Virulent strain of F. oxysporum isolated from diseased tomato plants growing in vegetable garden of University of the Punjab, Lahore was cultured on potato dextrose agar (PDA difco) for 10 days. Conidia were harvested by gentle scraping in sterile water and pathogen inoculum was prepared by adjusting the concentration to 10⁵conidia/ml using haemocytometer. P. aeruginosa (PM12) was grown on LB broth medium (100 ml) for 24 h at 35°C. For collecting extracellular metabolites culture was pelleted by centrifugation at 4000 g for 15 min, and the supernatant obtained was processed for ISR assay. Intracellular metabolites were extracted using sonication. Bacterial cell lysis was performed six times through sonication at resonance amplitude for 15 s at 4°C.

This study was performed to identify the involvement of intracellular metabolites and cell-free culture filtrates (CFCF) of P. aeruginosa for inducing systemic resistance against fungal wilt in tomato. Two-weeks old seedlings of Fusarium wilt susceptible tomato variety “Rio-Grande” seeded in sterilized pot media. In total, 50 ml of both extracts (intracellular and extracellular) were supplied to the allocated plastic pots containing 0.5 kg sterilized soil and after a period of 3 days these pots were inoculated with 50 ml of pathogen @ 10⁵conidia/ml. Positive control consisted of P. aeruginosa PM12 suspension made in water by adjusting the concentration to 10⁴ cfu/ml. Fifty milli liter of sterile distilled water was provided to the pots designated as untreated control. All the pots were incubated for 14 days under greenhouse conditions. There were ten replicates against each treatment and experiment was conducted twice. To determine the disease index (DI), wilting was scored based on the criteria developed by Epp (1987) (0 = no wilt symptoms; 1 = less than 25% of the plant turned yellow; 2 = yellowing and browning covered nearly 50% of plant; 3 = whole plant turned brown and died). The equation described by Cachinero et al., 2002 was used to calculate the DI.

where, ni = the number of tomato plants with wilt symptoms, si = value of the symptom score, N = the total number of tested plants, and S = the highest value of the symptom score.

Methodology proposed by Sumayo et al. (2013) was used to isolate bioactive compound (s) from extracellular metabolites of P. aeruginosa (PM12). Extracellular metabolites of P. aeruginosa PM12 were obtained as described previously and extracted twice with double volumes of organic solvents such as ethyl acetate, chloroform, n-hexane and n butanol. All the organic extracts were dried in a rotary evaporator at 50°C, mixed in 10% DMSO and subjected to ISR experimentation.

Bacterial metabolite exhibiting highest ISR activity was further fractioned through silica gel column chromatography. Column was washed with double volumes of methanol and ethyl acetate. The extract was fractioned using stepwise elution method with increased concentration of methanol in ethyl acetate. Obtained eluates were evaporated and mixed in 10% DMSO and checked for ISR activity in a test tube assay. Negative control received pathogen inoculum with 10% DMSO. Seeds of the variety “Rio-Grande” were surface sterilized with 1% NaOCl and sown in culture tubes containing Murashige and Skoog (MS) medium. Tubes were incubated in a growth chamber at 25°C for seedling development. Seedlings were then provided with different bioactive fractions for ISR activity separately. After 2 days of seedlings treatment, 10 μl of pathogen inoculum was provided to each tube @ 10⁵condia/ml. After 1 week of incubation data was recorded regarding DI. Experiment was repeated twice and there were ten replicates for each treatment.

Induced systemic resistance active sub-fraction obtained from silica gel chromatography was further subjected to instrumental analysis for identification of potential ISR determinant/s. This study was executed in Agilent GC/MS apparatus including capillary column (0.25 ID × 30 M × 0.25 μM film thicknesses) in which electron ionization was used as ion source. Helium with flow rate of 1.0 ml/min was supplied as carrier gas. Column temperature of apparatus was maintained at 30°C for 3 min which was increased at 50°C/min to 180°C and by 40°C/min to 200°C.

Another independent experiment was performed to screen ISR active biochemicals from P. aeruginosa. Biochemicals found in ISR active sub-fraction were purchased from the market. Three concentrations, i.e., 0.1, 1.0 and 10 mM of each of the pure biochemicals were applied as soil drench method, application of inoculum (bacterial/pathogen origin) to the soil surrounding the roots of transplanted seedlings of tomatoes, at the rate of 50 ml/pot (Algam et al., 2005). Data regarding DI was taken as described earlier after 15 days of treatment applications.

It was found in the previous experiment that HMB acted as the most active compound involved in elicitation of ISR in tested tomato plants therefore another independent experiment was carried out to study metabolic transitions brought about in the tomato plants under the influence of HMB elicitor.

