Introduction

The application of plant growth promoting rhizobacteria (PGPR) on horticulture tree species is gaining importance (Aslantas et al. 2007) as it is a widely accepted fact that PGPR play a key role in plant health and nutrition. Apple (Malus domestica Borkh.) is one of the important temperate fruit crops of Himachal Pradesh as well as trans Himalayan region of India.

The application of bio-fertilizers containing beneficial microorganisms instead of synthetic chemicals is known to improve plant growth through the supply of plant nutrients and may help to sustain soil health and productivity (O’ Connell 1992). Therefore, more recently, there has been a resurgence of interest in environmental friendly, sustainable and organic agricultural/horticultural practises (Karlidag et al. 2007). The mechanisms by which PGPR promote plant growth are not fully understood but are thought to include the following: (1) the ability to produce plant hormones, such as auxins (Egamberdiyeva 2005), cytokinins (Garcia de Salamone et al. 2001), gibberellins (Gutierrez-Manero et al. 2001); (2) asymbiotic N2 fixation (Sahin et al. 2004); (3) solubilization of inorganic phosphate and mineralization of organic phosphate and/or other nutrients (Jeon et al. 2003); and (4) antagonism against phytopathogenic microorganisms by production of siderophores, the synthesis of antibiotics, enzymes and/or fungicidal compounds and competition with detrimental microorganisms (Ramos-Solano et al. 2010; Pliego et al. 2011).

Phosphorus is one of the essential macro-nutrients for plant growth and development. It is generally present in soil in fixed forms. The P content in average soils is about 0.05 % (w/w), but only 0.1 % of the total P is available to plants (Scheffer and Schachtschabel 1992). Phosphate-solubilizing bacteria (PSB) play a significant role in making phosphorus available to plants by bringing about favourable changes in soil reaction and in the soil micro-environment leading to solubilization of inorganic phosphate sources (Perez et al. 2007). Microbial-mediated solubilization of insoluble phosphates through release of organic acid is often combined with production of other metabolites (siderophores, phytohormones and lytic enzymes), which take part in biological control against soil-borne pathogens (Vassilev et al. 2006). Soil inoculation with PSB improves P-solubilization of fixed soil phosphate and results in higher crop yields (Naik et al. 2008).

Though much information is available on the plant growth promoting activities of PSB for annual crops, a limited information is available in respect of PSB associated with perennial crops such as apple. Previous research in our laboratory has described beneficial effects of rhizobacteria on growth and nutrient uptake by apple seedlings (Shirkot and Sharma 2005). However, the studies on plant growth promoting traits (PGPTs) of PSB associated with apple trees have not been conducted.

The perennial crop plant root system of apple may constitute an important niche of microorganisms that may serve as an unexplored reservoir of P-solubilizing PGPRs. Investigations on bacterial communities in the rhizosphere soil (RS) and roots of many plant species have shown that PGPR can produce direct and indirect beneficial effects on plant’s growth and health, as well as the structure and activities of those communities are greatly influenced by the plant species (Ahn et al. 2007; Jung et al. 2008; Aranda et al. 2011). In this context, cultivated apple plants might constitute an ideal niche for exploring new P-solubilizing PGPR isolates harbouring some of the earlier mentioned PGPTs. Therefore, in this study, PSBs were isolated from root endosphere (ER) and RSs associated with apple trees at four apple growing locations of Himachal Pradesh. The focus was to test the hypothesis whether root exudates of apple plant exert selective pressure on the proliferation of specific kind of rhizobacteria on the surface of the root as well as within the root (endorhizosphere). The indole acetic acid (IAA) productivity, antifungal activity against Dematophora necatrix, siderophore and hydrocyanic acid (HCN) synthesis ability were evaluated, and the major factors influencing the ecological distribution and physiological characterization of PGPT-possessing PSB were also discussed.

