1 Introduction

Plant growth promoting rhizobacteria (PGPR) are free living rhizobacteria which colonize plant roots in the competitive environment and enhance crop productivity by exerting beneficial effects through various mechanisms of action (Mrkovacki and Bjelic, 2011). Capsicum is a remunerative crop to the hill farmers and is grown in open fields and under protective structures. The higher nutrient requirements of the crop and heavy diseases incidence in natural growing agro-climatic conditions has resulted in sizeable yield losses. The indiscriminate use of the chemical inputs has deleterious effects on agro-ecosystem as well as productivity of the crop (Gupta et al., 2015a). Further, the ever increasing cost of pesticides or fertilizers coupled with inclination of high profile society towards organic food and concern towards environmental degradation has forced agriculture scientists to have an eco-friendly, affordable alternate production system which may reduce the dependence on chemical inputs without lowering the productivity of the crop. Also, the integrated use of different organics not only increase the nutrient status of the agricultural soil but also help to improve the various physical, chemical and biological properties of soil leading to improved soil fertility and fertilizer use efficiency (Mishra et al., 2012). Further, these rhizobacteria due to versatile plant beneficial traits are promising the environmental friendly tools for sustainable agriculture (Ahemad and Kibret, 2014). Due to great variation in soil ecology of different regions no single PGPR can be used universally as a bio-inoculant. Hence, the present investigations were carried out to explore the rhizobacteria possessing multiple growth promoting traits and able to flourish well in most soils of the geographical niches of the state.

2 Materials and Methods

The experiment was conducted at laboratory of Department of Basic Sciences, Dr. YS Parmer University of Horticulture & Forestry Sloan, Himachal Pradesh (India) during 2013-2015. Indigenous PGPR were isolated, characterized and screened for various plant growth promoting activities. Effect of PGPR bacterisation on growth and yield of capsicum variety California Wonder selected by Dr YSPUHF, Nauni and recommended by Himachal Pradesh government to state farmers, was studied.

2.1 Isolation and screening of PGPR isolates

Capsicum roots along with the adhered rhizospheric soil were collected from different locations of Chamba, Kullu and Shimla districts of Himachal Pradesh (Agro-climatic zone-III, with altitude - 1,700 to 2,633 m above sea level) representing the high hill conditions of India. Bacterial isolates persisting in the rhizospheric soil samples and roots were obtained by serial dilution and plate count technique by using nutrient agar medium. Enumeration on Jensen’s (Jensen, 1987) and Pikovskaya’s medium (PVK) (Pikovskaya, 1948) was done using replica plate technique. Twelve most predominant, morphological distinct, isolates that were able to form clear halo zone on the PVK and grow on Jensen’s media, were selected for further screening. Phosphate solubilization index (PSI) was measured as per (Edi-Premono et al., 1996) and the quantitative estimation of phosphate solubilization was carried out as per method described by Bray and Kurtz (1945). The ability to fix nitrogen on nitrogen free Jensen media and to produce indole-3-acetic acid (IAA) on Luria Bertani Broth were determined by the method of Jensen (1987) and Gorden and Palleg (1957), respectively. The ability of isolates to use ACC (1-aminocyclopropane-1-carobylic acid) as a sole nitrogen source in minimal salts medium, produce siderophore and hydrocyanic acid (HCN) was assessed by Dworkin and Foster (1958), Schwyn and Neilands (1987) and Bakker and Schipper (1987), respectively.

2.1 Net house studies

Among twelve, best two isolates (JHA₆ and ROH₁₄) on basis of various PGP traits were selected for pot experiment under net house condition. Capsicum seeds were surface sterilized with 0.2% HgCl₂ were sown in a seed bed and 45 days after sowing the seedlings were carefully uprooted and the root portion was dipped into individual culture broth of selected isolates (cell density about 10⁸ cells/ml) for four hours. The following seven treatments were laid out in Completely Randomized Block Design, replicated thrice: T₁- 100% Recommended Dose (RD) of NPK; T₂- 100% RD of NPK +JHA₆; T₃- 100% RD of NPK +ROH₁₄; T₄- 80% RD of NPK + JHA₆; T₅- 80% RD of NPK + ROH₁₄; T₆- 60% RD of NPK + JHA₆; T₇- 60% RD of NPK + ROH₁₄. Uninoculated seeds with 100% recommended dose of chemical fertilizers (NPK) were designated as control (T₁). Recommended dose of N, phosphorus pent-oxide (P₂O₅) and potassium oxide (K₂O) was 254, 77 and 350 kg ha^-1, respectively. Urea (46% N), single super phosphate (16% P₂O₅) and muriate of potash (60% K₂O) were used as source of chemical fertilizers. The potting mixture was prepared by mixing sand, soil and farm yard manure (FYM) in a ratio of 1:1:2 and sterilized by autoclaving for three subsequent days at 121.5 ºC for 30 min. Soil mixtures used for experimentation was slightly acidic in reaction with pH (6.10), normal in soil electrical conductivity (EC) (0.42 to 0.47 dSm^-1) and high in organic carbon (OC) (0.89%), having available N, P and K contents in medium range. The seedlings were subsequently transferred into pots and allowed to grow for 135 days. Plant growth parameters like shoot oot length (cm) and biomass (g), fruit weight (g), number of fruits per plant and yield were recorded. Plant N, P and K content were determined as per Jackson (2005). N, P and K uptake (mg plant^-1) was calculated by multiplying total N, P and K content (root, shoot, leaf and fruit) of plant with total dry matter content. Available N, P and K content of soil were determined following standard procedures (Tandon, 2009). The data were statistically analyzed by analysis of variance (Gomez and Gomez, 1976) using critical difference at 5% level of significance.

