*3.5. Fourier Transform Infrared Spectrometry (FTIR) of Tomato Leaves*

The data presented in Table 2 revealed that control plant (uninfested and uninoculated) leaves extract had shown the presence of amines and amides with N-H stretch and bend, aliphatic amines stretching with C-N, alkenes with C-H bend and alkyl halides with C-Cl stretch. Plants inoculated with *Pseudomonas aeruginosa* exhibited an additional bonding indicating the presence of alkynes (at frequency of 638.51 with –C≡C–H: C–H bend) which were absent in uninoculated un-infested plants. While extract of plant leaves infested with *S. litura* exhibited alkanes with C–H stretch. In addition to amines and amides with N-H stretch, aliphatic amines stretching with C-N, alkenes with C-H bend and alkyl halides with C-Cl stretching. This was in contrast to plant leaves extract previously inoculated with *Bacillus endophyticus* (T4) or *Pseudomonas aeruginosa* and infested with *S. litura* (T5) which exhibited higher frequency of N-H stretch and =C-H bend and additional bonding indicating the presence of aldehyde and amine with H–C=O: C–H stretch, N–H bend (at frequencies of 2827.91 and 1630.78) as compared to plant extract infested with *S. litura* (T5).


**Table 2.** Fourier-transform infrared spectroscopy (FTIR) of thin-layer chromatography (TLC) eluent of tomato leaves under different treatments.

Values are mean of 4 replications per treatment. Small amount of TLC eluent corresponding to the Rf-value of key bands were placed directly on the germanium piece of the infrared spectro-meter with persistent pressure and the infrared absorbance was collected over the wave number ranged from 4000 cm–1 –675 cm–1 and computerized for analyses by using the Omnic software.

#### **4. Discussion**

This paper evaluates the effect of PGPR as a growth promoter as well as a biocontrol agent. Although the study deals with tomatoes only in one season and also limited by the lack of behavioral study of the insect.

The growth parameters of tomato were considerably amplified after PGPR inoculation, under uninfested condition; the *Pseudomonas aeruginosa* being more effective. Of note, the effectivity of PGPR were higher under infested conditions and produce higher proline as osmoregulant, more defense hormone e.g., SA and higher level of growth promoting hormone, e.g., IAA contents and for inducing antioxidant enzyme, SOD. The PGPR inoculation not only overcame the infestation induced decrease in root and shoot weight but also increased the root and shoot weight. The PGPR effect was more pronounced on shoot dry weight. Similar results were reported by Avis et al. [43] and Babalola [44]. Shannag and Abadneh [45] reported that fresh and dry weight of shoot and root were decreased by *Aphis fabae Scopoli* in Faba Bean as compared to its respective control. Yadav et al. [46]

reported a marked increase in shoot and root dry weight in chickpea treated with PGPR. The increased weight of tomato fruit was correlated with the number of flowers, branches, plant biomass concomitant with the osmotic balance and alleviating oxidative stress. Both the PGPR were effective and significantly enhanced (≥35%) the fruit fresh weight. This increase in fruit fresh weight by the PGPR may be attributed to the fact that PGPR significantly improves the root growth and plant vigor which lead to enhanced fruit production. Fabro et al. [47] reported that tomato plants treated with PGPR showed the highest number of branches when compared to infested control. PGPR has positive effects on tomato fruit quality attributes, particularly on size and texture [48]. Widnyana [49] reported that inoculation of tomato plants with *Pseudomonas* and *Bacillus* sp. speeded up the plant growth and yield and protection against plant pathogens. Similar results that PGPR inoculation enhances the plant growth, yield and fruit weight were also reported by Almaghrabi et al. [50] and Murphy et al. [51].

Results revealed that PGPR alleviated the osmotic imbalance by increasing, proline content in the insect infested plants. The different PGPR behaved differently, both for proline production and antioxidant enzymes. It is demonstrated that *Pseudomonas aeruginosa* combats osmotic stress in infested plants through increase in sugar content as osmoregulant whereas, *Bacillus endophyticus* enhances proline content to combat osmotic imbalance. The osmotic stress is one of the secondary stresses caused by insect infestation. Proline is revealed to be an osmoregulant that accumulates in plants under a wide range of stress conditions [52,53]. It is well known that free proline accumulation in vascular plants demonstrated stresses including pathogen attack [54,55]. The accumulation of cellular osmolytes such as proline, sugar alcohols, glucosinolates etc. and soluble sugars and the expression of antioxidant systems help plants in sustaining cellular function, crucial for physiological stability of plants under stress. Ullah et al. [56] indicated that application of PGPR to plants displayed substantial increase in proline content as compared to untreated plants. Phenolics are produced by many plant species for protection against biotic or abiotic stress growth conditions and their accumulation correlates with antioxidant capacity of plants in a number of species [57,58].

