Antagonistic Effect of Eight Sri Lankan Isolates of Pseudomonas fluorescens on, Meloidogyne incognita in Tomato, Lycopersicon esculentum

The study was conducted to determine the efficacy of Pseudomonas fluorescens isolates collected from eight locations in the Central Province of Sri Lanka against Meloidogyne incognita in tomato. Isolates were tested under laboratory conditions to determine the efficacy on egg hatchability and mortality of second stage juveniles. A planthouse experiment was conducted using potted tomato plants to determine the potential of P. fluorescens isolates and effective application technique. All tested isolates have significantly inhibited egg hatchability and increased the juvenile mortality after 72 hours. P. fluorescens isolate from Kangkung field in Pallekelle (PK) and tomato field in Udispattuwa (UT I) recorded 95% and 95.5% inhibition of egg hatchability after 72 hours. P. fluorescens isolates collected from tomato fields in Bopane (BT II) and Udispattuwa (UT II) and from Kangkung field in Pallekelle recorded the higher mortality of second stage juveniles 93%, 87% and 83.3% respectively. The highest reduction in the root knots (96.8%, 96.3%), egg masses (98.5%, 98.2%) and lower root galling index (1 and 1) were recorded in tomato plants treated as soil drench with UT II and PK isolates respectively.The root dipping technique gave higher reduction in the number of root knots (47.4%), egg masses (44.9%) and lower root galling index (3.75) were recorded from BT II, UT II and tomato fields in Nugethenna (NT) isolates respectively. UT II and PK found to be the most effective isolates and most effective application technique determined as soil drenching ten days after transplanting under plant house conditions.

induction of systemic resistance to minimize the populations of plant parasitic nematodes [11]. Tomato is an attractive cash crop that provides a source of income to the rural population in the central region of Sri Lanka. Nematodes of the genus Meloidogyne, is known to cause more than 50% crop losses to tomato [4]. This study was conducted to determine the antagonistic properties of eight Sri Lankan isolates of Pseudomonas fluorescens against Meloidogyne incognita in Tomato (L. esculentum) in vitro and in vivo and to select the effective P. fluorescens isolate(s) for the control the Meloidogyne spp.

II. METHODOLOGY 2.1. Preparation of water cultures of P. fluorescens
Soil samples collected from a Kang kung crop from Pallekelle, tomato crops from Nugethenna (one sample), Bopane (2 samples) and Udhispaththuwa (3 samples) and from a maize crop from Sooriyawewa were used for the experiments. One gram each of the above 8 samples was diluted in 100 ml of sterilized phosphate buffer solution and shake for 2 h. A series of 10-fold dilutions was prepared by repeating 6 times under aseptic condition. The diluted soil supernatants (0.1 ml) was spread on king's medium B agar plates and incubated at 28 °C for 48 h in an incubator. Culture plates were observed under ultraviolet trans-illuminator at 366 nm for few seconds and colonies with green fluorescence were streaked on King's medium B agar plates to get pure colonies. Well-grown 48 h old uncontaminated single colonies were used to prepare water cultures of P. fluorescens and cell density were estimated for all the isolates.

Effect of P. fluorescens on egg hatchability of M.
incognita Sterilized Petri dishes were filled with the eight P. fluorescens suspensions at the rate of one isolate per five Petri dishes. Similarly five Petri dishes were filled distilled water (as control). Egg masses of M. incognita were placed on the micro sieve (75-µm aperture, 20 mm diameter) at the rate of 10 egg mass per sieve. These micro sieves were placed in the Petri dishes to touch the egg masses with P. fluorescens isolates or water. The experimental set-up was kept at room temperature and number of emerged juveniles was counted at 24, 48 and 72 h after inoculation. At the end of the experiment, the egg masses were treated with 1% sodium hypochlorite to dissolve the gelatin matrix around the eggs and the unhatched eggs were counted. Percentage egg hatchability: (mean number of emerged juveniles in each treatment / Total number of juveniles and eggs in treatment) x 100 were calculated.The treatments were replicated 5 times in a Randomized Complete Block Design.

