Bioresource Technology 98 (2007) 1346–1352

InXuence of soil reaction on diversity and antifungal activityof Xuorescent pseudomonads in crop rhizospheres

Rajni Verma, Ajit Singh Naosekpam, Sanjay Kumar, Ramdeen Prasad, V. Shanmugam ¤

Floriculture Division, Institute of Himalayan Bioresource Technology, Palampur 176 061, Himachal Pradesh, India

Received 1 September 2005; received in revised form 20 May 2006; accepted 22 May 2006Available online 14 July 2006

Abstract

The diversity and antifungal activity of Xuorescent pseudomonads isolated from rhizospheres of tea, gladiolus, carnation and blackgram grown in acidic soils with similar texture and climatic conditions were studied. Biochemical characterisation including antibioticresistance assay, RAPD and PCR–RFLP studies revealed a largely homogenous population. At soil pH (5.2), the isolates exhibitedgrowth with varying levels of siderophore production, irrespective of crop rhizospheres. Two isolates with maximum chitinase productionshowed antagonism. The bacterial populations in general lacked the ability to produce deleterious traits such as cellulase, pectinase andhydrogen cyanide. However, increased pH levels beyond 5.2 caused reduction in metabolite production with reduced antifungal activity.The homogeneity of the bacterial population irrespective of crop rhizospheres together with decreased secondary metabolite productionat higher pH levels reinstated the importance of soil over host plant in inXuencing rhizosphere populations. The studies also yielded acidtolerant chitinase producing antagonistic Xuorescent pseudomonads.© 2006 Elsevier Ltd. All rights reserved.

Keywords: Soil reaction; Fluorescent pseudomonads; Rhizospheres; Diversity; Antifungal activity

1. Introduction

Pseudomonas spp. make up a diverse group of bacteriathat are generally found in all geographical conditions.Fluorescent pseudomonads are ubiquitous bacteria that arecommon inhabitants of rhizospheres, and are the moststudied group within the genus Pseudomonas. These Xuo-rescent pseudomonads stimulate plant growth by facilitat-ing either uptake of nutrients from soil or producingcertain plant growth promoting substances. Besides, thesebacteria also prevent proliferation of phytopathogens andthereby support plant growth (Weller, 1988). However,their eYcacy often varies since the populations are not dis-

* Corresponding author. Tel.: +91 1894 233339x314; fax: +91 1894230433.

E-mail address: shanpatho@yahoo.com (V. Shanmugam).

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biortech.2006.05.030

tributed at random. Apart from host plants, soil factorssuch as composition, organic matter, pH, water and oxygenavailability play signiWcant role in selection of natural Xora(Ross et al., 2000). Survival of Xuorescent pseudomonadsupon introduction varies widely in soils of diVerent pH andsalinity especially in rhizospheres (Rangarajan et al., 2001).The prevailing soil and/or climatic conditions greatly inXu-ence the performance of an introduced eVective strain iso-lated elsewhere (Capper and Higgins, 1993). It is thereforeessential to isolate indigenous organisms that can be usedin same ecological niche. For these, the structure and diver-sity of bacterial community in relation to environmentalfactors and ecosystem functions are to be understood(Torsvik et al., 1996). The present study was, therefore,aimed to assess the eVect of soil reaction on diversity andantifungal activity of rhizosphere populations of Xuores-cent pseudomonads associated with the host plants, tea,gladiolus, carnation and black gram grown in acidic soilsunder similar climatic conditions.

R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352 1347

2. Methods

2.1. Site description and sample collection

Soil samples were collected from seven diVerent loca-tions (32°6�N, 76°3�E, 1300 m above msl) representing rhi-zospheres of mainly tea (Camellia sinensis Linn.), gladiolus(Gladiolus hortulanus L.H. Bailey), carnation (Dianthus car-yophyllus Linn.) and black gram (Vigna mungo Linn.),around Palampur, the site of the institute. The soil texturewas silty clay loam (30% clay, 53% silt and 17% sand) withan average pH of 5.2 and the climate is wet temperate withan average annual rainfall of 2491 mm. Rhizosphere soilsfrom 15 healthy and mature plants were collected at regularintervals from each plot, to represent the entire area of cul-tivation, and then pooled before bacterial isolation.

2.2. Isolation of Xuorescent pseudomonads

Rhizoplane-colonising Xuorescent pseudomonads wereisolated from fresh intact root systems of all four-hostplants. The root samples collected from the gardens werevigorously shaken to remove the soil loosely adhered to theroots. A soil suspension was obtained by shaking 10 g ofroot plus lightly adhering soil in 90 ml sterile water for30 min at 180-rev min¡1 in a rotary shaker. The suspensionsfrom all four rhizospheres were diluted up to 104 and oneml of the suspension was plated onto King’s B agar (Kinget al., 1954). Single colonies that Xuoresced under UV light(366 nm) after incubating at 28§ 2 °C for 2 days wereselected and further cultured on same medium to establishpure cultures.