Compatible solutes are used for osmotic adjustment under adverse environmental conditions [59,60]. The soluble carbohydrates in plants attacked by a fungal pathogen, as well as proportions of individual sugars, may be variously modified, both by plant regulatory mechanisms and by pathogen interference. There are several causes for quantitative and qualitative changes of sugars at the infection site. The level of sugars is reduced by their consumption for both energy and structural purposes, their uptake by the pathogen, while in autotrophic tissues it happens due to the inhibition of photosynthesis [61]. The results further demonstrate the PGPR induced changes in chlorophyll and carotenoids in normal and insect infested plants and the *P. aeruginosa* being most effective. Similar results were reported by Wang et al. [62] that PGPR isolates increased chlorophyll content significantly in tomatoes. Inoculation with *Pseudomonas* B-25 resulted in greater synthesis of chlorophyll than the diseased control. Botha et al. [63] showed that *Diuraphis noxia* feeding caused decrease in chlorophyll content in Tugela and decreased levels of chlorophyll a upon infestation [64,65].

It was demonstrated that the *P. aeruginosa* adjust osmotic stress following infestation by stimulation in antioxidant SOD activity. The PGPR effectively enhanced the SOD activity to scavenge the ROS and prevent oxidative stress in plant cells. The observed enhancement in PGPR induced SOD activity in infested plants is a mechanism to combat insect induced oxidative stress. Recently it has been reported by Sharma and Mathur [66] that PGPR alone and/or in association with fungi significantly enhanced the antioxidant enzyme activities in *Brassica juncea* infested with *Spodoptera litura* that lead to enhanced immune system against herbivory. Similarly, Zhao et al. [67] reported Aphid resistance in plants infested with *B*. *tabaci* nymphs, associated with enhanced antioxidant activities. They concluded that this resistance probably acted via interactions with SA-mediated defense responses.

PGPR promoted growth by nutrient acquisition and by producing bioactive compounds [68,69]. They also improve the nutrient uptake in plants by modulating plant hormones level, thereby increasing root proliferation [70]. However, response of the 2 PGPR differed substantially; *Bacillus endophyticus* exhibited lower IAA but higher GA than that of *Pseudomonas aeruginosa*. PGPR can control plant disease

directly, through the production of antagonistic compounds, and indirectly, through the elicitation of a plant defense response [71]. Fernandez-Aunion et al. [72] also reported that PGPR enhances plant growth by synthesis of bioactive compounds and activating plant defense system.

While the PGPR-elicited ISR has been studied extensively in the model plant *Arabidopsis*, it is not well characterized in crop plants. The induction of ISR was investigated by *Bacillus cereus* strain BS107 against *Xanthomonas axonopodis* pv. *vesicatoria* in pepper leaves. Choudhary and Jobri [73] demonstrated the induction of ISR elicited by *Bacillus* spp. against several fungal bacterial and viral pathogens including root knot nematodes. Yang et al. [74] reported genetic evidence of the priming effect of a *rhizobacterium* on the expression of defense genes involved in ISR in pepper. A stronger negative effect of the PGPR on the performance of leaf folder larvae was noted in rice and found that combined treatment of PGPR is more effective than individually. Several plant secondary compounds such as glucosinolates and cyanogenic glycosides yield toxic products after hydrolysis by enzymes stored and liberated during attack by chewing insects [75,76].

It is demonstrated from the present findings that SA and ABA are both involved in inducing tolerance to plants, but the mechanism of inducing tolerance against the insect varied among the PGPR used. For example, *Bacillus endophyticus* inoculation ameliorated the adverse effects of insect infestation by significantly increasing SA and ABA many folds higher than infested plants whereas, *Pseudomonas aeruginosa* ameliorated the infestation by increasing SA higher than the former strain. Salicylic acid is the integral part of signal transduction pathways initiating resistance to disease and infection [77–79]. Plant defense in response to microbial attack is controlled by signaling molecules including SA, JA and ethylene [80]. SA is an important director of pathogen stimulated systemic acquired resistance (SAR), whereas JA and ET are compulsory for rhizobacteria-mediated induced systemic resistance (ISR) [81]. Branch et al. [82] found that SA is a vital constituent of motioning the induced resistance to root-knot nematodes [83].