Materials and methods

Site characterization and sampling

Samples (soil and roots) were collected from the rhizosphere of the planted population of apple trees growing at four different locations viz., Chamba, Shimla, Kinnaur and Kullu of Himachal Pradesh, India, at an elevation ranging 1,257–3,628 m above mean sea level. Five sites within each location based on elevation above mean sea level were selected purposely (Fig. 1). Ten trees were selected randomly from different apple orchards of each site. A total of 200 soil and root samples were used for isolation of total bacterial population and PSB population. Sampling was done in the month of March and April of the year 2009. Samples were immediately stored at 4 °C in plastic bags loosely tied to ensure sufficient aeration and to prevent moisture loss until assaying of bacterial community structure. The remaining RS was air-dried and sieved (2 mm) prior to soil physicochemical analyses.

Fig. 1
figure 1

Selected 20 sites of apple growing locations sampled in Himachal Pradesh

Full size image

Climatic characteristics (precipitation, mm; minimum and maximum temperature, °C) of each sampling site were obtained from different research centres of Dr. Y.S. Parmar University of Horticulture and Forestry located at Mashobra (Shimla), Bajaura (Kullu), Saru (Chamba) and Sharbo (Kinnaur). Altitudes of the sampling sites were determined using global positioning system.

Soil characterization

Physicochemical characteristics, that is, pH was determined by a pH metre (soil: water ratio, 1:2.5) and organic matter by the chromic acid filtration method (Walkley and Black 1934). Electrical conductivity (EC) and moisture content were determined by the procedures outlined by Jackson (1973). Available nitrogen, potassium and phosphorus were determined by the methods as standardized by Subbiah and Asija (1956), Merwin and Peach (1951) and Olsen et al. (1954), respectively.

Extraction of RS and ER PSB communities

Rhizobacteria were isolated from the RS and root samples of apple trees by serial dilution using standard spread plate technique. Total bacterial population was determined using nutrient agar (NA) medium. PSBs were enumerated on Pikovskaya’s (PVK) medium (Pikovskaya 1948) supplemented with tri-calcium phosphate as insoluble inorganic phosphate source. Populations were expressed as colony forming unit (cfu) per gram of dry soil weight. Endorhizobacteria residing inside the roots of apple trees were isolated using surface sterilization, aseptic trituration and dilution plating assay (Shishido et al. 1999) and expressed as cfu per gram of root weight. After cultivation, distinct PSB morphotypes differentiated by their colony morphology, pigmentation and growth rate were selected from the total colonies obtained on NA medium and restreaked on PVK medium. These isolated colonies were maintained on PVK medium with 30 % glycerol on cryopreservation tubes at −20 °C.

Screening for potential plant growth promoting attributes

Mineral phosphate solubilization

Isolates were screened on Pikovskaya’s (PVK) agar plates. Each isolate was inoculated on to the plate and incubated at 37 °C for 48 h. The P-solubilization was exhibited with a clear yellow zone formed around the colony.

Siderophore production

The ability of the isolates to produce siderophore(s) was determined using blue agar plates containing chrome azurol S (Schwyn and Neilands 1987). Each isolate was inoculated on to the plate and incubated at 37 °C for 48 h. Orange halos around the isolate on the blue agar served as indicators of siderophore(s) excretion.

IAA production

For the production of auxins, each isolate was grown in Luria–Bertani broth (amended with 5 mM l-tryptophan, 0.065 % sodium dodecyl sulphate and 1 % glycerol) for 72 h at 37 °C under shake conditions. Colorimetric estimation of IAA-like auxins was done using Salkowski reagent (Glick 1995).

HCN production

To qualitatively determine HCN production, PSB isolates were streaked on King’s B agar medium with 4.4 g glycine L−1. Filter paper, Whatman no. 1, was cut into uniform strips, 8 cm long and 0.5 cm wide, saturated with an alkaline picrate solution (0.5 % picric acid + 0.2 % sodium carbonate; pH 13), and placed inside the lid of a Petri dish. The plates were then sealed airtight with parafilm and incubated at 37 °C for 48 h. Thereafter, a colour change in the sodium picrate present in the filter paper from to reddish brown was considered to be an indication of HCN production (Baker and Schippers 1987).

