Bioactive metabolite from Pseudomonas

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Applied Biochemistry andBiotechnologyPart A: Enzyme Engineering andBiotechnology ISSN 0273-2289 Appl Biochem BiotechnolDOI 10.1007/s12010-015-1593-3

Characterization of the BioactiveMetabolites from a Plant Growth-Promoting Rhizobacteria and TheirExploitation as Antimicrobial and PlantGrowth-Promoting AgentsEmrin George, S. Nishanth Kumar,Jubi Jacob, Bhaskara Bommasani, RaviS. Lankalapalli, P. Morang & B. S. DileepKumar

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Characterization of the Bioactive Metabolites from a PlantGrowth-Promoting Rhizobacteria and Their Exploitationas Antimicrobial and Plant Growth-Promoting Agents

Emrin George1 & S. Nishanth Kumar1 & Jubi Jacob1&

Bhaskara Bommasani1 & Ravi S. Lankalapalli1 &

P. Morang2 & B. S. Dileep Kumar1

Received: 8 July 2014 /Accepted: 17 March 2015# Springer Science+Business Media New York 2015

Abstract A plant growth-promoting bacterial strain, PM 105, isolated from a tea plantationsoil from the North Eastern region of India was identified as Pseudomonas aeruginosa throughclassical and 16S ribosomal DNA (rDNA) gene sequencing. Further studies with this strainconfirmed broad spectrum antifungal activity against ten human and plant pathogenic fungalpathogens viz. Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, Aspergillustubingensis, Candida albicans, Colletotrichum gloeosporioides, Fusarium oxysporum,Pencillium expansum, Rhizoctonia solani, Trichophyton rubrum besides growth-promotingproperty in cowpea (Vigna unguiculata) and pigeon pea (Cajanus cajan). However, noantibacterial property was exhibited by this strain against the four test bacterial pathogenstested in agar overlay method. The crude bioactive metabolites produced by this strain wereisolated with three different solvents that exhibited significant antimicrobial and plant growth-promoting activity. Chloroform extract recorded significant antimicrobial and plant growth-promoting activity. Three major compounds viz. 1-hydroxyphenazine, pyocyanin, andphenazine-1-carboxamide were purified and characterized from crude extracts of this strainby various spectral data. The purified compounds recorded prominent antimicrobial activitybut failed to establish the plant growth promotion activity in test crop plants under gnotobioticconditions. Pyocyanin recorded significant antimicrobial activity, and best activity was

Appl Biochem BiotechnolDOI 10.1007/s12010-015-1593-3

Electronic supplementary material The online version of this article (doi:10.1007/s12010-015-1593-3)contains supplementary material, which is available to authorized users.

* B. S. Dileep [email protected]; [email protected]

1 Agroprocessing and Natural Products Division, National Institute for Interdisciplinary Science andTechnology (NIIST), Council of Scientific and Industrial Research (CSIR), Thiruvanathapuram 695019 Kerala, India

2 Department of Ecology and Environmental Science, Assam University, Silchar 788 011 Assam, India

Author's personal copy

http://dx.doi.org/10.1007/s12010-015-1593-3

recorded against T. rubrum (29 mm), followed by P. expansum (28 mm). These results suggestthe use of PM 105 as plant growth-promoting agent in crop plants after successful field trials.

Keywords Antimicrobial activity . Bioactivemetabolites . Crop plants .Pseudomonas . PGPR .

Structural elucidation

Introduction

Agricultural land reduction, soil pollution, environmental degradation, and diseases areamong the many factors hindering agriculture productivity around the globe. Skyrocketingprices and excessive use of synthetic chemicals to mitigate pests and diseases have alsoemerged as increasing concerns to the present day agricultural practices [1]. Hence, theinterest has been shifted to an environmentally safe and economically viable alternative forcrop improvement and disease control. Use of naturally occurring free-livingrhizobacterial strains, named plant growth-promoting rhizobacteria (PGPR), which canprotect and promote plant growth by colonizing and multiplying along the surface orcortex of the root of the introduced plant is found to be a safe and viable alternative choice.The species belonging to Pseudomonas and Bacillus is reported to be a promising agent,and their exploitation as PGPR was reported in the recent years. In addition to this, manyof these organisms also induced systemic resistance in host plants against many agricul-turally important diseases [1–3]. Antibiosis, parasitism, predation, competition and scav-enging nutrients and elements, particularly iron, production of hormones, andenhancement of defense system are some of the mechanisms these organisms directly orindirectly employ to achieve these targets.

Among fluorescent Pseudomonas, Pseudomonas aeruginosa is one of the free-livingbacterial species, which is commercially exploited as an eco-friendly viable plant growth-promoting rhizobacteria as well as a biocontrol agent in recent years [4]. Fluorescent pseudo-monads, including P. aeruginosa, produce a variety of metabolites that have a major role ingrowth promotion and disease control. Production of iron chelating compounds such assiderophore and many antibiotics with potential application in crop improvement and diseasecontrol are reported from P. aeruginosa species [5]. The ability to resist agroclimatic changes,capacity of persistent colonization, and multiplication on the root or root cortex region of theintroduced plants is considered to be an advantage to explore these organisms over the rest ofthe flora. Extraction, purification, and characterization of bioactive metabolites produced bythese organisms could aid in understanding the modus operandi of organisms’ chemicaladvantages, possibly providing insight into ways of controlling plant pathogens.

