Microbiol. Res. (1998) 153, 11 3-117

© Gustav Fischer Verlag

Effect of plant growth promoting rhizobacteria on competitiveability of introduced Bradyrhizobium sp. (Vigna) for nodulation

Alka Gupta*, A. K. Saxena, Murali Gopal*, K. V. B. R. Tilak

Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India* Present address: Central Plantation Crops Research Institute, Regional Station, Kayangulam, Krishnapuram - 690533,Kerala, India

Accepted : April 7, 1998

Abstract

Four plant growth promoting rhizobacterial (PGPR) isolatesbelonging to genera Bacillus and Enterobacter and two Bra­dyrh izobium spp. (Vigna) strains Cog 15 and S 24 were select­ed for competition studies. PGPR isolates did not antagonizeBradyrh izobium strains Cog 15 and S 24 on plates. In unsterilesoil, co-inoculations of PGPR and Bradyrhizobium strainsfailed to show any conclusive influence on nodulation andARA at 50 days of plant growth. PGPR isolates had a directeffect on shoot biomass development, N content and grainyield when tested with Bradyrhizobium strain S 24, after 50and 80 days of plant growth.

Single inoculations of PGPR isolates significantly increasedgrain yield over uninoculated control. The influence of PGPRisolates on the ability of Bradyrhizobium strains to competewith indigenous population of bradyrhizobia was studied interms of nodule occupancy. Bradyrhizobium strains weregenetically marked by studying their intrinsic Antibiotic Re­sistance pattern. The nodule occupancy of Bradyrhizobiumstrain S 24 increased from 60% (treatment with single inocu­lation of S 24) to 81% in presence of an Enterobacter isolateEG-ER-2. Another Enterobacter isolate KG-ER-I increasednodule occupancy of bradyrhizobial strain Cog 15 from 77%(treatment with single inoculation of Cog 15) to 88%.

Key words: Plant Growth Promoting Rhizobacteria (PGPR)­greengram - competitive ability - Bradyrh izobium sp. (Vigna)- nodule occupancy.

Introduction

Legume inoculation in soils containing indigenous po­pulations frequently results in only small proportion ofnodules produced by introduced strains (McLoughlin

Corresponding author: Alka Gupta

et 01., 1985). The potential value of superior introducedstrain is often decreased by the competing ineffectivenative rhizobial population, and other rhizospheric bac­teria. The process of nodulation involves signal ex­change between the plant and bacterium. The bacterialgenes involved in nodulation are activated by phenoliccompounds exuded by the plant (Firmin et al ., 1986; Pe­ters et al., 1986; Redmond et al., 1986). Rhizobacterialisolates of chickpea have been shown to produce fla­vonoid like substances and in tum induced nod gene ex­pression (Singh and Gaur, 1995). There are reports ofvarious bacterial and fungal species promoting plantgrowth (Grimes and Mount, 1984; Hicks and Loyna­chan, 1989) and nodulation by rhizobia (Azcon-Aguilarand Barea, 1981).

Wehave isolated plant growth promotingrhizobacte­ria (PGPR) from the rhizotic zones of greengram. In thepresent study, the primary objective was to determinewhether PGPR isolates could help Bradyrhizobium sp.(Yigna) to overcome the stress of interstrain competitionand significantly affect the ability of introduced strainsof Bradyrhizobium to dominate nodulation in a multistrain environment.

Material and methods

Organisms and media. Bradyrhizobium sp. (Vigna)strains Cog 15and S 24 were obtained from Division ofMicrobiology, Indian Agricultural Research Instituteand were maintained on Yeast extract mannitol agar(YEMA) medium. Intrinsic resistance levels to variousantibiotics were determined for both strains followingthe procedure of Josey et 01., (1979) and used for its

Microbial. Res. 153 ( 1998) 2 11 3

Table 1. Intrinsic Antibiotic Resistance (IAR) pattern of Bra­dyrhizobium sp. (Vigna) strains

Strain Tolerance to antibiotics (cone. mg/ml)

Kan Str Amp Cam Tet Nal Gen Tmp

identification. The intrinsic antibiotic resistance patternof both the strains is given in Table 1. Four PGPR iso­lates EG-ER-2, KG-ER-l, EG-RS-3 and EG-RS-4 se­lected on the basis of earlier studies (Gupta, 1995) weremaintained on nutrient agar (NA) medium.