Antifungal activity

Antagonistic activity of the culture filtrate (10 %) of PSB isolates was tested against D. necatrix (the fungal pathogen causing white root rot of apple) on potato dextrose agar using agar dilution technique (Warnock 1989). Percentage of growth inhibition was calculated using the formula given by Vincent (1947).

$$ I = \frac{C - T}{C} \times 100, $$

where I is the percentage of growth inhibition; C, the growth of fungus in control; T, the growth of fungus in treatment.

Statistical analysis

All the experiments were conducted under statistical framework in triplicates along with equal number of appropriate controls. Appropriate statistical/mathematical tools were utilized as per the objectives and cross-section variation of the data.

The data of soil physicochemical and climatic characteristics of the sampled sites were subjected to cluster analysis with Pearson correlation moment coefficients. To know the influence of RS and roots on distribution of PGPT-possessing PSB, principal component analysis was carried out. Dendrograms were also constructed using the unweighted pair-group method with arithmetic mean. All statistical procedures were performed using SPSS 16.0 (Analytical Software).

The diversity of P-solubilizing PGPR in the RS and ER of different locations was evaluated using diversity indices. Shannon–Weaver index of diversity (H m ) and Simpson’s index of dominance (D) are, in general, the measures of diversity that accounts for both richness and proportion of individual (Atlas and Bartha 1998). The diversity and dominance indexes were calculated by using the following formulas (Ventorino et al. 2007).

  1. 1.

    Shannon–Weaver index of diversity (H m )

$$ H_{m} = - \mathop \sum \nolimits P_{i} {\text{ln }}P_{i} , $$

where P i is the relative abundance of isolates calculated according to the following equation:

$$ P_{i} = \frac{{n_{i} }}{N} $$

where n i is the number of isolates with their respective activities and N, is the total amount of individuals considered

  1. 2.

    Simpson’s index of dominance (D)

$$ D = \mathop \sum \nolimits P_{i}^{2} $$

Results

Physicochemical properties of experimental soils

Twenty composite soil samples from each site of four locations were tested for physicochemical properties. The result on analysis of physicochemical properties of soil from different sites is presented in Fig. 2. Most of the soils under study were neutral with pH varying from 6.5 to 7.3. The EC values from different sites ranged 0.32–0.71 dS cm−1. The organic carbon content in soil was very high and ranged from 5.5 to 10.9 %. The highest organic carbon content (10.9 %) was found at site Naggar (Kullu) and minimum (5.5 %) at site Bharmour (Chamba). The phosphorus (P) and nitrogen (N) availability in the soils were very low to high, while the availability of potassium (K) was low to medium. The highest available P (133.3 kg ha−1) was found at site Rohru (Shimla), while minimum available P (11.9 kg ha−1) at site Kotkhai (Shimla). The highest available N (657 kg ha−1) was found at site Chopal (Shimla) and maximum available K (299.4 kg ha−1) at Manali site (Kullu). The minimum available N (222.5 kg ha−1) and available K (139.5 kg ha−1) were found at site Ribba (Kinnaur). Tissa (Chamba) had highest moisture content (81.9 %), while minimum (8.5 %) moisture content was found in Matiana (Shimla).

Fig. 2
figure 2

Cluster analysis of combined data from climatic and physicochemical soil characteristics of selected 20 sites

Full size image

Climatic characteristics of experimental sites

Twenty sites from four apple growing locations were studied for climatic parameters viz., temperature, rainfall and altitude. Data were recorded in the month of March 2009, and the results are shown in Fig. 2. Average maximum temperature ranged from highest (22.1 °C) to minimum (15.26 °C) and average minimum temperature varied between highest (7.9 °C) to minimum (1.8 °C) amongst the twenty sites of Himachal Pradesh. Nako (Kinnaur) was found to be coldest area amongst twenty sites with highest altitude (3,628 m) and minimum temperature (1.8 °C). The maximum temperature (22.1 °C) was found at site Sewbagh (Kullu) with lowest altitude (1,257 m). Rainfall ranged from high value of 59.0 mm at site Kotkhai (Shimla) to low value of 0.3 mm at site Tissa (Chamba).

