Application of potassium-solubilising Proteus mirabilis MG738216 inhabiting cattle dung in improving nutrient use efficiency of Foeniculum vulgare Mill.
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The present research is aimed to evaluate buffalo dung inhabiting potassium solubilising bacteria (KSB) comprising characteristics of plant growth promotion to improve growth and yield of Foeniculum vulgare Mill. As buffalo dung has been used from ancient time in agricultural practices, it was selected as a source to harness the KSB that may enhance vegetative and reproductive parameters of F. vulgare. In total 15 KSB isolates were selected for solubilizing insoluble potassium in the form of mica powder. Four isolates BUFF12, BUFF14, BUFF23 and BUFF38 having abilities of plant growth promotion such as IAA production, HCN production, P solubilisation and phytase production performed better (after seed bacterization) in terms of plant growth. On the basis of 16S rRNA gene sequencing, the most potential KSB isolate was identified as Proteus mirabilis. Selected isolate P. mirabilis increases vegetative and reproductive parameters of F. vulgare. It increased grain yield by 55.03%, biological yield by 39.06% and harvest index by 11.49% over control. It exhibited the most pronounced advantageous effect on the growth and nutrient uptake by F. vulgare. P. mirabilis proved as a bio-inoculant and can be used as substitute of K fertilizer for raising crop of F. vulgare in sustainable manner.
KeywordsBuffalo dung Potassium solubilisation Proteus mirabilis Foeniculum vulgare
Potassium (K) is considered as an essential macronutrient for organisms living on earth (Bahadur et al. 2019). It shows an important part in overall growth and development of plant. Although, in most of the soils major K pools are silicate minerals viz., biotite, orthoclase, muscovite, feldspar, mica, vermiculite, illite, smectite, etc. (Pramanik et al. 2019), but these reserves are considered to be non-available for plant as replaceable and non-replaceable K establishes a very small percentage of mineral K in soils hence. Further, absence of effective K replenishment protocols causes enhancement of K deficiency in soils coupled with a quick decline in the nutrient in demanding crop cultivation systems (Sattar et al. 2018).
Zeng et al. (2012) and Sarikhani et al. (2018) identified bacterial genera of Pseudomonas sp. having tendency to solubilize minerals of K and convert them into soluble forms by releasing organic acids. Therefore, use of these K solubilising microbes (KSM) is considered as an attractive and bold strategy. To improve agricultural productivity, these microbes serve as biofertilizer in lieu of chemical fertilizers by converting insoluble K in the soil into soluble form that is easily available to the plants. Vasanthi et al. (2018) studied a wide variety of bacterial species including Bacillus edaphicus, Bacillus mucilaginosus, Burkholderia sp., Bacillus circulans, Acidithobacillus ferrooxidans, Paenibacillus mucilaginosus and Paenibacillus glucanolyticus to solubilise K, as they produce high amount of organic acids like oxalic, tartaric and citric acids. Release of these acids by bacteria is considered as an important mechanism for the solubilization of K minerals through acidification and protonation by microbes into plant available form (Parmar and Sindhu 2019).
Buffalo is considered as a part of the property, possession and profession by rural farmers in many countries and occupied a central role to complete the development of society by contributing to milk, meat, hides, draft power and even dung for agricultural operations. Certain dung inhabiting bacteria and fungi are reported to enhance the plant growth (Dhiman et al. 2019). Because dung is used as a fertilizer, its feasibility as a source of beneficial bacteria with K solubilizing activity may be an extra benefit for sustainably growing crops. Hence, buffalo dung was selected and aimed to harness the potassium solubilising bacteria (KSB) to observe their effects on different growth parameters of Foeniculum vulgare.
