Ajay Kumar, Hariom Verma, Akhilesh Yadav, Waquar Akhtar Ansari, Prem Pratap Singh, Sandeep Kumar Singh, P.K. Singh and K.D. Pandey*

Centre of Advance study in Botany, Banaras Hindu University, Varanasi – 221 005, India.

Abstract

In this study,31 bacterial strains were isolated from the rhizospheric soil of Curcuma amada (mango ginger) and theirplant growth promotion potential, salinity tolerance, antibiotic sensitivity, antimicrobial propertieswere evaluated. Eight bacterial strains namely AzotobacterchroococcumKCA1, PseudomonasfluorescensKCA2, Bacillus subtilisKCA3, Bacillus sp. KCA4, Agrobacterium tumifaciensKCA5, Bacillus cereusKCL7,Pseudomonas putidaKCA8 andPaenibacillus sp. KCA9 have been identified on the basis of biochemicals and 16S rRNA 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 solaniand Alterneriaalternata during antimicrobial properties.

Keywords: Curcuma amada,PGPR, PGP traits, Antimicrobial properties, Salinity tolerance.

Introduction

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 inmorphologicalgrowth, enhancement in the secondary metabolite production of the plants(Kumar et al., 2016b). PGPR enhanceplant growth directly through the production ofsiderophore, N2 fixation, phosphate solubilization,synthesis ofplant growth hormones like auxin, cytokinin, gibberellins (Lucy et al.,2004)and alsobyincreasing the uptake of water andminerals (Perez-Montano et al., 2014).Recently, PGPR along with certain level of fertilizers have beenbroadly used as plantsor soil inoculants in sustainable agriculture because of their less adverse impact on thesoil, 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 plantsinoculantsfor the growth, yields and disease management(Goswami et al.,2013;Perez-Montano et al., 2014; Kumar et al., 2014,2015,2016b).
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 rhizomeofCurcuma 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 themost important constituent (Gupta et al.,1999). The researches carried out in the past half centuryin 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 amadaand to access their plant growth promotion potential, salinitytolerance as well as antimicrobial properties.
Materials and Methods
Soil sampling and bacterial isolation
Bacterial strain were isolated from the rhizospheric soil of young and healthy mangoginger (Curcuma amada)grown inthe Botanical garden of Banaras Hindu University, India (20˚ 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) fortotal bacterial counts usingKing’S B medium for Pseudomonas sp. and N2 free agar medium for Azotobacter sp. The plates were incubated (48h) at 30ºC and colonies showing morphological difference, separatelyisolated for further analysis (Kumar et al., 2016b). Furthercharacterizations 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).
Identification of bacterial isolates by 16 S rRNA amplification
16S rDNA sequenceamplification and sequencing
Genomic DNA was isolated using GeneiPureTM bacterial DNA purification kit (GeNeiTM, Bangaluru, India) according to the manufacture’s protocol. Universal eubacterial primers F-D1-5′-ccgaattcgtcgacaacagagtttgatcctggctcag-3′ and R-D1- 5′-cccgggatccaagcttaaggaggtgatccagcc-3′ (Kumar et al., 2015, 2016a, b), were used to amplify the1500bp region of 16S rRNA gene using a thermal cycler (BioRad, USA). Amplified products were resolved by electrophoresis in agarose (1%), and visualized in the gel documentation system (Alfa Imager, Alfa InfoTech Corporation, USA). The amplicons were purified using GeneiPureTM quick PCR purification kit (GeNeiTM, Bangaluru, India) and quantified at 260 nm taking calf thymus DNA as control. The purified partial 16S rDNAamplicon was sequenced in Applied Biosystems 3130 Genetic Analyzer (Applied Biosystems®, CA, and USA).
Analysis of 16S rRNAgene sequences
The 16S rRNA gene partialsequences of the isolated strains were sequenced and compared with the RNA databases. The resulting nucleotide sequence were assignedfor bacterial taxanomic affiliation based on the closest match to the sequences available at National Center for Biotechnology Information (NCBI) BLASTserver (www.ncbi.nlm. nih.gov/BLAST) using Nucleotide Basic Local Alignment Search Tool (BLAST) program (Kumar et al., 2015,2016a,b)
Plant growth promoting (PGP) traits of bacterial isolates
The bacterialisolates 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) andNH3production (Cappuccino and Sherman, 1992) as per the standard protocols.
2.5 Antibacterial activity
All the rhizospheric strainswere screened for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa andKlebsiellapneumoniae. 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 rhizosphericstrains were streaked on the test organisms toobserve their growthor inhibition after 24-48 h of incubation (30ºC). The diameters of the inhibition zone weremeasured in mm (Kumar et al., 2015, 2016 a, b).
Antifungal activity
All theeightrhizospheric strains were tested against fourmouldsFusarium solani, Alterneria alternata, Byssochlamysfulva andAspergillusfumigates to checktheir 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, 2016a, b).
Results and discussion
In the rhizospheric region of mango-ginger 31 bacterial isolates were isolated, all the strains were rod shapedand most of themwere 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), andBacillus(14)of phyla γ-Proteobacteria, α-Proteobacteriaand Firmicutes on the basis of morphology and biochemical characteristics (Table1).Further confirmations of the species levels of the isolates werecarried out through16S rRNA gene sequence analysis. Rhizobacterial isolates were identified as 8 different bacterial strains and their sequence were deposited in the NCBI and got accession no. namely AzotobacterchroococcumKCA1 [accession no. KM043465], PseudomonasfluorescensKCA2[accession no. KMO43460], Bacillus subtilisKCA3 accession no. KM043461Bacillussp.KCA4 [accession no.KM043462],Agrobacterium tumifaciensKCA5 [accession no.KMO43463], Bacillus cereusKCA7 [accession no. KMO43464], Pseudomonas putidaKCA8 [accession no.KM043466] andPaenibacillus sp. KCA9 [accession no. KMO43467].