Combined analyses of soil physicochemical and climatic characteristics of sites sampled in the study

Combined analyses of soil physicochemical and climatic characteristics grouped the twenty sampled sites into four main clusters (Fig. 2). The first cluster with only 9.0 % similarity amongst them consisted of four sites, two each from location Chamba (Tissa and Bharmour) and from location Kinnaur (Kilba and Spello).

The second cluster comprised of seven sites viz., Holi and Kundi (Chamba), Kotkhai and Chopal (Shimla), Manali and Lag valley (Kullu), and Ribba (Kinnaur). In this cluster, 9.0 % similarity was observed amongst six sites viz., Holi, Kotkhai, Kundi, Ribba, Chopal and Lag Valley, and two sites viz., Ribba (Kinnaur) and Chopal (Shimla) showed another sub-group at 18 % similarity.

Seven sampling sites were grouped together in third cluster in which Salooni (Chamba), Nako (Kinnaur) and Theog (Shimla) showed 9.0 % similarity; Matiana (Shimla), Sewbagh and Naggar (Kullu) showed 14.0 % similarity, and 18 % similarity was observed between Matiana (Shimla) and Sewbagh (Kullu).

The fourth cluster clearly separated out two sites of Manikaran (Kullu) and Pooh (Kinnaur) from the rest of the eighteen sites, and these two sites had 9.0 % similarity between them. Maximum dissimilarity was found between Tissa (Chamba) and Manikaran (Kullu). Comparison of RS and ER PSB isolates possessing PGPTs (IAA productivity, antifungal activity against D. necatrix, siderophore and HCN synthesis ability) were done.

Two hundred and six PSB were isolated from the RS and ER of four different apple growing locations. Fifty-five PSB colonies were selected from the RS of Chamba and 48 colonies from the ER of Chamba; 20 P-solubilizing colonies from the RS and 14 colonies from the ER of Shimla; 21 P-solubilizers from the RS of Kinnaur and 18 colonies from the ER of Kinnaur; and 20 P-solubilizing colonies from the RS and 10 P-solubilizers from the ER of Kullu, respectively. IAA production was detected in 50 isolates (24.2 %), siderophore synthesis in 53 isolates (25.7 %), HCN in 40 isolates (19.4 %) and percentage growth inhibition against D. necatrix in 61 isolates (29.6 %). The colony morphology of the isolates varied from circular to flat, convex, or raised and entire or undulating margins. The majority of the PSB isolates (116) was from RS, and 90 isolates were endophytes isolated from internal root tissues. Microscopic observation showed majority of the isolates to be Gram-positive and rod-shaped bacteria.

Table 1 shows the results of the PGPT tests for the PSBs from the RS and ER associated with apple trees. Percentages of PSB not having PGPT were relatively higher in the RS of Kullu (85 %) and ER of Chamba and Kinnaur (66.6 %). The lower percentages of bacteria, not having PGPT, were found in the RS of Chamba (43.6 %) and ER of Shimla (57.1 %), respectively. Overall, 63 (54.3 %) of the total 116 PSB isolates from the RS samples lacked PGPT, and 58 (64.4 %) of the total 90 PSB from the ER samples did not display PGPT.

Table 1 Characterization of RS and root endophytic P-solubilizing rhizobacterial isolates for multifarious PGPTs
Full size table

Among four locations, percentages of PSB having IAA production were highest in RS of Kullu (5 %) and ER of Kinnaur (5.6 %). Maximum percentages of siderophore producing PSBs were found in RS of Shimla and Kullu (5 %) and ER of Kinnaur (5.6 %). PSB percentages having antifungal activity against D. necatrix were highest in RS (5 %) and ER (14.3 %) of Shimla. Percentages of PSB showing HCN production were absent in all samples except RS of Chamba (3.6 %). Overall, percentages of PSB with single PGPT viz., IAA production (2.6 %), siderophore synthesis (3.4 %) and HCN production (1.7 %) were highest in RS, whereas PSB having antifungal activity against D. necatrix was found maximum in ER samples.