3 Results and Discussion

3.1 Isolation and screening of PGPR isolates

The microbial counts in rhizosphere and plant roots varied significantly with location (Table 1). Among various locations, on nutrient agar medium highest rhizospheric bacterial count (95.5×10⁵ cfu/g soil) was recorded from Theog region whereas maximum endophytic bacterial population (94.0×10² cfu/g root) was recorded from Bharmour region of Chamba district.

The nitrogen fixers and P-solubilisers count, in general, was more in rhizosphere as compared to roots. Capsicum rhizospheric soil of Banikhet was found to harbor maximum population (50.5×10⁵ cfu/g soil) of N-fixers whereas Sarahan rhizospheric soil was found to be dominated by P-solubilisers population with highest count of 68.0 ×10⁵ cfu/g soil. Among endophytes, maximum N-fixers (38.0×10² cfu/g root) were recorded from Patlikhul, whereas, Theog had the highest count (51.5×10² cfu/g root) of P-solubilisers. This variation in rhizosphere and roots may be attributed due to positive influence exerted by root exudates, environmental conditions, age of plant, variety/cultivar type, time of sampling and physico-chemical properties of soil (Ahemad and Khan, 2011). 

Table 1 Enumeration of rhizospheric and endophytic bacterial population associated with capsicum

All the selected twelve isolates possess two or more plant growth promoting (PGP) traits (Table 2). Phosphorus and nitrogen are among the essential nutrients of the crop. Available form of phosphorus to plants is phosphate anions, which are mostly trapped via precipitation with cations such as Mg²⁺, Ca²⁺, Al³⁺ and Fe³⁺ and so become insoluble and unavailable to plants in these forms. The plants depend on biological nitrogen fixation (BNF) for available nitrogen as plant species is capable for fixing atmospheric dinitrogen into ammonia and expend it directly for its growth.

Table 2 Screening of selected isolates for their multifarious plant growth promoting traits

All the selected isolates were able to solubilise the tri-calcium phosphate with phosphate solubilisation index (PSI) and solubilisation efficiency ranging from 1.27 to 1.95 and 94.87% to 26.74%, respectively and able to grow on N-free media. Maximum Phosphate solubilisation index (PSI) and solubilisation efficiency (1.95 and 94.87%) was noted for isolate JHA₆, however, was on a par with isolate BHAR₄, PAT₉, SARA₉, THE₁₇, ROH₆ and ROH₁₄. Singh et al. (2013) has also reported similar release of phosphate by different isolates.  

Iron is a vital nutrient for almost all forms of life. In the aerobic environment, iron occurs principally as Fe³⁺ and is likely to form insoluble hydroxides and oxy-hydroxides, thus making it generally inaccessible to both plants and microorganisms. Commonly, bacteria acquire iron by the secretion of low-molecular mass iron chelators referred to as siderophores which have high association constants for complexing iron (Ahmed and Kibret, 2014). Siderophore production ranged from 87.62% to 29.85%. Maximum siderophore unit was produced by isolate THE₁₈ (87.62%), which was statistically at par with BHAR₄, BAJ₁₆, PAT₉, PAT₁₃, SARA₉ and ROH₁₄.

PGPR are well known to stimulate the plant growth by acting as phytostimulators. IAA has been implicated in virtually every aspect of plant growth and development, as well as defense responses. This diversity of function is reflected by the extraordinary complexity of IAA biosynthetic, transport and signaling pathways (Pratap and Ranjitha Kumari, 2015). Out of twelve isolates, 66.66% (8/12) exhibited IAA production activity on Luria Bertani Broth. The level of IAA production in the selected isolates ranged from 13.00 to 31.33 µg ml^-1. The isolate SARA₉ produced maximum concentration of IAA (31.33 µg ml^-1) followed by BHA₂₂ (25.67 µg ml^-1). Comparable amount of IAA and siderophore for Bacillus and Pseudomonas sp. have been reported by Jarak et al. (2012).