Throughout the ages, natural products have been the most consistently successfulsource of lead compounds that have many applications in the fields of medicine, pharma-cy, and agriculture [6]. Microbial natural products have been the source of most of theantibiotics in current use for the treatment of various infectious diseases. Many terrestrialbacteria are known to produce secondary metabolites that can suppress microorganismscompeting for the same resources [6]. However, bacterial resistance emerges when anantimicrobial agent is introduced into the market or just after its introduction [7]. So, thereis a perpetual need for new antimicrobial agent to compete with the pathogens. In thisperspective, microbial secondary metabolites remain the most important source for dis-covery of new and potential drug molecules.

Appl Biochem Biotechnol


With a view of the above, the present investigation was undertaken to study the role of afluorescent Pseudomonas strain, designated as PM 105, and its bioactive metabolites onantimicrobial activity against ten fungal and four bacterial strains besides growth-promotingability in two economically important crop plants.

Materials and Methods

Ethical Statement

Field studies in the present study did not involve any endangered or protected species. Thepresent study was conducted in pots and is carried in NIIST campus.

Seeds and Soil

Cowpea (Vigna unguiculata) seed was procured from the Kerala Agricultural University,Thiruvanathapuram, Kerala, India, whereas the pigeon pea (Cajanus cajan) seeds wereobtained from a local seed supplier. The experiments were conducted on sandy-loam soil withpH 7.4, total nitrogen of 0.02 %, organic carbon 0.27 %, and having no previous history of anypesticide or synthetic agrochemical application.

Fungal Pathogens

The fungal pathogens used in this investigation, Aspergillus flavus (MTCC 183), Asper-gillus fumigatus (MTCC 3376), Aspergillus niger (MTCC 282), Aspergillus tubingensis(MTCC 2425), Candida albicans (MTCC 277), Colletotrichum gloeosporioides (MTCC2151), Fusarium oxysporum (MTCC 284), Penicillium expansum (MTCC 2006), Rhi-zoctonia solani (MTCC 4634), and Trichophyton rubrum (MTCC 296), were procuredfrom the Microbial Type Culture Collection and Gene Bank (MTCC) of Institute ofMicrobial Technology (IMTECH), Council of Scientific and Industrial Research (CSIR),Chandigarh, India.

Bacterial Pathogens

Bacterial pathogens used in this investigation that are Bacillus subtilis (MTCC 2756),Escherichia coli (MTCC 2622), P. aeruginosa (MTCC 2642), Staphylococcus aureus(MTCC 902) were also procured from the MTCC Division of CSIR-IMTECH,Chandigarh, India.

Beneficial Bacteria

A fluorescent Pseudomonas strain, designated as PM 105, that was isolated from tea plantationsoil of the Barak Valley region of Assam, India, was used in this investigation. The region inAssam (northeastern part of India) where tea is cultivated is generally known as Barak Valley.The confirmatory identification of this strain was done by the MTCC Division of CSIR-IMTECH, Chandigarh, India. The strain was identified as P. aeruginosa, and its accessionnumber is MTCC 10658.



16S rDNA Gene Sequencing and Phylogenetic Analysis

Genomic DNA of PM 105 was extracted through enzymatic hydrolysis [8]. The complete 16Sribosomal DNA (rDNA) gene (1.4–1.5 kb) was amplified via PCR, using universal bacterialprimers 27 F (5′-AGA GTT TGATCC TGG CTC AG-3′) and 1541 R (5′-AAG GAG GTGATC CAG CCG CA-3′). The amplification was carried out on a DNA thermal cycler(BIORAD, USA). The 50 μl PCR reactions contained 4 μl of 2.5 U/μl Taq DNA polymerase(Genei, Bangalore, India), 5 μl of 10× buffer (Genei), 1 μl of 20 mM dNTPs (Genei), 37 μl ofSDW, 1 μl of 50 μM each primer, and 1 μl of template.

The PCR conditions were maintained for initial denaturation at 94 °C for 4 min, 30amplification cycles of 94 °C for 1 min, 56 °C for 1 min, and 72 °C for 2 min, and finalelongation at 72 °C for 10 min. The PCR product was purified using a QIAquick Gelextraction kit (QIAGEN, Tokyo, Japan) and sequenced in both directions using the sameprimers used for the PCR amplification. The nucleotide sequence obtained was processed toremove low-quality reads, transformed into consensus sequences with Geneious Pro softwareversion 5.6. The resulted high-quality sequences were analyzed with BLASTn (NCBI) toconfirm the authenticity of the bacterium. The sequences of related species and genus weredownloaded from the Genbank database, and a phylogenetic study was carried out with theprogram MEGAversion 5 [9]. Sequences were aligned using the computer package ClustalW[10] and were analyzed to determine the relationships between isolates by the neighbor-joiningmethod [11] using the maximum composite likelihood model. Bootstrap values were generatedusing 2000 replicates.

