Rhizosphere mediated nutrient management in Allium hookeri Thwaites by using phosphate solubilizing rhizobacteria and tricalcium phosphate amended soil

ABSTRACT This work describes integrated nutrient management for cultivation of Allium hookeri by using phosphate solubilizing bacteria (PSB) applied in rhizosphere, along with tricalcium phosphate (TCP). Arthrobacter luteolus S4C7, Enterobacter asburiae S5C7, Klebsiella pneumoniae S4C9, S4C10 and S6C1, and K. quasipneumoniae S6C2, were isolated from rhizosphere of Allium hookeri Thwaites, and were found to release substantial amount of soluble phosphate (124.8–266.4 μg/mL) from TCP in vitro conditions. These isolates were experimented for plant growth promoting attributes, including IAA, siderophore, and nitrogen-fixation. Treatment with PSB resulted in enhanced growth of A. hookeri Th., which was even better with TCP amendment with PSB. K.quasipneumoniae S6C2 resulted in 39.1% and 533.3% increase (p ≤ 0.05) of root length and weight respectively. The treatment with these isolates, in TCP amended soil also resulted in 200–250% increase in available P in soil, which was maximum for K. quasipneumoniae (1.866 mg/g).


Introduction
Phosphorus (P) is the second most important macro-nutrient and an essential growth limiting nutrient for plant growth, as it is required in important metabolic pathways like photosynthesis, biological oxidation, nutrient uptake and cell division (Epstein 1972;Illmer and Schinner 1992;Holford 1997). P is present in soil in abundance, in both organic and inorganic forms, but the majority of P is immobilized and rendered unavailable for plant uptake as it complexes with cations of Ca, Fe, and Al, depending on the type of soils. Thus, only the phosphate in a soluble ionic form (Pi) is effective as a mineral nutrient (Kucey et al. 1989;Ae et al. 1991). Plants are unable to utilize precipitated form of phosphorous. However, organic matter, on the other hand, is an important reservoir of immobilized phosphate that accounts for 20-80% of soil phosphorous (Richardson 2001) and only a small portion (∼0.1%) is available to plants. Conversion of the insoluble forms of phosphorous to a form accessible by plants, like orthophosphate, is an important trait of phosphate solubilizing rhizobacteria (PSRB) in increasing growth and yield of crop plant. Application of phosphate solubulizing bacteria increases soil fertility due to their ability to convert insoluble P to soluble P by releasing organic acids, chelation and ion exchange (Omar 1998;Narula et al. 2000;Whitelaw 2000). The main active strains in this conversion belong to a range of genera, including Pseudomonas, Mycobacterium, Arthrobacter, Serratia, Chryseobacterium, Gordonia, Phyllobacterium, Delftia, Enterobacter, Pantoea, Klebsiella, Micrococcus, Bacillus, Flavobacterium, Rhizobium, Mesorhizobium and Sinorhizobium (Asea et al. 1988;Salih et al. 1989;Rodríguez and Fraga 1999;Chen et al. 2006;Chung et al. 2005).
Rhizospheric bacteria are known to play a very significant role in plant growth promotion by different mechanisms, one of which is the ability to dissolving poorly soluble fixed P, and such bacteria are known as phosphate-solubilizing bacteria (PSB). Their role is to convert these insoluble phosphates into soluble forms through the process of acidification, chelation, and production of low molecular weight organic acids such as acetic, gluconic, 2-ketogluconic, glycolic, isobutyric, isovaleric, lactic, malonic, oxalic, fumaric and succinic by lowering the p H of the surrounding (Bolan et al. 1994;Goldstein 1995;de Freitas et al. 1997;Sharma et al. 2005). These acids are produced in the periplasm of many gram-negative bacteria through a non-phosphorylated direct oxidation pathway of glucose (Matsusshita et al. 2002). Plant growth promoting rhizobacteria (PGPR) are a heterogeneous group of bacteria that can be found in the rhizosphere, at root surfaces and in association with the roots that benefit the plant growth with direct and/or indirect mechanisms (Glick 1995;Ahmad et al. 2008). The mechanisms of PGPR includes fixation on atmospheric nitrogen to ammonia, production of IAA, solubilization of phosphorus, siderophore production, antibiotics, lytic enzymes etc. (Compant et al. 2005).
Allium hookeri Th., is a wild plant that grows in countries including Greece, Yunnan, Southern China, Bhutan, Sri Lanka and India (Hooker 1892). Besides having medicinal properties, it is used for seasoning ethnic cuisines, while its leaves and fleshy roots are also consumed as vegetables and also it has good marketability in areas of its common occurrences (Singh et al. 2003).
With the emphasis on the isolation and characterization, (Rodriguez and Fraga 1999;Harris et al. 2006;Perez et al. 2007) PSB are isolated and characterized for their ability to solubilize unavailable reduced phosphorus (P) to available forms. Such transformations increase P availability and promote plant growth (Rodriguez and Fraga 1999;Whitelaw 2000;Rudresh et al. 2005;Harris et al. 2006). However, to date, investigations on isolation, characterization and the positive effects of PSB strains on A. hookeri Th. is not reported in the literature. In present study, application of efficient PSB, isolated from A. hookeri rhizosphere, has been described for integrated nutrient management with tricalcium phosphate and examine their effect on growth of host plants. Further, effect on availability of soluble phosphate in soil and uptake of P by plants was also estimated with bacterial augmentations and TCP amendments.