Materials and methods
Analysis of dung
N content was determined by the methodology given by Bremner (1960). In this method, dung was treated with 10 g K2SO4, 1 g CuSO4·5H2O, 0.1 g Se and 30–40 ml H2SO4 and the mixture was heated until it cleared after boiling gently for 5 h. The digest was diluted with distilled water to make the final volume 50 ml as indicated by graduation on digestion tubes. An aliquot of the diluted digest containing from 0.2 to 0.5 mg of N was transferred to a 100-ml steam distillation flask. Steam distillation was achieved and ammonium was estimated by using 0.007N H2SO4 for titration of distillate.
Available P was monitored by the methodology of Olsen (1954). In this method, 20 ml extracting solution (0.5 M NaHCO3, pH 8.5) was mixed with 1 g dung sample and shook at 200 rpm for 30 min at room temperature. Charcoal (200 mg) was added to obtain a colourless filtrate. Extract was filtered through Whatman No. 42 filter paper and P was analysed by calorimeter.
K was determined by flame photometer according to method of Jadoon et al. (2015). Dung sample (5 g) was taken and 50 ml ammonium acetate solution was added to it. The mixture was shaken for 30 min and then left for a while. The sample was then filtered and the filtrate was read by flame photometer to determine the K content.
Isolation of potassium solubilising microorganisms (KSM) from cattle (buffalo) dung
Serial dilution plate method was used to isolate KSM by using Aleksandrov’s agar medium amended with insoluble source of potassium (mica powder) (Setiawati and Mutmainnah 2016). The pH of the medium was stabilised to 7.5 and after inoculation plates were incubated at 37 °C for 24–72 h. K solubilisation by different bacterial isolates was detected on the basis of ability to form zone of solubilisation. Colonies showing clear zone were selected and the solubilisation index (SI) was calculated.
Evaluation of PGP characteristics
All the isolates were screened for different plant growth promoting attributes. The ability of the isolates for phosphate solubilisation was measured qualitatively and quantitatively. The qualitative assessment was done on Pikovskaya’s medium as described by Pikovskaya (1948) and quantitative analysis by following the method of Olsen (Olsen 1954). For phytase activity screening medium amended with calcium and sodium phytate as sole source of organic phosphate was taken and inoculated by the isolates. Solubilisation efficiency was determined as described by Kumar et al. (2012). The qualitative and quantitative determination of IAA production was done by using Salkowaski reagent (Gordan and Weber 1951). Further, isolates were also analysed for production HCN (Baliyan et al. 2018).
Optimization of growth conditions for efficient solubilisation of potassium
Potassium solubilisation was checked at neutral pH and 28 ± 2 °C in Aleksandrov’s broth. The effect of different temperatures as well as pH along with amendments of various sugars was evaluated to determine optimum conditions for efficient solubilisation.
Assessment of impact of different sugars on the solubilisation of mica
To measure the effect of carbon sources, bacterial isolates were inoculated in Aleksandrov's broth wherein glucose was replaced with different carbon sources (galactose, xylose and arabinose) and incubated at 28 ± 1 °C. The amount of released K was assessed in comparison to un-inoculated control (Parmar and Sindhu 2013).
Assessment of potassium solubilisation ability
After incubation (for 7 days), available K was estimated in cell free-culture supernatant obtained after centrifugation (at 10,000 rpm for 10 min). The available K content was estimated in the collected supernatant at regular intervals (Zhang and Kong 2014). The broth without bacterial inoculant acted as control. The standard curve was prepared by using KCl solution.
Detection of organic acid
The organic acid detection was done in cell free culture supernatant. Isolate BUFF14 was inoculated in Aleksandrov’s broth and incubated for 7 days. After incubation broth was centrifuged at 10,000 rpm for 15 min and the resultant supernatant was collected. Supernatant (20 μl) was then passed through C-18 columns of high performance liquid chromatography (HPLC) machine (model Shimadzu 10A) at 0.6 ml min−1 flow rate. Methanol: Water solution (30:70) was used as mobile phase (Suleman et al. 2018). The process was monitored at 210 nm. Peak area and retention time were compared with the standards used for the quantification of organic acids as per Wang et al. (2017).