Apart from being a plant growth regulator, ethylene has also been established as a stress hormone, as the endogenous level of ethylene is significantly increased which negatively affects the overall plant growth. (Bhattacharyya and Jha, 2012). Plant growth promoting rhizobacteria which possess the enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, facilitate plant growth and development by decreasing ethylene levels, as take up the ethylene precursor ACC and convert it into 2-oxobutanoate and NH₃ inducing salt tolerance and reducing drought stress in plants (Nadeem et al., 2007; Zahir et al., 2008). Except, three isolates i.e. BHAR₄, PAT₁₃ and THE₁₇, all isolates were able to grow on minimal (M9) medium having 1-aminocyclopropane- 1-carboxylic acid (ACC) as sole source of nitrogen.

Microbial production of HCN has been suggested as an important antifungal feature to control root fungi pathogen (Junaid et al. 2013). Only three (BHAR₄, JHA₆ and JHA₈) isolates were HCN producers. The host plants are generally not harmfully affected by inoculation with HCN production bacteria and host specific rhizobacteria can operate as biological control agents (Saharan and Nehra, 2011).

3.2 Net house studies

A perusal of data (Table 3) revealed maximum shoot height and biomass (79.0 cm and 39.75g) under T₂, which was statistically on a par with T₃. Maximum fruit yield (1.31 kg plant^-1) was noted under T_2, which was significantly on a par with T₃ (1.28 kg plant^-1). Maximum plant height and dry matter accumulation in T₂ and T₃ may be attributed to increased root length and biomass (32.92 cm and 2.53 g; 30.51 cm and 2.63 g) as a result of biofertilization (increasing availability of the soil nutrients particularly N (asymbiotic N-fixation), P (P-solubilisation) and Fe (siderophore production)) to plant by the direct mechanisms of application of PGPR inoculum in the rhizosphere (Datta et al. 2011). Higher yield under these treatments was attributed to more number of fruits per plant (13.86 and 13.82) and maximum average fruit weight (98.00 and 93.67 g).

The data (Table 4) indicate maximum N uptake (548.69 kg ha^-1) in T₂ which was statistically on a par with T₃ (518.93 kg ha^-1) and T₄ (524.68 kg ha^-1). Maximum P uptake (59.80 kg ha^-1) was recorded in T₂, which was statistically on a par with T₁ and T_5. Higher N and P uptake in PGPR treated plants may be explained in light of the observations made by Kaushal and Kaushal (2013) who reported increased N and P fertilizers use efficiency when applied along with PGPR strains in cauliflower. Application of PGPR could not exert a significant impact on K uptake of capsicum as compared to control.

The available soil nutrient status (Table 4) revealed maximum available N (408.24 kg ha^-1) in T₂, which was 36.67 per cent higher over initial soil test value. Such an increase in T₃ was 32.42 per cent. The differences between T₂ and T₃ were statistically non-significant. Maximum available P (51.67 kg ha^-1) was observed in T₂ followed by T₃.  The higher available N in 100% RD of chemical fertilizers in combination with either JHA₆ and ROH₁₄ isolate may be attributed to more asymbiotic nitrogen fixation by bacterial isolates. Higher available P in PGPR treatments may be attributed to phosphate solubilising activity of microorganisms, which might have brought some P from unavailable pool to available pool. The enhanced nutrient availability especially N and P in the presence of PGPR has also been reported by Gupta et al. (2015b). Further, treatment T₂ registered maximum available K (277.83 kg ha^-1), which was, however, statistically at par with T₃ (274.73 kg ha^-1). Since, available K was comparable between T₁, T₂ and T_3, it is inferred that K content of soil is independent of PGPR activities.

Table 3 Effect of liquid bacterial inoculum on the growth and yield of capsicum

Table 4 Effect of liquid bacterial inoculum on Nutrient content of capsicum and nutrient status of the soil at the end of the experiment

4 Conclusions

Both isolated PGPR (JHA₆ and ROH₁₄) increased availability and uptake of N and P. Available K content of soil and its uptake was independent of PGPR activity. The productivity of capsicum with 80% RD of chemical fertilizers plus PGPR and sole application of 100% RD through chemical fertilizers was statistically on a par, thereby, saving about 50 kg N ha^-1 and 15 kg P₂O₅ ha^-1 fertilizers. The study, therefore, indicates the potential of isolated JHA₆ and ROH₁₄ PGPR strains in partial replacement of N and P (~20%) applied through chemical fertilizers, besides higher productivity of crops.

5 Acknowledgements

Financial support from “Innovation in Science Pursuit for Inspired Research (INSPIRE) Programme”, Department of Science and Technology, New Delhi is duly acknowledged.

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