2.3. IdentiWcation of Xuorescent pseudomonads

Total genomic DNA was isolated from cultures grownin nutrient broth at 28§2 °C for 16 h employing standardprocedures (Graves and Swaminathan, 1993). To conWrmthe bacterial isolates as Xuorescent pseudomonads, 16S-23SrRNA intervening sequence-speciWc primers ITS1F (AAG-TCGTAACAAGGTAG); ITS2R (GACCATATAACCC-CAAG) were used to get an amplicon size of 560 bp(Kumar et al., 2002). The products were subjected to elec-trophoresis on 1.4% agarose gels containing 0.5 �g ml¡1

ethidium bromide. The identiWed isolates were subjected tofurther characterisation.

2.4. Biochemical characterisation

The isolates were phenotypically characterized for thefollowing reactions: indole formation, nitrate reduction,arginine dihydrolase, gelatin hydrolysis, levan production,casein and urea hydrolysis, Tween 80 hydrolysis, lysinedecarboxylase, catalase, oxidase, utilisation of carbonsources such as glucose, cellobiose, L-arabinose, rhamnose,mannose, lactose, inulin, sucrose, galactose, sorbitol, xylose,inositol, mannitol, maltose, fructose, citrate and trehalose

(Stanier et al., 1966; Bossis et al., 2000). Results of thesetests were scored as either positive or negative and the bac-terial isolates were distinguished into diVerent biovars (Sta-nier et al., 1966; Krieg and Holt, 1984; Bossis et al., 2000).

2.5. Antibiotic resistance

The isolates along with the reference strain (PAPF-Nag)were screened for intrinsic antibiotic resistance to nine anti-biotics: penicillin (50 �g/ml); ampicillin (75 �g/ml), tetracy-cline (50�g/ml); rifampicin (80 �g/ml); streptomycin (75 �g/ml); kanomycin (30�g/ml); neomycin (10 �g/ml); carbeni-cillin (100�g/ml) and choramphenicol (30 �g/ml). Antibi-otic discs (Himedia, India) for Pseudomonas were used forthe purpose. The diameters of inhibition zones were mea-sured and correlated to the zone size interpretative chartsupplied by the manufacturer.

2.6. RAPD Wngerprinting

The extent of molecular diversity of isolates from each ofthe crop rhizospheres was analysed along with the referencestrain. Fifty primers from the kits OPA, OPAA, OPB, OPGand OPH, each consisting of 20 random decamer primerswere tested. The primers were supplied by Operon technol-ogies, CA, USA, and the PCR reagents by BangaloreGenei, India. AmpliWcations were carried out in 25 �l reac-tion volume consisting of 10£ buVer with 1.5 mM MgCl2,2.5�l; 2 mM dNTPs, 2.5 �l; 3 U/�l Taq DNA polymerase,0.33 �l; 20 pM primer, 2 �l; 45 ng template DNA, in aBiorad (USA) thermalcycler using the PCR conditions94 °C for 3 min (initial denaturation), 94 °C for 1 min (dena-turation), 37 °C for 1 min (annealing) and 72 °C for 2 min(extension). The reaction control consisted of all compo-nents, except the genomic DNA. The total number of cycleswas 40, with the Wnal extension of 72 °C for 10 min. TheampliWed products (25 �l) were size separated on 1.4% aga-rose gel containing 0.5�g ml¡1 ethidium bromide and pho-tographed in a gel documentation system (Alpha Imager2200). A 100 bp DNA ladder (GeneRuler plus, MBI, Fer-mentas) was used as molecular weight size markers. Theanalysis was repeated at least three times, Wngerprints werecompared and the bands which appeared consistently wereevaluated. The pair-wise coeYcient similarity based onpresence and absence of bands and cluster analysis withunweighed pair group method arithmetic mean (UPGMA)were used to generate similarity matrix.

2.7. Restriction analysis of 16S rDNA

The PCR ampliWed 16S rDNA product (560 bp) wasrestriction digested for 3 h at 37 °C in 25 �l reaction mixturecontaining 8 �l (300 ng) of PCR product, 2.5 �l of 10£PCRbuVer, and 10 U of one of the following restriction enzymes,HindIII, BamHI, PstI, EcoRI, AluI, RsaI and MboI.Restriction digestion was then analysed by agarose electro-phoresis (1.4%) containing 0.5 �g ml¡1 ethidium bromide

1348 R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352

(Sambrook et al., 1989). A 100 bp ladder (MBI, Fermentas)was used as molecular size marker. The analysis was doneat least twice with each enzyme.