The rhizobacterial isolates were subjected to standardbacteriological tests and classified to genera Enterobac­ter (EG-ER-2 and KG-ER-l) and Bacillus (EG-RS-3and EG-RS-4) (unpublished). These isolates did notaffect the growth of both bradyrhizobial strains in vitro.

Effect ofPGPR isolateson plant growth, yield and com­petitive ability ofbradyrhizobialstrains. A green houseexperiment was conducted to evaluate the influence ofPGPR isolates on growth and yield of greengram andnodulation competitiveness of test strains S 24 and Cog15. Four greengram cv. Pusa 105 seeds were sown inearthern pots filled with 12 kg farm soil (unsterilized;sandy loam, pH 7.2) after imbibing in water for 4 h. Theplants were later thinned to two per pot. Bradyrhizobiumsp. (Vigna) strains Cog 15 and S 24 were inoculatedalone (l08 cells/ml) and in combination with PGPRisolates in a ratio of I : 1. One ml of each strain/isolate(l08 cells/ml) was added over the sown seeds andcovered with soil. All treatments were replicated tentimes and arranged in a completely randomized blockdesign. Uninoculated controls were maintained and tapwater was added to plants as needed.

The plants were harvested from five replications after50 days of seedling emergence. The root portion withintact nodules was excised. Excess soil from roots wasremoved by gentle shaking and root-nodule system wasplaced in glass tubes stoppered with seals and 2 ml of theair phase was replaced with equal volume of acetylenegas. The tubes were incubated at 28 DC for 30 min andthe gas phase was analyzed for acetylene reductionusing a Gas Chromatograph (Shimadzu GC 14 A)(Hardy et al., 1968), fitted with flame ionization detec­tor (FID) and Porapak N 80 column. The injector temp­erature was maintained at 110DC; detector temperature110 DC; column temperature 80 DC; flow rate of carrier

gas (N2) at 35 ml min-I; flow rate of fuel gas (H2) at25 ml/min, From each tube, 1 ml gas sample was in­jected to determine the amount of ethylene produced.The extent of acetylene reduced was determined byusing standard ethylene (lOS vpm) obtained from EDTresearch, London, UK. The nitrogenase activity was ex­pressed as n moles of C2H4 formed per hour per plant.

After determination of nitrogenase activity, numberof nodules on tap and lateral roots was recorded sepa­rately. The shoot portions were oven-dried at 80 DC for48 h and the dry weight of shoot was determined. Nodu­les were carefully removed from the root portion andsurface-sterilized by immersing in 70% alcohol for30 sec followed by 0.1% mercuric chloride solution for3 min and washed repeatedly six times with sterile dis­tilled water. A total of sixty nodules per treatment, se­lected randomly, were aseptically crushed and streakedin duplicate on YEMA containing congo-red dye andtryptone yeast-extract (TY) plates supplemented withappropriate concentration of marker antibiotics. To pre­vent fungal contamination, 100 flg/IOO ml of cyclo­heximide was also incorporated into the TY medium.The plates were incubated at 28 ± 1DC and scored forgrowth by comparing the control plates devoid of anti­biotics. The competitive ability of a strain was deter­mined by calculating the per cent nodule occupancy asfollows:

Number of nodules occu­pied by inoculant strain

% Nodule occupancy = x 100Total number of nodules

tested

An arc sine transformation of the data (Gomez andGomez, 1984) was used for statistical analysis and dif­ferences were assessed for significance by analysis ofvanance.