The data was analyzed by performing ANOVA, and the significance of difference between the treatments was determined by DNMRT at P < 0.05 using software DSASTAT (Onofri, 2007).

Extracellular metabolites of P. aeruginosa (PM12) were found to be actively involved in suppressing Fusarium wilt of tomato under greenhouse conditions (Table 1). Significant reductions in the DI up to 73% were recorded in case of the extracellular metabolites that was almost equal to the efficacy of live cells, i.e., 75.86%. Intracellular metabolites were not found capable in suppressing the disease to a significant level. Resultantly, it was concluded that extracellular metabolites are involved in disease suppression and contains ISR determinant/s (Figure 1).

In this experiment extracellular metabolites/CFCF of P. aeruginosa PM12 were examined for the presence of ISR determinant/s. Extraction of extracellular metabolites was done by using different organic solvents and then applied to the plants as soil drench method and was challenged with the pathogen afterward. Compound/s present in ethyl acetate fraction of extracellular metabolites significantly reduced wilt disease in tomato plants when observed on visual grounds (Figure 2).

Ethyl acetate fraction of extracellular metabolites exhibiting maximum ISR activity was further processed via column chromatography using step wise elution method and ISR experiment was performed in a test tube assay. Sub-fraction showing maximum disease suppression was then analyzed through GC/MS. Chromatogram obtained from GC/MS analyses showed three peaks that were identified as 3-hydroxy-5-methoxy benzene methanol (HMB), eugenol and tyrosine, respectively (Figure 3).

Biochemicals obtained were again tested for ISR activity in their pure form. The ISR bioassay showed that only HMB significantly (P ≤ 0.05) reduced disease severity to 76.25% at 10 mM concentration. Whereas eugenol and tyrosine were not found efficient in reducing disease severity at the same concentration (Figure 4).

This study explored metabolic transitions observed in the tomato variety “Rio-Grande” under the stimulus of ISR elicitor (HMB) using GC/MS analysis. Whole metabolome of plants inoculated with HMB and pathogen in either combination were studied to elucidate ISR mechanism. In four different treatments, 94 metabolites were detected some of which were up regulated and some were down regulated as shown by different color schemes in heat map (Figure 5). Metabolite values obtained using Mzmine software were log2-transformed (Steinfath et al., 2008). Heat map was generated using online ClustVis tool with row wise scaling and correlation-based clustering. Most pronounced changes were depicted in metabolite levels of the plants receiving elicitor + pathogen making plants more resistant against pathogen attack by timely accumulation of defense related chemicals (Figure 5). Changes brought about in central metabolites under the stimulus of different treatments were normalized to respective control and were expressed in fold change values. Most metabolic fluctuations were documented in primary and secondary metabolism, signaling and defense pathways. Treatment receiving elicitor and pathogen showed variation in primary metabolism with different intensities. Upregulation of various metabolites involved in phenylpropanoid pathway was documented in case of the elicitor, e.g., 4-hydroxybenzene, cinnamate and tryptophan were increased to 3.19-, 2.31-, and 1.24-folds, respectively (Figure 5). Similarly plants treated with elicitor and pathogen significantly enhanced levels of most of the metabolites taking part in amino acid metabolism and TCA pathway (Figure 5). These findings illustrate that synergistic effect of the elicitor and the pathogen in inducing systemic resistance was more pronounced due to the active participation of the pathogen in some cases or elicitor in other.

Plants treated with the elicitor (HMB) showed enhanced levels of fructose, glucose and sucrose to 3.4-, 3.1-, and 1.2-folds, respectively. Whereas concentration of trehalose was found to be 0.9-fold less as compared to the untreated control (Figure 5).

In case of phosphorylated metabolites there was an increasing trend in the concentration of fructose-6- phosphate, glucose-6-phosphate and myo-inositol-phosphate to 3.9-, 3.3-, and 1.9-folds, respectively. Whereas concentration of glycerol-3-phosphate (0.9-fold) was decreased to 0.9-fold in the presence of the elicitor in comparison to the untreated control.

Plants treated with the elicitor showed increased levels of organic acids including α ketoglutarate (2.7-fold), malate (2.8-fold), oxaloacetate (2.3-fold), citrate (2.1-fold) and succinate (1.4-fold). Whereas low levels of fumarate (0.4-fold) and cis- aconitate (0.3-fold) were recorded in comparison to the untreated control.

Elicitor treated plants also showed raised quantities of polyamines like picolinic acid (7.7-fold), pipecolic acid (4.7-fold), putrescine (2.8-fold), and spermidine (1.4-fold).

