Indian Journal of BiotechnologyVol 2, April 2003, pp 246-250

Detection of Azospirillum and PSB in Rice Rhizosphere Soil by Protein andAntibiotic Resistance Profile and Their Effect on Grain Yield of Rice

M R Khan, N C Talukdar* and 0 ThakuriaSoil Microbiology Laboratory, Department of Soil Science, Assam Agricultural University, Jorhat 785013, India

Received 23 April 2002; accepted 25 October 2002

Two biofertilizer agents, Azospirillum amazonense A10 and Bacillus megaterium P7, alone and in combinationincreased the grain yield of rice in autoclaved soil by 103-256% over control. Colonies of A, amazonense and B.megaterium were distinguished from the contaminant phosphate solubilizing bacteria (PSB) and Azospirilla coloniesin the respective media based on colony morphology, colour and thickness of clearing zone, Population densities of A,amazonense and B. megaterium were found to be 2.6 x 108 and 2.9 x 108 CFUg .] soil, respectively at 48 day posttransplanting. Protein and antibiotic resistance profiles of the inoculated and reisolated A. amazonense, B.megaterium and the contaminant colonies with the pure cultures indicated that the isolates were able to establish inthe rice rhizosphere with the resultant enhancement of plant growth and yield.

Keywords: rice, Azospirillum, PSB, protein profile, antibiotic resistance profile

IntroductionAzospirilla and phosphate solubilizing bacteria

(PSB) are two known groups of soil bacteria, whichimprove Nand P nutrition of crops. Local isolates ofthese microorganisms have been exploited inbiofertilizer programmes (Okon et al, 1988; Talukdaret al; 2001). In a successful biofertilizer programme,it is often necessary to ascertain that the introducedbiofertilizer agent establishes and persists in the rootrhizosphere of the crop beyond the time ofapplication. Conventionally used morphological andbiochemical properties of bacteria alone are not usefulto confirm identity of the introduced agents after theirreisolation, because native population of Azospirillaand PSB also inhabit the crop rhizosphere (Rosswell,1973). Tagging the introduced bacteria with a reportergene like gfp, gus and Tn5-iacZ is useful inmonitoring introduced bacteria (Saha et al, 2001;Tchebotar et al, 1998; Pal et al, 2000). Among theDNA based techniques, DNA restriction patternanalysis (Giovannetti et al, 1992), AFLP (Avrova etal, 2002) and RAPD (Fancelli et al, 1998; Gupta et al,2001) are more useful in establishing the identity ofthe introduced bacteria from the background bacterialpopulation. These methods require sophisticatedlaboratory facilities. However, methods based on

*Author for correspondence:Tel & Fax: (91) 0376-23340044E-mail: nct@aau.ac.in

protein profile and antibiotic resistance pattern arealso useful in distinguishing closely related bacteria(Kersters & De Ley, 1975; Biavati et al, 1982; Ni &Boone, 1991; Kluepfel, 1993; Mowade &Bhattacharya, 2000) and can be used with minimumlaboratory facilities. These two methods were usedalong with the conventional methods to determinewhether two promising biofertilizer agents, A.amazonense and B. megaterium establish and survivein rice crop rhizosphere after their inoculation.Growth parameters and grain yield of rice due toinoculation with the biofertilizer agents in differenttreatment combinations were also determined.

Materials and MethodsEffect of A. amazonense Ai 0 and B. megaterium P7on Rice Crop and Their Detection in RiceRhizosphere

In order to study the establishment of A.amazonense A10 and B. megaterium P7 in the ricerhizosphere and their effect on rice, a pot experimentwas conducted during the year 2000 in sali season. Toreduce the load of indigenous soil microflora, the soil(sandy loam texture, pH-4.5, %OC-0.61, total N-2399kg/ha, av P20s-24.3 kg/ha and av K20-119 kg/ha) wasautoclaved before growing rice. Concrete pots werefilled with 6 kg sterile soil and puddled by addingsterile distilled water. The bacterial isolates wereobtained from the collection of agriculturallyimportant microbial isolates maintained in soil