In Vitro Antibiosis Test with Live Organism

In vitro antifungal activity of PM 105 was done by dual culture technique according to DileepKumar and Dube [12] against ten test fungi. For this, an actively growing fungal mycelial disc(approx. 6 mm2) was placed on one side of the petri plate containing potato dextrose agar(PDA), 2.0 cm inside the periphery, and a loopful of PM 105 was streaked in a line, on theopposite side at a distance of 5.0 cm from the mycelial disc. The plates were incubated at 28±2 °C, and inhibition zone was measured, as the distance (in mm) between the PM 105 andfungal pathogen after 7 days of growth. Plates without PM 105 served as control.

In vitro antibacterial activity of PM 105 was done by agar overlay method. In agar overlaymethod, PM 105 was streaked in a line on nutrient agar plates and incubated for 2 days at30 °C and then overlaid with 10 ml of nutrient agar with a standardized suspension of testbacteria and incubated at 37 °C for 24 h. The zone of inhibition (in mm) around the colonieswas measured. Each experiment was repeated three times, and the data was expressed as mean±standard deviation.

Production and Extraction of Bioactive Metabolites

The production and extraction of bioactive metabolites by PM 105 was examined according toDileep Kumar and Bezbaruah [13]. For this, a lawn of PM 105 was prepared in King’s Bmedium (KB) (Hi-Media, India) at 28±2 °C for 7 days. Then, the green bacterial lawn grownmedium was cut into small pieces (≈2 cm2) and extracted with 90 % aqueous acetone till thebluish medium turned into colorless. The extract was then filtered through a double layer ofcheesecloth, and the acetone was removed under vaccum pressure through a rota evaporator



(Buchi Rotavapor R-215). The resultant aqueous part was then treated with sodium chloride(50 g/l) and centrifuged at 10,000 rev/min for 20 min. The collected aqueous part was thentreated with sodium chloride (50 g/l) and centrifuged at 10,000 rpm for 20 min. Thesupernatant was collected and extracted with diethyl ether (3:1v/v) in a separating funnel,and the diethyl ether fraction was collected. The resultant aqueous fraction was then treatedwith chloroform (3:1v/v), and the chloroform fraction was collected. The remaining aqueouswas finally treated with ethyl acetate (3:1v/v) and collected. All the three fractions obtainedwere concentrated and dried in a rotary evaporator for further studies.

Antimicrobial Activity of Crude Extracts

The crude extracts obtained were screened for their antifungal activity against testfungi by disc diffusion method [14, 15]. The fungal cultures were grown on potatodextrose broth for 7 days, and the mycelial mat was collected and resuspended innormal saline solution. Hundred microliters of the test fungi (adjusted to have 1.5×105

colony forming unit (CFU)/ml) was applied on the surface of the media and spread byusing a sterile cotton swab. Subsequently, filter paper discs (6 mm in diameter)containing 100 μg/ml concentration of crude extracts were placed on the agar platesand incubated at 30 °C, and the zone of inhibition was measured after 72 h. Theexperiments were conducted in triplicate set, and the data were expressed as mean±standard deviation.

The antibacterial activity of the crude extracts was also determined by the disc diffusionmethod [16]. For this, 100 μg/ml of crude extract were incorporated in a 6-mm sterile disc andplaced on Mueller Hinton (MH) agar (Hi-Media, India) plates swabbed with 0.1 ml of 18 h oldeach bacterial strain (1×106 CFU/ml) and incubated at 37 °C, and the zone of inhibition wasrecorded after 18 h. Each experiment was repeated three times, and the data was expressed asmean±standard deviation.

Purification of Bioactive Compounds

The crude chloroform and diethyl ether extracts (250 mg) were purified by column chroma-tography on a silica gel column which is equilibrated with hexane and eluted successivelyusing a linear gradient hexane/ethyl acetate (100:0 to 0:100) and finally with ethyl acetate/methanol (19:1). Diethyl ether crude extract was as such having large amount of yellowneedle-like crystals that was separated in pure form by filtration. These crystals were dissolvedin methanol, and TLC was performed in 9:1 ethyl acetate/methanol to check the purity of thecompound.

Structure Elucidation of Bioactive Compounds

The structure elucidation of the compounds was carried by NMR spectroscopy (Bruker DRX500 NMR instrument, Bruker, Rheinstetten, Germany) using CDCl3 as the solvent.

1H and 13Cspectra were recorded at ambient temperature at 500 and 125 MHz, respectively. Chemicalshifts are given in parts per million and coupling constants in hertz. Chemical shifts arereported relative to the solvent peaks δ 7.24 and δ 77.2 for 1H and 13C, respectively. Massspectrometry was carried out using electrospray ionization mode of a Thermo ScientificExactive Orbitrap LC-Mass Spectrometer with ions given in m/z.