2.8. Selection for acidic pH

Pure culture of each of the isolates was plated in tripli-cates on KB (King et al., 1954) medium with the pHadjusted to 5.2, 5.8 and 6.4. The plates were incubated at28§2 °C for 2 days.

2.9. Test for in vitro antagonism

The Xuorescent pseudomonads were tested for in vitroantagonism at 5.2, 5.8 and 6.4 pH levels against the testpathogen, Fusarium oxysporum f.sp. dianthi (Prill. &Delacr.) W.C. Synder & H.N. Hans (MTCC 6659) causingvascular wilt in carnation. The test pathogen was pre-viously isolated, characterized and its pathogenicity wasestablished. The screening was performed on potato dex-trose agar (PDA) medium in triplicates. An agar plug(5 mm diameter) taken from an actively growing fungal cul-ture was placed at one side on the surface of the PDA plate.After 48 h, the Xuorescent pseudomonads were streakedperpendicular to the agar plug on the opposite side towardsthe edge of plates. Plate inoculated with fungal agar plugsalone was used as control. The plates were incubated at28§2 °C until fungal mycelia completely covered the agarsurface in control plate. Isolates that inhibited the mycelialgrowth of the pathogen were identiWed.

2.10. Hydrolytic activity and hydrogen cyanide (HCN) production

Cellulase and pectinase production were determined asdescribed earlier (Cattelan et al., 1999). M9 medium agar(Na2HPO4 6.0 g; KH2PO4 3 g; NaCl 0.5 g; NH4Cl 1.0 g;MgSO4 0.5 g; glucose 2 g; CaCl2 0.015 g; distilled water 1 L)(Miller, 1974) amended with 10 g of cellulose and 1.2 g ofyeast extract per litre of distilled water was used to test thecellulase activity. The isolates were plated and incubated at28 °C for 8 days. Development of halos was considered aspositive.

For determining the pectinase activity, 10 g pectin and1.2 g yeast extract were amended in M9 medium agar andplated. After incubating at 28 °C for 2 days, the cultureswere Xooded with 2 M HCl. Clear halos around the colo-nies were considered as positive for pectinase production.

Test for HCN production was carried out as describedearlier (Meena et al., 2001). The bacterial isolates weregrown at 28 °C on a rotary shaker in tryptic soy broth. Fil-ter paper (Whatman no. 1) was cut into uniform strips of10 cm long and 0.5 cm wide, saturated with alkaline picratesolution and placed inside the conical Xasks in a hangingposition. After incubation at 28 °C for 48 h, the sodium pic-rate present in the Wlter paper was observed for a change incolor.

These assays were carried out at three pH levels, 5.2, 5.8and 6.4 and three replications were maintained for eachstrain in every assay.

2.11. Siderophore and chitinase assays

Siderophore production was quantiWed as suggested(Reeves et al., 1983). The bacterial isolates were grown inKB broth of 5.2, 5.8 and 6.4 pH and incubated at 28§2 °Cfor 3 days and the culture supernatants were collected bycentrifugation at 3000g for 10 min and passed through bac-terial proof Wlters 0.22 �m (Millipore, USA). The pH of thesupernatant was adjusted to 2.0 with 1 N HCl and equalquantity of ethyl acetate was added in a separating funnel,mixed well and ethyl acetate fraction was collected. Fivemilliliters of ethyl acetate fraction was mixed with 5 ml ofHathway’s reagent (1.0 ml of 0.1 M FeCl3 in 0.1 N HCl to100 ml distilled water + 1.0 ml of 0.1 M potassium ferricya-nide). The absorbance for dihydroxyl phenols was read at700 nm in a spectrophotometer. A standard curve was pre-pared with dihydroxy benzoic acid and the quantity of sid-erophore produced was expressed as �mol of benzoic acid/ml of culture Wltrate.