Plant species
A. hookeri Th. (Alliaceae family) is a non-bulbous liliaceous plants and growing as a wild herb in a wide range of soils. The leaves are thick evergreen, linear with prominent midribs, basal leaves membranous and shorter than the subtrigenous scape. Mainly, the freshly leaves and roots of this species are consumed as vegetables. Therefore, it has good marketability in areas of its common occurrence. Because of its therapeutic properties, it is used in excessive body temperature and vertigo and reducing blood pressure, stomach ulcer, hypertension, coronary heart diseases, etc. Due to its many benefits, it is used by the ethnic people of North East India as one of the important plants (Kala 2005;Pandey et al. 2008).

Screening and Isolation of phosphate solubilizing bacteria
Rhizospheric soils were collected from the A. hookeri Th. from different parts of Manipur, India. Manipur lies in the Eastern Himalayan region of India at latitude of 23°83 ′ N-25°68 ′ N and longitude of 93°03 ′ E-94°78 ′ E. The soil samples were collected using sterilized forceps and kept in the sterilized plastic bags. 1.0 g of soil sample were suspended in 9.0 ml of phosphate buffer saline with p H 7.2. Suitable dilution of soil samples was plated on yeast extract mannitol agar (YEMA) medium, and incubated at 30°C for 3 days. Bacterial colonies appearing on the medium were isolated and sub-cultured for further analysis. PSB strains were selected on the basis of clear zone of phosphate solubilization around the colony on Pikovskaya's (PVK) agar having compositionglucose (10 g L −1 ), Ca 3 (PO 4 ) 2 (5 g L −1 ), (NH 4 ) 2 SO 4 (0.5 g L −1 ), K 2 SO 4 (0.2 g L −1 ), MgSO 4 .7H 2 O (0.1 g L −1 ), yeast extract (0.5 g L −1 ), MnSO 4. H 2 O (0.002 g L −1 ) and FeSO 4 (0.002 g L −1 ) (Nautiyal and Mehta 2001). Once purified, each strain was stored in a glycerol stock (2%) at −20°C.
Morphological, biochemical, and molecular identification of bacteria PSB isolates grown on YEMA plates were characterized for phenotypic and physiological characteristics according to methods described in Bergey's Manual of Systemic Bacteriology (Holt et al. 1994). The PSB isolates were identified on the basis of 16S rRNA gene sequence homology. 16S rRNA gene was amplified using universal eubacterial primers FD1 5 ′ CCG AAT TCG TCG ACA ACA GAG TTT GAT CCT GGC TC AG 3 ′ and RD1 5 ′ CCC GGG ATC CAA GCT TAA GGA GGT GAT CCA GCC 3 ′ (Bazzicalupo and Fani 1995). The PCR reaction conditions were: initial denaturation of 7 min at 94°C followed by 29 cycles of denaturation of 1 min at 94°C, annealing temperatures 54°C for 7 cycles, 53°C and 52°C for one cycle each, 51°C for 20 cycles and extension of 1 min at 72°C and a final extension of 10 min at 72°C. Further, all the 16S rRNA sequences were compared with sequences available showing above 95% in GenBank using BLAST program (http://blast.ncbi.nlm.nih.gov/Blast. cgi). The 16S rRNA sequences was deposited to NCBI to obtain GenBank accession number. Multiple alignment and neighbor joining phylogenetic tree was constructed using Kimura 2 parameter, MEGA version 4.0 software (Tamura et al. 2007).

Phosphate solubilization
For the quantitative estimation of P solubilization, PSB were grown in NBRIP broth glucose (10 g L −1 ), (NH 4 ) 2 SO 4 (0.1 g L −1 ), MgSO 4 .7H 2 O (0.25 g L −1 ), KCl (0.2 g L −1 ), MgCl 2 .6H 2 O (5.0 g L −1 ), Ca 3 (PO 4 ) 2 (5 g L −1 ) (Jackson 1973) in sterile conditions at 30°C. Amount of phosphorus in culture supernatant was measured by vanado-molybdateyellow color method. To a 0.5 ml aliquot of the supernatant, 2.5 ml Barton's reagent was added and volume was made to 50 ml with de-ionized water. The absorbance of the resultant color was read after 10 min at 430 nm in UV/Visible Spectrophotometer. The total soluble phosphorus was calculated comparing with the standard curve. The values of soluble phosphate liberated were expressed as μg/mL over control. The p H of culture supernatants was also measured for each sample.
Indole-3-acetic acid assay IAA produced by the cultures was estimated by growing the isolates in YEM broth amended with 0.2% of L-tryptophan. The cultures were incubated in a shaker at 120 rpm at 30°C and harvested the cultures by centrifugation at 8000 rpm for 15 min. The production of IAA was observed after every 24 h interval. 1 ml of supernatant was mixed with 2 ml of Salkowski reagent (50 ml, 35% perchloric acid with 1 ml, 0.5 M ferric chloride [FeCl 3 ]) (Gordon and Weber 1951). The optimal density (OD) was measured at 530 nm and the amount of IAA produced was calculated by comparing with IAA standard curve.

Siderophore production
Siderophore was determined on Chrome-azurol S (CAS) with the formation of orange to yellow medium following the incubation at 28°C for 48 h (Schwyn and Neilands 1987). Quantitative estimation of siderophores was performed by CAS-shuttle assay (Payne 1994). 1 ml of culture supernatant was mixed with 1 ml of CAS reagent, and absorbance was measured at 630 nm against a reference consisting of 1 ml of uninoculated broth and 1 ml of CAS reagent. Siderophore content in the aliquot was calculated in percentage siderophore units (SUs) by using the formula where Ar = absorbance of references at 630 nm (uninoculated broth + CAS reagent) and As = absorbance of sample at 630 nm (culture supernatant + CAS reagent).