Antimicrobial Assay with Identified Pure Compounds

Antifungal and antibacterial activities of pure compounds against ten fungal and four bacterialpathogens were done according to disk diffusion method as earlier described with 50 μg ofpure compound loaded on sterile 6 mm diameter paper disk (Hi-Media, India). Ciprofloxacinserved as the control for test bacteria, and bavistin and amphotericin B served as the control forthe test fungi. Each experiment was repeated three times, and the data was expressed as mean±standard deviation.

Seed and Plant Bacterization Studies

Seed bacterization studies were done according to Dileep Kumar et al. [17]. For this, cowpeaand pigeon pea seeds were surface sterilized by placing in 1 % mercuric chloride solution for1 min followed by rinsing in 1:29 mixture of hydrogen peroxide and distilled water for 30 minand dried under a sterile air stream. The strain PM 105 grown in KB for 48 h was harvestedwith a sterile glass rod and suspended in 20 ml of sterile 1 % carboxymethylcellulose (CMC)solution. Five grams of surface-sterilized seeds was steeped into this bacterial suspension for1 h and then surface dried overnight under a sterile air stream. The treated seeds wereexamined for CFUs on KB, and the bacterial suspension was adjusted to give 1.2×107 CFU/seed for each treatment. Seeds treated with only 1 % CMC served as the control.

To study the effect of different crude extracts in plant growth, 200 μg of extract dissolved in200 μl of methanol was added to soil prior to planting the seeds. In control set, 200 μl ofaqueous methanol (25 %) was added to soil prior to planting seeds. The seeds were planted inthe plastic pots (9.0×9.0 cm), filled with a mixture of soil and farmyard manure (FYM) in 3:1ratio, and the percent seed germination was noted up to 7 days. The plant growth in terms ofshoot height was recorded at every 14 days, and root length and fresh and dry weight wereobserved after 28 days of growth.

Growth Promotion Studies in Gnotobiotic System

Gnotobiotic system consisted of a glass tube (2.5 cm inner diameter, 38 cm length) connectedto a 100 ml conical flask at the bottom (Fig. 1). A sand column of 20 cm height wasconstructed within the glass tube using sand of 0.1 to 0.3 mm particle size. The tube wasthen fixed tightly to the conical flask containing Hoffland’s plant nutrient solution (PNS),consisting of 5 mM Ca(NO3)2, 5 mM KNO3, 2 mM MgSO4, 1 mM KH2PO4, andmicronutrients (g/l of MnSO4—0.61, ZnSO4·7H2O—0.1, H3BO3—1.27, Na2MoO4·2H2O—0.40, and CuSO4—0.04). The bottom portion of the tube was immersed in the nutrientsolution to keep the sand column moist. The system was autoclaved and cooled; then,bacterized cowpea and pigeon pea seeds were aseptically transferred 5 mm below the surfaceof the sand column. One percent CMC alone treated seeds served as the control.

To study the effect of pure compounds in plant growth, 200 μg of pure compound inmethanol was added to sand column prior to the introduction of the seeds. Sand column treatedwith the same amount of methanol served as the control. After 7 days of growth, three numbersof representative plants from each set were selected randomly and uprooted with utmost care tokeep the roots intact and then washed gently under running tap water to remove the adheringsoil particles. Data on growth promotion in terms of increase in shoot height, root length, andfresh and dry weight of plant were recorded over the non-treated control plants.



Statistical Analysis

All statistical analyses were performed with SPSS (version 17.0; SPSS, Inc., Chicago, IL,USA). Data for plant growth study was presented as means±standard deviation. Statisticalsignificance was defined as p<0.05.

Results

16S rDNA Sequencing and Phylogenetic Analysis

In addition to the classical identification from IMTECH, Chandigarh, molecular identificationof PM 105 was also done based on the 16S rDNA gene sequencing. PCR amplification yielded

Fig. 1 Glass tube system used for gnotobiotic study



~1500 bp amplicon. Blast analysis showed 99 % similarity to P. aeruginosa sequence availablein the Genbank database, and thus, the bacteria was identified as P. aeruginosa. Partial 16SrRNA sequence data have been deposited in the GenBank (NCBI) nucleotide database underthe Accession No. KF241279. The phylogram clearly portrayed the relationships of theisolates used in the analysis. The present bacterial isolate (P. aeruginosa strain) was success-fully grouped along with other P. aeruginosa isolates obtained from the GenBank databaseconfirming the authenticity of the isolate (Fig. 2).

In Vitro Antibiosis Test with Live PM 105

The strain PM 105 exhibited in vitro antagonism against all the fungal pathogens tested(Table 1). Maximum inhibition was recorded against A. fumigatus (20 mm) followed byC. gloeosporioides (18 mm) (Fig. 3). C. albicans exhibited the lowest inhibition among thetest strains. However, antagonism is not exhibited by PM 105 against any of the test bacterialstrains screened.

