Allelopathic effect of .lcaciaflnilotica on growth, nodulation and nitrogen fixation by Rhizobium (cowpea) spp. was studied. In pots with tree rhizospherc soils,.
0971 4693t94 Allelopathy Journal | (l) : 47-52 (1994)
A International Allelopathy Foundation1994
Allelopathiceffectof Acacia niloticaon nodulationand nitrogen fixation by Rhizohium(cawpea) J. S. DUHAN, P. K, SHARMA AND K. LAKSHMINARAYANA* Department of Microbiology CCS Haryana Agricultural University, Hisar-I25 004, India
I
(Receivedin,revisedform : July 21,1993)
ABSTRACT Allelopathic effectof .lcaciaflniloticaon growth, nodulation and nitrogen fixation by Rhizobium (cowpea) spp. was studied. In pots with tree rhizospherc soils, nodulation of clusterbean(Cyamopsistetragonoloba)by three rhizobial isolateswas l0tol5l lowerthanincontrol. Inchillumjar experinrents, l0 and2OYolllelopathic) soil extracts (ASE) inhibited nodulation of clusterbeanby 4l and54olo, respectively, while with l0 and 201(combined) plant extracts (CPE) inhibition of nodulation was 46 and 65olo'respectively. Inhibition of in plant nitrogenase activity varicd from 12 to t9l with both types of extracts. The use of chillum jars can be a convenient techniqueto test allelopathicnature of plants in a Rhizobium-hostsymbiosis. Key words :
Acacia nilotica, allelopathy,N2 fixation, nodulation, Rhizobium(cowpea) spp'
INTRODUCTION Acacia nilotica L. is rvidely planted tree in Haryana, India and is prominently seenon road-sides. It is commonly observed that large area around the tree remains barren, free from any plant growth. rn areas adjoining the trees, growth of important summer and winter crops such as clusterbean,pearlmiliet, cotton, mustard and chickpea is jnhibited upto 7-8 m from the stem of the tree but the reasons are not known. Allelopathic inhibition of seedgermination and plant growth is known to occur near many plants (8, l0) and is shown to be due to the harmful effectsof phytotoxins releasedfrom the roots, leavesand other plant parts. Microorganisms also play an important role in the liberation of phytotoxic compounds from plant residues during decomposition (13, l4). Allelopnthic effect has becn shown to extend from producer plants to variotis agriculturally important bacteria such as rhizobia. Murthy and Ravindra (7) demonstrated that root and shoot extracts of Aristida adscensionishad an inhibitory effect on the growth of Rhizobium,and its nodulation of Indigofera corddolia. Root and leaf extracts of Parthenium 'hysterophorus was similarly sho,vn to inhibit the growth of Rhizobium phaseoli (4). The objective of tCorrespondenceauthor.
48
Duhan, Sharma and Lakshninarayana
the present inv:stigrtiun was to study the allelopathic effect of A. nilotica on nodulation in clusterbean(Cyamopsis tetragonolobaL.) and in vivo nitrogen fixation in Rhizobium (cowpea). MATERIALS AND METHODS Rhizobium(cowpea)strains IC-3100, G-3, PS-l and G-23 were isolated from the nodules of field grown clusterbean plants (6) and maintained on yeast extract mannitol agar (YEMA) slants (12). Clusterbean seeds(Cyamopsis tetragono'HG-75' were from the Ioba) variety Department of plant Breeding, Haryana Agricultural University, Hisar. For preparing allelopathic soil extract (ASE), 500 g of soit, taken from the rhizosphere of at least six A. nilotica trees, was mixed in 1.0 I tap water, sterilized at l5lbs/sq inch pressurefor I h, cooled and filtered. Non-allelopathic soil extract (SE) (control) was similarly prepared using unaffectedsoil from the same fields. For preparing combined plant extract (cpE), I00 g of loots, shoots, leaves, bark, flowers and seedsof fewtrees lvere taken togetherin equal quantity and soaked in 100 ml water for 24 h. The extract was sterilized at l5 lbs for 20 min, cooled and filtered. Surfacesterilizationof healthy seedsof clusterbeanwas done by immersing in 960/'ethanol for 5 min followed by treatment with 5% commercial sodium hypochlorite for l0 min. Seedswere then washedthoroughly atleast ten times with sterile tap water before use. The sterilized seeds were treated with I ml of 4-5 day old Rhizobium (cowpea) isolate containing about 106to 107cells ml-r. For pot culture studies,allelopathic and control soils were brought from the same field and used to fill the washed pots (3 kg pot-r). ..Treated and untreated seeds rvere sown and the plants were irrigated with water once in 2-3 days. Nitrogerr (urea) was applied @ 20 mg kg-r of soil once in 20 days. Three replicateswere kept for each isolate alongwith control. The crop was harvcsted after 60 days. Data were recorded for nodules plant-l, shoot dry weight plant-r, nodule fresh weight plant-r and nitrogenase activity of nodules as describeCbv Hardy et al. (3) and Lakshminarayanaet al. (5). For conducting nodulation studiesin clusterbean,chillum jars were used as describedby Dahiya and Khurana (l). Jars containing thoroughly washedcoarse sand were autoclaved for 3 h at 15lbs. Treatments given werel (i) l0 and 2O/" allelopathic soil extract, (ii) l0 and 20llcombined plant extract and (iii) nitrogen in the form of urea (20 mg kg-r sand). Each of these treatments was repeated thrice once in 20 days during a total period of 60 days. Jars containing four treated or untreated seedswere kept in the pot house. The plants were supplied with sterile tap water on alternatedays and Sloger'snitrogen free nutrient (ll) medium once a
Allelopathiceffectof Acacia
49
week. Data wererecordedfor nodulesplant-r, shoot dry weightplant-r, nodule freshweightplant-r and nodulenitrogenase activityas describedby Harclyet at. G) et al. (5). and Lakshminarayana RESULTS AND DISCUSSION The resultsobtainedwith threeRhizobium(cowpea)isolates(G-3, IC-3100 and PS-I) and on the numberof nodules,nodulefresh weight, acetylene reducing activities(ARA) and shootdry weightin allelopathic and non-allelopathic soils are shownin Table l The inhibitionvariedbetweenl0 to 2Al. TABLE t.