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 α-proteobacteria, β-proteobacteria, γ-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 isolatesproduced IAA, ammoniaand solubilized tri-calcium phosphate on Pikoskaya nutrient agar petriplates, whereasonly 50% strainsproduced siderophores(Table1). Phytohormones produced by plant–associated bacteria are mainly indole-3-acetic 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 aniron-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. Plantswhich 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,Pseudomonassp. (Maheshwari et al., 2012; Laslo et al., 2012; Kumar et al.,2016b), Agrobcteriumtumifaciens, Bacillus sp. and Pseudomonas sp. by (Zhao et al., 2014; Kumar et al.,2016b).
Most of the rhizobacterialstrainsshowntolerance to salinity upto 4% of NaCl except A. tumifaciensKCA5 (1-2% NaCl). Strains Paenibacilus sp. KCA9 shown tolerance upto 5% of NaCl, Strains P. fluorescensKCA2, B. subtilisKCA3, Bacillus cereusKCA7 tolerated salinity upto 6 % of NaCl, whereasP. putidaKCA8 shows maximum salinity tolerance (7% of NaCl) (Table1).
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. putidaexhibit maximum salinity tolerance contrary to the minimum (1-2% NaCl) in A. tumifaciens. Already more or less similar trends of salinity tolerance was reported by Kumar et al.(2015, 2016b). In a study Rashid et al.(2012) reported differential salinity tolerance pattern of the strains P. fluorescens(4% NaCl), Bacillus sp. (3.5% NaCl) and A. tumifaciens(0.5-1% NaCl). Bacillus sp. isolated from marshy areas had tolerance upto 10% of NaCl (Gayathri et al., 2010). Singh et al. (2013) also reported tolerance 4–10 %of NaClin case of Momordica charentiabacterial isolates.
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. Strainswere mostly sensitive to chloramphenicolfollowed by erythromycin while some strains were resistant to rifampicin and polymixin-B. Strains P. fluorescensKCA2, B. subtilisKCA3andP. putidaKCA9showed high resistance against two antibiotic (rifampicin and polymixin-B) whereas strains A. tumefaciensKCA5, and A.chroococcumKCA1were highly susceptible to all the antibioticstested.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).
Antibacterialand antifungal activity
All therhizobacterial strainswere assessed forantibacterial property againstE. coli, P. aeruginosaand 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. tumefaciensKCA5. StrainsP.fluorescensKCA2, B. subtilisKCA3andP.putidaKCA8were antibacterial againstK. pneumonia, whereas none of the strains inhibited growth of P. aeruginosa.
During antifungal activity, all therhizobacterial strains exhibited antifungal property with the exception of A. chroococcumKCA1,A. tumefaciensKCA5and Paenibacillus sp. KCA9 thatdid not show fungistatic activity against B. fulvaandA. 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)formedinhibition zones against most of the fungal strains. In antibacterialactivity tests, all thestrains inhibited growth of E. coli except strains A. chroococcumKCA1, A. tumefaciensKCA5 and Paenibacillus sp. KCA9. These threestrains also showed poor activity during antibiotic tests.
The fungal strains used during the antifungal activitytest are the potent pathogens and causes severe infection to plant speciesas well as on theliving 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. fulvais 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 strainsisolated is attributed to the secretionof lytic enzymes, chitenase andcertain antibiotics. Our finding show that all the bacterial isolates possess antifungal characteristics against above fungal strains except the strain A. chroococcumKCA1and A. tumefaciens KCA5 that did not from inhibition zone againstA. NigerorB. 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 inoculantsand considered promising for managing soil fertility and plant growth (Aarons et al., 2000). This study shown that strains P.fluorescensKCA2,P.putidaKCA8 and Bacillusstrains may be proven asbetter choice in the sustainable agriculture as plant or soil inoculants.
Acknowledgement
AuthorAjay 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 providinglab facilities.
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Table 1. Biochemical characterization of selected bacterial isolates from C.amada rhizosphere. (+ve-Positive,-ve-Negative)
Bacterial strain Gram staining Shape Catalase Oxidase Nitrate reduction H2S Production Starch hydrolysis Salinity tolerance Phosphate solubilization IAA Production Ammonia production Siderophore production
KCA1 – Rod + + + _ + 2% + + + –
KCA2 – Rod + + + – + 6% + + + +
KCA3 + Rod + + – + + 6% + + + –
KCA4 + Rod + + + – + 5% + + + +
KCA5 – Rod + + + + + 1% + + + –
KCA7 + Rod + + + – + 5% + + + +
KCA8 – Rod + + – – + 7% + + + +
KCA9 – Rod + + – – + 5% + + + –

Table 2.Antibiotic sensitivity, antibacterial activity and antifungal activity of rhizospheric strainsof C. amada.
Bacterial strain Antibiotic sensitivity
(antibiotics inhibition zone in mm) Antibacterial activity
(inhibition zone in mm) Antifungal activity
(fungal growth inhibition)
Chloramphenicol Erythromycin Rifampicin Polymixin-B Escherichia coli P.aeruginosa K.pneumoniae F.solani A. alternata B. fulva A. fumigatus
KCA1 28 25 15 11 _ – – + – – –
KCA2 19 16 – – +11 – + 9 + + + +
KCA3 22 19 9 – +9 – +7 + + + +
KCA4 26 21 10 8 +7 – – + + + +
KCA5 24 22 12 10 – – – + – – –
KCA7 26 20 10 9 +9 – +8 + + + +
KCA8 22 16 – – +12 – +10 + + + +
KCA9 25 18 – – – – – + + – –