For seed bacterization, healthy seeds were procured from Vegetable Research Centre (VRC), G. B. Pant University of Agriculture and Technology, Pantnagar, Uttarakhand. The healthy seeds of fennel variety, Pant Madhurika (Pantnagar, Uttarakhand) were selected to conduct the trials. Seed bacterization was done according to Weller and Cook (1983) and modified by Dhiman et al. (2019). The seeds were surface sterilised by treating with 95% ethanol for 1 min followed by 4% NaOCl treatment for 3 min and then washed with sterile distilled water. Cell biomass of 24 h old culture of bacterial isolates BUFF12, BUFF14, BUFF23 and BUFF38 was used to obtain a population density of 108 cfu/ml. and Cell suspension of bacterial isolates was mixed with 1% carboxy methyl cellulose (CMC) to make slurry for the adhesion of bacteria on the surface of seeds. The coating of seeds without bacteria served as control.
Pot trials were performed by using random block design in triplicates. The sandy + clay soil was used to conduct the experiment and pots (12″ dia) were kept in green house conditions. The sets of treatments of bacterized seeds were prepared and sown in pots as follows: T1-BUFF12, T2-BUFF14, T3-BUFF23, T4-BUFF38, T5-control. Effect of all the isolates on seed germination was recorded 30 days after sowing (DAS) along with other parameters such as shoot and root length, fresh and dry weight and vigour index after harvesting of crop (60 DAS).
Crop raising in field
Nutrient use efficiency of F. vulgare
The plants raised were categorised and the seeds were grinded very finely after drying at 65° C for 48 h. The seed powder was used to determine the N, P and K contents according to Wang et al. (2003). Kjeldahl method was used to detect the total N (Bremner 1960) while sodium carbonate fusion method was used to determine P concentration. K concentration was analysed by flame photometric method (Knudsen et al. 1982).
On the basis of observations (for maximum biological yield and harvest index) in comparison to other isolates, isolate BUFF14 was identified by 16S rDNA sequence analysis. The genomic DNA was isolated according to Sambrook and Russel (2001). The 16S rRNA gene was amplified by using PCR (MJ Research PTC-100) using primers 27F 5′ (AGA GTT TGA TCM TGG CTC AG) 3 and 1492R 5′ (TAC GGY TAC CTT GTT ACG ACT T) 3′. By using BLAST programme of GenBank database (NCBI), 16S rRNA gene sequence was aligned and identified. The obtained sequences were aligned by Clustal W using MEGA7 software. Phylogenetic tree was created by using neighbour-joining method to confirm the identity of these isolates (Kumar et al. 2016).
The data recognized during the study was exposed to one-way analysis of variance (ANOVA) to estimate the significance by the magnitude of the F value (Gomez and Gomez 1984).
Dung sampling and analysis
The buffalo dung (pH 7.2) contained different amount of nutrients. On an average the values were: 2.4% N, 1.4% K, 0.5% total P, 37.11% organic matter and 71% moisture content.
Isolation of potassium solubilising microorganisms (KSM)
Plant growth promoting characteristics
The isolates were screened for plant growth promoting (PGP) characteristics and the results explained that the potent isolate BUFF14 was found to show best PGP activities such as phosphate solubilization; phytase production and cyanogen production. The isolates showed IAA production (by forming pink colour in culture filtrate). Along with the incubation, the strength of colour in culture supernatants got enhanced, and reached maximum after 120 h. After 72 h of incubation, BUFF14 produced maximum amount of IAA followed by BUFF12, BUFF23 and BUFF38. The isolates were further screened for phosphate solubilisation monitored up to 5 days in Pikovskaya’s medium at 37 ± 1 °C. The maximum solubilisation of phosphate was observed after 3 days of incubation. Quantitative estimation revealed BUFF14 as maximum phosphate solubilizer (among all isolates) followed by BUFF12, BUFF38 and BUFF23. The potent isolates BUFF12, BUFF14, BUFF23 and BUFF38 were observed to solubilize calcium and sodium phytate and formed halo zone around colonies. BUFF14 showed maximum phytase production and BUFF23 showed least. BUFF12, BUFF14 and BUFF38 were effective HCN producers as they changed colour of filter paper from yellow to moderate brown. Maximum colour change was shown by BUFF14.