Effect of P. fluorescens on mortality of juvenile M.
incognita Forty, sterilized 60 mm diameter watch glasses were filled separately with 3 ml of P. fluorescens isolates and similarly five watch glasses were filled with 3 ml distilled water. Newly hatched second-stage juveniles of M. incognita were added to the bacteria suspensions at the rate of ten per watch glass . After 24, 48 and 72 h the numbers of dead juveniles were counted under a stereomicroscope. The treatments were replicated 5 times in a Randomized Complete Block Design. Percentage mortality: (Mean number of dead juveniles in the treatment / Total number of juveniles in treatment) x 100 were calculated.

Efficacy of P. fluorescens isolates for the control of
M. incognita on tomato A pot experiment was conducted in the plant house using the tomato variety KWR, a variety susceptible to Meloidogyne spp., to determine the efficacy of P. fluorescens isolates on the root damage using two application techniques.

Soil drenching of P. fluorescens isolates to potted tomato plants
Two-week-old tomato plants potted in 15 cm dia. plastic pots at the rate of 1 plant per pot were used for the experiment. About 2 cm of top soil layer was removed near the root system and the soil were drench with 50 ml P. fluorescens isolates separately. In addition, two sets of plants were treated with distilled water as an untreated control. The plants were then covered with 2 cm sterilized soil layer. After 24 h each pot was inoculated with 1,000 juveniles except one set of plants treated with distilled water. The experiment was arranged in a Randomize Complete Block design with 5 replicates.

Root dipping of tomato plants with P.
fluorescens isolates Roots of another set of tomato plants were dipped separately with P. fluorescens isolates for one minute before planting in sterilized potting media. Another set of plants were treated only with the nematodes + distilled water. The experiment was arranged in Randomize Complete Block Design with 5 replicates. Sixty days after inoculation of nematodes, tomato plants were uprooted and the nematode damage was assessed by the number of egg masses per root system. The intensity of root damage was determined through the diagrammatic root knot scoring chart (John and Sam, 1980). Plant height, shoot fresh weight, shoot dry weight, root fresh weight were also measured.

Data analysis
Proc Catmod was performed to check for normality and homogeneity, if the results were significant, numerical data were square root-transformed prior to analysis. The data were analyzed using analysis of variance and treatment means were compared by Duncan's Multiple Range Test at P<0.05 level. Data were subjected to analysis using Statistical Analysis Software (SAS) package version 8.2.

III. RESULTS
The densities of P. fluorescens isolates in the 8 samples ranged from 1.01 x 10 8 to 2.00 × 10 8 specifying that the bacterial colonies falled within a narrow range suitable for pathogenicity estimates (Table 1).  Table 2). The lowest egg hatchability was observed in PK (Pallekele -Kangkung), NT (Nugethenna -Tomato) and UT 1 (Udhispaththuwa -Tomato) isolates

Effect of P. fluorescens on mortality of juvenile M. incognita
The mortality of M. incognita juveniles has shown significant effect compared to the controls from 24 to 72 h after treatment at chi-square < 0.05 level. The suppressive activity of P. fluorescens increases gradually with the increased exposure time. BT II (Udispattuwa isolate 2 from tomato crop) exhibited the highest mortality of juveniles after 72 h (Table 3).  We observed that the tested P. fluoresces isolates and application technique significantly influenced on the root knots per root system and the number of egg masses per root; chi-square < 0.05 probability level according to Proc Catmod and significant difference between application methods at P< 0.05 level of probability according to Duncan's multiple range test except the NT and po sitive control (Fig 1 and 2). P. fluorescens is olates from PK (Pallekele/ Kangkung crop); UT II (Udhispaththuwa/ Tomato crop 2) and BT II (Bopane /tomato crop 2) recorded a significantly low number of root knots when the isolated were soil drenched. It was observed that root knot count per root significantly influenced by the application technique; and soil drenching as the most effective application technique for all tested isolates.

