Similarity Analysis of Fluorescent Pseudomonas from Apple and Pear Rhizosphere

Molecular markers have been found useful in assessing the genetic diversity in a precise manner, which is based on naturally occurring DNA polymorphism. Random Amplified Polymorphic DNA analysis was performed using four arbitary decamer primers to observe genetic homogeneity/ polymorphism among 10 isolates of fluorescent Pseudomonas spp. isolated from apple and pear rhizosphere from different locations of Mandi and Chamba districts. Out of 52 bands generated with 4 RAPD primers, 13.46% were monomorphic bands, while 86.33% were polymorphic bands. The result showed that the isolates of fluorescent Pseudomonas sp. were divided into 2 clusters by RAPD primers. Among all the combinations maximum similarity (90.48%) was found between PN-7-Cha and PN-2-San isolates of Chamba and Mandi districts. However, minimum similarity (36.7%) was observed in PN-11-San and PN-7-Cha isolates of Mandi and Chamba districts. This technique is highly beneficial for testing similarity analysis among fluorescent Pseudomonas spp. isolated from different locations.

Rapidly evolving technologies in molecular biology and genetics have provided new insights into the underlying mechanisms by which biocontrol agents' function in their natural environments to a degree not previously possible. The application of the polymerase chain reaction (PCR), in particular, have greatly facilitated genomic analyses of microorganisms, provide enhanced capability to characterize and classify strains, and facilitate research to assess the genetic diversity of populations (Louws et al., 1999).
The aim of the present study was to standardize simple, reproducible RAPD-PCR protocol for PGPR especially fluorescent Pseudomonas species growing in replant and normal sites of apple and *Corresponding author email : Sheetal.rana1987@gmail.com pear orchards of Mid-Himalayan ranges of Himachal Pradesh to check the genetic diversity of native fluorescent Pseudomonas species.

Collection of soil samples
Rhizospheric soil samples along with roots were collected from different plants of apple and pear from the five normal sites of Chamba and Mandi districts of Himachal Pradesh and also from the five sites of replant problem areas of these two districts.

Isolation, enumeration and identification
The media employed for the isolation of Pseudomonas spp. were nutrient agar (NA) and selective King's B medium supplemented with 3 antibiotics i.e., Penicillin-G (75,000 units/l), Cycloheximide (75 mg/l) and Novobiocin (45 mg/l). Plates were incubated at 28 ± 2ºC for 24 -48 h. Ten isolates were classified on the basis of colony characteristics such as size, colour, shape, texture and type of fluorescent pigment production. The most predominant Pseudomonas spp. isolates showing greenish/yellowish fluorescent pigments at 302 nm wavelength in BIO-RAD gel doc system XR were assumed to be fluorescent colonies.
Then they were characterized on the basis of morphological and biochemical tests as per their genera as prescribed in Bergey's Manual of Systematic Bacteriology. The pure culture of ten selected strains were maintained on the nutrient agar slants at 4ºC and were subcultured periodically on the same media at 28 ± 2ºC. They all were also maintained and preserved in 20% glycerol at -20ºC. All the further experiments were conducted after raising fresh cultures.

In vitro screening of bacterial isolates for plant growth promoting traits
All isolates were screened for the ability for phosphate solubilisation and siderophore HCN and ammonia production.
Available phosphorous (Olsen et al., 1954) was quantified. Bacterial cultures grown in PVK broth supplemented with 5.0 g/l tri -calcium phosphate (TCP) and solubilization of phosphorous was calculated using standard curve of KH2PO 4 (100-1000 μg/ml). Siderophore production in liquid culture of Pseudomonads in Chrome-azurol-S (CAS) broth was carried out and change in the colour of reaction mixture was observed from dark blue to orange or pink (Schwyn and Neilands, 1987).For estimation of hydrocyanic acid (HCN) production by Pseudomonas spp., a color change in the sodium picrate containing filter paper strip was observed from yellow to orange brown to dark brown (Bakker and Schippers, 1987). For ammonia production, the assay method of Lata & Saxena (2003) was followed. Pseudomonas isolates were grown in 5ml of peptone water in tubes at 28±2ºC for 4 days. After 4 days, 1ml of Nessler's reagent was added to each culture. Presence of very light brown color (+) indicates small amount of ammonia production and light brown (++) to orange brown color (++++) indicates large amount of ammonia production.