Optimization of growth conditions for efficient solubilisation of potassium
In the present research, the optimum conditions for K solubilisation (in terms of sugars, pH and temperature) were studied for selected potent isolates BUFF12, BUFF14, BUFF23 and BUFF38. Optimization of conditions for efficient K solubilisation by the bacterial isolates showed that maximum was achieved in glucose amended broth. Solubilisation was observed less in galactose, xylose and arabinose amended medium. Maximum K solubilization in case of all the sugars was observed with BUFF14 followed by BUFF12, BUFF38 and BUFF23.
Assessment of potassium solubilisation ability
The capability of all the 15 KSB isolates to solubilize K was evaluated. The activity to solubilize K (by the isolates) ranged from 0.55 to 4.6 mg l−1 (Fig. 1). Out of 15 isolates, BUFF14 exhibited maximum K-solubilisation in comparison to other isolates.
Detection of organic acids
HPLC analysis of the P. mirabilis culture filtrate was done for qualitative and quantitative analysis of the organic acids produced during the process of solubilisation of insoluble form of K, i.e., mica. The HPLC profiling of crude samples showed the presence of organic acids. The HPLC elution profile of standard (gluconic acid, malic acid and citric acid) showed major peaks at 6.660, 2.816 and 3.815 and that of supernatant showed peak around 6.660 suggesting the abundance of gluconic acid. Other acids detected were citric acid and malic acid but their presence was only in traces.
Growth parameters of F. vulgare Mill. under pot trial (60 DAS) when inoculated with different isolates
Seed germination (%)
Seedling weight (g)
Crop raising in field
Plant growth promotion of F. vulagre Mill. in field when inoculated with different isolates (180 DAS)
Grain yield (kg/ha)
Biological yield (kg/ha)
Nutrient use efficiency of F. vulgare
Molecular characterization and phylogenetic analysis of isolate BUFF14
K is a macro nutrient limiting the plant growth if it is deficient. In K deficient conditions the crop productivity is improved due to extensive use of chemicals which adversely affect soil properties (Qureshi et al. 2018). Parida et al. (2019) reported some of the soil microorganisms as active K solubilizers which play key role in cycling of this key nutrient. From ancient time, buffalo dung is a potent source of both micronutrients and macronutrients and it has been used as an organic compost (Dhiman et al. 2019). But maximum beneficial activity (of dung) is due to the presence of beneficial microorganisms (Meena et al. 2018). Besides this buffalo dungis also a rich source of cellulose, hemicellulose, crude fibre, crude protein along with several minerals such as N, K, S, and traces of P, Fe, Co, Mg, Cl, Mn, etc. Dung inhabiting microbes play an important role in agriculture by solubilising minerals and making them available to the plants.
Earlier studies have showed that KSB can enhance plant growth and act as PGP microbes (Basak and Biswas 2010). Present study indicated that KSB isolates can increase growth of plant by increasing K content. It was studied that soil inoculated with the isolates can boost the growth of F. vulgare along with the absorption of N, P and K content. By observing the effects of isolates on the growth of F. vulgare, it was concluded that all four potent isolates promoted the growth of plant to different grades. These isolates were found to have tendency to improve the crop productivity by persuading PGP attributes like production of IAA and by solubilising minerals.
The isolates showed their constitutive and induced nature in IAA production. Isolates not only produced IAA in medium amended with tryptophan but also were found to be capable to produce the phytohormone without addition of tryptophan (Baliyan et al. 2018). Highest amount of IAA production was observed in case of BUFF14 (74 µg/ml) in comparison to other isolates. Early seed germination and seedling parameters were observed due to higher IAA production by PGPBs as it is an efficient plant growth hormone. Current research also observed morphological and physiological changes in the young seedlings due to the production of sufficient amount of IAA (Masciarelli et al. 2013).
