Plant Growth Promoting Rhizobacteria of Curcuma amada (Mango ginger)

In this study, 31 bacterial strains were isolated from the rhizospheric soil of Curcuma amada (mango ginger) and their plant growth promotion potential, salinity tolerance, antibiotic sensitivity, antimicrobial properties were evaluated. Eight bacterial strains namely Azotobacter chroococcum KCA1, Pseudomonas fluorescens KCA2, Bacillus subtilis KCA3, Bacillus sp. KCA4, Agrobacterium tumifaciens KCA5, Bacillus cereus KCL7, Pseudomonas putida KCA8 and Paenibacillus sp. KCA9 have been identified on the basis of biochemicals and 16S r RNA gene sequence analysis. All the strains solubilized tri-calcium phosphate and produced IAA, ammonia but only 50% of the strains produced siderophores during PGP traits analysis. Strains KCA8 tolerated maximum NaCl (7%) relative to strain KCA5 (1-2%). The strains were sensitive to the antibiotic chloromphenicol followed by erythromycin and most of these effectively inhibited growth of Escherichia coli, Fusarium solani and Alterneria alternata during antimicrobial properties.

Plants growth promoting rhizobacteria (PGPR) are the heterogeneous group of bacteria that effectively colonize roots and exerting plants growth promotion.These PGPR directly or indirectly involved in morphological growth, enhancement in the secondary metabolite production of the plants (Kumar et al., 2016b).PGPR enhance plant growth directly through the production of siderophore, N 2 fixation, phosphate solubilization, synthesis of plant growth hormones like auxin, cytokinin, gibberellins (Lucy et al.,2004) and also by increasing the uptake of water and minerals (Perez-Montano et al., 2014).Recently, PGPR along with certain level of fertilizers have been broadly used as plants or soil inoculants in sustainable agriculture because of their less adverse impact on the soil, plants and environment (Lucy et al., 2004;Perez-Montano et al., 2014).Numerous studies has been carried out from last two decades in the field of isolation, characterization of PGPR and their uses as soil and plants inoculants for the growth, yields and disease management (Goswami et al.,2013;Perez-Montano et al., 2014;Kumar et al., 2014Kumar et al., ,2015Kumar et al., ,2016b)).
Curcuma amada (mango ginger) member of family Zingiberaceae is a spice and well known traditional medicine in the Ayurveda since ancient time in the Indian sub-continent (Sasikumar,2005).Plant extensively used in foods as prickles, salad etc (Shankaracharya,1982) and in pharmacology broadly used as antioxidant, anti-inflammatory, treatment of flatulence, jaundice, menstrual difficulties, hemorrhage and colic (Mujumdar et al.,2000;Prakash et al.,2007;Policegoudra et al.,2007).The rhizome of Curcuma amada have also cancer preventive or therapeutic capabilities.It has been shown to suppress multiple signaling pathways and inhibit cell proliferation, invasion, metastasis and angiogenesis (Policegoudra, 2008).Curcuminoids are the important constituents of mangoginger.Amongst curcuminoids, curcumin is the most important constituent (Gupta et al.,1999).The researches carried out in the past half century in this field clearly indicated the importance of curcumin in pharmacology.
The objective of this study is to isolate bacterial strains from the rhizospheric soil of Curcuma amada and to access their plant growth promotion potential, salinity tolerance as well as antimicrobial properties.

Soil sampling and bacterial isolation
Bacterial strain were isolated from the rhizospheric soil of young and healthy mangoginger (Curcuma amada) grown in the Botanical garden of Banaras Hindu University, India (20Ú 18 ' N and 80° 36 ' E, elevation 80.71m) using standard microbiological techniques (soil serial dilutions or spread-plate methods).Rhizospheric soil (1g) was dissolved in 10 ml of sterile distilled water, making 10 -1 dilution.This dilution was further diluted to 10 -7 . 1 ml of each dilution 10 -6 and 10 -7 was placed on nutrient agar, in triplicate (Kumar et al.,2016b) for total bacterial counts using King'S B medium for Pseudomonas sp. and N 2 free agar medium for Azotobacter sp.The plates were incubated (48h) at 30ºC and colonies showing morphological difference, separately isolated for further analysis (Kumar et al., 2016b).Further characterizations of bacterial isolates were performed on the basis of morphology and biochemical screening according to Bergey's manual of systematic bacteriology (Holt et al., 1994).