Percentages of PSB displaying binary activities of IAA production and antifungal activity against D. necatrix were highest in the Shimla samples (5 % and 7.1 % for RS and ER, respectively). Percentages of PSB having siderophore synthesis and antifungal activity against D. necatrix were the highest in RS (5.5 %) and ER (2.1 %) of the Chamba samples. PSB percentages having IAA and siderophore production ability were highest in RS of Chamba (3.6 %) and ER of Kullu samples (10 %). The highest percentages of PSB with antifungal activity and HCN production were found in RS of Shimla (5 %) and ER of Chamba samples (2.1 %). Percentages of PSB showing combination of binary activities of IAA and HCN production; and siderophore and HCN synthesis ability were absent in all the samples of RS and ER collected from different apple growing locations. Overall, the ratio of PSB having antifungal activity against D. necatrix and siderophore synthesis ability in RS was relatively higher (4.3 %). Similarly, ratios of PSB having IAA and siderophore synthesis ability in ER were relatively higher (4.4 %), whereas the ratios of PSB in RS possessing IAA and siderophore production capability were relatively lower (1.7 %). Only 1.1 % PSB having combination of binary activities of antifungal and siderophore synthesis; and antifungal activity with HCN production was found in ER.

The ratios of PSB showing combination of triple activities of IAA production, siderophore synthesis and antifungal activity against D. necatrix were the highest in RS of Shimla samples (10 %) and ER of Kullu (10 %). Percentages of PSB displaying triple activities of IAA, HCN production and antifungal activity were highest in RS of Shimla (5 %) and ER of Chamba samples (6.3 %). PSB percentages having siderophore synthesis, antifungal activity and HCN production ability were higher in RS of Chamba (5.5 %) and ER of Kullu samples (10.0 %). Ratios of PSB showing combination of triple activities of antifungal activity, HCN and IAA production abilities were highest in Shimla (10 %) and ER of Kinnaur (5.6 %), respectively. Overall, the ratios of PSB showing IAA, siderophore synthesis and antifungal activity were relatively higher in RS (5.2 %). Highest percentages of PSB displaying siderophore, antifungal activity and HCN production abilities were found in ER (4.4 %), respectively.

The ratios of PSB exhibiting four PGP activities (antifungal activity, IAA, siderophore and HCN production) were highest in RS of Kinnaur samples (9.5 %) and ER of Shimla samples (7.1 %). On the basis of total PSB, the ratios of PSB showing four PGP activities were (6.9 %) and (4.4 %) in the RS and ER samples, respectively.

Percentages of PSB having IAA, siderophore, HCN synthesis and antifungal activity against D. necatrix from the four apple growing locations (Fig. 3) depicted that the ratios with IAA production activities were arranged in the order of Shimla (29.4 %) > Chamba (26.2 %) > Kinnaur (20 %) > Kullu (13.3 %). In particular, 35 % of the PSB isolated from the RS of Shimla sample had IAA producing activity, even though only 10 % of PSB from RS of Kullu samples could display this activity.

Fig. 3
figure 3

Graphical representation of percentages of RS and root endophytic P-solubilizing rhizobacterial isolates of four locations for multifarious PGPTs: IAA production (a), siderophore synthesis (b), HCN production (c) and antifungal activity against D. necatrix (d)

Full size image

Phosphate-solubilizing rhizobacteria (PSB) ratios showing siderophore synthesis ability were in the order of Shimla (29.4 %) > Chamba (27.2 %) > Kinnaur (25.6 %) > Kullu (16.7 %). The highest percentage of PSB possessing siderophore synthesis ability was found in RS of Shimla (35 %) followed by RS of Chamba (34.5 %), respectively. Percentages of PSB having HCN production activity were arranged in the order of Chamba (22.3 %) > Shimla (20.6 %) > Kinnaur (20.5 %) > Kullu (6.7 %). The highest percentages of PSB having HCN synthesis were found in RS of Chamba (27.3 %), and lowest percentages of HCN producers were observed in RS of Kullu (5.0 %), respectively. PSB ratios showing antifungal activity were in the order of Shimla (38.2 %) > Chamba (31.06 %) > Kinnaur (25.6 %) > Kullu (13.3 %). The highest P-solubilizers with antifungal activity were recorded in samples collected from RS of Shimla (40.0 %) followed by RS of Chamba (38.2 %) and Kinnaur (38.1 %). The lowest percentages of PSB with antifungal activity against D. necatrix were found in RS of Kullu.