Dried shoot samples were powdered with pestle andmortar and known quantity was taken for Kjeldahl di­gestion. The samples were analyzed on Tecator KjeltecAuto 1030 Analyzer. The plants from remaining five re­plicates were harvested at the grain ripe stage (80 DAS).The yield of straw (roots and shoots) and grains wererecorded and the samples were processed for per centnitrogen estimation as described earlier.

Statistical analysis: The data were statistically ana­lysed using the one way analysis of variance (ANOVA)and differences were assessed for significance (Gomezand Gomez, 1984).

Results

The four PGPR isolates EG-ER-2, KG-ER-I, EG-RS-3and EG-RS-4, isolated from field-grown greengramshowed neutralistic response towards Bradyrhizo-

20100

5100

5050

20 ­100 5

Str =StreptomycinCam = ChloramphenicolNal = Nalidixic acidTmp =Trimethoprim

100100

20 75100 5

Cog 15S 24

Kan = KanamycinAmp = AmpicillinTet = TetracyclineGen =Gentamycin

114 Microbiol. Res. 153 (1998) 2

Table 2. Influence of the interaction of PGPR and Bradyrhizobium sp. (Vigna) strains S 24 and Cog 15 on various plant para-meters (50 DAS; unsterile pot culture experiment)DAS =days after sowing

Treatment plant top weight N Tap root Lateral root ARAt(mg) % nodulation* nodulation**

Control (Uninoculated) 300 1.95 0.2 0.4 18

BradyrhizobiumCog 15 496 2.51 1.0 0.8 156.3S 24 340 2.38 0.8 0.4 165

PGPREG-ER-2 429 2.3 0.0 0.0 4KG-ER-l 315 2.46 0.0 0.0 11EG-RS-3 343 2.2 0.0 0.4 3EG-RS-4 390 2.22 0.0 0.4 14

Bradyrhizobium+ PGPREG-ER-2 + Cog 15 260 2.05 0.8 0.2 35EG-ER-2 + S 24 540 2.62 0.4 0.2 134KG-ER-I + Cog 15 452 3.2 1.0 0.6 351KG-ER-I + S 24 542 2.54 0.8 0.4 127EG-RS-3 + Cog 15 500 2.7 1.4 0.8 133EG-RS-3 + S 24 350 2.71 1.0 0.4 174EG-RS-4 + Cog 15 431 2.8 1.2 1.0 54EG-RS-4 + S 24 589 2.73 0.4 0.6 28LSD (p =0.05) 21.4 0.49 11.4LSD (p =0.01) 28.9 0.66 15.4

Results are an average of ten plants* no nodule, 0; 1-5 nodules, 1; 6-10 nodules, 2 ** no nodule, 0; medium nodulation, 1; good nodulation, 2t n mol CZH 4 formed h' plane]

Table 3. Microbial effects on yield and N content of plant root, shoot and yield (80 DAS; unsterile pot experiment)

Treatment Dry wt. (mg) Grain yield N%

Shoot Root(mg/pot)

Shoot Root Grain

Control (Uninoculated) 420 90 80 2.02 0.906 3.6

BradyrhizobiumCog 15 1190 150 210 2.03 1.4 4.0S 24 285 50 130 2.06 1.8 4.3

PGPREG-ER-2 480 100 280 1.96 1.37 3.98KG-ER-l 190 50 180 1.26 0.98 4.02EG-RS-3 470 70 250 1.79 0.94 4.45EG-RS-4 580 160 257 1.75 1.44 4.33

Bradyrhizobium+ PGPREG-ER-2 + Cog 15 510 90 110 1.6 0.84 4.29EG-ER-2 + S 24 660 90 250 2.07 U6 4.21KG-ER-l + Cog 15 150 30 170 1.93 1.21 4.18KG-ER-l + S 24 780 50 330 2.1 U6 4.29EG-RS-3 + Cog 15 1010 170 330 2.17 1.1I 4.35EG-RS-3 + S 24 680 80 170 1.81 1.14 4.17EG-RS-4 + Cog 15 530 150 80 2.33 1.01 3.83EG-RS-4 + S 24 420 100 300 1.54 U4 4.52LSD (p =0.05) 275 8.5 I I 0.16 0.62 0.12LSD (p =0.01) 371 11.5 14.9 0.22 0.84 0.16

Results are an average of ten plants.

