Systemic Resistance in Tomato against Meloidogyne incognita Induced by Plant Growth Promoting Rhizobacterium, Pseudomonas fluorescens

Resistance-related enzymatic activities due to selected strains of Pseudomonas fluorescens were assayed by split root bioassay in tomato under glass house conditions. Application of P. fluorescens as seedling root dip increased the level of peroxidase (PO), polyphenol oxidase (PPO) and phenylalanine ammonia lyase (PAL) in tomato roots indicated the systemic resistance induced by the bacterial formulations against root knot nematode, Meloidogyne incognita . Among the four P.fluorescens isolates tested alone and in consortium with Bacillus subtilis , Bbv 57, consortial application of P. fluorescens , Pf 128 with B. subtilis, Bbv 57 recorded the highest enzymatic activity in tomato roots compared to other strains either alone or in consortium. Results indicated that P. fluorescens was capable of inducing systemic resistance against M. incognita in tomato by the accumulation of the defense enzymes like PO, PPO and PAL.

Tomato (Lycopersicon esculentum Mill.) is one of the most important vegetables across the globe seriously affected by root-knot nematode, Meloidogyne incognita (Kofoid and White) Chitwood.Economic loss in India due to this nematode was estimated to be 27.21 per cent (Jain et al., 2007).Pseudomonas fluorescens, a promising rhizo bacterium was reported to have the ability to colonize the roots of wide range of crops (Kloepper et al., 1980) and induce systemic resistance against the plant pathogens by producing defense enzymes (Leeman et al., 1995).The defense enzymes were reported to reinforce the cell wall structure and cause biochemical and physiological changes in the plant system (Chen et al., 2000), which are directly inhibitory to nematodes (Paul and Kumar, 2003).P. fluorescens, in consortium with Bacillus subtilis was reported to be effective against root-knot nematode, M. incognita in tomato (Jonathan et al., 2009).Banana plants treated with P. fluorescens enhanced the production of defense enzymes which are responsible for induced systemic resistance and suppression of nematode infestation (Senthilkumar et al., 2008).In the present study, induction of defense enzymes by P. fluorescens against M. incognita infestation on tomato was studied by split root bioassay under glass house conditions.

Materials and Methods
Split root bioassays were performed for a better understanding of the mechanism of nematode suppression by the bacteria.In this method, a single root was split into equal halves and the treatments were given to one half of the root and the other half was maintained as control.About 30 days old healthy and uniform sized tomato cv.Co3 seedlings were uprooted carefully from the seedling pan and their root system was split into equal halves and one half of the root was dipped in 100 ml of the bacterial suspension (2.5 x 109 cfu/ml) for about 30 min.Other half of the root was maintained as control without any treatment.The seedlings were planted in a polybag containing 250 g of sterilized pot mixture in such a way that each half of the root system was planted in a separate polybag (Plate 1) apposed to each other.Carbofuran treated (1 kg a.i/ha) and a control plant were also maintained for comparison.Care was taken that the separated halves of the root system do not come into contact with each other.Watering was done using rose can in order to avoid cross contamination between the two separate root portions.Five days after planting, both the halves of the root system were inoculated with the second stage juveniles of M. incognita @ 250 nematodes per root system.The treatments were arranged in a completely randomized design (CRD) with ten treatments and three replications and seedlings were maintained for two months.
After two months, the plants were uprooted and about one gram of root sample was collected from the two split halves of the root system and homogenized in 1 ml of 0.1 M phosphate buffer (pH *Corresponding author email: meenaa5@rediffmail.com7.0) at 4°C for 15 min.and the supernatant was collected.It served as an enzyme source.Peroxidase, Polyphenol-oxidase and Phenylalanine ammonia lyase activities were analysed from the samples spectrophotometrically.Nematode incidence in the treated and control plants was recorded by taking gall index.The gall indices were graded on 0 to 5 scales (Taylor and Sasser, 1978).

Assay of Peroxidase (PO)
Reaction mixture consisted of 1.5 ml of 0.05 M pyrogallol, 0.5 ml of enzyme extract and 0.5 ml of one per cent H202.Reaction mixture was incubated at room temperature (28±2°C).Changes in absorbance at 420 nm were recorded at 30 sec intervals for 3 min.Enzyme activity was expressed as changes in the absorbance min -1 g -1 (Hammer schmidt et al., 1982).

