Comparative Effect of Pseudomonas aeruginosa, Pseudomonas fluorescens and Pseudomonas putida on the Growth of Replanted Apple

Fluorescent Pseudomonas species have a number of traits like antifungal, siderophores, phosphate solubilization, lytic enzymes, HCN, ammonia and plant growth regulators that allow them to act as plant growth promoting and biocontrol agents. They competitively colonize plant roots, play important role in enhancing the growth of plants and in plant disease control. These may be used for soil improvement and to increase soil fertility hence for management of replant problem of apple in Himachal Pradesh. To ensure the sustained availability of PGP and biocontrol agent’s in soil formulation development protocol has to be standardized. On the basis of different PGPR activities three indigenous strains viz., Pseudomonas aeruginosa C, Pseudomonas fluorescens M and Pseudomonas putida L were selected for field studies. In all the treatments with individual (L, M and C) and consortia strains (L+M, L+C, M+C, L+M+C) there was an 8.0 to 86.5 per cent increase in plant height as compared to control. The performance of replanted apple was much better in terms of root colonization capacity, plant establishment and increase in plant growth in terms of plant height, number of nodes and branches over their respective control after fifteen months of plantation.

Apple (Malus domestica Borkh.) is one of the most important fruit crop grown and consumed all around the world and has been distributed almost across the whole temperate region in the Northern and Southern hemispheres 1 .
Replant problem refers to the poor growth of replanted young trees on the old sites. It is distributed worldwide and is often encountered in establishing new orchards 2 . The disease is a complex syndrome that reduces growth, survival and yield of replanted tree. Replant problem is caused by biotic and abiotic factors 3 . The biotic factors includes the rhizosphere microflora (bacteria, fungi, actinomycetes, nematodes and their interactions) and abiotic factors includes phytotoxins, nutrient imbalance, low or high pH, soil structure and damage, and lack of excess of moisture 4 .
Fluorescent Pseudomonas species are the most diverse and versatile group of plant growth promoting rhizobacteria. Their potential to synthesize different secondary metabolites with diverse biological activities is the important function of soil fertility and sustainability of crops 5 . The integration of their important traits like production of antifungal antibiotics, iron chelating siderophores, lytic enzymes, plant growth regulators, phosphate solubilization, ammonia and HCN production with ecological fitness of the strains will be prerequisite for designing useful, efficient and effective novel bioagent. The large scale application of indigenous plant growth promoting fluorescent Pseudomonas sp. may be able to manage replant problem of fruit crops especially apple.
The main objective of this research was to exploit the indigenous potential fluorescent Pseudomonas strains viz., P. aeruginosa, P. fluorescens and P. putida in individual and consortial combination to act as biofertilizer which can become one of the most promising biotechnologies to solve apple replant disease and can also improve production with low inputs in fertilizer. The aim of the study was to select more effective combination of fluorescent Pseudomonas sp. which can be used for biofertilizer development for management of apple replant problem.

Isolation of fluorescent Pseudomonas sp. from apple rhizosphere
The fluorescent Pseudomonas sp. was isolated from the rhizosphere of apple plants in normal and replant sites of Shimla district (Himachal Pradesh). The rhizosphere soil loosely adhering to apple roots were gently teased out with small root pieces in polythene bags and immediately transported to laboratory under cold conditions (4ºC) for further process. The serial dilution agar plate method was used to isolate Pseudomonas sp. on King's B medium 6 . The composition of the medium was (g/l -1 ): Peptone, 20.0; K 2 HPO 4 , 1.5; MgSO 4 .7H 2 O, 1.5; Glycerol, 15.0 ml. All the twelve isolates from apple rhizosphere were morphologically, physiologically and biochemically characterized so as to select the isolates belonging to P. aeruginosa, P. putida and P. fluorescens group.

Molecular characterization and in-vitro screening of fluorescent Pseudomonas sp. for plant growth promoting activities
All the twelve fluorescent Pseudomonas sp. were characterized for plant growth promoting activities viz., phosphate solubilization 7 , siderophore production 8 , HCN production 9 , ammonia production 10 and plant growth regulator production 11,12,13 according to their respective methods. Three potential fluorescent Pseudomonas isolates L, M and C were selected and molecularly characterized by 16S rRNA technique using Pseudomonas specific primers viz., FP-1 (GGTCTGAGAGGATGATCAGT) and RP-1 (TTAGCTCCACCTCGCGGC) in MJ Mini BIORAD personal thermal cycler-100 (PTC-100) 14 . The PCR amplification was as follows: denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and elongation at 72°C for 2 min with a total of 35 cycles. For DNA sequencing, eluted amplified DNA products of fluorescent Pseudomonas isolates L, M and C was purified followed by sequencing in Bioserve Private limited (Hyderabad, India). After sequencing the obtained sequence were analyzed by Basic local alignment search tool (BLAST) for their identification with the NCBI data base 15 . On submission of partial sequence of Pseudomonas putida L, Pseudomonas fluorescens M and Pseudomonas aeruginosa C to GenBank database (NCBI) the accession no. assigned was KF751235, KF751236 and KJ871666

Replant sites for field experiment
The field experiment was conducted with apple rootstocks (MM793 and MM111) in ARD (apple replant disease) infected sites of Shimla distt. (Himachal Pradesh). The two sites at Maggota and Sharontha were selected for conducting field experiment on the bases of age of orchard and symptoms of ARD. A total of eighty apple rootstocks (MM793 and MM111) were purchased from KVK (Rohru) by farmers. The experimental data was analyzed statistically using ANOVA.