The antagonistic bacterial isolates were evaluated fortheir chitinolytic ability employing colloidal chitin, pre-pared from crab shell chitin (Sigma) (Berger and Reynolds,1958). A loopful of 48 h old cultures was streaked in tripli-cates on water agar medium incorporated with 0.4% colloi-dal chitin and pH adjusted to 5.2, 5.8 and 6.4. Developmentof clear halos around the colonies was recorded after 5 daysof incubation at 28§2 °C. Chitinase production was alsoquantitatively evaluated by colorimetric method (Bollerand Mauch, 1988). The reaction mixture consisted of 10�lof 1 M sodium acetate buVer (pH 4.0), 0.4 ml supernatantfrom culture Wltrate of isolates grown separately in KBbroth of 5.2, 5.8 and 6.4 pH and 0.1 ml colloidal chitin(10 mg) in triplicates. After incubating for 1 h, the resultantchitin oligomers were treated with 2 ml dimethyl aminobenzaldehyde (DMAB) for 20 min at 37 °C and the absor-bance was measured at 585 nm. N-acetylglucosamine (Glc-NAc) served as the standard. The enzyme activity wasexpressed as nmoles GlcNAc equivalents/min/ml.

3. Results

3.1. Isolation and identiWcation of Xuorescent pseudomonads

Twenty-eight Xuorescent pseudomonads were isolatedfrom the rhizosphere soil samples. PCR ampliWcation of theITS (16S-23S) region from all 28 isolates gave a singleamplicon of 560 bp size, conWrming the identity of theseisolates as P. Xuorescens.

3.2. Biochemical characterisation

Substrate utilisation pattern showed that the isolatesfrom all four rhizospheres were gram-negative, positive for

R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352 1349

catalase, oxidase, casein, urea and gelatin hydrolysis, indoleformation and citrate utilisation with varied patterns ofcarbon utilisation. None of them could use lactose, rham-nose, galactose and mannose. The isolates from carnationrhizosphere utilized lesser number of carbon sources thanthe ones from other rhizospheres. The characteristics of theisolates in general barring few exhibited a similar patternwithin each rhizosphere (data not shown). Interpretation ofthese substrate utilisation patterns distinguished Xuorescentpseudomonads into four biovars. The rhizosphere sampleswith the exception of black gram were predominated bybiovar 1, typical of P. Xuorescens. Whilst biovar II wasobserved only in carnation and black gram samples, biovarIII was predominant in black gram rhizosphere. Maximumpopulations of biovar V were observed in carnation andblack gram rhizospheres (Table 1).

3.3. Antibiotic resistance

The isolates in general showed variation in antibioticresistance. Among them, only those from tea rhizosphereshowed cent per cent resistance to penicillin, ampicillin,tetracycline, rifampicin, streptomycin, kanamycin andneomycin. However, irrespective of rhizosphere sources,the isolates showed maximum resistance to penicillin andampicillin. Very few isolates showed resistance to testedconcentrations of carbenicillin and chloramphenicol(Table 2).

Table 1Diversity of Xuorescent pseudomonads in crop rhizosphere

Putativecharacterisationof P. Xuorescens

Percentage of occurrence in crop rhizosphere

Tea (7) Gladiolus (7) Carnation (7) Black gram (7)

Biovar I 71.5 71.5 42.9 28.6Biovar II 0 0 14.3 14.3Biovar III 14.3 14.3 14.3 28.6Biovar V 14.3 14.3 28.6 28.6

3.4. RAPD analyses

Among the tested primers, only two primers viz. OPA09(5�-GGGTAACGCC) and OPB07 (5�-GGTGACGCAG)were observed to be useful for RAPD typing of the isolates.The non-selected primers mostly generated non-reproduc-ible banding patterns. The size of the ampliWed fragmentsranged from 0.3 to 3 kb, and the number of fragments variedfrom 1 to 7 per primer per sample. Of the total 170 bandsscored, 91.2% were polymorphic. The RAPD proWles gener-ated by these selected primers were highly discriminativeand reproducible with consistent fragment patterns. Thepolymorphic bands in total distinguished the species fromeach other. RAPD proWle of the isolates, analysed withprimer OPA 09 is represented in Fig. 1. The per centagepolymorphism was 61%, 58%, 48% and 60% in tea, gladio-lus, carnation and black gram rhizospheres, respectively, andthe inter-host polymorphism was 91.2%. At 75% dissimilar-ity level, the isolates were grouped into two clusters (Fig. 2).