Analysis of 16S r RNA gene sequences
The 16S r RNA gene partial sequences of the isolated strains were sequenced and compared with the RNA databases.The resulting nucleotide sequence were assigned for bacterial taxanomic affiliation based on the closest match to the sequences available at National Center for Biotechnology Information (NCBI) BLAST server (www.ncbi.nlm.nih.gov/BLAST) using Nucleotide Basic Local Alignment Search Tool (BLAST) program (Kumar et al., 2015(Kumar et al., ,2016a,b) ,b)

Plant growth promoting (PGP) traits of bacterial isolates
The bacterial isolates were screened for their plant growth promoting potential including phosphate solubilization (Laslo et al., 2012), Indole acetic acid production (IAA) (Brick et al., 1991), siderophore production (Schwyn and Neilands, 1987) and NH 3 production (Cappuccino and Sherman, 1992) as per the standard protocols.

Antibacterial activity
All the rhizospheric strains were screened for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae.The bacterial strains were grown on nutrient agar plates and inoculated with single streak at the center of the petri-plate and incubated at 30ºC for 2-4 days.24 h old growth cultures of the isolated rhizospheric strains were streaked on the test organisms to observe their growth or inhibition after 24-48 h of incubation (30ºC).The diameters of the inhibition zone were measured in mm (Kumar et al., 2015(Kumar et al., , 2016 a, b) a, b).

Antifungal activity
All the eight rhizospheric strains were tested against four moulds Fusarium solani, Alterneria alternata, Byssochlamys fulva and Aspergillus fumigates to check their fungistatic activity.A drop of the 24 h old cultures of selected bacterial strains grown in nutrient broth were placed separately in a row on the fungal test cultures prepared in Potato Dextrose Agar (PDA)medium.The plates were incubated at room temperature (3-5 days) and inhibition zone exhibit the fungistatic activity of the bacterial strains (Kumar et al., 2015(Kumar et al., , 2016a, b), b).

RESULTS AND DISCUSSION
In the rhizospheric region of mangoginger 31 bacterial isolates were isolated, all the strains were rod shaped and most of them were Gram negative (62.5%).All the rhizospheric isolates were positive for catalase and oxidase test.The isolated bacterial strains belongs to different genera namely Azotobacter (6), Pseudomonas (8), Agrobacterium (3), and Bacillus (14) of phyla ã-Proteobacteria, á-Proteobacteria and Firmicutes on the basis of morphology and biochemical characteristics (Table1).Further confirmations of the species levels of the isolates were carried out through 16S r RNA gene sequence analysis.Rhizobacterial isolates were identified as 8 different bacterial strains and their sequence were deposited in the NCBI  Plant interacts with diverse communities of microorganism, rhizosphere is the most prominent zone for growth and interactions among the microbes mainly due to the secretion of root exudates.In the previous study it is reported that in the rhizospheric zone the predominant bacterial species belong to phyla á-proteobacteria, âproteobacteria, ã-proteobacteria and firmicutes, rhizodeposition predominantly colonized the Gram negative microbial community (Ambardar and Vakhlu, 2013;Kumar et al., 2016b).In this study, mango-ginger rhizome was also dominated by Gram negative (62.5%) and proteobacteria phyla.

Plant growth promoting (PGP) traits
Plant growth promoting traits mainly includes synthesis of phytohormones, phosphate solubilization, production of siderophores, these attributes helps in plant growth.All the bacterial isolates produced IAA, ammonia and solubilized tri-calcium phosphate on Pikoskaya nutrient agar petriplates, where as only 50% strains produced siderophores (Table1).Phytohormones produced by plant-associated bacteria are mainly indole-3acetic acid (IAA), cytokinin and gibberellins which frequently stimulate plant growth and protect against biotic and abiotic stresses (Taghavi et al., 2009).All the eight rhizobacterial strains synthesized IAA in presence of tryptophan.Siderophore is an iron-chelating compounds secreted by certain PGPR strains.The presence of iron-chelating compounds makes the bacteria better competitors for iron that prevents growth of pathogenic microorganisms.Plants which unable to uptake sufficient amount of iron benefitted from siderophore producing bacteria that chelate Fe +3 and make it available to plants for growth.In a previous study, phosphate solubilization, IAA and siderophore production are already reported in A. chroococcum, Pseudomonas sp. ( Salinity is one of the most severe abiotic stresses that limits crop growth and productivity.Under salt stress, PGPR have positive effects on plants or parameters like germination rate, tolerance to draught and development of shoots and roots.
In the present study, P. putida exhibit maximum salinity tolerance contrary to the minimum