Microbiol. Res. 153 (1998) 2 115

Table 4. Nodule occupying ability of Bradyrhizobium sp.(Vigna) strains Cog 15andS 24as influenced bycoinoculationwithPGPRisolates (50 DAS; unsterile pot experiment)

a =Cog 15takenas control for statistical analysis.b = S 24 takenas control for statistical analysis.* =significant at 5% level; ** =significant at 1% level.

Uninoculatedcontrol 70

Cog 15 150 115 76.60

KG-ER-l + Cog l5a 150 132 88.40*EG-ER-2 + Cog IS" 150 115 76.60EG-RS-3 + Cog 15a 150 119 79.00EG-RS-4 + Cog IS" 150 119 79.00

LSD (p = 0.05) 10.18

S 24 150 90 60.29

KG-ER-1 + S 24b 150 97 64.60EG-ER-2 + S 24b 150 121 80.80**EG-RS-3 + S 24b 150 98 65.40EG-RS-4 + S 24b 150 99 66.10

LSD (p = 0.01) 13.36

bium sp. (Vigna) strains Cog 15 and S 24 (Gupta,1995).

PGPR isolates EG-ER-2, KG-ER-l, EG-RS-3 andEG-RS-4, When added to non-sterile soil in combina­tion with Bradyrhizobium sp. (Vigna) strains, Cog 15and S 24, showed a differential response in growth,nodulation and N-content of greengram at 50 days ofplant growth. All four isolates significantly increasedshoot dry weight and per cent N when tested with Bra­dyrhizobium strain S 24 where as only isolate EG-RS-3could significantly enhance shoot dry weight and percent N when coinoculated with Bradyrhizobium strainCog 15 (Table 2).

Bacillus isolate (EG-RS-3) enhanced nodulation incombination with Bradyrhizobium strains Cog 15 andS 24 with a corresponding increase in per cent shoot N.Isolates KG-ER-I and EG-RS-4 did not show signifi­cant increase in nodulation over single inoculation ofboth bradyrhizobial strains. However, isolate EG-ER-2reduced nodulation by both bradyrhizobial strains Cog15 and S 24. Greengram plants inoculated with Entero­bacter isolate (KG-ER-l) and Bradyrhizobium strainCog IS exhibited significantly enhanced ARA activity(125% increase over single inoculation of Cog 15)which was reflected in terms of increase in per centshoot N (28% increase). A Bacillus isolate (EG-RS-4)

Discussion

drastically reduced nitrogen fixation by strains Cog 15and S 24 although nodulation was at par with single in­oculation of Cog 15 and S 24. In contrast to this, KG­ER-l isolate (Enterobacter sp.) significantly increasednitrogen-fixation by bradyrhizobial strain Cog 15 without any increase in nodulation over single inoculation ofCog 15 (Table 2).

After 80 days of plant growth, the PGPR isolates ex­cept EG-RS-4 significantly increased total biomass andgrain yield when coinoculated with Bradyrhizobiumstrain S 24. In comparison to this, only one isolate EG­RS-3 could significantly increase grain yield of green­gram when inoculated with Bradyrhizobium strain Cog15. However, all four isolates increased grain yield overuninoculated control, when inoculated alone (Table 3).Two Bacillus isolates EG-RS-3 & EG-RS-4 and oneEnterobacter isolate EG-ER-2, when inoculated alone,showed significantly higher grain yield as compared touninoculated control and inoculation of bradyrhizobialstrains Cog 15 and S 24 alone. EG-RS-3 increased grainyield by 19% and 92%; EG-RS-4 increased grain yieldby 31% and 112%; whereas isolate EG-ER-2 increasedgrain yield by 33% and 115%, as compared to singleinoculation of bradyrhizobial strains Cog 15 and S 24,respectively.