Nitrogenase activity
Nitrogenase activity of the isolates was determined in nitrogen free medium (Burk's medium) by the acetylene reduction assay (ARA) (Hardy et al. 1968). Pure cultures of all the isolates were inoculated in the Burk's medium and were grown for 48 h at 30°C on a rotatory shaker at 120 rpm. The vials were inoculated with each isolates (OD 600 of 1) and incubated at 30°C until exponential phase. Following the incubation, the gas phase of each vial was replaced with acetylene (10% v/v) and again incubated at 30°C for 6 h. Ethylene production was measured using a gas chromatography (Ceres 800 plus, Thermoscientific) using flame ionization detector (GC-FID) and a Porapak T stainless steel column. After the completion of the ARA, the free-living cultures, the cells were collected and broken by sonication. Protein concentration in the resulting mixture of the suspension was also determined (Lowry et al. 1951).

Detection of organic acid
The analysis of organic acids produced by PSB cultures was studied. Briefly, the late log phase cultures of bacteria were filtered through 0.2 µm Whatmann No.1 filter membrane and 20 µl of filtrates were injected to HPLC (Model: Perkin-Elmer Series 200, USA) equipped with UV/Vis Detector. The mobile phase consisted of 0.1% phosphoric acid at a flow rate of 1 ml/min. Retention time of each signal was recorded at a wavelength of 210 nm and compared the peaks with standard organic acids. Organic acid standards including acetic acid, citric acid, gluconic acid, lactic acid, malic acid, propionic acid, succinic acid and oxalic acid were run in parallel.

Plant growth experiment
Each PSB isolate was grown separately in YEM broth at 30°C in a rotatory shaker at 120 rpm. The cultures were centrifuged at 6000 rpm for 15 min. The pellets thus obtained were resuspended in sterile distilled water, to obtain OD 600 of 1, and used as inoculum for pot trials.
Plant growth experiment was conducted in a completely randomized block design. The pots were disinfected with ethanol and filled with sterilized soil (about 4.0 kg per pot). Roots of tissue cultured plantlets were sterilized by dipping in 2% NaOCl solution for 10 min and then washed three times with sterile distilled water. Roots were dipped into the selected bacterial suspension of (OD 600 of 1). The plantlets were planted under two different soil conditions (with or without TCP, added 1 g/kg soil) and 14 individual treatments. (i) soil (control 1); (ii) soil+TCP (control 2); (iii) soil+A. luteolus S4C7; (iv) soil+K. pneumoniae S4C9; (v) soil+ K. pneumoniae S4C10 (vi) soil+E. asburiae S5C7; (vii) soil+ K. pneumoniae S6C1; (viii) soil+ K. quasipneumoniae S6C2; (ix) soil+TCP+A. luteolus S4C7; (x) soil+TCP+K. pneumoniae S4C9; (xi) soil+TCP+K. pneumoniae S4C10; (xii) soil+TCP +E. asburiae S5C7; (xiii) soil+TCP+K. pneumoniae S6C1; (xiv) soil+TCP+K. quasipneumoniae S6C2. The experiment was conducted in three independent trials. The plants were harvested after 120 days, and plant growth parameters of each plant were recorded, i.e. shoot length, root length, number of leaves, fresh shoot weight, fresh root weight, dry shoot weight, dry root weight, moisture content were measured and compared with the control plants.
Determination of available P content in soil, and uptake of P by host plants The available P content in soil was determined by ascorbic acid method (Bray and Kurtz 1945). 5 g soil samples were extracted with the 0.5M NH 4 F (ammonium fluoride) at p H 7.0 using 1:5 dilution, with shaking for 1 h followed by 0.03N NH 4 F + 0.025N HCl using 1:10 dilution, with shaking for 1 min. The extract was filtered with Whatmann No.1 filter paper. The phosphate in the solution was determined by the molybdate blue method using ascorbic acid as a reducing agent. The color developed after 20 min was measured at spectrophotometer at 882 nm.
Total phosphorus uptake in A. hookeri Th. plants was determined by using vanadomolybdo phosphoric yellow color method (Jackson 1973). 1 g sample (roots or shoot tissues) was taken in Coors alumina crucibles and were placed into a warm muffle furnace, ignited at 550°C for 3-4 h, which was then removed and allowed to cool at room temperature. The ashes were extracted with 2N HCl of 50 mL were added to the flasks with 1 h shaking. The extract was filtered with Whatmann No.1 filter paper. 5 mL filtrate were added with 5 mL of ammonium vanadate solution and the total phosphorus content was determined after 10 min by reading absorbance at 420 nm.

Phosphorus use efficiency
The relative efficiency of phosphorus use (REP%) was calculated as the ratio between the plant DM (dry mass) under low Pi (Phosphate) and DM (dry mass) under high Pi, as described (Ozturk et al. 2005): The agronomic P use efficiency (APE, g DM g −1 Pi) was obtained by expression adapted from Oliveira et al. (1987). APE = DM high Pi − DM low Pi difference in the total available P between high Pi and low Pi treatments

Statistical analysis
Statistical analysis was conducted by using Analysis of Variance (ANOVA) statistical package for social sciences (SPSS) software, version 21 followed by comparison of multiple treatment levels with the control, using the poshoc at P ≤ 0.05 and Tukey's test.

