estimation

The colorimetric determination of total sugar (simple sugar, oligosaccharides and reducing sugar) was done following the method of Dubois et al. [32]. Fresh tomato leaves (500 mg) were grinded with 10 mL of distilled water in autoclaved mortar and pestle, centrifuged at 3000 rpm for 5 min. To the supernatant (100 μL), 1 mL of 80% (w/v) phenol and 5 mL concentrated sulfuric acid was added. The mixture was heated in a water bath till boiling and then incubated for 4 h at room temperature. The absorbance of each sample was finally measured at 420 nm.

$$Sugar\left(\frac{mg}{g}\right) = \frac{K - value \times diffusion\ factor \times absorber}{leaf\ weight}$$

Value of K = 20 Dilution factor = 10 Weight of leaf sample = 500mg

#### 2.5.5. Superoxide Dismutase (SOD) Assay

The SOD activity was estimated following the method of Beauchamp and Fridovich [33]. The activity of Superoxide dismutase was expressed as units/100 g F.W. Superoxide dismutase was calculated by the following formula:

R4 = R3-R2

#### *Agronomy* **2020**, *10*, 778

SOD activity = R4/A R1 = O.D of Reference, R2 = O.D of Blank, R3 = O.D of Sample A = R1 (50/100)