Effect of P. fluorescens on root knot score on tomato:
The mean value of root knot scores in P. fluorescens isolates. Moreover, there is significant difference between control and all other isolates in soil drench techniques at chi-square < 0.05 probability level of according to Proc Catmod. There is no significant difference between two application techniques at P< 0.05 level of probability according to Duncan's multiple range test. According to table 5, Bacteria isolates of PK, UT II and shows the significant low root knot score (score =1) compared to control treatment in soil drenching technique. In root dipping technique, NT isolate showed the low mean number of root knot score (score =3.75).

Fig.4: Root fresh weight in different P. fluorescesisolations applied with two different soil application techniques
Fresh root weight was significantly lower in bacterial isolates treated treatments than nematode present treatment at P< 0.05 level of probability. Highest fresh root weight was recorded in nematode present treatment in both soil application techniques. The lowest fresh root weight was recorded in UT II (5 g) bacterial isolate treatment in soil drenching technique and BT II (9 g) bacteria isolate treatment in root dipping technique respectively ( Figure 5).Tomato plants treated with the isolates as soil drench technique recorded the lowest root fresh weight than root dipping technique.

Effect of P. fluorescens on plant growth parameters:
Tomato plants treated with the different isolates as soil drenching and root dipping, shows variations in shoot length, shoot fresh weight and shoot dry weight. There were no significant difference among treatments in dry shoot weight at P< 0.05 level of probability according to Duncan's multiple range test ( Table 5 and 6). Root knot nematodes are soil pathogen. They directly attack to the root system of plants and main symptoms were occurred in the below ground parts of the plant. Secondary symptoms will have occurred in the above ground plant parts. Alsothe plants were maintained under plant house conditions and because of that, the effect of bacterial treatments on above ground plant parameters were not clearly expressed.   DISCUSSION Some reports says that, P. fluorescens has nematicidal activity to root knot nematode, M. javanica, juveniles and nematicidal activity against potato cyst nematode, Globodera rostochiensis eggs as the same might contribute as mechanism of M. incognita mortality and egg hatchability [9]. P. fluorescens can produce large number of toxic secondary metabolites such as; phenazine, indole, compounds, phenyl-pyrroles and pterines [1]. These metabolites might also be toxic to M. incognita juveniles [1]. The production of the metabolite 2, 4diacetylphloroglucinol (2, 4-DAPG) by P. fluorescens strain CHA0 induced mortality in juvenile of root-knot nematodes [13]. Some P. fluorescens strains are known to contain 1aminocyclopropane-1carboxylic acid which inhibits ethylene production in roots and henceminimizes colonization of root knot nematodes and root knot development [9]. Also reported that application of P. fluorescens bacteria led to reduce the number of egg masses of nematodes [4]. Some articles described DAPG produced form P. fluorescens strains, reduced the mobility and survival the second stage juveniles, the infective stages of some plantparasitic nematodes [10]. The production of this antibiotic in the rhizosphere of plants suppress nematode penetration of roots [12]. In addition, it is known that DAPG affect root morphology and such changes in root architecture that may alter the number of available infection sites and, therefore, lead to a complex response with regards to nematode suppression [10]. The percentage of gall formation and root gall index found to decrease when P. fluorescens were introduced prior to M. incognita infestationon tomato plants [1]. Similar results were observed that the highest reduction in the numbers of second-stage juveniles in soil, host root galls and egg mass indices when P. fluorescens was drenched before planting [5]. However, it was reported that, strains CHA805 and CHA89 had no significant impact on nematode population densities in soil and rootknots in tomato and soybean crops [10].

V. CONCLUSION
The Pseudomonas fluorescens isolated from tomato rhizosphere from Nugethenna (NT) and Udhispaththuwa (UT I) effectively minimize egg hatchability of Meloidogyne incognita. P fluorescens isolated from tomato rhizosphere from Bopane (BT II) and Udhispaththuwa (UT II) effectively controlled juveniles of M. incognita in tomato. The tomato plants treated with P. fluorescens isolated from Kang Kung from Pallekele (PK) and UT II had lower number of root knots and egg masses. These experiments indicated that the tomato rhizosphere from Udhispaththuwa and Kang-Kung rhizosphere from Pallekele contained effective P. fluorescens isolates that can be used for the management of M. incognita. The effective application technique determined as soil drenching ten days after transplanting under plant house conditions .