Plant phenolics comprises a wide array of secondary metabolites including flavonoids, Cinnamic acid, Kaempferol, Coumaric acid as well as salicylic acid synthesized to provide resistance to plants. Their number, type and concentration increase under insect attack [84] and appear to be stimulated following PGPR application. *Pseudomonas aeruginosa* produced both flavonoid glycoside and kaempferol in addition to coumaric acid whereas, *Bacillus endophyticus* had only myricitin and lack kaempferol and coumaric acid but on infestation both produced similar bioactive metabolites in plant. The chromatographic separation of leaf extract also revealed the presence of bands corresponding to Rf value of SA as well as phenolic compounds e.g., Kaempferol and coumaric acid thereby demonstrating the induction of ISR by PGPR in the inoculated plants infested with *S. litura.* Generally, the role of phenolic compounds in defence is related to their antibiotic, antinutritional or unpalatable properties. Besides their involvement in plant- animal or plant-microbe interactions; plant phenolics also play a key role as antioxidants and stress signaling [85–87]. Hammerschmidt [88] reported that phenolic metabolites are related to the resistance phenomenon of plants against their enemies.

The FTIR spectrum was used to identify the functional group of the active components based on the peak value in the region of infrared radiation. Production of alkynes and aldehyde in plants inoculated with *Bacillus endophyticus* and *Pseudomonas aeruginosa* on infestation with *S. litura* demonstrate the PGPR induced defense strategy against insects. Similar results reported by Panda and Khush [89] that chemical derived substances e.g., alkanes, aldehydes, ketones, waxes are involved in host-plant resistance to insects' function as a protective layer to save the plant. Whereas, Shavit [90] reported that inoculation of tomato plants with *P. fluorescens* WCS417r enhanced the performance of the phloem feeding insect *Bemicia tabaci.* Previously, the FTIR has been applied to classify the actual structure of certain plant secondary metabolites [91]. FTIR is one of the extensively used approaches to categorize the chemical ingredients and clarify the compounds structures [92]. The FTIR of the leaves extract revealed the presence of additional peaks of aldehyde in the FTIR of leaves of infested plants pretreated with PGPR. Chehab et al. [93] reported that aldehydes play a positive role in plant defense. Plants defend themselves from pathogens attack by producing secondary metabolites and proteins [94,95].

### **5. Conclusions**

The *Bacillus endophyticus* and *Pseudomonas aeruginosa* can be used to combat oxidative and osmotic stresses induced by *S. litura* infestation. Both the PGPRs combat insects induced adverse effects on plant growth and productivity through the production of phenolics, SA and ABA. *Bacillus endophyticus* was more effective in the improved defense strategy induction through the modulation of phytohormones and secondary metabolites. These PGPR are more effective under uninfested conditions and can be implicated as bioinoculant to endure the plants to cope better with insect infestation. Since there is alteration in the functional group and presence of aldehyde predominantly detected in plants treated with PGPR and infested with the insect armyworm. Further investigations using nuclear magnetic resonance (NMR) and liquid chromatography-mass spectrometry (LC-MS) are needed to unveil the secondary metabolites produced in PGPR inoculated plants versus uninoculated insect infested plants. Finally, an integrated approach of molecular mechanism of PGPR induced defense in plants against pests and parasites needs thorough investigation.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4395/10/6/778/s1, Table S1: Results of the statistical analysis (ANOVA) for the performed parameters.

**Author Contributions:** Conceptualization, A.B.; Methodology, B.K. and N.K.; Software, B.K. and N.K.; Validation, B.K., A.B. and N.K.; Formal Analysis, N.K.; Investigation, B.K. and A.B.; Resources, A.B.; Data Curation, N.K.; Writing—Original Draft Preparation, B.K.; Writing—Review & Editing, A.B. and N.K; Visualization, B.K.; Supervision, A.B.; Project Administration, A.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