Results
Isolation and identification of PSB from the rhizosphere soil A total of 97 bacterial strains were isolated from rhizosphere of A. hookeri, and six PSB (S4C7, S4C9, S4C10, S5C7, S6C1 and S6C2) were selected on basis of better phosphate solubilization activity on Pikovskaya's (PVK) agar plate amended with TCP. The morphological, physiological and biochemical characteristics of the bacterial isolates are given in Table 1. The selected six PSB showed optimum growth at pH 7.0, 35°C, and 0.5% salt concentration. The result of the BLAST search of the 16S rRNA gene sequences indicated S4C7 and S5C7 are closely related to Arthrobacter luteolus and Enterobacter asburiae respectively; S4C9, S4C10, S6C1 to Klebsiella pneumoniae and S6C2 to Klebsiella quasipneumoniae (Table 2). Based on the neighbor joining phylogenetic tree constructed with the 16S rRNA similarity (%), the nearest taxas of PSB isolates were identified as Arthrobacter luteolus LNR3 for S4C7, Klebsiella pneumoniae S-49 for S4C9, Klebsiella pneumoniae F3 for S4C10, Enterobacter asburiae M16 for S5C7, Klebsiella pneumoniae SR-143 for S6C1 and Klebsiella quasipneumoniae 07A044 T for S6C2 ( Figure 1). Though the isolate S6C2 (K. quasipneumoniae) belongs to the genus Klebsiella, it clustered closely with Enterobacter.

Phosphate solubilization
The P-solubilizing ability of selected isolates was estimated quantitatively. The maximum amount of available phosphate varied from 124.8 to 266.4 µg/mL in TCP amended NBRIP broth for selected isolates. The maximum amount of P was recorded after 96 h of incubation which was 266.4 µg/mL by S4C7 with decreased in p H to 3.77 ( Figure 2). Amount of soluble phosphate with S4C9 was 236.8 µg/mL, while with S6C1, it was 228.4 µg/mL. In all isolates, it was invariably observed that P-solubilization was maximum when p H of the culture filtrate was minimum. The initial p H of medium was 6.8 that reduced below 5 within 24 h for all isolates except A. luteolus S4C7. For S4C9, S4C10, S5C7, S6C1, S6C2, the P solubilization activity gradually decreased with incubation, with successively increase in the p H , however, in A. luteolus S4C7, increase in phosphate solubilization was recorded with decreased in p H .

Siderophore production assay and influence of iron concentration
Siderophore production was confirmed by the development of an orange halo zone around the bacterial colonies on the CAS agar plate inoculated with PSB, observed after 24 h. All the PSB isolates produced siderophore, except A. luteolus S4C7. Siderophore released was further confirmed by quantitative CAS test where instant decolorization CAS reagent from blue to orange was observed. E. asburiae (S5C7) and the three K. pneumoniae -S4C9, S4C10, S6C1and K. quasipneumoniae S6C2 released appreciable amount of siderophore within 24 h of incubation in YEM broth. Maximum siderophore was released by S5C7 after 72 h of incubation (89.772 units), followed by S4C9, S6C2, S6C1 and S4C10 ( Figure  4A). Siderophore production was considerably affected by the presence of iron in medium. Initial increase in iron concentration induced siderophore production. Increase in iron concentration resulted in successive decrease of siderophore production by all PSB isolates. Maximum siderophore (86.172%) unit was observed with S5C7, at 1 µM concentration of iron. However, siderophore release decreased gradually corresponding to that of increase in iron concentration ( Figure 4B).
HPLC analysis confirmed the presence of organic acids in the broth culture in TCP amended liquid medium by all six PSB. The peak of strains S4C7 and S6C1 were identified as acetic acid; while S4C9, S4C10, S5C7 and S6C2 showed the peak for citric acid when compared with the retention time with the authentic standards. Catalase

Effect of PSB on plant growth
With the inoculation of PSB, the growth of A. hookeri was enhanced significantly in unamended TCP soil. The best growth parameters were recorded with treatment of K. pneumoniae S6C1 where fresh shoot weight was 365.5% higher as compared to control, followed by A. luteolus S4C7 (227.58%). However, in fresh shoot length, the highest length were recorded with the treatment of K. pneumoniae S6C1 (32.1 cm), followed by A. luteolus S4C7 (31.5 cm).
Amendment of TCP did not affect the growth of A. hookeri Th. in comparison to un-amended control, as all the growth parameters were in similar range except moderate increase in root fresh weight. However, treatment of PSB with TCP enhanced the growth of A. hookeri Th. better than treatment with respective 'PSB only' in case of S5C7+TCP, and S4C10 +TCP. There was 24.57% increase in shoot length with the treatment of S5C7+TCP as compared to S5C7 treatment alone. Similarly, with the treatment of S4C10+TCP, there was increased in fresh shoot weight by 409.09% as compared to S4C10 treatment alone (Table 3, Figure 5).

Effect of PSB on P content of soil, and plant tissues
Inoculation with PSB individually increased the available P content of the soil. The results revealed the maximum available of P by the K. quasipneumoniae S6C2 treatment in the soil. Un-amended soil with the inoculated K. quasipneumoniae S6C2 had 0.056 g available P content 64.7%, followed by A. luteolus S4C7 at 0.053 g (55.882%), higher than the control, ( Table 4). The TCP amended soil, K. quasipneumoniae S6C2 had 1.866 g of P available an increase of 261.6%, followed by K. pneumoniae S4C10 at 1.818 g (252.32%), compared to the TCP amended soil control. Further, P uptake by plants was increased, as higher P content was recorded in the leaves and roots. The maximal increase in P uptake was shown by the shoots and roots inoculated with K. quasipneumoniae S6C2 (235.2% in shoots and 202.5% in roots, in un-amended soil; 403.9% in shoot and 294.9% in roots, in TCP amended soil, as compared to control) (Table 4). Inoculation with other PSB showed increase in P uptake in shoots and roots in both the TCP un-amended and amended soil, when compared to the uninoculated PSB soil as control. Overall, the amount of uptake of P by the shoots in TCP amended soil is higher than the roots and unamended TCP soil of shoots and roots of the plants.