Yield of Crude Extracts

An average of 270, 243, and 321 mg crude extract was obtained from 1 l of medium,respectively, in diethyl ether, chloroform, and ethyl acetate.

Fig. 2 Phylogenic relationships of P. aeruginosa strain isolated and known bacterial relatives based on 16SrRNA gene sequences (neighbour-joining method)



Antifungal Activity of the Crude Extracts

It is evident from the data that the two of the crude extracts (chloroform and diethyl ether)recorded significant antifungal activity against all the test fungal strains (Table 2). The bestinhibition was exhibited by the chloroform extracts followed by diethyl ether extract. Highestactivity of 24 mm was recorded against P. expansum by chloroform crude extract. It is alsoevident from this table that among the extracts, ethyl acetate extract recorded least inhibitionagainst all the test pathogens.

Antibacterial Activity of Crude Extracts

The antibacterial activity of crude extract against test bacteria is given in Table 3. Chloroformextract exhibited maximum inhibition followed by diethyl ether extract. So, chloroform anddiethyl ether extracts were taken for further purification of secondary metabolite using columnchromatography.

Purification of the Crude Extract

From the chloroform fraction crude extract, two pure fractions, compound A (11 mg), a yellowpowder with Rf 0.68 (EtOAc 100 %), and compound B (3 mg), a blue powder with Rf 0.60(EtOAc/MeOH 19:1), were isolated. The diethyl ether crude extract was found to have a

Table 1 Antifungal activity of livePM 105 Fungal pathogen Zone of inhibition (mm)

A. flavus 13±0

A. fumigatus 20±1

A. niger 10±0.52

A. tubingensis 14±0.77

C. gloeosporioides 18±1.52

C. albicans 5±0

F. oxysporum 11±0.52

P. expansum 13±1

R. solani 12±0

T. rubrum 15±1

Fig. 3 In vitro antifungal activity of PM 105 against A. fumigatus (a) and C. gloeosporioides (b)



yellow needle-like crystal which was separated in pure form by filtration. These crystals weredissolved in methanol, and TLC was performed in ethyl acetate/methanol (9:1) which showeda single UV-visible spot (Rf 0.76). These crystals yielded 13.6 mg of compound C.

Identification of Isolated Secondary Metabolites

Structures of all three compounds A, B, and C were determined by NMR and by comparisonwith reported compounds from Pseudomonas species. Compounds A, B, and C were identi-fied as phenazin-1-ol, pyocyanin, and phenazine-1-carboxamide, respectively (Fig. 4).

Compound A: 1-Hydroxyphenazine. Yellow powder; Rf 0.68 (EtOAc 100 %).1H NMR(CDCl3): δ 8.28–8.26 (m, 1H), 8.24–8.22 (m, 2H), 7.88–7.82 (m, 2H), 7.80–7.75 (m, 2H),7.25–7.23 (dd, J=6.5, 1.9 Hz, 1H). 13C NMR (CDCl3): δ 151.7, 144.1, 143.8, 141.2, 134.7,131.8, 130.7, 130.5, 129.7, 129.2, 119.9, 108.8 (Supplementary Fig. 1). Negative HR-ESI-MSm/z: 195.05623 [M–H]– (calcd. for 195.05584). Positive HR-ESI-MSm/z: 197.07139 [M+H]+

(calcd. for 197.07149) (Supplementary Fig. 2).Compound B: Pyocyanin. Blue powder; Rf 0.60 (EtOAc/MeOH 19:1). 1H NMR (CDCl3):

δ 8.35 (d, J=8 Hz, 1H), 7.93–7.90 (m, 1H), 7.79–7.75 (m, 2H), 7.61–7.58 (m, 1H), 6.60 (d, J=

Table 2 Antifungal activity of the crude extract

Test fungus Diameter of zone of inhibition (mm)

Diethyl ether Chloroform Ethyl acetate

A. flavus 11±0 14±0.52 –

A. niger 13±1 16± –

A. fumigates 10±0 15±0 –

A. tubingensis 9±0.52 18±1.52 –

C. gloeosporioides 14±1.53 21±0.53 –

C. albicans – 19±0 –

F. oxysporum 17±0.52 23±1 2±1

P. expansum 16±0 24±0.72 4±2.2

R. solani 20±1 20±1 3±0

T. rubrum 19±0 22±0.52 –

– No activity

Table 3 Antibacterial activity of the crude extract

Bacteria Diameter of zone of inhibition (mm) Chloroform Ethyl acetate

Diethyl ether Chloroform Ethyl acetate

B. subtilis 12±0 22±1 4±0

E. coli 18±1.72 20±0.77 –

P. aeruginosa 8±1.15 16±0.52 2±1.72

S. aureus 7±0 23±0 3±1.15

– No activity



9 Hz, 1H), 6.18 (d, J=7.5 Hz, 1H), 4.03 (s, 3H) (Supplementary Fig. 3). Positive HR-ESI-MSm/z: 211.08585 [M+H]+ (calcd. for 211.08714) (Supplementary Fig.4).