The experimental studies showed that the isolates were capable to solubilise phosphate (organic and inorganic) and BUFF14 proved to be an outstanding phosphate solubiliser possibly due to production of organic acid thus lowering the pH. BUFF14 was found to solubilise maximum amount of phosphate (16.8 μg ml−1) as compared to other isolates. In addition of this, BUFF14 was also found to be able to produce phytase and carry out calcium phytate solubilisation. These isolates observed to upsurge the accessibility of minerals into the soil and stimulate the plat growth. For sustainable agriculture, use of KSB as biological fertilizers can usher a new era along with other plant growth promoting rhizobacteria (PGPR) (Dhiman et al. 2019).
Sheng et al. (2008 ) reported that organic acid production is a chief mechanism of solubilisation of K minerals due to acidification and protonation making it available to plants. Various soil microbes excrete organic acids that lowers the pH of surrounding soil thus facilitating the solubilization of insoluble minerals into soluble form, easily taken up by plants. Other than the release of organic acids, increase in carbonic acid concentration due to degradation of particulate and dissolved organic carbon, leads to weathering by proton-promoted dissolution mechanisms and influences release of K from unavailable forms (Sattar et al. 2018). In the present research, conditions for efficient K solubilization were also checked and optimised. Earlier, some workers also studied for the optimization of different conditions for efficient K solubilisation (Parmar and Sindhu 2013; Sheng and Huang 2002). They reported that pH, dissolved oxygen and bacterial strain used affects the release of K from minerals. These researchers, as also is the finding of the current study, reported that the optimum conditions for K solubilization are 30 °C, pH 7 and glucose as preferable carbon source.
Isolate BUFF14 identified as P. mirabilis, by molecular characterization, enhanced both vegetative and reproductive growth of F. vulgare. Other BUFF isolates promoted the plant growth to different degrees and enhanced nutrient use efficiency of F. vulagare as well but were not as effective as P. mirabilis BUFF14. P. mirabilis BUFF14 exhibited most efficient K solubilization. The potent isolate BUFF14 was selected for the analysis of organic acid production in Aleksandrov’s liquid medium by HPLC. Gluconic acid was detected as the major organic acid secreted by BUFF14 which can be responsible for the K solubilisation. In this study, the kind of organic acid secreted by the isolate was found to be differed from that reported in earlier studies by the KSB; such as tartaric acid and oxalic acid produced by Bacillus edaphicus (Sheng and He 2006) and malic, oxalic and tartaric acid secreted by some strains as reported by Prajapati and Modi (2012).
The potassium solubilising P. mirabilis BUFF14 contributed in boosting growth of F. vulgare. BUFF14 was best among all the other isolates used in the experiment that displayed increase in germination and growth parameters of seedling. Under field conditions also P. mirabilis BUFF14 was found to be the best which effectively increased all the vegetative and reproductive plant parameters and showed potency to improve the quality of crop. BUFF 14 is thus a potential isolate with multiple PGP charactersistics, including ability to solubilize K, which can be used as a biostimulant for sustainable agriculture, particularly in nutrient deficient soils.
The study showed the presence and isolation of excellent KSB from cattle dung. The work presented here showed that isolate Proteus mirabilis BUFF14 was most efficient K solubilizer and also proved to be best in improving the yield of F. vulgare. Inoculation of P. mirabilis BUFF14 enhanced germination, vegetative parameters, yield and nutrient efficiency of F. vulgare in field conditions and thus could be a promising substitute to supply nutrients, particularly K to the crops in a sustainable and eco-friendly manner.
The authors are grateful to the Department of Botany and Microbiology, Gurukula Kangri University, Haridwar for providing essential services and conveniences to carry out this research.
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