3.5. PCR–RFLP analyses

The restriction digestion patterns of the isolates wereanalysed using selected enzymes. The restriction patterns of

Table 2Antibiotic resistance of Xuorescent pseudomonads

S. no. Antibiotics Crop rhizosphere

Tea(7)

Gladiolus(7)

Carnation(7)

Blackgram (7)

1 Penicillin (50 �g) 7 7 7 62 Ampicillin (75 �g) 7 7 7 63 Tetracycline (50 �g) 7 6 7 54 Rifampicin (80 �g) 7 5 4 55 Streptomycin (75 �g) 7 7 5 76 Kanamycin (30 �g) 7 6 7 67 Neomycin (10 �g) 7 7 5 48 Carbenicillin (100 �g) 2 0 2 19 Chloramphenicol (30 �g) 1 3 4 1

Fig. 1. Representative RAPD proWle of Xuorescent Pseudomonads (Primer OPA09). M: 100 bp DNA ladder plus; 1: PAPF-Nagrota (R); 2: TEPF-Banuri;3: TEPF-Sungal; 4: TEPF-Bir; 5: TEPF-Holta; 6: TEPF-Raipur; 7: TEPF-Darang; 8: TEPF-Nagrota; 9: GLPF-Banuri; 10: GLPF-Sungal; 11: GLPF-Bir;12: GLPF-Holta; 13: GLPF-Raipur; 14: GLPF-Darang; 15: GLPF-Nagrota; Lane 16: CAPF-Banuri; 17: CAPF-Sungal; 18: CAPF-Bir; Lane 19: CAPF-Holta; 20: CAPF-Raipur; 21: CAPF-Darang; 22: CAPF-Nagrota; 23: BGPF-Banuri; 24: BGPF-Sungal; 25: BGPF-Bir; 26: BGPF-Holta; 27: BGPF-Rai-pur; 28: BGPF-Darang; 29: BGPF-Nagrota; 30: HindIII digested DNA ladder.

3000

100200 300400500

700

900800

15002000

1200

600

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

bp

1350 R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352

four hexa cutter enzymes were monomorphic across theisolates. Among tetra cutters, only MboI and AluI showeddigestion, yielding monomorphic and polymorphic bandingpatterns, respectively. AluI produced two patterns amongsixteen isolates from all four-crop rhizospheres exhibitingbanding patterns similar to that of the reference strain(Fig. 3).

3.6. Acidity tolerance

All isolates irrespective of crop rhizospheres toleratedthe pH levels of 5.2, 5.8 and 6.4 (data not shown).

Fig. 2. UPGMA dendrogram based on the DICE similarity index (SD)illustrating the genetic relationships of Xuorescent pseudomonads. 1:PAPF-Nagrota (R); 2: TEPF-Banuri; 3: TEPF-Sungal; 4: TEPF-Bir; 5:TEPF-Holta; 6: TEPF-Raipur; 7: TEPF-Darang; 8: TEPF-Nagrota; 9:GLPF-Banuri; 10: GLPF-Sungal; 11: GLPF-Bir; 12: GLPF-Holta; 13:GLPF-Raipur; 14: GLPF-Darang; 15: GLPF-Nagrota; Lane 16: CAPF-Banuri; 17: CAPF-Sungal; 18: CAPF-Bir; Lane 19: CAPF-Holta; 20:CAPF-Raipur; 21: CAPF-Darang; 22: CAPF-Nagrota; 23: BGPF-Banuri; 24: BGPF-Sungal; 25: BGPF-Bir; 26: BGPF-Holta; 27: BGPF-Raipur; 28: BGPF-Darang; 29: BGPF-Nagrota.

Coefficient0.00 0.25 0.50 0.75 1.00

1 6 3 18 4 11 14 16 21 5 15 27 28 7 23 17 8 10 13 24 25 19 12 26 29 22 20 2 9

3.7. Test for in vitro antagonism

Among the bacterial isolates, only two isolates viz.,TEPF-Sungal and BGPF-Nagrota exhibited antagonismagainst the test pathogen both in presence and absence ofFeCl3. TEPF-Sungal showed maximum inhibition of themycelial growth. However, the antifungal eYcacy asobserved by per cent mycelial growth inhibition declined atincreased test pH levels of 5.8 and 6.4 (Fig. 4).

3.8. Hydrolytic activity and HCN production

All the isolates were tested to be negative for cellulase,pectinase and HCN production (data not shown).

Fig. 4. Antagonism of Xuorescent pseudomonads in presence and absenceof FeCl3 at diVerent pH levels.