Correlation analysis

Table 2 shows the result of the correlation analysis among the PGPT-possessing PSB. There was a negative correlation (−0.99, p < 0.05) between HCN producers isolated from root samples and total IAA producers, without considering the RS or ER samples. A positive significant correlation (0.99, p < 0.05) was found between total PSB possessing antifungal activity without considering RS or ER samples and IAA producers from ER samples. A positive correlation (0.99, p < 0.05) was also observed between HCN producers and siderophore producers from RS samples. A positive significant correlation (0.94, p < 0.05) was also observed between PSB possessing antifungal activity isolated from roots and total PSB having HCN synthesis ability, without considering the RS or ER samples.

Table 2 Correlation matrices showing relationship amongst various PGPTs of the P-solubilizing rhizobacterial isolates
Full size table

The relationship between the physicochemical properties of the soil samples (pH, EC, organic carbon, available N, P, K, moisture content) and PSB possessing IAA, siderophore, HCN and antifungal activity against D. necatrix was analysed. Significant correlation (0.471, p < 0.05) between available P content in soil and IAA producers was observed. Non-significant correlation existed between physicochemical properties and rest PGPT-possessing PSB (data not shown). Correlation analysis between climatic parameters (temperature, altitude and rainfall) and PGPT-possessing PSB revealed a negative correlation (−0.45, p < 0.05) between HCN producers and rainfall. A significant positive correlation (0.42, p < 0.05) was observed between HCN producers and altitude. Non-significant correlation existed between climatic parameters and rest PGPT-possessing PSBs (data not shown).

Cluster analysis

Cluster analysis was carried out through principal components with the ratios of PSB having IAA, siderophore, HCN and antifungal activity against D. necatrix in the RS or ER of each apple growing locations (Fig. 4). The summary of principal component analysis (Table 3) of the gathered data based on abundance of PGPT-possessing PSBs shows that two principal components (PC1 and PC2) out of eight principal components had eigen value greater than one and were retained for the analysis. PC1 had eigen value of 4.99 and explained 62.48 % of total variation. PC2 with eigen value of 2.38 explained 29.84 % of total variation. Together they accounted for 92.33 % of variation of original variables.

Fig. 4
figure 4

Principal component analysis of the percentages of PGPTs possessing P-solubilizing rhizobacteria considering apple growing locations, sampling position and PGPT types

Full size image
Table 3 Loading of coefficients of percentages of PGPTs possessing P-solubilizing rhizobacterial isolates for the first two principal components
Full size table

In the PC1, ER of location Shimla (SER) had highest coefficient value (0.194) followed by rhizosphere of location Chamba (CRS) with coefficient value of 0.193. In PC2, maximum coefficient value (0.399) was found in case of RS of Kullu (K2RS).

Diversity indices

The diversity of the two sampling positions (RS and ER) according to the physiological PGPR traits shown by PSB was calculated by the Shannon–Weaver algorithm. The results (Fig. 5) showed that higher Shannon–Weaver diversity index (1.40) was found in RS as compared to less value of 1.29 in ER. Similarly, Simpson’s index of dominance of 0.31 for RS and 0.18 for ER was observed.