KHAN et al: DETECTION OF AZOSPIRILLUM AND PSB IN RICE RHIZOSPHERE SOIL

microbiology laboratory, Assam AgriculturalUniversity. To A. amazonense inoculated pot, 0.014 gurea per kg soil @ half the recommended dose (20kg/ha) was added. Similarly, to B. megateriuminoculated soil, rock phosphate (RP) was applied as'P' source. To eliminate microbial contaminants,twice sterilized compost was added @ 4.5 g/kg soil toeach pot (recommended practice for rice in Assam).Other nutrient sources (SSP and MOP) were added asper the following treatment combinations: 1. Compost+ N + P as SSP + K (Control); 2. Compost +A. amazonense + V2 N + P as SSP + K; 3. Compost +B. megaterium + N + P as RP + K, and 4. Compost +A. amazonense and B. megaterium + V2 N + P as RP +K. Each treatment was replicated thrice. Twentyfive-day-old seedlings of rice (var. Luit) were uprootedand the roots were thoroughly washed with tap waterfollowed by sterile distilled water.

Mass Multiplication, Inoculation and Enumerationof A. amazonense AIO and B. megaterium P7

A. amazonense did not grow well in liquid Nfbmedium and its growth as pellicle in semi-solid Nfbdid not serve as a homogeneous inoculum forapplication to rice seedlings. Therefore,A. amazonense was grown in liquid Nfb mediumsupplemented with (NH4hS04 (Subba Rao, 1999) andB. megaterium grown in Pikovskaya's broth for 72 hrsin an environmental shaker (Rotek-LES) at 182 rpmand 30°C. Inoculation was carried out by dipping theroots of 25-day-old seedlings in different liquid brothmixtures depending upon treatment combinations for2 hrs. Two seedlings were planted in respectivetreatment pots. Sterile water was used to water theplants (water was maintained at 2 em above the soilsurface). Populations of A. amazonense and B.megaterium in the inoculated roots at the time oftransplanting were determined by serial dilution pourplate technique (Halvorson & Zeigler, 1933) on rojocongo (CR) and Pikovskaya's agar plates,respectively. Similarly, Azospirilla and PSBpopulations were also determined in the ricerhizospheric soil of respective treatments after 48days of inoculation. Morphology of colonies of A.amazonense and B. megaterium was recorded andcompared with the laboratory maintained purecultures. The colonies of A. amazonense and B.megaterium on the respective media resembling thoseof their pure cultures were stored at 4°C forverification of their identity using protein andantibiotic resistance profile. The representatives of

247

other colonies of the Azospirilla and PSB, whichappeared as contaminants, were also stored forcomparison.

Analyses of Protein and Antibiotic ResistanceProfiles for Verification of Identity of A.amazonense Al 0 and B. megaterium P7

Soluble proteins were extracted from the purecultures of A. amazanense, B. megaterium and theirrepresentative colonies after reisolation from ricerhizosphere. Soluble proteins of contaminantAzospirilla and PSB colonies were also extracted. Thesoluble proteins were electrophoresed in 12%separating gel following the technique described byMoore et at (1980). Similarly, antibiotic resistanceprofiles were determined following paper disc method(Cremer, 1984). Six antibiotics, viz. Ampicillin,Chloramphenicol, Gentamycin, Kanamycin,Rifampicin and Streptomycin were used to test theresistance level of the pure and reisolated cultures ofA. amazonense and B. megaterium as well as thecontaminants. Graded concentrations (5, 10, 15, 20,3~ 50, 100, 15~ 200, 250, 30~ 350, 400, 450, 500ppm) of antibiotic solutions were prepared in theirrespective solvents and filter sterilized by using 0.22urn nitrocellulose filters. Resistance level of eachisolate was determined thrice at each concentrationsof the antibiotics used.

