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Microbiological Research 163 (2008) 173—181

0944-5013/$ - sdoi:10.1016/j.

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www.elsevier.de/micres

Screening of free-living rhizospheric bacteria fortheir multiple plant growth promoting activities

Farah Ahmad, Iqbal Ahmad�, M.S. Khan

Department of Agricultural Microbiology, Aligarh Muslim University, Aligarh 202002, India

Accepted 10 April 2006

KEYWORDSPGPR;Indoleacetic acid;Siderophores andantifungal activity

ee front matter & 2006micres.2006.04.001

ing author. Tel.: +91 941ess: iqbalahmad8@yaho

SummaryPlant growth promoting rhizobacteria (PGPR) are known to influence plant growth byvarious direct or indirect mechanisms. In search of efficient PGPR strains withmultiple activities, a total of 72 bacterial isolates belonging to Azotobacter,fluorescent Pseudomonas, Mesorhizobium and Bacillus were isolated from differentrhizospheric soil and plant root nodules in the vicinity of Aligarh. These test isolateswere biochemically characterized. These isolates were screened in vitro for theirplant growth promoting traits like production of indoleacetic acid (IAA), ammonia(NH3), hydrogen cyanide (HCN), siderophore, phosphate solubilization and antifungalactivity. More than 80% of the isolates of Azotobacter, fluorescent Pseudomonas andMesorhizobium ciceri produced IAA, whereas only 20% of Bacillus isolates was IAAproducer. Solubilization of phosphate was commonly detected in the isolates ofBacillus (80%) followed by Azotobacter (74.47%), Pseudomonas (55.56%) andMesorhizobium (16.67%). All test isolates could produce ammonia but none of theisolates hydrolyzed chitin. Siderophore production and antifungal activity of theseisolates except Mesorhizobium were exhibited by 10–12.77% isolates. HCNproduction was more common trait of Pseudomonas (88.89%) and Bacillus (50%).On the basis of multiple plant growth promoting activities, eleven bacterial isolates(seven Azotobacter, three Pseudomonas and one Bacillus) were evaluated for theirquantitative IAA production, and broad-spectrum (active against X three test fungi)antifungal activity. Almost at all concentration of tryptophan (50–500 mg/ml), IAAproduction was highest in the Pseudomonas followed by Azotobacter and Bacillusisolates. Azotobacter isolates (AZT3, AZT13, AZT23), Pseudomonas (Ps5) and Bacillus(B1) showed broad-spectrum antifungal activity on Muller-Hinton medium againstAspergillus, one or more species of Fusarium and Rhizoctonia bataticola. Furtherevaluation of the isolates exhibiting multiple plant growth promoting (PGP) traits onsoil–plant system is needed to uncover their efficacy as effective PGPR.& 2006 Elsevier GmbH. All rights reserved.

Elsevier GmbH. All rights reserved.

2371170; fax: +91 571 2703516.o.co.in (I. Ahmad).

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Introduction and exert their effect on the plant. The environ-

Plant growth promoting rhizobacteria (PGPR) area heterogeneous group of bacteria that can befound in the rhizosphere, at root surfaces and inassociation with roots, which can improve theextent or quality of plant growth directly and orindirectly. In last few decades a large array ofbacteria including species of Pseudomonas, Azos-pirillum, Azotobacter, Klebsiella, Enterobacter,Alcaligens, Arthobacter, Burkholderia, Bacillusand Serratia have reported to enhance plantgrowth (Kloepper et al., 1989; Okon and Laban-dera-Gonzalez, 1994; Glick, 1995). The directpromotion by PGPR entails either providing theplant with a plant growth promoting substancesthat is synthesized by the bacterium or facilitatingthe uptake of certain plant nutrients from theenvironment. The indirect promotion of plantgrowth occurs when PGPR lessen or prevent thedeleterious effect of one or more phytopathogenicmicro-organisms.