Compound C: Phenazine-1-carboxamide. Yellow powder; Rf 0.3 (Hexane/EtOAc1:1). 1H NMR (CDCl3): δ 10.72 (s, 1H), 9.00 (dd, J=7, 1.5 Hz, 1H), 8.42 (dd, J=9,1.5 Hz, 1H), 8.28–8.19 (m, 2H), 7.97–7.88 (m, 3H), 6.62 (s, 1H); 13C NMR (CDCl3): δ166.7, 143.4, 143.1, 141.5, 140.8, 135.9, 134.3, 131.7, 131.1, 129.8, 129.7, 129.1,128.8 (Supplementary Fig.5). Positive HR-ESI-MS m/z: 246.06448 [M+Na]+ (calcd. for246.06433) (Supplementary Fig. 6).

Plant Growth Promotion Study Under Nursery Condition

Pigeon pea and cowpea seeds bacterized with live PM 105 showed significant plant growth-promoting activity with respect to shoot and root elongation (Fig. 5). No increase in thepercentage of seed germination rate was observed for treated seeds when compared withcontrol. All the seeds germinated within 3 days. All the three crude extracts obtained werefound to enhance the growth of pigeon pea and cowpea under nursery conditions (Table 4).Chloroform extract recorded significant plant growth promotion activity in pigeon pea andcowpea in all the parameters observed followed by diethyl ether extract (Table 4).

Plant Growth in a Gnotobiotic System

The gnotobiotic studies with seeds bacterized with live PM 105 also showed enhancedplant growth compared with control (data not provided). Crude extracts showed plantgrowth promotion in both cowpea and pigeon pea. Purified phenazine compounds did

Fig. 4 Structures of isolated bioactive compounds. a 1-Hydroxyphenazine, b pyocyanin, and c phenazine-1-carboxamide





not show any plant growth promotion (data not included), but the crude extractsparticularly chloroform extract was found to be best in plant growth promotion withrespect to shoot height, root length, and wet and dry weight (Table 5).

Antifungal Activity of Pure Compounds

The antifungal activity of pure compounds is as shown in Table 6. C. albicans was found to bethe most susceptible organism for the purified compounds. 1-Hydroxyphenazine and pyocya-nin recorded maximum fungal inhibition. It is evident from the results that some compoundscould only inhibit the spore formation in tested fungal pathogens. Antifungal activities observedwith pyocyanin were less pronounced with detectable activity against C. albicans only.

Antibacterial Activity of Pure Compounds

The antibacterial activity of the pure compounds is as shown in Table 7. The maximumantibacterial property was recorded by the pyocyanin. The maximum zone of inhibition wasalso recorded by pyocyanin against S. aureus (25 mm), and this test pathogen was found to bemost susceptible to all compounds.

Discussion

The search for safer technologies that do not pose risks to the environment along with betterhuman health has achieved increasing interest from all agriculturists. One viable alternate is theuse of beneficial microorganisms as an alternative to various synthetic fertilizers and pesticidesas seed or plant inoculants [18]. Plant growth-promoting rhizobacteria (PGPR) can benefitplant development through multiple mechanisms of action, exercised directly, through theproduction of substances which promote growth and increase nutrient availability in soil or,indirectly, through the suppression of plant pathogens in the rhizosphere [19]. Most of themexhibited the ability to colonize the root surface or cortex region of the introduced plants [20].

Selection of bacterial strains for plant growth promotion studies based on their in vitroantagonism is one of the common practices adopted in recent times by many researchersworldwide [20, 21]. Many of these bacterial strains will also exhibit induction of systemicresistance as seed inoculants [22]. In the present study, P. aeruginosa PM 105 exhibitedsignificant in vitro antifungal activity and plant growth promotion activity in cowpea andpigeon pea. In recent years, P. aeruginosa strains of plant rhizosphere soil origin such asP. aeruginosa 7NSK2 from barley rhizosphere and P. aeruginosa PNA1 from chickpearhizosphere have been used as effective biocontrol agents [23–25]. Gupta et al. [26] alsoreported P. aeruginosa GRC1 as an effective biocontrol agent against S. sclerotium causingstem rot of peanut. In our earlier investigation, this strain PM 105 recorded plant growthpromotion in tea plants as plant root inoculants [27]; hence, we selected this strain for thedetailed investigation in crop plants.