Fig. 5
figure 5

Representation of abundance of P-solubilizing rhizobacteria based on PGPTs in the RS and ER according to the Shannon–Weaver index (H m ) and Simpson index of dominance (D)

Full size image

Discussion

The use of PGPR including PSB is currently increasing worldwide as a promising alternative to the use of chemicals in the environment mainly in the sustainable and organic system of cultivation. PSBs are an essential and important component of the microbial community in natural and cultivated woody trees habitats (Dash and Gupta 2011). Moreover, PGPR are largely responsible for functioning of ecosystems because of their role in transformation of most of nutrients, their beneficial effects on plant growth and health and as a source of potential bio-control agents of soil-borne fungal and other plant pathogens (Verma et al. 2010). In this context, the cultivated long-living trees, such as apple, might constitute an ideal niche for exploring new PSB isolates with diverse mechanisms of PGPTs. Therefore, in this study, to compare PGPTs of PSBs, 206 PSBs were isolated from RS and ER sampled from sites of four different locations of apple growing region of Himachal Pradesh.

The present study is one of the first to explore environmental and physicochemical properties of soil interacting with apple-associated PSBs. In this study, results of cluster analysis of soil physicochemical and climatic characteristics indicated that each sampled site differ in soil type and climate (Fig. 2). Accordingly, a pattern of high variation among twenty apple growing sites of four locations was revealed. Interestingly, the cluster analysis also revealed the occurrence of sites from different locations in one cluster. For example, in cluster, one four sites comprised of two sites each from location Chamba and Kinnaur. This may be attributed to high similarity in the climatic factors and physicochemical properties of soil.

Both RS and the roots of apple trees seem to be the preferential sites for PSB suggesting that PSB proliferates both in RS and ER. To the best of our knowledge, this is the first report of PSB endophytes residing within apple root tissues.

The percentages of PSB not having PGPTs were relatively higher (Table 1) in ER, thus indicating that the ratio of PGPT-possessing PSB was higher in RS (45.7 %) than ER (35.6 %). These results are in accordance with earlier reports that Pseudomonas sp. having PGPTs were present in high proportions in all the RS samples associated with Chamaecytisus proliferus (Donate-Correa et al. 2004). The higher occurrence of P-solubilizers in rhizosphere is of direct significance to the plants as it helps in mobilization of insoluble phosphorus near the root, especially in phosphorus deficient soils.

The tested PSB isolates could simultaneously exhibit two, three or four PGPR traits, which may promote plant growth directly, indirectly or synergistically, suggesting that the application of PSBs with multifaceted traits for plant growth promoting activity is more beneficial. The exhibition of multiple PGPTs by a single strain of PGPR has also been reported earlier (Mehta et al. 2010).

The ratio of PSB to show binary activities of siderophore synthesis and IAA production to be 1.7 % (2/116) is more than the ratio of 0.9 % (1/66) reported for leguminous plants (Cattelan et al. 1999), but is much less than the 34.8 % (8/23) in the RS of Carex leiorhyncha (Koo et al. 2010). The siderophores play important role in the growth of plants with their ability to supply iron (Ramos-Solano et al. 2010). Proposed role for exogenous production of IAA by PGPR also include the determination of rooting capacity (Fogaca and Fett-Neto 2005), the stimulation of release of plant metabolites (Lambrecht et al. 2000) and/or the promotion of root elongation and shoot growth of inoculated plant.

It is also worth mentioning that PSB isolates from RS and ER of apple trees also show combination of three PGPTs, that is, PGPR ratio 3.4 % (4/116) and 5.2 % (6/116); and four PGPR traits, that is, PGPR ratio of 6.9 % (8/116), probably because more bacterial genes are expressed simultaneously. However, the PGPR ratio having triple PGPT was 2 % (2/84) (Dell’Amico et al. 2005), and there was no PGPR having triple activities in isolates from soya bean (Cattelan et al. 1999).

Binary activities in combination of IAA and HCN, and siderophore and HCN were not found in the PSB tested in the present study, suggesting that apple rhizosphere may not be potential niche for PSB with these binary activities. This reveals how accurate and selective a plant can be, so that the bacterial population in the rhizosphere may be different, or behave metabolically differently, by dependence on the plant’s physiological status.