The exact mechanisms by which PGPR promoteplant growth are not fully understood, but arethought to include (i) the ability to produce orchange the concentration of plant growth regula-tors like indoleacetic acid, gibberellic acid, cyto-kinins and ethylene (Arshad and Frankenberger,1993; Glick, 1995), (ii) asymbiotic N2 fixation(Boddey and Dobereiner, 1995), (iii) antagonismagainst phytopathogenic microorganisms by pro-duction of siderophores (Scher and Baker, 1982),antibiotics (Shanahan et al., 1992) and cyanide(Flaishman et al., 1996), (iv) solubilization ofmineral phosphates and other nutrients (De Freitaset al., 1997; Gaur, 1990). Most popular bacteriastudied and exploited as biocontrol agent includesthe species of fluorescent Pseudomonas and Bacil-lus. Some PGPR may promote plant growth indir-ectly by affecting symbiotic N2 fixation, nodulationor nodule occupancy (Fuhrmann and Wollum,1989). However, role of cyanide production iscontradictory as it may be associated with deleter-ious as well as beneficial rhizobacteria (Bakker andSchippers, 1987; Alstrom and Burns, 1989).

In addition to these traits, plant growth promot-ing bacterial strains must be rhizospheric compe-tent, able to survive and colonize in therhizospheric soil (Cattelan et al., 1999). Unfortu-nately, the interaction between associative PGPRand plants can be unstable. The good resultsobtained in vitro cannot always be dependablyreproduced under field conditions (Chanway andHoll, 1993; Zhender et al., 1999). The variability inthe performance of PGPR may be due to variousenvironmental factors that may affect their growth

mental factors include climate, weather condi-tions, soil characteristics or the composition oractivity of the indigenous microbial flora of the soil.To achieve the maximum growth promoting inter-action between PGPR and nursery seedlings it isimportant to discover how the rhizobacteria exert-ing their effects on plant and whether the effectsare altered by various environmental factors,including the presence of other micro-organisms(Bent et al., 2001).

Therefore, it is necessary to develop efficientstrains in field conditions. One possible approach isto explore soil microbial diversity for PGPR havingcombination of PGP activities and well adapted toparticular soil environment. So keeping in view theabove constrains, the present study was designedto screen certain rhizospheric bacterial isolatesbelonging to Azotobacter, Mesorhizobium ciceri,fluorescent Pseudomonas and Bacillus for theirmultiple plant growth promoting activities.

Materials and methods

Isolation and characterization

All the isolates of Azotobacter, Pseudomonas andBacillus were isolated from the rhizospheric soil ofdifferent crops (mustard, barseem, wheat, sugar-cane, brinjal, onion, cauliflower, cabbage and chickpea) grown in vicinity of Aligarh, UP, India.Mesorhizobium was isolated from the nodules ofchickpea on yeast extract mannitol agar containingper liter of distilled water: 10 g mannitol, 0.5 gK2HPO4, 0.2 g MgSO4 � 7H2O, 0.1 g NaCl, 1.0 g yeastextract, 3.0 g CaCO3, 15ml Congo red (1:400aqueous solution), 20 g agar, pH 6.8–7.0. M. ciceriisolates were confirmed by nodulation assay understerile pot condition as described by Vincent(1970).

Whereas other bacteria were isolated on theirrespective media, Azotobacter on Jensen’s mediumcontaining per liter of distilled water: 20 g sucrose,1 g K2HPO4, 0.5 g MgSO4 � 7H2O, 0.5 g NaCl, 0.1 gK2SO4, 0.005 g Na2MoO4, 20 g agar, pH 6.9, Pseudo-monas on King’s B medium containing per liter ofdistilled water: 10 g proteose peptone, 10mlglycerol, 1.5 g K2HPO4, 1.5 g MgSO4, 20 g agar, pH7.2 and Bacillus on nutrient agar (NA) containingper liter of distilled water: 5.0 g peptone, 1.5 gyeast extract, 1.5 g beef extract, 5.0 g NaCl, 20 gagar, pH 7.2. Bacterial cultures were maintained onthe respective slants. Fluorescence of Pseudomo-nas colonies was observed on King’s B medium

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under UV exposure. All the microbiological mediaand media ingredients were purchased from Hi-Media Lab. Pvt. Ltd., Mumbai, India.

The bacterial isolates were characterized bytheir cultural conditions, morphological and bio-chemical characteristics (hydrolysis of starch, lipidand chitin, utilization of glucose, sucrose, lactose,mannitol, citrate and catalase reactions) usingstandard methods (Cappuccino and Sherman,1992). Biotin prototrophy was determined bygrowing the isolates on Bacto biotin assay medium(Difco Laboratories) for 48–72 h at 2872 1C.