0

10

20

30

40

50

60

70

80

TEPF- Sungal onPDA with FeCl3

TEPF- Sungal onPDA without FeCl3

BGPF- Nagrota onPDA with FeCl3

BGPF- Nagrota onPDA without FeCl3

Isolates

Per

cen

t m

ycel

ial g

row

th in

hib

itio

n

pH 5.2 pH 5.8 pH 6.4

Fig. 3. Restriction digestion of 560 bp PCR amplicon for Xuorescent Pseudomonads. M: 100 bp DNA ladder plus; 1: PAPF-Nagrota (R); 2: TEPF-Banuri;3: TEPF-Sungal; 4: TEPF-Bir; 5: TEPF-Holta; 6: TEPF-Raipur; 7: TEPF-Darang; 8: TEPF-Nagrota; 9: GLPF-Banuri; 10: GLPF-Sungal; 11: GLPF-Bir;12: GLPF-Holta; 13: GLPF-Raipur; 14: GLPF-Darang; 15: GLPF-Nagrota; 16: CAPF-Banuri; 17: CAPF-Sungal; 18: CAPF-Bir; 19: CAPF-Holta; 20:CAPF-Raipur; 21: CAPF-Darang; 22: CAPF-Nagrota; 23: BGPF-Banuri; 24: BGPF-Sungal; 25: BGPF-Bir; 26: BGPF-Holta; 27: BGPF-Raipur; 28:BGPF-Darang; 29: BGPF-Nagrota.

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 M

R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352 1351

3.9. Siderophore and chitinase activity

Siderophore production was highly variable as deter-mined by quantitative assay. Though all isolates grown attest pH levels produced siderophores, the production grad-ually decreased with increasing pH (Fig. 5). Assessment forchitinolytic ability of Xuorescent pseudomonads on chitinagar showed that only TEPF-Sungal and BGPF-Nagrotawere able to form lytic zones. Among them, TEPF-Sungalproduced larger clearing/lytic zone (data not shown). Simi-larly, enzyme assays also showed that both the isolates wereable to produce chitinases. However, the enzyme produc-tion drastically reduced at 5.8 and 6.4, when compared tosoil pH (5.2) (Fig. 5).

4. Discussion

Plant rhizosphere is a versatile and dynamic ecologicalenvironment of intense microbe–plant interactions for har-nessing essential micro- and macro-nutrients from a limitednutrient pool (JeVries et al., 2003). Soil has been known toplay dominant role in inXuencing rhizosphere populationsthan the host plant. Endophytic populations were greatlyinXuenced by plants rather than rhizosphere ones (Latouret al., 1996). Screening of natural populations could providevaluable isolates, with multiple stress tolerance coupledwith potential for use as eYcient biocontrol agents. Abioticstresses like soil pH being important limiting factor for theeYciency of many strains (Miller and Woods, 1996), thebacterial populations from rhizospheres of four predomi-nantly cultivated crops grown in similar soil type and cli-matic conditions were assessed for the inXuence of soilreaction on diversity and antifungal activity.

Fig. 5. Siderophore and chitinase production by Xuorescent pseudomo-nads at diVerent pH.

0

2

4

6

8

10

12

14

16

18

pH 5.2 pH 5.8 pH 6.4 pH 5.2 pH 5.8 pH 6.4

TEPF- Sungal BGPF- Nagrota

Strains

µmo

l ben

zoic

aci

d e

qiu

vale

nt/

ml

0

1

2

3

4

5

6

7

8

9

nm

oles G

lcNA

c eqiu

valent/m

in/m

lSiderophore production Chitinase production

Both phenotypic and molecular characterisations of theisolates from diVerent rhizospheres indicated a predomi-nantly homogenous population suggesting non-inXuence ofhost exudates. Whilst host-mediated selection by diVerentcrop plants were known to cause diVerentiation amongpopulations of Xuorescent pseudomonads, diVerences insoil type were proposed to be the dominant selective factor(Lemanceau et al., 1995; Achouak et al., 2000; Mazzola andGu, 2000). Miller and Woods (1996) reported the possiblerole of soil reaction (pH) in inXuencing microbial selectionprocess as environmental stress was shown to reduce bacte-rial diversity. In the present studies, the bacterial growthobserved at soil pH (5.2) indicated their ability to tolerateacidity in rhizosphere soil to undergo natural selectionresulting in homogeneity. Similar observation on microbialselection in crop rhizosphere was observed by Rangarajanet al. (2001), who reported the inXuence of soil salinity onthe diversity of Pseudomonas populations.