Amino acids like arginine, glutamine and proline levels were significantly increased to 3.04, 2.7 and 2.32-folds in case of the elicitor whereas leucine, tyrosine and threonine were decreased in comparison to the untreated control (Figure 5). Methionine acts as a precursor for ethylene biosynthesis through S-adenosyl methionine (SAM) (Bleecker and Kende, 2000). Methionine levels were significantly raised in plants under the influence of elicitor and elicitor + pathogen treatments.

Levels of salicylic acid were significantly upregulated in all the treatments in comparison to the untreated control. Plants treated with the elicitor and the pathogen showed increased levels of phenylalanine to 2.8-folds whereas application of the elicitor resulted in an increase of 1.2-folds as compared to the non-treated control. Metabolic pathways were drawn to show transitions in various metabolites in the presence of elicitor HMB (Figures 6–8). Metabolites belonging to the phenylpropanoid, glycolysis, photosynthesis and signal transduction pathways were significantly increased by the elicitor (HMB) in treated tomato plants.

Alterations in the metabolites of the plants receiving elicitor and elicitor + pathogen revealed that HMB elicitor from P. aeruginosa is responsible for inducing systemic resistance against Fusarium wilt in tomato.

Recent attempts in exploiting non-pathogenic rhizospheric microbes in biocontrol programs have shown their involvement as antagonists and plant defense stimulators. However, the exact nature of biochemicals imparting systemic resistance in plants against diseases remains poorly understood. This research was performed to isolate potential ISR determinant/s from P. aeruginosa (PM12) against F. oxysporum f. sp. lycopersici in tomato plant.

Pseudomonas aeruginosa PM12 is a non-pathogenic strain that was isolated from tomato rhizosphere. To avoid the use of whole bacterium as it is categorized as opportunistic pathogen we focused on the signal compound/s involved in eliciting defense mechanisms of the plant and identified the ISR determinant from extracellular metabolites of P. aeruginosa PM12. Moreover opportunistic pathogens are microbes that are capable of causing disease only in people who are immunocompromised or are otherwise especially susceptible. A critical issue concerns the pathogenic strains proposed for registration as biopesticides, since these strains are typically isolated from the environment, for example agricultural fields, rather than as clinical specimens. As such, these strains have no history of actually causing disease and may not be able to do so (GPO, 1999).

Extracellular metabolites and live cells of P. aeruginosa PM12 showed significant (P ≤ 0.05) protection against Fusarium wilt whereas the intracellular metabolites were unable to provide any considerable control (Table 1). The ethyl acetate fraction of extracellular metabolites of the bacterium exhibited maximum ISR activity and was sub fractioned using column chromatography. The active sub fraction was then analyzed through GC/MS. The chromatogram obtained identified three compounds, i.e., HMB, tyrosine and eugenol. Reconfirmation of active biochemical was achieved using pure compounds which revealed HMB as the most capable component in mitigating Fusarium wilt in tomato.

The elicitor compound, HMB, isolated from P. aeruginosa (PM12), is not yet reported for its biological activity against Fusarium wilt so this is the first finding describing its involvement in systemic resistance in tomato. Colonization of plant roots with P. fluorescens enhanced plant defenses against pathogen by sensitizing the plant through signal compounds (Verhagen et al., 2004). Literature showed the use of benzene in making pesticides, detergents, drugs, dyes, rubbers, explosives and lubricants (Ceresana, 2014). Furthermore antifungal and antimicrobial activity of poly substituted benzene derivatives has been reported. The substituent attached to the benzene ring and its position affects the activity of the compound against pathogen (Uyanik et al., 2009). Similarly elicitor benzothiazole isolated from Pseudomonas sp. has shown inhibition of mycelial growth and germination from ascospores and sclerotia of Sclerotinia sclerotiorum (Fernando et al., 2005). Aldehydes, ketones and benzenes from Bacillus amyloliquefaciens have been known to restrict mycelial growth and spore germination of F. oxysporum (Yuan et al., 2012). Bacterial volatile compounds help in maintaining plant health through ISR and production of antifungal compounds (Weisskopf, 2013). As benzene is involved in protection of plants against different pests so it is concluded that benzene is the actual molecule in elicitor compound HMB for imparting resistance against FOL in tomato.

A considerable diversity of the elicitors involved in ISR has been observed in case of Pseudomonads. They include the building blocks of bacterial cells as well as extracellular compounds synthesized by the microbes (Jankiewicz and Kol-tonowicz, 2012). In current investigation HMB was discovered as an ISR determinant from P. aeruginosa PM12. Nowadays awareness regarding concept of immunity development in plants by the use of beneficial microbes is gaining momentum. In conventional agriculture system the identification of MAMPs from beneficial microbes and their use in formulation stimulates resistance in plants against pest attack.