Nodulation and nitrogen fixation of Rhizobium (cowpea) strains G-3, IC-3100 and PS-l in allelopathic and non-allelopathicsoils in pot culture
Culturc and
Number of nodulesplant-r
Nodule fresh weight plant-r (mg)
treatmcnt
NonAlleloallelopa- pathic thic soil soil
Allelopa- NonNonAllelopa- Nonallelopathic allelopaallelopathic thic soil thic soil thic soil soil soil
ARA* (pr,moles CzHz reduced g-l h-r)
G-3 G-3+N*r
42 29
36 24
445 331
3r9
2.096 r.928
rc-3t00
30
27
394
323
IC-3100+N**25
2r
,72
294
3l 23
27 20
265 215
29 Control control+N*r 1l
t6
139
C. D. at 5o/o 8.36
5.97
PS-t PS-t+N**
9
lll
28.34
386
Shoot dry weight plant-I (B) Allelopathic soil
t.653 t.385
1.876 t.991
1.538 t.721
t.433
1.292
1.762
1.342
1.254
1.002
1.834
1.398
221 198
1.204 1.142
r.674 t.813
1.211 r.460
t29
0.706 0.553 o.246
0999 0.686 o.57r0.282
0.873 0.989
0.417
0.284
0.613 0791 0.970
t7 49.82
*ARA -Acetylcne reduction assay. **N-Nitrogcn @ 2Omg kg-r of soil.
The reductionin nodulenumberin isolatesG-3, IC-3100and PS-I in allelopathicsoil was 15, l0 and l3/o,respectivelyas comparedto non-allelopathic soil. Reductionwas more in the sampleswhere nitrogen treatment was given in all the three strains The decrease in nodule fresh weightof G-3, IC-3100and PS-I in allelopathic as comparedto non-allelopathicsoil. Nitrosoil was 14, 19 and l7f , respectively, genaseactivity of G-3, IC-3100 and PS-I in allelopathicsoil was reducedby 20, l0 and l8%, respectively.
Duhan, Sharma and LakshmindraYana
50
IC-3100 and The shoot dry weight decreasedby 19, 24 and 28\inG-3' PS-I, respectively in allelopathic soil than in control soil. In treatments where nitrogen was included, the decreasein shoot dry weight was less. Results obtained in chillum jars with two Rhizobium (cowpea) isolates are shown in Table 2. Treatment with both the isolates showed good nodulation (49 and 46 plant-r), nodule fresh weight (408 and 465 mg plant-r), high ARA (2.963 and2.270p moles g-rh-r) and high shoot dry weight (761 and677 mgplant-r). Ten and 20% ASE,reduced nodulatioh (34 to 54%), nodule fresh weight (30 to 6l%), ARA (22 to 30%) and shoot dry weight (23 to 50%)- Similarlv, CPE also causeda significantreduction in each ofthe above parameters. soilextracton nodulation TABLE 2. Effectof combinedplantextractof A. niloticaandnllelopathic (cowpea) strainsin chillumjar and nitrogenfixationby Rhizobium Treatment
Number of nodulesplant-r
Nodule fresh weight Plant-r (mg)
ARA (pr,moles C*H, reducedg-lh-r)
Shoot dry weight plant-r (mg)
G-3
G-23
G-3
G-23
G-3
G-23
G-3
c-23
Control
49
46
408
465
2.963
2.270
761
677
Allelopathicsoil cxtract
29
28
286
239
2.318
1;746
5tl
4t9
23
22
224
183
2.080
1.610
427
340
Nitrogen*
36
4t
368
4r4
2.602
813
650
Nitrogen** Allelopathic soil extract (10%)
31
32
297
269
2.567
2.075 -t.9r6
66r
495
Nitrogenr * Allelopathic soil extract (201)
24
29
287
247
2.481
1.888
557
451
Combined plant extract
2l
)