Assay of Polyphenol oxidase (PPO)
Reaction mixture consisted of 200 μl of the enzyme extract and 1.5 ml of 0.1 M sodium phosphate buffer (pH 6.5).To start the reaction, 200 μl of 0.01 M catechol was added and the activity was expressed as changes in absorbance at 495 nm min -1 g -1 (Mayer et al., 1965).

Assay of Phenylalanine ammonia lyase (PAL)
Samples (1g) were homogenized in 3 ml of icecold 0.1 M sodium borate buffer (pH 7.0) containing 1.4 mM of 2-mercaptoethanol and 0.1 g of insoluble polyvinylpyrrolidine.The extract was filtered through cheese cloth and the filtrate was centrifuged at 16,000 g for 15 min.The supernatant was used as enzyme source.PAL activity was determined at the rate of conversion of L-phenylalanine to trans-cinnamic acid at 290 nm.Samples containing 0.4 ml of enzyme extract were incubated with 0.5 ml of 0.1M borate buffer, pH 8.8 and 0.5 ml of 12 mM L-phenylalanine in the same buffer for 30 min at 30° C. The amount of Transcinnamic acid synthesized was calculated.Enzyme activity was expressed as nmol trans -cinnamic acid min -1 g -1 (Dickerson et al., 1984).

Results and Discussion
In the split root bioassay, enhanced activity of peroxidase in tomato roots was observed in P. fluorescens treated root half than the control root half portion.Among the treated root portions, high level of PO activity was observed in the plants treated with consortium, Pf 128 + Bbv 57 (0.737 min -1 g -1

Treatment Peroxidase (Change in
Poly Phenol Oxidase (Change Phenylalanine Ammonia Lyase absorbance min -1 g -1 ) in absorbance min -1 g -1 ) (Change in cinnamic acid min -1 g -1 ) root).Its untreated root half portion also recorded the PO activity of 0.592 min -1 g -1 root.Per cent increase of PO activity of treated portion over untreated control portion was recorded as 24.49 per cent.It is followed Pfbv 22 + Bbv 57 treated root portion (0.688 min -1 g -1 root) with 25.55 per cent increase over its untreated root portion.
Lowest PO activity was recorded in carbofuran treated plants and control plants (Table 1).
Similarly, polyphenol oxidase activity was also observed to be higher in Pf 128 + Bbv 57 treated root half (0.088 min -1 g -1 root) over its untreated control (0.071min -1 g -1 root) with 23.94 per cent increase over control.It was followed by Pfbv 22 + Bbv 57 treated root half (0.087 min -1 g -1 root) which recorded 29.85 per cent increase over its control.PPO activity was in a negligible level in carbofuran treated and untreated control plants (Table 1).
Increased activity of PAL was observed in Pf 128 + Bbv 57 (0.519 min -1 g -1 root) treated root half over its untreated control (0.451) with significant increase of 28.38 per cent over control root portion.It was followed by Pfbv 22 + Bbv 57 applied root halves (0.458 min -1 g -1 root) with 29.75 per cent increase over its control root portion.Carbofuran treated and untreated control plants recorded the lowest PAL activities when compared to other treatments (Table 1).
A significant decrease in the nematode population in the soil and root ultimately resulted in the lowest gall index (1.3) in the Pf 128 + Bbv 57 treated plants followed by Pfbv 22 + Bbv 57 which recorded (1.4).In contrast the highest gall index (4.3)was recorded in the control.
This study was in accordance with the findings of Siddiqui and Shaukat (2004) who observed the application of bacterial cell suspension to one-half of the root system lowered the populations of rootknot nematode in non-bacterized nematode-treated sections indicating enhanced defense in the nonbacterized half.It is ascertained that fluorescent pseudomonads induce systemic resistance against root-knot nematode viz., a signal transduction pathway, which is independent of SA accumulation in roots.These results were also in confirmation with the findings of Niknam and Dhawan (2002), who documented P. fluorescens and B. subtilis induced systemic resistance in tomato against R. reniformis in split root bioassay.