Use of biocontrol agents in elicitation of resistance in plants is gaining importance these days. Some strains of Pseudomonas are clearly potent inducers of systemic resistance in plants (Van Wees et al., 1999; Meena et al., 2000).

Biological inducers have been known to elicit metabolites involved in different pathways like pentose pathway, signaling transduction and tricarboxylic acid cyclic, etc. (Verhagen et al., 2004; Shoresh and Harman, 2008; Damodaran et al., 2010; Brotman et al., 2012). In second phase of this study heat map was developed to show variations in metabolites under the influence of HMB and accentuated in physiological pathways. In this investigation upregulation of metabolites involved in phenylpropanoid pathway may lead to resistance development in tomato plants treated with the elicitor HMB.

A significant decrease in fumaric acid was recorded in the elicitor and elicitor + pathogen treated plants. These results are in connection with the findings of Doornbos et al. (2009) who inoculated Arabidopsis plants with the PGP and ISR-inducing bacterium WCS358r and observed decrease in intermediates of Krebs cycle that showed an increased demand for carbon respiration.

Brotman et al. (2012) reported that putrescine induction by Trichoderma spp. was related to growth promotion in Arabidopsis thaliana. Polyamines were also involved in various defense responses (Tonon et al., 2004) similarly in this study upregulation of putrescine was noted.

In plant microbe interactions, sugars serve as signaling molecules for resistance induction against the pathogens and this resistance is termed as “sweet immunity” (Bolouri Moghaddam and Van den Ende, 2013). Like sucrose is involved in activating plant immune responses against pathogens (Tauzin and Giardina, 2014). Sucrose is known to trigger the production of isoflavonoids against F. oxysporum in lupine (Morkunas et al., 2011). Trehalose stimulates the activity of phenyl ammonia lyase and peroxidase against powdery mildew in wheat (Reignault et al., 2001). Fructose, sucrose and glucose elicited PR-protein transcripts in tobacco in a SA-independent pathway (Herbers et al., 1996). Moreover Kawano and Shimamoto (2013) and Yamaguchi et al. (2013) found that chitin is responsible for stimulation of Mitogen-Activated Protein Kinase (MAPK) cascade, lignin and phytoalexins production in rice. In this study significant increase in quantities of some carbohydrates like fructose, glucose, mannose, etc., were also documented that are reportedly involve in plant growth and development.

Salicylic acid is involved in activation of defense responses in plants after challenging with a pathogen (Klessig et al., 2000). In a study enhanced levels of salicylic acid were documented when plants were challenged with pathogen or with P. fluorescens (Kumar and Sharma, 2013). Our results also showed upregulation of salicylic acid in the presence of the bacterial elicitor HMB.

Glutamate levels were significantly raised in treatments receiving the elicitor or elicitor and pathogen in combination. These results are in line with findings of Brotman et al. (2012). Glutamate acts as signaling compound in nitrogen signaling pathway (Forde and Lea, 2007).

Biochemicals like polyamines are involved in different physiological processes of the plants (Tonon et al., 2004). In this study plants primed with the elicitor (HMB) showed significant increase in polyamine levels. Likewise an increasing trend was also observed in organic acids in the HMB + pathogen treated plants. Shikimate pathway is stimulated by the synthesis of aromatic compounds which is responsible for defense response of plants (Tzin and Galili, 2010). Moreover shikimic acid concentration was also found to be enhanced under the effect of the bacterial elicitor HMB. Shikimic acid and malonic acid pathways are involved in phenolics synthesis leading to plant defense against pathogens. Phenolics include a variety of defense related compounds like anthocyanins, phytoalexins, flavonoids, furanocoumarins tannins and lignin (Freeman and Beattie, 2008). Present study illustrated the significance of metabolic dynamics engaged in defense mechanisms of the tomato plants infected with F. oxysporum ensured triggering of ISR by the bacterial elicitor HMB.

Phenylpropanoids like lignin, phytoalexins, flavonoids, tannins, etc., are the secondary metabolites of the plants produced under stress conditions (Vogt, 2010). They provide resistance in plants against phytopathogens through accumulation of lignin that acts as a barrier for penetration in plant tissues. Enzymes involved in phenylpropanoid pathways viz; PPO, PO and PAL impart rigidity and mechanical strength to the plant cell walls through production of lignin metabolites that act as barrier for invading fungal pathogens (Mohammadi and Kazemi, 2002; Anjum et al., 2012). Metabolic dynamics engaged in defense mechanisms of the tomato plants infected with F. oxysporum ensured elicitation of ISR by the bacterial elicitor HMB. Enhanced metabolite upregulation under influence of the elicitor + pathogen treatment may be due to the synergistic effect of ISR and SAR that may lead to better cope with the stress condition of the plant by activation of defense machinery.