RESULTS AND DISCUSSION
Tw e l v e p l a n t g r o w t h p r o m o t i n g rhizobacteria were isolated from apple rhizosphere and characterized as belonging to fluorescent Pseudomonas sp. on the bases of biochemical and physiological characteristics. Reynolds 17 (2004) also characterized isolates on the bases of biochemical tests including oxidase, catalase, gelatin hydrolysis, nitrate reduction, growth at 4 and 41°C and identified them as P. fluorescens.
The screening strategy was carried out to find out an effective PGP fluorescent Pseudomonas strain that act through the combination of several different mechanisms. The screening resulted in a group of bacteria able to produce phosphate solubilizing activity, siderophore production, HCN production, ammonia production and growth hormone production thus allowing us to select Pseudomonas strains showing multifarious plant growth promoting activities. All the fluorescent Pseudomonas sp. showed significant production of phosphate solubilizing activity, siderophores, HCN, ammonia, and growth hormones (Table 1). Overall result showed that fluorescent Pseudomonas strains L, M and C produced maximum number of plant growth promoting activities in-vitro and also inhibited major fungal pathogens of apple.
Plant growth promoting effect of PGPR strains in different crops were clearly   19 . This present investigation confirms the earlier work. In this study, inoculation of fluorescent Pseudomonas sp. increased all growth and soil parameters as compared to control plants. The performance of replanted apple rootstocks after fifteen months of cyclic treatment with individual and consortium strains of P. putida, P. fluorescens and P. aeruginosa along with control at two different replant sites is detailed in Table 3. The details of replant site, age of orchard and rootstocks used for field experiment were presented in Table 2. The data was presented as per cent increase over control for plant height, number of nodes and available NPK content of soil.  Table 3. The maximum number of branches was observed in consortium of three strains viz., L+M+C at both the replant sites. The present experiment revealed that rhizosphere inoculation with Pseudomonas sp. resulted in an increased plant height, number of nodes, branches and available NPK content   [21][22][23][24] .
The chlorophyll content of leaves of replanted apple rootstocks was also estimated. Results in Table 4 showed that there was not a significant increase in chlorophyll content (a, b and total) in all the treatments as compared to control. The available N, P and K of rhizosphere soil were also assessed before and after the cyclic application of fluorescent Pseudomonas sp. ( Table 5).The results showed that after application of Pseudomonas sp. in the field, there was a considerable increase in available NPK content of rhizosphere soil as compared to control plants. Our results were supported by the findings of Verma et al., (2014) 20  After cyclic application of liquid formulations of both individual and consortial strains (L, M, C, L+M, L+C, M+C, L+M+C) in field the decrease in total bacterial and fungal population was observed with a gradual increase in fluorescent Pseudomonas population ( Figure  1). After fifteen months of plantation, there was an increase of 20 to 46 cfu/g of soil in total fluorescent Pseudomonas sp. count in all the bioformulation treated rootstocks at both the sites. The results indicated towards the possibility of sufficient increase in number and establishment of Pseudomonas species in rhizosphere of apple plants planted in replant sites of apple orchards. They also might have decreased the deleterious microflora up to some extent. Burd et al., (2000) 25 reported that plant growth promoting rhizobacteria might enhance plant height and productivity by synthesizing phytohormones, increasing the local availability of nutrients, facilitating the uptake of nutrients by the plants decreasing the heavy metal toxicity in the plants antagonizing plant pathogens. The increased plant height and number of nodes/leaves as compared to control plants clearly showed the beneficial role of Pseudomonas as a rhizobacteria. Such an improvement might be attributed to phosphate solubilising capacity of bacteria as well as the ability of these microorganisms to produce growth promoting substances 26 .
The results of this study suggest that plant growth promoting fluorescent Pseudomonas sp. isolated from apple rhizosphere has potential to be used successfully for replant problem of apple. There was a considerable increase in various plant and soil parameters after fifteen months of cyclic treatment of fluorescent Pseudomonas formulation. So it can be concluded from the present study that the individual and consortium of fluorescent Pseudomonas strains can be further exploited for bio fertilizer development to overcome the replant problem in apple orchards.