Statistical AnalysisData were analyzed by separate one way analysis

of variance. When F values were significant, the leastsignificant difference (LSD) test was used to separatemeans (Gomez & Gomez, 1976). The treatmentdifferences for all parameters are reported at P= 0.05.

Results and DiscussionPlant Growth and Grain Yield due to DifferentTreatment Combinations

Inoculation of rice with A. amazonense and B.megaterium in sterilized soil increased the grain yield;it was highest in pots inoculated with B. megaterium(Table 1). In dual inoculated pots, where N fertilizerwas reduced to half, yield increase was lesser thanthat in pots inoculated with B. megaterium along withcompost and full dose of Nand P fertilizer: This resultindicated that the amount of N fixed byA. amazonense was not enough to produce effectsimilar to that of full dose of N fertilizer applied inB. megaterium inoculated pot. Nevertheless, theA. amazonense influenced growth and yield of rice

24~ INDIAN J BIOTECHNOL, APRIL 2003

positively, In B. megaterium inoculated pots, plantstook less time to flower than those in dual inoculatedpots (Table 1). Improvement of P nutrition byB. megaterium resulted in hastening of flowering andincreased grain yield. The P content of theexperimental soil was low and the PSB had morepronounced effect on the crop. Grain quality was alsofound to be better in B. megaterium inoculated pots asreflected in their 1000 grain weight (Table 1). Such aneffect of PSB and Azospirilla on the growth of ricewas also observed in earlier studies (Subba Rao et al,1979; Mohmd et al, 1991; Talukdar et al, 2001).Grain yield in the autoclaved soil under recommendedlevel of nutrient was strikingly low (Control pot).Autoclaving reduced or completely eliminated soilmicrobial flora and also brought about changes inphysio-chemical properties of soil (Rovira & Bowen,1966) and this could be a reason for low grain yield.Inoculation with the beneficial bacteria might haveovercome the adverse effect of autoclaving. However,

contaminant Azospirilla and PSB population was alsodetected in the rice rhizosphere of control pot. Thesecontaminants originated either from rice seedlings orfrom air. If the contaminants established from airtowards later part of plant growth, they might nothave any role on elimination of adverse effect ofautoclaved soil. It is possible that in the absence ofinoculated bacteria in the rhizosphere, the rice plantsin the control pot took longer time to establish andcomplete its life cycle. It was evident from theduration of time to flowering (Table 1). However,future research is required to throw more light on thisobservation.

Identification of Introduced A. amazonense AIO andB. megaterium P7 Among the Bacterial IsolatesCollected from Rice Rhizosphere

Colony morphology of the introduced bacterialisolates was compared with that of their putatives andit was observed that the bacterial colonies on the

Table I-Effect of inoculation of A. amazonense AlO and B. megaterium P7 in combination with fertilizers and compost on sali ricegrown in autoclaved soil

Treatment

Compost + N + P as SSP + K (Control)A. amazonense + Compost + Y2N + P as SSP + KB. megaterium + Compost + N + Pas RP + KA. amazonense and B. megaterium+Compost+Y2 N + P as RP + KLSDo.05

Days to flowering Total grain wt (g/pot) 1000 grain wt (g )

78 10.1 19.868 20.5 20.852 35.9 24.858 29.4 23.5

4.71 2.29 0.46

Table 2--Population dynamics of the introduced A. amazonense AlO and B . megaterium P7 in the rice rhizosphere

Treatment A. amazonense Azospirilla at 48 DAT (Log CFU/g soil) B. megaterium PSB at 48 DAT (Log CFU/g soil)at transplant Total A. Contaminant at transplant Total PSB B. Contaminant(Log CFUI Azospirilla amazonense Azospirilla (Log CFUI megaterium PSBseedling) seedling)

Compost + N + P as ND 8.42 ND 8.42 ND 8.85 ND 8.85SSP + K (Control)

A. amazonense + 9.57 8.50 8.41 7.40 ND 7.23 ND 7.23Compost + Y2N + P asSSP+KB. megaterium + ND 8.15 ND 8.15 8.08 8.48 8.46 7.14Compost + N + P asrock phosphate + K