In vitro screening of bacterial isolates fortheir plant growth promoting (PGP) activities

Assay for indoleacetic acid (IAA) productionIAA production was detected by the modified

method as described by Brick et al. (1991).Quantitative analysis of IAA was performed usingthe method of Loper and Scroth (1986) at differentconcentrations of tryptophan (0, 50, 150, 300, 400and 500 mg/ml). Bacterial cultures were grown for72 h (Azotobacter) and 48 h (Pseudomonas andBacillus) on their respective media at 2872 1C.Fully grown cultures were centrifuged at 3000 rpmfor 30min. The supernatant (2ml) was mixed withtwo drops of orthophosphoric acid and 4ml of theSalkowski reagent (50ml, 35% of perchloric acid,1ml 0.5M FeCl3 solution). Development of pinkcolour indicates IAA production. Optical densitywas taken at 530 nm with the help of spectro-photometer Spectronic 20 D+. Concentration of IAAproduced by cultures was measured with the helpof standard graph of IAA (Hi-media) obtained in therange of 10–100 mg/ml.

NH3 productionBacterial isolates were tested for the production

of ammonia in peptone water. Freshly growncultures were inoculated in 10ml peptone waterin each tube and incubated for 48–72 h at 2872 1C.Nessler’s reagent (0.5ml) was added in each tube.Development of brown to yellow colour was apositive test for ammonia production (Cappuccinoand Sherman, 1992).

HCN productionAll the isolates were screened for the production

of hydrogen cyanide by adapting the method ofLorck (1948). Briefly, nutrient broth was amendedwith 4.4 g glycine/l and bacteria were streaked onmodified agar plate. A Whatman filter paper no. 1soaked in 2% sodium carbonate in 0.5% picric acidsolution was placed in the top of the plate. Plates

were sealed with parafilm and incubated at2872 1C for 4 days. Development of orange to redcolour indicated HCN production.

Siderophore productionBacterial isolates were assayed for siderophores

production on the Chrome azurol S agar medium(Sigma, Ltd.) described by Schwyn and Neilands(1987). Chrome azurol S agar plates were preparedand divided into equal sectors and spot inoculatedwith test organism (10 ml of 106 CFU/ml) andincubated at 2872 1C for 48–72 h. Development ofyellow–orange halo around the growth was con-sidered as positive for siderophore production.

Phosphate solubilization by test bacteriaAll isolates were first screened on Pikovskaya’s

agar plates for phosphate solubilization as de-scribed by Gaur (1990). Quantitative analysis ofsolubilization of tricalcium phosphate in liquidmedium was made as described by King (1932).Briefly, the test isolates were inoculated in 25mlPikovskaya’s broth and incubated for 4 days at2872 1C. The bacterial cultures were centrifugedat 15,000 rpm for 30min. Supernatant (1ml) wasmixed with 10ml of chloromolibidic acid and thevolume was made up to 45ml with distilled water.Cholorostannous acid (0.25ml) was added and thevolume was made up to 50ml with distilled water.The absorbance of the developing blue colour wasread at 600 nm. The amount of soluble phosphoruswas detected from the standard curve of KH2PO4.

Antifungal assayThe agar well diffusion method as adopted

earlier (Mehmood et al., 1999) was used. Thebacterial isolates tested for their antifungal activ-ity were fully grown in the respective broth media.Test fungi were grown on Sabaroud dextrose agar(SDA), (per liter of distilled water: 40 g dextrose,10 g peptone, 20 g agar) slants. The spores werescraped and suspended in 10ml of sterile normalsaline solution (NSS). Diluted spore suspension(0.1ml, 105 CFU/ml) of the fungi was spread onMuller Hinton agar (per liter of distilled water:300 g beef infusion, 17.5 g casein acid hydrolysate,1.5 g starch, 20 g agar, pH 7.2), NA and SDA plates.Wells of 8mm diameter were punched into the agarmedium and filled with 200 ml (2� 107 CFU/ml) ofbacterial culture. Nutrient broth was taken asnegative control and 100 mg/ml antifungal antibio-tic, nystatin was used as positive control. Theplates were incubated for 5–6 days at 2872 1C. Theantifungal activity was evaluated by measuring thegrowth inhibition zone against test fungi.