RAPD Analysis
RAPD analysis was performed on ten different isolates of fluorescent Pseudomonas. The DNA was extracted according to manufacturer's (Banglore GeNei's) specifications using DNA isolation kit. Eluted DNA was quantified spectrophotometrically at 260 nm and stored at -20oC for future use.
RAPD analysis were carried out in 25 ìl reaction, contained 25 ng of genomic DNA , 18.8 μl sterilised distilled water, 0.20 μl Taq DNA polymerase, 2.50 μl Taq buffer A, 1.50 μl dNTPs mixture, 1.0 μl random primer, and 1.0 μl genomic DNA. The PCR was carried out in a thermal cycler with a total of 45 cycles. Each cycle consisted of 1 minute denaturation at 94ºC, 1 minute annealing at 36ºC, and 2 minute extension at 72ºC. All the PCR samples were given 3 minutes pre-amplification at 94 ºC and 10 minutes post-amplification at 72 ºC, PCR products were analysed in 1.2% agarose gels stained with ethidium bromide and were visualized and photographed using a BIO-RAD (USA) Gel Documentation System. The primers used were All isolates were used for cluster analysis based on RAPD results. Each amplification band was scored as 1 (present) or 0 (absent) for all isolates. The isolates were clustered by the unweighted average pair group method (UPGMA). Differentiation between the isolates was done by observing readily discernible band patterns.

Results and Discussion
Pseudomonas spp, the most diverse, versatile and indigenous group of microorganisms may have wide applications in agriculture. The development of different strategies using a mixture of PGPR strain especially fluorescent Pseudomonas spp. would be an important emerging area in crop growth promotion, protection and also in establishment of newly planted trees on replant sites of apple and pear. It may reduce the losses caused by replant problem in economically important fruit crops especially apple. Hence the genetic diversity of indigenous fluorescent Pseudomonas species of those true species is very important. They all produced fluorescent pigment.
Biochemical studies showed that all the isolates were positive for gelatin liquification and lecithinase activity while four isolates i.e. AN-1-Cha, AN-3-Cha, PN-7-cha and PN-8-Cha were found negative for tween hydrolysis. Optimum growth was recorded at 250C. PN-7-Cha and PN-12-San showed growth at 4oC while all strains showed growth at 420C except one strain i.e. PR-2-San. Phosphorus is one of the major essential macronutrients for biological growth and development (Fernandez et al., 2007). The concentration of soluble phosphate in soil is usually very low. This pool of immediately available phosphate is extremely small and must be replenished regularly to meet plant requirements. Fluorescent Pseudomonas spp. are prevalent in the rhizosphere of plants (Lamanceau et al., 1995). Certain members of this group are called plant growth-promoting rhizobacteria (Schroth and Hancock, 1982) because they are able to promote plant growth through solubilization of inorganic phosphates present in soil and through protection of plants against diseases caused by phytopathogenic fungi.
RAPD-PCR is a genotypic identification and characterization system that has shown great specificity and sensitivity to define bacterial isolates. Marques et al. (2008) studied the phenotypic characteristics and genetic fingerprints of a collection of 120 bacterial strains, belonging to Pseudomonas syringae sensu lato group, P. viridiflava and reference bacteria were evaluated, The maximum solubilization of inorganic phosphorus and maximum release of ortho phosphate (Pi) in supernatant was shown by PR-2-San (355μg/ml) while minimum was showed by PN-2-San (195 μg/ml) shown in Table 1.
There are number of reports suggesting that plant species are capable of obtaining iron from some microbial siderophores (Park et al., 2005). Cline et al. (1984) demonstrated that the iron from the microbial hydroxamates siderophores may become available to plants both in nutrient solution and in soil. Siderophores from fluorescent Pseudomonas species have also been implicated in iron uptake by tomato plant (Dass et al., 1986).
Microbial production of HCN has been reported as an important antifungal trait to control root with the aim of species identification. Babalola et al. (2002) analysed three rhizosphere bacteria, Pseudomonas sp., Enterobacter sakazakii and Klebsiella oxytoca, for genetic variation. Cluster analysis indicated that E. sakazakii and K. oxytoca are the most closely related of the three.
Total 52 scoreable bands were amplified with 4 primers of these 45 bands were found to be polymorphic (86.53%) and 7 monomorphic bands (13.46%) were found shown in Fig 1 & 2. The similarity coefficient value ranged from 0.36-0.90. Among all the combinations, maximum similarity was found between PN-7-Cha and PN-2-San isolates of Chamba and Mandi district (90.48%) followed by 84.4% between PN-13-San and PR-2-

Conclusion
The present RAPD profiles of phylogenetic analysis suggest that different isolates of Pseudomonas have significant inter and intra species differences and can be of great significance for their exploitation as plant growth promoting bioagents.