P-use efficiency
The values of P-use efficiency were higher for TCP amended soil with PSB inoculation in A. hookeri Th. plants. It was observed that P use efficiency, as calculated by the ratio between the plant DM under low Pi and DM under high Pi (APE, g DM/g P), was 0.892 for control (without bacterial inoculation) and 1.848 with K. quasipneumoniae S6C2 treatment (Table 5), which corresponds to a 107.17% increase of P-use efficiency. A. luteolus S4C7 also resulted in 92.60% increase of P-use efficiency.

Discussion
A. hookeri Th. is a wild herb abundantly available in wide range soils found in North eastern region under Himalayan range of India. The leaves and its fibrous roots riched in carbohydrate, minerals, vitamins, are consumed as vegetables in soups and pickles. Because of its medicinal properties, and undefined agri-practices, need of an eco-friendly organic cultivation strategy was identified, and therefore role of PSB in its growth was studied. The rhizosphere soil of A. hookeri Th., which is used as condiment of intrinsic Manipuri cuisines, was selected for the isolation of PSB. Out of the 97 rhizobacteria that grew on the PVK medium plate, only 6 PSB isolates are selected based on their excellent ability of P solubilization on Pikovskaya agar plate medium. Based on 16S rRNA sequence analysis, the bacterial isolates were identified as A. luteolus, K. pneumoniae, E. asburiae, and K. quasipneumoniae. Klebsiella sp. and Enterobacter sp. are recognized as phosphate solubilizing bacteria from the rhizosphere of crop plants as reported from Korea (Chung et al. 2005). The different strains of phosphate solubilizing bacteria Proteus vulgaris, K. pneumoniae, E. aerogens, Burkholderia cepaciae, Citrobacter freundii, A. lwoffi and Pseudomonas fluorescens isolated from the rhizosphere of different plants has been reported as efficient for phosphate solubilization by forming halozones on agar plates of Pikovskaya growth medium (PVK) by solubilizing tricalcium phosphate of the medium (Sadiq et al. 2013). The plant growth-promoting P-solubilizing bacteria such as Bacillus circulans has been investigated as the potential strain from the rhizospheric region of apple plant, Himachal Pradesh, India (Mehta et al. 2015). In several reports, the efficient PSB Bacillus spp. able to convert insoluble forms of phosphorus to accessible forms and found to mobilize P efficiently in the sunflower and improved the plant's activities (Ekin 2010). Similarly, Elkoca et al. (2007) reported phosphate solubilizing bacteria Rhizobium, B. subtilis and B. megaterium as the efficient strains for phosphate solubilization. Several strains PGPR genera Acetobacter, Acinetobacter, Methylococcus, Bacillus, Micrococcus, Planococcus from the rose plant rhizosphere had the capacity to solubilize phosphate when the solid medium was supplemented with tricalcium phosphate. (El-Deeb et al. 2012). Enterobacter sp., isolated from nodules of Arachis hypogea L. was reported to solubilize tricalcium phosphate in unbuffered or buffered medium (Anzuay et al. 2013). In fact, these bacteria had been reported as biofertilizers for other plants such as Arthrobacter sp. for Zea mays L. (Arruda et al. 2013) and Glycine max L. (Kloepper et al. 1992); Enterobacter sp. for Phaseolus radiates (Zhao et al. 2011) and Arachis hypogaea L. (Anzuay et al. 2013) and Klebsiella sp. for Brassica campestris (Ahemad and Khan 2011) and Oryzae sativa (Chaiharn and Lumyong 2011). Chen et al. (2006) reported phosphate solubilization ability of Arthrobacter sp. isolated from sub-tropical soil. However, reports on K. quasipneumoniae as PSB are scanty in literature. Only recently, K. quasipneumoniae AG5-3 has been reported for their ability to solubilize and fix the free nitrogen from the acid sulphate soil rhizosphere of rice grown of Mekong Delta, Vietnam (Xuan et al. 2016). In this work, A. luteolus, K. pneumoniae, E. asburiae, and K. quasipneumoniae are being reported as PSB from rhizosphere of A. hookeri Th, which has not been explored previously for this purpose.
It is known that PSB populations are largely found in agricultural soils (Yahya and Al-Azawi 1989), but A. hookeri Th. is generally not grown in agricultural soils for large scale harvesting. The amount of soluble P released was different in NBRIP broth for different isolate. A. luteolus (S4C7) was observed as an excellent solubilizer, as free P level varied from 124.8 to 266.4 µg/mL, which was followed by two K. pneumoniae strains -S4C9 and S6C1 that solubilized 236.8 and 228.4 µg/mL P respectively. Previously, NBRIP broth has been used for estimation of P solubilization in Arthrobacter sp., Enterobacter sp. and Klebsiella sp. (Chung et al. 2005;Chen et al. 2006), without quantification. Lowering of p H was found to be correlated with P solubilization ability of all six rhizobacterial strains. Walpola and Yoon (2013) also reported that the P-solubilizers Pantoea agglomerans and Burkholderia anthina solubilized the P at the range of 600 µg/mL in the NBRIP medium with lowering the p H . Earlier, Enterobacter sp. isolated from non-rhizospheric soil of cowpea (Vigna unguiculata (L.) Walp.) has been reported to solubilize the phosphate at 58.5-79.5 µg/mL in NBRIP medium, which was substantially less than the values reported in present work for E. asburiae S5C7 . Mamta et al. (2010) reported the P solubilization by Enterobacter sp. at