�Fig. 5 Effect of PM 105 on the plant growth promotion in pigeon pea and cowpea. Pigeon pea and cowpeatreated with live Pseudomonas aeruginosa PM 105 by immersing seeds in bacteria suspension to determine theeffect of plant growth promotion. Differences between treatment means were determined using the leastsignificant difference (Duncan’s) test at a probability level of 0.05 (p=0.05). a Pigeon pea and b cowpea



In pot experiment, it was observed that seed bacterization with PM 105 significantly (p=0.05)promoted the growth of cowpea and pigeon pea. In general, seed bacterization resulted inincreased seed germination, leaf area, shoot length, root length, and fresh and dry weight ofseedling and yield. Similarly, promotion in growth parameter and yield of various crop plants inresponse to inoculation with rhizobacteria were reported by Gravel et al. [28] and Shaharoonaet al. [29]. In the present study, also seed bacterization with PM 105 significantly enhanced shootlength, root length, and fresh and dry weight of cowpea and pigeon pea. Haas and De´fago [30]had reported certain strains of fluorescent Pseudomonas as PGPR as they promote plant growthby secreting auxins, gibberellins, and cytokinins. Fluorescent pseudomonads like Pseudomonasfluorescens, Pseudomonas putida, P. aeruginosa, and Pseudomonas aureofaciens suppressed thesoil-borne pathogens through different proposed mechanisms including rhizosphere colonization,antibiosis, and iron chelation by siderophore production [31].

Table 4 Growth promotion studies under nursery condition with crude extract

Extract Shoot height (cm) Root length (cm) Wet weight (g) Dry weight (g)

Pigeon pea

Control 19.00±1.32a 9.17±0.76a 1.85±0.16a 0.29±0.02a

Diethyl ether 26.5±0.50c 17.8±0.76b 3.02±0.18b 0.47±0.04b

Chloroform 35±0.50d 23±1.00c 4.32±0.19c 0.80±0.04c

Ethyl acetate 24.33±1.53b 16.33±2.08b 2.24±0.36a 0.36±0.05a

Cowpea

Control 19.00±1.00a 14.67±1.53a,b 4.56±0.41a 0.53±0.05a

Diethyl ether 24.50±1.80b 18.47±0.55b 6.02±0.82b 0.67±0.07b

Chloroform 27.67±2.08c 22.00±2.0c 10.76±0.58c 0.92±0.04c

Ethyl acetate 22.17±0.76b 14.86±1.85a 6.28±0.29b 0.51±0.03a

All values are the mean of three replicates. Means with same letter within columns are not significantly differentat p<0.05

Table 5 Growth promotion studies under gnotobiotic system with crude extract

Extract Shoot height (cm) Root length (cm) Wet weight (g) Dry weight (g)

Pigeon pea

Control 18.17±1.04a 4.13±0.21a 0.30±0.02a 0.08±0a

Diethyl ether 20.93±0.40b 6.28±1.69b 0.60±0.06b 0.11±0.01b

Chloroform 26.33±0.58c 4.5±0.50a 0.65±0.08b 0.11±0.02b

Ethyl acetate 21±1.00b 4.23±0.25a 0.55±0.03b 0.12±0.02b

Cowpea

Control 18.0±0.50a 6.33±0.35a 0.86±0.05a 0.06±0.02a

Diethyl ether 25.67±0.58c 8.0±0.50b 1.22±0.13b 0.11±0.02b

Chloroform 28.33±1.53d 8.07±0.49b 1.56±0.31c 0.22±0.06c

Ethyl acetate 22.6±1.35b 5.70±1.21a 0.87±0.17a 0.11±0.0b

All values are the mean of three replicates. Means with same letter within columns are not significantly differentat p<0.05



OG talc-based formulation has successfully enhanced growth promotion in green gram andchickpea [32]. Increased root length, shoot length, biomass, and various other vegetativeparameters of plants treated with OG-based bioformulation that were due to the bacterialstrains were clearly demonstrated by them in both in vitro as well as in vivo conditions. Thepresent study also support this as PM 105 recorded significant enhancement of root length,shoot length, and biomass under in vitro as well as in vivo condition. The crude extract of PM105 also recorded significant plant growth-promoting property. However, plant growth-promoting experiments with the isolated pure compounds in gnotobiotic and nursery conditionrecorded no activity. Moreover, the plant growth-promoting property of the crude extract maybe due to the synergistic effect of the compounds present in the extract.

Seed bacterization by fluorescent Pseudomonas proved to be a potential method for theenhancement of growth and suppression of plant pathogenic fungi in seedlings of R. solani andS. rolfsii [29]. The positive effect of seed bacterization in the suppression of percent diseaseincidence in the Macrophomina phaseolina-introduced soil suggests the possibility to usethese strains as a biocontrol agent against this most destructive pathogen affecting crop. RM-3inoculation reduced charcoal rot disease in M. phaseolina-infested soil when compared withthe control by 82.8 %, making the organism a potential biocontrol agent of charcoal rot of

Table 6 Antifungal activity of pure compounds

Test fungus Diameter of zone of inhibition (mm)

Compound A Compound B Compound C Bavistin Amphotericin B

A. flavus 15±0 24±2.1 13±1 21±1.72 –

A. niger 18±0.52 22±0 23±0.72 24±0.52 –

A. fumigatus 15±0.52 20±0.52 14±1.72 23±0 –

A. tubingensis 21±1 20±0.77 15±1 26±1 –

C. gloeosporioides 22±0 26±0 11±2.1 25±0 –

C. albicans 5±1 24±0 10±0 – 26±1

F. oxysporum 19±1 26±1.1 16±1.72 27± –

P. expansum 20±1.72 28±0 17±1 27±1.17 –

R. solani 22±1.52 22±1.77 13±0 28±0.72 –

T. rubrum 26±0 29±0 18±0 – 27±0

– No inhibition

Table 7 Antibacterial activity of isolated pure compounds

Bacteria Inhibition zone (mm)