The results of functional diversity analysis indicate the presence of more variable PSB population. PSB producing various PGP properties (IAA, siderophore, HCN and antifungal activity and/or fungicidal compounds against D. necatrix) varied significantly amongst the sites of four locations as compared to those from ER (Fig. 3). However, PSB from the rhizosphere showed larger variation in functional properties. The existence of such large variation among the different locations could either be plant or soil type dependent (Mittal and Johri 2007). Thus, in the present study, soil texture can affect the rhizosphere PSB by limiting the availability of root exudates. The dominance of some PGPT-possessing PSB population in the rhizosphere can be explained on the basis of rhizodeposition. Variety of environmental factors may also contribute for the variation in rhizobacterial community at different locations. A study by Germida et al. (1998) strongly supports the present observation wherein rhizobacterial population associated with roots of canola and wheat plants differed significantly in two different climatic zones.

The ecological and physiological properties of rhizobacteria in soils are directly or indirectly affected by abiotic parameters such as soil texture, temperature, pH, salinity (Lebeau et al. 2008; Weyens et al. 2009). The results revealed that among physicochemical properties (pH, EC, organic carbon, moisture, available N, P, K) of soil and PGPT (IAA, siderophore, HCN, antifungal activity) possessing PSB, significant correlation (r = 0.471) existed only between available P content of soil and PSB having IAA producing ability. However, no significant correlation existed between physicochemical properties and rest PGPTs (siderophore, HCN, antifungal activity) possessing PSB. There is a report wherein no significant correlation existed between physicochemical properties of soil and PGPR with IAA production, ACC deaminase activity and siderophore synthesis activity (Koo et al. 2010). Also, positive significant correlation (r = 0.42) existed between HCN producers and altitude. Thus, indicating that population of PSB with HCN activity may increase with increase in altitude.

The positive significant correlation (r = 0.9) between PSB having antifungal activity against D. necatrix and IAA producers; HCN producers and siderophore producers; HCN producers and PSB with antifungal activity (Table 2) is a significant observation since IAA is a growth promoting hormone, and HCN and siderophore production have been proposed as a possible line of defence against soil-borne plant pathogens (Principe et al. 2007; Selvakumar et al. 2008).

Cluster analysis through principal component (Table 3; Fig. 4) reveals that sampling position RS and ER are important factors on distribution rates of PGPT-possessing PSBs. The distribution rates of PGPT-possessing PSB were affected by RS or rhizoplane of six different plants species grown in contaminated soil with petroleum and heavy metal (Koo et al. 2010). SER is important factor on distribution rates of PGPT-possessing PSBs. Since ER is the inner portion of the plant’s root, bacteria inhabiting on and/or in the ER are closely associated with a host plant. This close interrelationship might influence the ecological distribution and physiological characterization of PGPT-possessing PSBs.

In view of the values (Fig. 5) from the Shannon–Weaver diversity index, the rhizosphere showed higher diversity of PGPT-possessing PSB than the ER, indicating that rhizosphere of apple trees may constitute an ideal niche for exploring PSB with diverse mechanisms of plant growth promotion. The low diversity of PGPT-possessing PSB is probably due to undisturbed nature of apple ER ecosystem that is supposed to be close to an equilibrium state or due to the marked environment fluctuations that take place in the ER caused by the plant’s physiological cycle. Our results are in agreement with previous study wherein rhizosphere was good reservoir of bacterial diversity (Mittal and Johri 2007; Joshi and Bhatt 2011).

In conclusion, we report for the first time a high diversity of PGPT-possessing PSBs in the RS and ER of apple trees, which to a large extent remains hidden and unexplored and represent excellent reservoirs of those PGPR. Significant variation exists in rhizospheric and endospheric PSB unequivocally suggests the hypothesis that root exudates of apple plant exert selective pressure on the proliferation of specific kind of rhizobacteria on the surface of the root as well as within the root (endorhizosphere). The present study has made its contribution by providing data for further studies on the dynamics of bacterial populations associated with apple rhizosphere. The isolates containing plant growth promoting related properties could probably be one of the most significant strategies for disease management and sustainable agriculture/horticulture in the near future.