the range of 200 µg/mL released of soluble-P of in TCP supplemented liquid medium from the rhizospheric soil of Stevia rebaudiana while we recorded values higher than 270 µg/mL for E. asburiae S5C7. The pH of the broth was found to decline, in each case, due to bacterial activity; lowering of pH coincided with increase in the efficiency of phosphate-solubilizing activity, due to the released of organic acid in the medium. In fact, microorganisms capable of producing a halo zone due to solubilization of organic acids in the surrounding medium (Singal et al. 1991) are selected as potential phosphate solubilizers (Das 1989) and are routinely screened in the laboratory by a plate assay method (Gerretsen 1948) using Pikovskaya agar (Pikovskaya 1948) or tricalcium phosphate medium (Nautiyal (1999)). The decrease in p H of the culture broth was attributed to the secretion of organic acids by PSB. The HPLC system was used to analyze the organic acid produced by PSB isolates. In this study, production of acetic acid by isolates A. luteolus S4C7 and K. pneumoniae S6C1 was observed. Further, production of citric acid was also observed by the isolates K. pneumonia-S4C9, S4C10, K. quasipneumoniae S6C2 and Enterobacter sp (S5C7). Similar observations were recorded by Chen et al. (2006) for Arthrobacter sp., CC-BC03 where two organic acidscitric and lactic acids were reported. Kim et al. (1997) also have evidenced of producing citric acid by phosphate solubilizing bacteria Enterobacter sp. from the rhizospheric soil of plant tomato. Klebsiella sp. isolated from the pigeon pea (Cajanus cajan) rhizospheric soil have been reported for the production of acetic acid (Patel et al. 2010), which is similar to results with rhizobacteria of A. hookeri Th. in present study. Shahid et al. (2012) have reported production of gluconic acid as well as malic acid by phosphate solubilizing Enterobacter sp Fs-11 in the culture  25.9 ± 1.5m 15.7 ± 3.3m 0.5 ± 0.3m 3.0 ± 1.6m 0.24 ± 0.07m 0.4 ± 0.25m 4.0 ± 1.0m S+ TCP+ S4C7 35.0 ± 2.6n 18.5 ± 2.2n 1.8 ± 0.9n 6.1 ± 1.0n 0.86 ± 0.13n 1.3 ± 0.33n 6.6 ± 0.57n S+ TCP+ S4C9 30.8 ± 1.9o 16.1 ± 2.0m 0.9 ± 0.1m 3.8 ± 0.4m 0.69 ± 0.05o 0.7 ± 0.06o 6.6 ± 0.5n S+ TCP+ S4C10 32.2 ± 3.4p 16.2 ± 1.2m 2.8 ± 1.6p 3.8 ± 1.3m 0.68 ± 0.2o 0.8 ± 0.25o 7.6 ± 1.1o S+ TCP+ S5C7 36.5 ± 1.7q 20.0 ± 0.5m 1.1 ± 0.6o 4.9 ± 0.5o 0.67 ± 0.07o 0.8 ± 0.09o 6.3 ± 0.5n S+ TCP+ S6C1 31.0 ± 1.1o 16.8 ± 1.9m 2.8 ± 0.4m 3.4 ± 0.5m 0.6 ± 0.07o 0.7 ± 0.03o 6.6 ± 0.5n S+ TCP+ S6C2 30.9 ± 3.4o 20.7 ± 2.0o 1.5 ± 0.8n 6.05 ± 0.8n 0.66 ± 0.13o 1.0 ± 0.12p 5.3 ± 1.5n a S-unamended soil. b Values are means of three replicates, Mean values (mean±S.D) sharing the same letter do not differ significantly by Tukey at P ≤ 0.05. c TCP-Tricalcium phosphate.
have pronounced effects on plant growth and development.
The results showed that the isolate A. luteolus S4C7 produced IAA in range of 52.9-186.65 µg/mL supplemented with different amount of L-tryptophan. Dastager et al. (2010) reported a plant growth promoting, phosphate solubilizing bacteria, NII 0909 Micrococcus sp. isolated from the Western Ghat forest soil produced IAA of 109 µg/mL. The attribute of IAA synthesis is considered essential for selecting favorable microorganisms as there have been reports suggesting that the phosphate solubilizing bacteria producing IAA at the range of 28.2 µg/mL have reflective effects to enhance the growth of Aloe barbadensis Miller (Gupta et al. 2012). Earlier, Ahmad et al. (2008) investigated the rhizobacteria like Azotobacter, fluorescent Pseudomonas, Mesorhizobium and Bacillus produced IAA at 22.02 µg/mL supplemented with different concentration of L-tryptophan and exhibited multiple plant growth promoting (PGP) traits on soil-plant system as effective PGPR. Another important trait of PGPR, that may indirectly influence the plant growth, is the production of siderophores. They bind to the available form of iron Fe 3+ in the rhizosphere, thus making it unavailable to the phytopathogens and protecting the plant health. In the present study, E. asburiae S5C7 and three K. pneumoniae -S4C9, S4C10, S6C1 and K. quasipneumoniae S6C2 were able to produce siderophore. A. luteolus S4C7 was unable to produce siderophore. Though in earlier reports, A. luteolus isolated from rare earth environment of Chavara, Kerala, India, was found to produce catechol-type siderophore (Emmanuel et al. 2012). The results showed that E. asburiae (S5C7) produced 89.77 unit of siderophore in YEM broth after 72 h of incubation and further followed by PSB K. pneumoniae -S4C9, S4C10, S6C1 and K. quasipneumoniae S6C2. Previously, Pseudomonas fluorescens NCIM 5096 and Pseudomonas putida NCIM 2847 has been reported to produce 87% and 83% units of siderophore (Sayyed et al. 2005). The influence of different iron concentration on siderophore production unit was conducted in succinic acid medium (SM). The most suitable iron concentration for siderophore production was 1 µM for all the isolates with the highest record of 86.172 unit of siderophore by E. asburiae S5C7 in SM. It was also reported that P. fluorescens NCIM 5096 produced 72% unit of siderophore at 1 µM of iron concentration. Earlier, Pandey et al. (2005) reported that the production of siderophore was induced by iron in P. aeruginosa GRC1 and was found maximum at 0.2 µM of iron concentration. Also, siderophore release was reported to decrease gradually with corresponding increase in iron concentration, which is similar to results obtained with E. asburiae S5C7, K. pneumoniae -S4C9, S4C10, S6C1 and K. quasipneumoniae S6C2 in present work. Siderophore releasing rhizobacteria have not been reported earlier from A. hookeri Th.