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Results

Isolation and biochemical characterization

On the basis of cultural, morphological andbiochemical characteristics a total of 66 soilisolates were grouped into Azotobacter, Bacillus,fluorescent Pseudomonas as described in Bergey’sManual of Determinative Bacteriology (Holt et al.,1994). Six nodule isolates of chickpea (Ciceraretinum) were characterized as M. ciceri (Nouret al., 1994). General features of the test isolatesare illustrated in Table 1. All the isolates of M.ciceri were prototrophic for biotin whereas 91.49%isolates of Azotobacter, 83.33% isolates of fluor-escent Pseudomonas and 80% isolates of Bacilluswere prototrophic for biotin.

Plant growth promoting traits of test isolates

Screening results of PGP traits are depicted inFigs. 1 and 2. IAA production was shown in all theisolates of fluorescent Pseudomonas followed byAzotobacter (83.3%), M. ciceri (83.3%) and Bacillus(20%). Phosphate solubilization was detected in 80%of isolates of Bacillus followed by Azotobacter(74.47%), fluorescent Pseudomonas (55.56%) and M.ciceri (16.67%). Production of siderophore andantifungal activity was simultaneously exhibitedby isolates of Azotobacter (16.22%), fluorescent

Table 1. Biochemical characterization of the test isolates

Biochemical characters Azotobacter species

Number of isolates 47Pigmentation Transparent, milky,

some becomes blackishbrown on aging

Colony morphology Watery, mucilaginousshrink, serratedmargins

Polysaccharide production +Gram reaction, cell shape � rodsGrowth on N2 free medium +Catalase, citrate test 100

HydrolysisStarch 68.09Lipid 48.94Biotin prototrophy 91.49

Carbohydrate utilizationGlucose 63.83Lactose 70.21Sucrose 78.72Mannitol 36.17

Pseudomonas (11.11%) and Bacillus (10%). Allisolates were negative for chitin hydrolysis whereaspositive for ammonia production.

Quantitative assay of IAA production byselected isolates

A total of 11 selected isolates of Azotobacter(seven), fluorescent Pseudomonas (three) andBacillus (one) were tested for the quantitativeestimation of IAA in the presence of differentconcentrations of tryptophan. With no addition oftryptophan, production of IAA was not observed.With the addition of tryptophan from 50 to 500 mg/ml the production of IAA was increased. Theproduction of IAA was highest in isolates offluorescent Pseudomonas, followed by Azotobacterand Bacillus, respectively. Amongst the Azotobac-ter, isolates AZT26 produced highest amount of IAAfollowed by AZT34AZT134AZT234AZT14AZT20 asdepicted in Table 2.

Antifungal activity of the test isolates

Antifungal activity of AZT1, AZT3, AZT9, AZT13,AZT20, AZT23, Ps5 and B1 was checked againstAspergillus sp., Fusarium solani, F. ciceri, F.oxysporum, Rhizoctonia bataticola using threedifferent media, MH, NA and SDA (Table 3). Theantifungal activity of strains tested varied with

Mesorhizobiumciceri

FluorescentPseudomonas

Bacillus species

6 9 10– Fluorescent

green–

Pin head,mucilaginouswhite

Button shaped Serratedmargins

+ � �

� rods � rods + rods� � �

100 100 100

– 55.56 8050 77.78 80100 83.33 80

83.33 55.56 8016.67 11.11 2083.33 33.33 8016.67 – 70

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0102030405060708090

100P

erce

nt is

olat

es

Azotobacter spp. Mesorhizobium

ciceri

Fluorescent

Pseudomonas

Bacillus spp.

IAA production

Phosphate solubilization

NH3 production

Figure 1. Direct PGP activities of test isolates.

0

10

20

30

40

50

60

70

80

90

Per

cent

isol

ates

Azotobacterspp.

Mesorhizobiumciceri

FluorescentPseudomonas

Bacillus spp.

Siderophore production

Antifungal activity

HCN production

Figure 2. Indirect PGP activities of test isolates.