Compound A Compound B Compound C Ciprofloxacin

B. subtilis 11±0 21±1.72 8±1.52 30±0

E. coli 10±1.15 18±1.52 10±0 31±1

P. aeruginosa 12±1 17±0 8±1 31±1.52

S. aureus 15±1.52 25±1.15 10± 30±0



moong beans (Vigna radiate). This study demonstrates the potential of exploiting indigenousfluorescent pseudomonads for biocontrol and plant growth stimulation.

The P. aeruginosa produce an array of secondary metabolites, which play a pivotal role inthe biological control of plant diseases caused by bacteria and fungal plant pathogens [33].P. aeruginosa was found to produce several antibiotics, which include phenazines, pyrolnitrin,pyoluteorin, and phloroglucinols that were reported earlier [34, 35]. The present investigationwas initiated to identify the antibiotic compound produced by PM 105 that was originallyisolated from a tea rhizosphere with acidic soil conditions. However, PM 105 did not producepyrolnitrin, pyoluteorin, and DAPG. Interestingly, other than the above three different groupsof compounds, probably phenazines would have mediated major role in the wide-spectrumbiocontrol activity of P. aeruginosa PM105.

Phenazines are N-containing heterocyclic-pigmented compounds produced by many spe-cies of the bacterial genera, Brevibacterium, Burkholderia, and Pseudomonas and also by theactinomycetes like Streptomyces [36]. The major phenazine synthesized by P. aeruginosa ispyocyanin (1-OH-5-methyl phenazine) [37]. Almost all phenazines exhibit broad-spectrumactivity against many bacteria and fungi [38], and the phenazine production by a biocontrolagent, P. aureofaciens, in the rhizosphere has been demonstrated [39]. In the current study, alsophenazine compounds recorded significant antimicrobial activity especially against humanpathogenic microbes. As P. aeruginosa PM105 exhibited wide-spectrum antimicrobial activ-ity, it can be concluded that the production of phenazines could be the biocontrol mechanismagainst the fungal and bacterial phytopathogens.

Impact of the Present Study

Phytopathogens can influence in the yield reduction of crop from 30 to 100 %. This is a hugeloss of productivity affecting the economy of many nation especially the developing countrieslike India and China. Phytopathogen damage to crops is generally controlled by the use ofchemical agents. The indiscriminate use of chemicals for controlling the plant diseases resultedin environmental pollution and health problems. Moreover, haphazard use of chemicals breaksdown the natural ecological balance by killing the beneficial soil microbes. Furthermore, thegrowing cost of pesticides especially fungicides and moreover consumer demand for organicfood has forced to search for substitutes. It is therefore cautious to explore cost-effective, lowrisky, non-synthetic chemical, eco-friendly methods for controlling various plant diseases.Biological control is thus being considered as an alternative viable way of reducing the use ofthe current practices in agriculture.

The secondary metabolites producing P. aeruginosa strain PM 105 exhibited inherentpotential of plant growth promotion and significant disposition in suppressing many soil-borne phytopathogenic and human pathogenic fungi. The in vivo experiments suggest thepossible use of the organism in agriculture for achieving greater growth and yield of crops.

Conclusions

PM 105 is a potent strain of P. aeruginosa having antagonistic effect against many pathogenicfungi and bacteria that was confirmed through this investigation. The molecular and conventionaltaxonomical identification reveals PM 105 as P. aeruginosa. From the results of gnotobiotic andnursery pot experiment, it can be concluded that this strain is plant growth promoting, and hence,



it can be used as a seed or root inoculants for growth promotion and disease control. These resultshighlight the importance of the secondary metabolites produced by the strain in enhancing plantgrowth and in controlling fungal and bacterial pathogen. The secondary metabolites that werepurified in the pure form were all phenazine derivatives, but they only showed antimicrobialproperty, and no plant growth promotion was shown by them. So, other crude extracts thatshowed plant growth promotion need to be analyzed, and the metabolites present in those need tobe investigated to exactly know the plant growth-promoting compound.

Acknowledgments We thank Director CSIR-NIIST for his kind permission to carry out and publish this work.The authors are also grateful to Kerala State Council for Science, Technology and Environment (KSCSTE) forfunding. Nishanth Kumar. S thanks Kerala State Council for Science, Technology and Engineering (KSCSTE)for PDF fellowship. Acknowledgement is due to Department of Science and Technology (DST) for providingINSPIRE fellowship to Jubi Jacob (IF 130648).

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http://dx.doi.org/10.4172/2157-7471.1000129

Bioactive metabolite from Pseudomonas

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