The ability to reduce acetylene is an indirect measure of N 2 -fixation, it is specific for monitoring functional nitrogenase activity and is indicative of N 2 -fixing potential. Park et al. (2005) reported the surveyed the rhizosphere soil of agriculturally important crops widely cultivated in Cheongju province, Korea for the presence of nitrogen fixing and plant growth promoting bacteria grown on Burk's N-free medium, from rhizosphere of different crops. The ARA of different isolates PM-3, PM-6, PM-7, PM9 and PM-23 were reported to be 23. 74, 15.52, 19.85, 20.19 and 14.10 nmol C 2 H 4 /µg/ protein/hour respectively. In the present study, PSB isolate K. quasipneumoniae S6C2 was found to have maximum 28.638 nmol C 2 H 4 /µg/protein/hour in Burk's N-free medium among the genus K. pneumoniae -S4C9, S4C10 and S6C1, accessed by acetylene reduction assay using GC-FID technique. The other isolates -A. luteolus and E. asburiae were not able to fix N 2 . Venieraki et al. (2011) isolated Azospirillum brasilensis from the rhizosphere of Mexicalli, Kopaida, Vietia, with the ability of fixation of N 2 of 9.9 nmol C 2 H 4 /µg/protein/ hour.
The inoculation of PSB isolates -A. luteolus S4C7, E. asburiae S5C7, K. pneumoniae-S4C9, S4C10, S6C1 and K. quasipneumoniae S6C2 enhanced the growth of A. hookeri Th. plants in TCP amended, as well as un-amended soil, putatively due to their plant growth promoting attributes, as detected in in-vitro experiments. The data presented in Table 3 and Figure 5 clearly suggests substantial (365.5%) increase in fresh shoot weight of A. hookeri Th. by treatment of PSB K. pneumoniae S6C1 in TCP un-amended soil. It was followed by treatment with A. luteolus S4C7 (227.58%). The fresh shoot length, was recorded highest with the treatment of K. pneumoniae S6C1 (32.1 cm), followed by A. luteolus S4C7 (31.5 cm). In a similar study, P-solubilizer P. synxantha was found to increase shoot length by 36.9 cm, and root length by 11.9 cm, of Aloe vera plant (Gupta et al. 2012). Yu et al. (2011) also reported that treatment with PSB -P. chlororaphis, P. fluorocens, B. cereus increased the height of walnut plant by 32.18, 32.06, 29.7 in un-amended TCP soil. The plant heights of tomato increased treated with Pantoea agglomerans (PSB-1) and Burkholderia anthina (PSB-2) by 134.01 and 139.67 cm respectively (Walpola and Yoon 2013). In this work, treatment of PSB, along with TCP enhanced the growth of A. hookeri Th. better than treatment with respective PSB alone. There was 24.57% increase in shoot length with the treatment of S5C7+TCP as compared to S5C7 treatment alone as evident from data given in Table 3. Similarly, with the treatment of S4C10+TCP, there was increased in fresh shoot weight by 409.09% as compared to S4C10 treatment alone. This is in accordance to earlier reports where treatment of PSB with TCP amendment in soil showed increased in all possible growth parameters of host plants (Yu et al. 2011;Gupta et al. 2012;Walpola and Yoon 2013). Kumari et al. (2008) have explained that the influence of PSB in TCP added soil could be due to its simple structure and the absence of any free carbonates when compared with the crystalline lattice structure of the other form of insoluble phosphates.
The inoculation of PSB provided increased the amount of available P in soil. The highest amount of available P in TCP un-amended soil was 56 mg/kg with the treatment with K. quasipneumoniae S6C2, which was 64.7% higher than control. A. luteolus S4C7 also resulted in improving the soluble P content by 55.882% (53 mg/kg) in TCP un-amended soil (Table 4). In previous report, treatment with the PSB from the calcareous cinnamon soil of China increased significantly the availability of P in reclaimed soil in coal mining subsidense region by 35.11% (Shi et al. 2017). Similar study also reported that inoculation with PSB-Erwinia tasmaniensis TP08, Pseudomonas aeruginosa TP16 and Pseudomonas aeruginosa TP373 increased the availability of P in the soil of Oryza sativa L. plant by 10%, 1.5% and 10% respectively (Duarah et al. 2011). In contrast, the PGPR strains Bacillus megaterium M3, Bacillus subtilis OSU-142, Paenibacillus polymyxa RC05 and Azospirillum brasilense Sp245 gave no significant increase in the availability of P (without PGPR application was higher than that of the treatments with PGPR treatment) to the soil of wheat plant (Turan et al. 2012), while in present study, the availability of soluble P increased by application of PSB along with TCP in A. hookeri rhizosphere. Earlier, Yu et al. (2011) reported that the amount of available P by treatment of P. chlororaphis, P. fluorocens and B. cereus was 32.72%, 24.08% and 9.68% higher respectively in TCP un-amended soil as compared to control. However, in this work, treatment with TCP amended soil plus K. quasipneumoniae S6C2 resulted in 1866 mg/kg of available P, which was 261.6% higher as compared to TCP amended soil without bacterial treatment. Shi et al. (2017) reported that treatment with PSB+TCP significantly enhanced the availability of P (28.5%) in the soil. Similarly, Qureshi et al. (2012) reported