Nodule Occupancy of introduced bradyrhizobial strains

Nodule occupancy was determined as the number ofnodules occupied by the test strain as a percentage of to­tal nodules. Nodules formed on uninoculated controlswhich is indicative of indigenous population of Brady­rhizobium sp. (Vigna). On coinoculation, an Entero­bacter isolate EG-ER-2 enhanced nodule occupancy ofBradyrhizobium strain S 24 from 60% (treatment withsingle inoculation of strain S 24) to 81% (21% increase) ;likewise another Enterobacter isolate KG-ER-l increas­ed nodule occupancy of Bradyrhizobium strain Cog 15from 77% (treatment with single inoculation of Cog 15)to 88% (11% increase). Other combinations of PGPRand Bradyrhizobium except EG-ER-2 + Cog 15 mar­ginally increased nodule occupancy (Table 4).

The results obtained in the present study clearly indicatethat all four PGPR isolates influenced the shoot biomassdevelopment and N-content when coinoculated withBradyrhizobium strain S 24, which perhaps had bettersurvival and colonization in rhizosphere than strain Cog15. Also, PGPR isolates produced indole-3-acetic acid(unpublished) which might have lead to increased plantsize and nutrient uptake and accounted for increasedshoot N.

No.of nodules Nodulefrom which occu­Bradyrhizobium pancystrainwas (%)recovered

No. ofnodulestested

Inoculant strain(isolate no.)

116 Microbial. Res. 153 (1998) 2

In our study, a Bacillus isolate enhanced nodulationand per cent shoot N. Halverson and Handelsman (1991)also found increased nodulation in soybeans by coinocu­lation of Bacillus cereus with Rhizobium japonicumunder green house as well as field conditions. In contrastto this, another Bacillus isolate drastically reduced nitro­gen-fixation though nodulation was at par with single in­oculations. The reason could be formation of ineffectivenodules that failed to fix nitrogen. Similarly, one Entero­bacter isolate showed increased nitrogen-fixation, ARAand per cent shoot N by Bradyrhizobium strain Cog 15,though nodulation was at par with single inoculation.Whereas another Enterobacter isolate showed reducednodulation by bradyrhizobial strains. This might haveled to reduced nitrogen-fixation by both the strains Cog15 and S 24.

Results from this study did illustrate differences ininteractions among Bradyrhizobium sp. (Vigna) strainsCog 15 and S 24 and the PGPR isolates. PGPR isolateswere able to increase grain yield of greengram when co­inoculated with Bradyrhizobium strain S 24 rather thanCog 15. This again confirm our results obtained withBradyrhizobium strain S 24 after 50 days of plantgrowth.

The Enterobacter isolates enhanced nodule occu­pancy of either of the bradyrhizobial strains Cog 15 andS 24. Both isolates produced antibiotics and sidero­phores (unpublished) which might have inhibited otherrhizospheric rhizobia enabling the inoculant bradyrhizo­bial strains to occupy successfully the nodulation sites.This is in agreement with the findings of Fuhrmann andWollum (1989) who reported that coinoculation of a si­derophore-producing pseudomonad with mixtures ofthe competing bradyrhizobia typically enhanced nodu­lation by Bradyrhizobium japonicum strain USDA 110.Rhizobacterial isolates have been reported to produceflavonoid like substances which are the key signals forinduction of rhizobial nod genes (Singh and Gaur,1995). Combined inoculations with Bradyrhizobiumand Enterobacter may be able to enhance nodulation insoils with bradyrhizobial strains that have proved un­responsive to the introduction of Bradyrhizobium in thepast.

Our results suggest that PGPR may affect different­ially, the in situ growth of Bradyrhizobium sp. (Vigna)strains, and additionally raise the possibility of alteringnodulation competition among strains through manipu­lation of the associated root microflora.

Acknowledgement

The first author is grateful to Director, IARI and the Dean, PGSchool, IARI, New Delhi for providing financial assistance tocarry out this study.

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