Table 2. Production of indoleacetic acid by selected bacterial isolates grown on their respective medium�

Isolate designation IAA production (mg/ml7SD) at different tryptophan concentrations (mg/ml)

0 50 150 300 400 500

AZT1 ND 1.4770.25 3.5370.32 6.5770.31 9.3770.15 11.8370.21AZT3 ND 1.7770.21 5.0070.20 8.9070.30 11.2770.12 13.4770.45AZT13 ND 1.6070.30 3.7770.25 7.0770.15 9.7370.25 12.8070.20AZT9 ND 1.2770.31 3.5370.25 6.6370.40 8.5770.25 10.1770.25AZT20 ND 1.2770.25 3.4070.20 6.4070.20 8.3370.25 10.4770.15AZT23 ND 1.5070.20 3.6070.30 6.4770.31 8.9370.25 11.9070.20AZT26 ND 2.1370.15 4.6770.15 7.6770.15 10.4370.47 15.0070.26Ps5 ND 3.0070.20 7.4770.25 13.3770.35 16.8070.20 18.7070.30Ps7 ND 2.6070.20 5.9070.20 11.3070.36 15.1770.25 18.0770.25Ps9 ND 3.6070.20 6.1070.20 11.4770.15 14.9370.15 22.0270.20B1 ND ND ND 3.4070.20 5.0370.15 7.0370.15

ND–not detectable.�For Azotobacter Jensen’s, Pseudomonas King’s B, Bacillus nutrient media was used.

Screening of free-living rhizospheric bacteria 177

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PRES

S

Table 3. Antifungal activity of the test isolates on different media

Test isolates Media used Zone size (mm 8 SD)

Aspergillus sp. Fusarium solani Fusarium ciceri Fusarium oxysporum Rhizoctonia bataticola

AZT1 MH 22.6770.58 ND ND 15.6770.58 NDNA 18.3370.58 ND ND 14.3370.58 NDSDA 13.0071.41 ND ND 16.0071.00 ND

AZT3 MH 30.5070.50 23.5370.50 17.6770.58 30.8370.76 15.0071.00NA 25.6770.58 18.6771.15 15.3370.58 22.3370.58 NDSDA 19.0071.00 25.8370.76 21.3371.15 24.1770.29 ND

AZT9 MH 25.0071.00 ND ND 13.3371.15 NDNA 21.0071.00 ND ND ND NDSDA 16.6770.58 ND ND ND ND

AZT13 MH 31.5070.50 21.0071.00 16.6770.58 17.6770.58 16.5070.50NA 26.3370.58 16.00 14.5070.71 16.00 13.8370.76SDA 22.0072.00 17.00 21.0071.00 17.5070.50 ND

AZT20 MH 19.0071.00 ND ND 15.6770.58 NDNA 16.00 ND ND ND NDSDA ND ND ND ND ND

AZT23 MH 17.5070.50 16.3370.58 ND 18.6770.58 NDNA 14.00 ND ND 15.3370.58 NDSDA ND ND ND 19.0071.00 ND

Ps5 MH 15.5070.50 15.3370.58 15.3370.58 19.0071.00 NDNA 13.8370.7 12.0071.00 12.0071.00 14.8370.76 NDSDA ND ND ND 12.0071.00 ND

B1 MH 15.5070.50 16.5070.50 16.5070.50 20.1770.76 NDNA 12.8370.29 13.3370.58 13.3370.58 15.5070.50 NDSDA 11.5070.71 14.2570.35 14.2570.35 12.6770.58 ND

ND–not detected.

F.Ahm

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Screening of free-living rhizospheric bacteria 179

inhibition zones in diameter from 11.50 to35.00mm. Strains AZT3 and AZT13 induced largerinhibition zones compared to the other strains.AZT1 showed activity against Aspergillus sp. and F.oxysporum on all three media. AZT3 and AZT13showed good antifungal activity against all thefungi but no activity was detected on SDA and or NAagainst R. bataticola. AZT9 and AZT20 isolateshowed activity against Aspergillus sp. on all threemedia whereas antifungal activity against F. oxy-sporum was observed on MH medium. AZT23 waseffective against Aspergillus sp., F. oxysporum andF. solani but results varied with different media.PS5 and B1 could also exhibit broad-spectrumactivities against test fungi. MH medium was mostappropriate medium for screening of antifungalactivity and Aspergillus sp. was most susceptibleorganism (Table 3).