treatment with phosphate solubilizing bacteria Bacillus sp. significantly enhanced the available P in the soil of cotton plant by 6.80%. It was observed that, K. pneumoniae S4C10 plus TCP resulted in 1818 mg/kg of available P that was 252.32% higher, compared to the TCP amended soil. This was in accordance to earlier report with Stevia rebaudiana Bertoni plant treated with TCP and PSB, where Burkholderia gladioli 10216 caused 332.02% increase of soluble P, and Enterobacter aerogenes resulted in 269.1% available P (Mamta et al. 2010). The importance of phosphate solubilizing microorganisms is also considered in terms of P immobilized in the biomass of host plants (Demetz and Insam 1999). The stimulatory effect of the inoculation of PSB was evident on P uptake by A. hookeri Th. plants, which might be due to the better availability of soluble P. Maximal P content in shoots and roots was shown by in treatments with K. quasipneumoniae (S6C2) in un-amended TCP soil, where P content was respectively 235.2% and 202.5% higher than control. Earlier, Kaur and Reddy (2013) reported the efficiency of Pantoea cypripedii PSB-3 to enhance the total P uptake in shoots by 20.0% and in roots by 22.22%, in maize plant. Another PSB, Pseudomonas plecoglossicida PSB-5 was reported to result in 29.92% and 41.73% higher P in shoot and root tissues respectively in maize plant. The P uptake was even better in bacterial treatments with TCP amended soil. In fact, the P content in shoot and root tissues were 403.9% and 294.9% higher, respectively, with E. asburiae S5C7 + TCP treatment. In earlier report, inoculation with bioinoculants of PSB (Bacillus polymyxa), showed the enhancement of nutrient (P) uptake by 31.61% by the plant pigeon pea (Cajanus cajan L.) over the control . The inoculation with the P-solubilizing bacteria Pseudomonas putida showed significantly increased P content of the plant Stevia rebaudiana Bertoni when comparison to the control (Vafadar et al. 2014). Similar work has been reported in maize plant that the inoculation with Enterobacter radicincitans and Pseudomonas fluorescens increased the P uptake by maize plant by 1.86% and 1.49% respectively (Krey et al. 2013). Duarah et al. (2011) investigated phosphate solubilizing bacteria Erwinia tasmaniensis TP08, Pseudomonas aeruginosa TP16 and Pseudomonas aeruginosa TP373 among the plant growth promoting rhizobacteria (PGPR) used as biofertilizers for plant growth and nutrient (P) use efficiency on yard-long bean (Vigna unguiculata) plant by 65.21%, 86.95% and 30.43% respectively. Some workers have reported similar observations in other plants with TCP (Mamta et al. 2010), such as, Walpola and Yoon (2013) reported that shoot and root had respectively 26.02% and 155.05% higher P with the treatment of Pantoae agglomerans in TCP amended soil. Gupta et al, (2012) also observed the similar results in Aloe vera plant, where 52.41% higher P was recorded in plant treated with TCP + Enterobacter hormaechei. The PGPR (Bacillus subtilis HJR3) associated with the maize (Zea mays L.) from the region of Himalayan region showed the maximum total P contents both in shoot and root by 488.57% (Zahid et al. 2015). Also, Turan et al. (2012) demonstrated a similar connection among the inoculation of PGPR's Bacillus subtilis OSU142 and Bacillus megaterium M3 has growth stimulatory effect that reflected by higher P concentrations in most plant organs-leaves (30% and 139% respectively) and roots (140% and 300% respectively). Similarly, treatment with rhizobacterial strain S7 from rhizospheric region of field grown runner bean (Phaseolus coccineus L.) significantly increased the grain yields with 15.03% and yield increase can be attributed with the nutrient (P) uptake by the plants and other plant activities (Stefan et al. 2013). Mäder et al.(2011) found that mutualistic root microorganisms such as plant growth promoting rhizobacteria (PGPR) Pseudomonas jessenii R62 and Pseudomonas synxantha R81 enhanced the P concentration of the plant by 45.32%, in this way, improves the plants fitness and growth responses to plants like Wheat (Triticum aestiva L.), with the inoculation of PGPR. The bacteria, by increasing the volume and root development, increased the plant's access to nutrients and water, thereby attracting the plant nutrients. Finally, the plants nutrients' uptake increased the plant shoot growth (Davoodifard et al. 2012).
Further, agronomic P-use efficiency and relative efficiency of phosphorus (APE and REP) was recorded maximum with the application of K. quasipneumoniae S6C2 with the proportionate amount of TCP (107.17%). Mäder et al. (2011) reported that the inoculation with Pseudomonas strains (P. jessenii and P. synxantha) increased the P-use efficiency of 95% which means efficiently taken up from the soils of the wheat plants. Kumar and Chandra (2008) obtained enhanced P uptake of 38% in lentil crops inoculated with Pseudomonas diminuta. Neto et al. (2016) reported that agronomic P-use efficiency varied among coffee cultivars, E16Shoa, E22Sidamo, Iêmen and Acaiá cultivars were classified as the most efficient and responsive to Pi supply. Enhancement of APE and REP with the application of PSB in TCP amended soil provide evidences that these PSB, including K. quasipneumoniae S6C2 and A. luteolus S4C7 have potential for commercialization and may be utilized in systematic cultivation of A. hookeri Th.