At soil pH, the bacterial isolates in general lacked theability to produce deleterious traits such as cellulase, pec-tinase and HCN. However, exhibition of antagonism byselected isolates among them in presence and absence ofFeCl3 indicated the production of antifungal metabolitesother than siderophores as well. Though the isolatesirrespective of host rhizospheres produced varying levelsof siderophores, only isolates producing detectable levels ofchitinases showed antagonism implying the signiWcance ofthe enzyme in antifungal activity. Interestingly, in eithercases, the production dipped with increasing pH beyond 5.2(soil pH) indicating the signiWcance of soil reaction.Though the possible role of inherent genetic property couldnot be negated, expression of such genetic properties isgreatly inXuenced by soil environment (Nowak-Thompsonet al., 1994). The roles of HCN for biocontrol activity in thesoil are not yet clear (Blumer and Haas, 2000). It has beendemonstrated that HCN is involved in the inhibition ofenergy metabolism of crop plants by interfering in thecytochrome oxidative respiration and thus decreased thegrowth and yield in potato. Other investigations alsorevealed that cyanogenic pseudomonads could inhibit thegrowth of bean and lettuce (Alstrom and Burns, 1989;Kremer and Souissi, 2001). Hence, Bakker and Schippers(1987) grouped HCN-producing Xuorescent pseudo-monads as deleterious bacteria for plant growth. Availabil-ity of siderophores in soil for microorganisms and plantsdropped dramatically with increasing pH above 6 (Leong,1986). Similarly, chitinolytic enzymes acting as potentialbioagents in suppressing fungal propagules (Frankowskiet al., 2001) were greatly inXuenced by acidic pH (El-Kat-atny et al., 2001).

In general, isolates that work well in one environmentmay fail to elicit any plant response in others due to condi-tions prevailing in new environment. Screening of naturalpopulation in this study provided antagonistic isolateslacking deleterious traits and producing plant growth pro-moting and biocontrol traits with acid tolerance. This con-formed with the objective of studies on diversity within a

1352 R. Verma et al. / Bioresource Technology 98 (2007) 1346–1352

targeted bacterial population, to screen genotypes that arebest adapted to particular environmental stress or ecologi-cal habitat (Achouak et al., 2000).

5. Conclusion

The forgoing studies concluded that the bacterial popu-lations in rhizospheric soil and their secondary metaboliteproduction were greatly inXuenced by soil reaction besidesproviding valuable stress tolerant antagonistic isolates.However, the variation in secondary metabolite productionimplied that a range of other factors in natural environ-ment might have their role as well, which are to be studiedin detail.

Acknowledgements

The authors are thankful to the Director, IHBT (CSIR),Palampur for support and encouragement during thecourse of this investigation. This work was supported bythe Council of ScientiWc and Industrial Research, Govern-ment of India (30-(2050)/SMM 02/2003-dated 19.1.2004)through co-ordinated network programme. IHBT Publica-tion Number: 0588.

References

Achouak, W., Sutra, L., Heulin, T., Meyer, J.M., Fromin, N., ChristenDegraeve, S., Gardan, L.R., 2000. Pseudomonas brassicacearum sp. nov.and Pseudomonas thivervalensis sp. nov., two root associated bacteriafrom Brassica sp. and Arabidopsis thaliana. International Journal ofSystematic Bacteriology and Evolutionary Microbiology 50, 9–18.

Alstrom, S., Burns, R.G., 1989. Cyanide production in rhizobacteria as apossible mechanism of plant growth inhibition. Biology and Fertilityof Soils 7, 232–238.

Bakker, A.W., Schippers, B., 1987. Microbial cyanide productions in therhizosphere in relation to potato yield reduction and Pseudomonasspp.-mediated plant growth-stimulation. Soil Biology and Biochemis-try 19, 451–457.

Berger, L.R., Reynolds, D.M., 1958. The chitinase system of a strain ofStreptomyces griseus. Biochimica et Biophysica Acta 29, 522–534.

Blumer, C., Haas, D., 2000. Mechanism, regulation, and ecological role ofbacterial cyanide biosynthesis. Archives of Microbiology 173, 170–177.

Boller, T., Mauch, F., 1988. Colorimetric assay for chitinase. Methods ofEnzymology 161, 430–435.

Bossis, E., Lemanceau, P., Latour, X., Gardan, L., 2000. The taxonomy ofPseudomonas Xuorescens and Pseudomonas putida: current status andneed for revision. Agronomie 20, 51–63.

Capper, A.L., Higgins, K.P., 1993. Application of Pseudomonas Xuorescensisolates to wheat as potential biological control agents against take-all.Plant Pathology 42, 560–567.

Cattelan, A.J., Hartel, P.G., Fuhrmann, J.J., 1999. Screening for plantgrowth promoting rhizobacteria to promote early soybean growth.Soil Science Society of American Journal 63, 1670–1680.

El-Katatny, M.H., Gudelj, M., Robra, K.H., Elnaghy, M.A., Gubitz, G.M.,2001. Characterisation of a chitinase and an endo-beta-1,3-glucanasefrom Trichoderma harzianum Rifai T24 involved in control of the phy-topathogen Sclerotium rolfsii. Applied Microbiology and Biotechnol-ogy 56, 137–143.