A. amazonense and 7.08 7.63 7.61 6.30 6.85 7.02 6.08 6.96B. megaterium +Compost + Y2N + P asrock phosphate + K

t (0.05)= 5.75 3.97 16.39 17.18

LSD (005) = 0.52 0.29 0.22 0.19

* DAT = Days after transplanting; *.cFU= Colony forming unit

KHAN et al: DETECTION OF AZOSPIRILLUM AND PSB IN RICE RHIZOSPHERE SOIL 249

Table 3------lntrinsicantibiotic resistance profiles (IARP) of the introduced and reisolated cultures of A. amazonense A I0, 8. megateriumP7 and the contaminant colonies

Minimum Inhibitory Concentration (ppm)Antibiotic Introduced PSB reisolated Introduced Azospirilla reisolated

8. megaterium P7 PCl* PC2 A. amazonense AlO ACI** AC2

Ampicillin 400 400 15 20 200 200 5 150Chloramphenicol 400 400 100 50 250 250 50 300Gentamycin 400 400 5 5 LOO 100 50 40Kanamycin 400 400 5 5 300 300 50 350Rifampicin 200 200 5 200 L50 150 5 40Streptomycin 400 400 L5 L5 300 300 50 350

*PC, Contaminant phosphate solubilizing bacterial isolates**AC, Contaminant Azospirilla isolates

serial dilution plates were a mixture of A. amazonenseand B. megaterium and contaminants (Table 2). At48th day after inoculation, a large population of theintroduced bacteria survived in the rice rhizosphere.The population of A. amazonense and B. megateriumwas smaller in dual inoculated pots as compared tothe single inoculated pots. However, the size of theirinitial inocula was also smaller in dual inoculatedpots.

The representative protein profiles of A.amazonense and B. megaterium are presented inFig. 1. Among the various bacterial isolates obtainedfrom the rice rhizospheric soil of A. amazonenseinoculated pots, protein bands of three coloniesresembling A. amazonense were exactly similar to one

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Fig. I-The representative protein profiles in 12% separating gel:lane L - Marker (BSA); PSB isolates: lane 2 - PC 1; lane 3 - P7(Reisolated); lane 4 - PC2; lane 5 - 8. megaterium P7 (Pureculture); Azaspirillum isolates: lane 6 -ACl; lane 7 - AlO(Reisolated); lane 8 - AC2; lane 9 - A. amazonense AIO (Pureculture) and lane 10 - A. brasilence CDJA (reference strain)PC 1 and PC 2= PSB ContaminantsACI and AC 2= Azospirilla contaminants.

another and with that of A. amazonense maintained inthe laboratory. Similarly, three colonies obtained fromB. megaterium inoculated pots, produced proteinbands, which were similar to B. megateriummaintained in the laboratory. The number of coloniesof A. amazonense and B. megaterium were higherthan the colonies of contaminant Azospirilla and PSB,which appeared on the serial dilution plates of therhizosphere soil samples (Table 2). Thus, it was foundthat the protein profiles of the inoculated isolates weresimilar with that of the reisolated cultures. However,there may be minor change in protein profile resultingfrom spontaneous, chemically induced, or transposonmediated mutation. Mot and Vanderhyden (1989)detected minor change in the protein profiles of theinoculated strains of Arospirillum brasilence in thewheat rhizosphere. This similarity between inoculatedand reisolated cultures of A. amazonense A10 and B.megaterium P7 was also reflected in their intrinsicantibiotic resistance profiles (IARP) against sixcommon antibiotics tested (Table 3). Unlike proteinprofile, IARP has been found to be very stable overtime in earlier studies (Moawad & Bohlool, 1992).Thus, it can be concluded that A. amazonense and B.megaterium established successfully in the ricerhizosphere and produced their growth promotingeffect on rice. The usefulness of this technique for theidentification of bacteria based on protein andantibiotic resistance profile should be used todetermine their persistence in field soil for severalyears following their inoculation.

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