Antibiotic sensitivity
The sensitivity pattern of antibiotic disc towards bacterial isolates were determined by disc diffusion method.All the four antibiotics showed differential pattern of inhibition as results showed in Table 2. Strains were mostly sensitive to chloramphenicol followed by erythromycin while some strains were resistant to rifampicin and polymixin-B.Strains P. fluorescens KCA2, B. subtilis KCA3 and P. putida KCA9 showed high resistance against two antibiotic (rifampicin and polymixin-B) whereas strains A. tumefaciens KCA5, and A. chroococcum KCA1 were highly susceptible to all the antibiotics tested.The antibiotic disc acted differentially on growth of the same bacterial strains of different isolation source (Arunachalam and Gayathri, 2010;Kumar et al., 2015;2016b).

Antibacterial and antifungal activity
All the rhizobacterial strains were assessed for antibacterial property against E. coli, P. aeruginosa and K. pneumoniae.The isolates which inhibited the growth of any test organism(s) was considered having antibacterial activity and the diameter of inhibition zone was measured in mm (Table2).All the strains showed antibacterial activity against E. coli except A.chrocoocum KCA1 and A. tumefaciens KCA5.Strains P.fluorescens KCA2, B. subtilis KCA3 and P.putida KCA8 were antibacterial against K. pneumonia, whereas none of the strains inhibited growth of P. aeruginosa.
During antifungal activity, all the rhizobacterial strains exhibited antifungal property with the exception of A. chroococcum KCA1, A. tumefaciens KCA5 and Paenibacillus sp.KCA9 that did not show fungistatic activity against B. fulva and A. fumigates (Table 2).In the previous study, it is reported that some of the bacterial strains are a rich source of bioactive natural compounds like Ecomycins, Pseudomycins and Kakadumycins, which contains antibacterial and antifungal properties (Christina et al., 2013) and play significant role in antibacterial and antifungal properties.In the present investigation, P. fluorescens (KCA2), B. subtilis (KCA3) B. cereus (KCA7) and P. putida (KCA8) formed inhibition zones against most of the fungal strains.In antibacterial activity tests, all the strains inhibited growth of E. coli except strains A. chroococcum KCA1, A. tumefaciensKCA5 and Paenibacillus sp.KCA9.These three strains also showed poor activity during antibiotic tests.
The fungal strains used during the antifungal activity test are the potent pathogens and causes severe infection to plant species as well as on the living organisms.Fusarium solani is a filamentous fungus commonly isolated from the soil and plant debris.A. alternate causes several diseases on the plants like leaf spot.It is an opportunistic pathogen on numerous hosts causing 'leaf spots', 'rots' and 'blights' on many plant parts.B. fulva is responsible for fruit rot in certain plants.A. fumigatus, a saprotroph, is widespread in nature, and typically found in soils and decaying organic matter (Kumar et al., 2016b).The antifungal activity of the strains isolated is attributed to the secretion of lytic enzymes, chitenase and certain antibiotics.Our finding show that all the bacterial isolates possess antifungal characteristics against above fungal strains except the strain A. chroococcum KCA1 and A. tumefaciens KCA5 that did not from inhibition zone against A. Niger or B. fulva.
Environment friendly sustainable agriculture is preferred to obtain the desirable yield.In this respect, PGPR are applied in a wide variety of agro and allied industries as inoculants and considered promising for managing soil fertility and plant growth (Aarons et al., 2000).This study shown that strains P. fluorescens KCA2, P. putida KCA8 and Bacillus strains may be proven as better choice in the sustainable agriculture as plant or soil inoculants.

ACKNOWLEDGEMENT
Author Ajay Kumar, thankful to University Grant Commission, New Delhi for financial assistance in the form of JRF and SRF and also to Head, Department of Botany for providing lab facilities.
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