Frankowski, J., Lorito, M., Scala, F., Schnid, R., Berg, G., Bahl, H., 2001.PuriWcation and properties of two chitinolytic enzymes of Serratiaplymuthica HRO-C48. Archives of Microbiology 176, 421–426.

Graves, L.M., Swaminathan, B., 1993. Universal bacterial DNA isolationprocedure. In: Persing, D.H., Smith, T.F., Tenover, F.C., White, T.J.(Eds.), Diagnostic Molecular Microbiology: Principles and Applications.American Society for Microbiology, Washington, DC, pp. 617–621.

JeVries, P., Gianinazzi, S., Perotto, S., Turnau, K., Barea, J.M., 2003. Thecontribution of arbuscular mycorrhizal fungi in sustainable maintenanceof plant health and soil fertility. Biology and Fertility of Soils 37, 1–16.

King, E.O., Ward, M.K., Rany, D.E., 1954. Two simple media for the dem-onstration of pyocyanin and Xuorescein. Journal of Laboratory andClinical Medicine 44, 301–307.

Kremer, R.J., Souissi, T., 2001. Cyanide production by rhizobacteria andpotential for suppression of weed seedling growth. Current Microbiol-ogy 43, 182–186.

Krieg, N.R., Holt, J.G., 1984, ninth ed. Bergey’s Manual of SystematicBacteriology (vol. I). The Williams and Wilkins Co., Baltimore.

Kumar, N.R., Arasu, V.T., Gunasekaran, P., 2002. Genotyping of anti-fungal compounds producing plant growth-promoting rhizobacteria,Pseudomonas Xuorescens. Current Science 82, 1463–1466.

Latour, X., Corberand, T., Laguerre, G., Allard, F., Lemanceau, P., 1996.The composition of Xuorescent Pseudomonad population associatedwith roots is inXuenced by plant and soil type. Applied and Environ-mental Microbiology 62, 2449–2456.

Lemanceau, P., Corberand, T., Gardan, L., Latour, X., Laguerre, G.,Boeufgras, J.M., Alabouvette, C., 1995. EVects of two plant species, Xax(Linum usitatissimum L.) and tomato (Lycopersicon esculentum Mill.),on diversity of soilborne populations of Xuorescent pseudomonads.Applied and Environmental Microbiology 61, 1004–1012.

Leong, J., 1986. Siderophores – their biochemistry and possible role in thebiocontrol of plant pathogens. Annual Review of Phytopathology 24,187–209.

Mazzola, M., Gu, Y.H., 2000. Impact of wheat cultivation on microbialcommunities from replant soils and apple growth in greenhouse trials.Phytopathology 90, 114–119.

Meena, B., Marimuthu, T., Vidhyasekaran, P., Velazhahan, R., 2001. Biolog-ical control of root rot of groundnut with antagonistic Pseudomonas Xuo-rescens strains. Journal of Plant Disease and Protection 108, 369–381.

Miller, J.H., 1974. Experiments in Molecular Genetics, second ed. ColdSpring Harbor, New York.

Miller, K.J., Woods, J.R., 1996. Osmoadaptation by rhizosphere bacteria.Annual Review of Microbiology 50, 101–136.

Nowak-Thompson, B., Gould, S.J., Kraus, J., Loper, J.E., 1994. CanadianJournal of Microbiology 40, 1064–1066.

Rangarajan, S., Loganathan, P., Saleena, L.M., Nair, S., 2001. Diversity ofpseudomonads isolated from three diVerent plant rhizospheres. Jour-nal of Applied Microbiology 91, 742–749.

Reeves, M., Pine, L., Neilands, J.B., Bullows, A., 1983. Absence of sidero-phore activity in Legionella sp. grown in iron deWcient media. Journalof Bacteriology 154, 324–329.

Ross, I.L., Alami, Y., Harvey, P.R., Achouak, W., Ryder, M.H., 2000.Genetic diversity and biological control activity of novel species ofclosely related pseudomonads isolated from wheat Weld soils in SouthAustralia. Applied and Environmental Microbiology 66, 1609–1616.

Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular cloning: A Lab-oratory Manual, second ed Cold Spring Harbor, New York.

Stanier, R.Y., Palleroni, N.J., DoudoroV, M., 1966. The aerobic pseudomo-nads a taxonomic study. Journal of General Microbiology 43, 159–217.

Torsvik, V., Sorheim, R., Goksoyr, J., 1996. Total bacterial diversity in soiland sediment communities – a review. Journal of Industrial Microbiol-ogy 17, 170–178.

Weller, D.M., 1988. Biological control of soilborne plant pathogens inthe rhizosphere with bacteria. Annual Review of Phytopathology 26,379–407.