Discussion

Plant rhizosphere is known to be preferredecological niche for various types of soil micro-organisms due to rich nutrient availability. It hasbeen assumed that inoculation with diazotrophicbacteria like Rhizobium, Azotobacter and Azospir-illum enhanced the plant growth as a result of theirability to fix nitrogen. However, despite of exten-sive research efforts only rhizobia have been shownto increase yields from dinitrogen fixation. Growthpromotion may be attributed to other mechanismssuch as production of plant growth promotinghormones in the rhizosphere and other PGPactivities (Arshad and Frankenberger, 1993; Glick,1995). In the present investigation 47 isolates ofAzotobacter and 25 isolates belonging to M. ciceri,fluorescent Pseudomonas and Bacillus species werescreened in vitro for PGP activities. IAA productionwas detected in all the test isolates of fluorescentPseudomonas, 83.3%, of both Azotobacter and M.ciceri isolates. Our findings of IAA production inAzotobacter isolates are in agreement with otherworkers (Gonzalez-Lopez et al., 1986; Jagnow,1987; Nieto and Frankenberger, 1989).

Phosphate solubilization was most frequentlyencountered by Bacillus isolates (80%), followedby Azotobacter, Pseudomonas and least by Mesor-hizobium isolates. However, production of ammo-nia was a common trait in all selected group ofbacteria. Siderophore production was detectedamong some isolates of Azotobacter (12.77%),followed by Pseudomonas and Bacillus isolates.However, 50% and 80% isolates of Bacillus andPseudomonas were detected positive for HCN

production. While antifungal activity was shownby 12.77% of Azotobacter isolates, followed byPseudomonas (11.11%) and Bacillus isolates (10%).

Some of the above-tested isolates could exhibitmore than two or three PGP traits, which maypromote plant growth directly or indirectly orsynergistically. Similar to our findings of multiplePGP activities among PGPR have been reported bysome other workers while such findings on indigen-ous isolates of India are less commonly explored(Gupta et al., 1998). On the basis of preliminaryscreening, quantitative analysis of IAA productionwas made on seven Azotobacter isolates, threefluorescent Pseudomonas and one Bacillus isolate.There was an increase in the level of IAA with theincreasing concentration of tryptophan (50–500 mg/ml). Similar trend of IAA production with theincreasing concentration of tryptophan was alsoreported by Barazani and Friedman (2000). IsolatesAZT26, AZT3, AZT13 could produce relatively highconcentration of IAA compared to other Azotobac-ter isolates. Production of high levels of IAA byfluorescent Pseudomonas is a general characteristicof our test isolates. Similar high level of IAAproduction was recorded by other workers (Xie etal., 1996). The production of IAA was founddependant upon bacterial isolates and concentra-tion of tryptophan. Such findings may have directpractical application, although intrinsic ability ofbacteria to produce IAA in the rhizosphere dependson the availability of precursors and uptake ofmicrobial IAA by plant (Arshad and Frankenberger,1993).

Another important trait of PGPR, that mayindirectly influence the plant growth, is theproduction of siderophores. They bind to theavailable form of iron Fe3+ in the rhizosphere, thusmaking it unavailable to the phytopathogens andprotecting the plant health. In the present inves-tigation six isolates of Azotobacter and the fluor-escent Pseudomonas strain Ps5 showed multiplePGP activities including siderophore production andantifungal activities against one or more test fungi.

On the basis of data obtained it could be statedthat: (i) sensitivity of test fungi was in order ofAspergillus sp.4F. oxysporum4F. solani4Rhizoc-tonia bataticola; (ii) Muller–Hinton medium wasbest out of the three tested media to detect the invitro antifungal activity which is probably due tothe non-interfering composition of this mediumwith the assay system; and (iii) isolate AZT3 andAZT13 demonstrated broad spectrum antifungalactivity against the five tested fungi. The anti-fungal activity of the test isolates indicated a closerelationship between production of HCN and side-rophores. The antifungal activity of the test

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isolates might be due to the production of side-rophore and HCN or synergistic interaction of thesetwo or with other metabolites. However, role ofchitinase was not expected as these isolates werenegative for chitin hydrolysis. Several studies havedemonstrated that production of siderophores,other secondary metabolites and lytic enzymes byPseudomonas strains was most effective in control-ling the plant root pathogens including F. oxyspor-um and R. solani (O’Sullivan and O’Gara, 1992;Nagrajkumar et al., 2004). Further studies on theperformance of these isolates and their mutants onthe growth of plant will uncover the mechanismand potential of these PGPR exhibiting multiplePGP traits.

Acknowledgements

We thank the Chairman, Department of Agricul-tural Microbiology for providing necessary facilitiesfor this work, and Mr. Farrukh Aqil for technicalhelp.

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