Indian Journal of Experimental Biology Vol. 41, April 2003, pp. 341-345
Associative diazotrophs of pearl millet (Pennisetum glaucum) from semi arid region - Isolation and characterization
Mala Tiwari, Shashi Parada & K R Dadarwal*
Department of Microbiology, CCS Haryana Agricultural University, Hisar 125004. India Received 21 October 2002; revised 9 January 2003
Diversity of the native diazotrophs associated with the rhizosphere of pearl millet (P. glaucum), grown in nutritionally poor soils of semi-arid regions was studied with a view to isolate effective nitrogen tixin~ and plant growth stimulating bacteria with root associative characteristics. The native population varied from 103_104 g' of rhizosphere soil after 40 d growth and belonged to genera Azospirillum, Azotobacter and Klebsiella. Another non-diazotrophic root associative group was Pseudomonas sp., which also produced IAA and enhanced plant growth. Some of these rhizobacteria showed high in vitro acetylene reduction activity along with production of indole acetic acid. Out of II selected diazotrophs used as seed inoculants, M lOB (Azospirillulll sp.), Mil E (Azotobacter sp.) and M 1204 (Klebsiella sp.) resulted in significant increase in total root and shoot nitrogen at 45 and 60 days of plant growth under pot culture conditions.
The rhizosphere of cereals and legumes harbours a variety of microorganisms. The microbiocoenotic in- teractions of these microorganisms influence plant growth in ways that may be beneficial, neutral or det- rimentall•2
• A large number of rhizosphere bacteria have been identified for use as inoculants in cereals and legumes. Such bacteria are referred to as plant growth promoting rhizobacteria (PGPR)3.
Inoculation of plants with PGPR, viz. the associa- tive diazotrophs is one of the novel approaches to provide cereals with biologically fixed nitrogen. Sub- stantial yield increase has been reported in various cereals on inoculation with these organisms, which is mainly due to nitrogen fixation, production of plant growth regulators like auxins and cytokinins, suppres- sion of pathogens and increase in nutrient uptake effi- cienc/. Experimental evidences showing significant amount of nitrogen fixation by diazotrophs in eco- nomically important crops like maize, sorghum, pearl millet, sugarcane, rice and forage grasses have gener- ated tremendous interest in biological N2 fixation in non-legumes5.6.
Pearl millet is one of the major grain crops in semi- arid tropics in India. It is mainly grown on marginal lands with minimum use of chemical fertilizers or organic manures. The main objective of the present study is to isolate, characterize and evaluate native
*Correspondent author:
Phone: 091-1662-231171 (0), 236341 (R) Fax: 091-1662-234952, 234613
E-mail: dadarwal@hau.nic.in
diazotrophs from pearl millet rhizosphere from semi- arid region of Gurgaon (Haryana) where it is mainly grown as a rain-fed crop.
Materials and Methods
Most probable number (MPN) counts of di- azotrophs in rhizosphere- Rhizosphere samples of pearl millet (Pennisetum glaucum) grown under field conditions in rain-fed areas of Gurgaon district (Hary- ana) were taken at 40 days of plant growth from 14 locations. The excess soil with roots was removed by gentle shaking and the closely adhered soil along with roots (10 g) was transferred to 90 ml sterilized water blanks. After vigorous shaking for 30 min on a rotary shaker, ten-fold serial dilutions were made and 0.1 ml aliquots from different dilutions were transferred to 5 ml semi-solid Dobereiner's medium6 modified by replacing half the quantity of sodium malate with mannitol (5 g )"1) and reducing the amount of yeast extract by half (0.25 g )"1), henceforth referred as MM medium and Jensen's7 medium tubes in triplicates.
The tubes were incubated at 28° ± 1°C for 5 days.
Most probable number (MPN) of diazotrophs were determined by acetylene reduction assay (ARA) method8.
Isolation of diazotrophs from ARA positive tubes- For isolation of diazotrophs from ARA positive tubes, growth in tubes was diluted by lO-fold serial dilution and plated on MM as well as Jensen's media. The plates were incubated for 4 days at 28° ± 1°C and typical colonies having colony characteristics as those
342 INDIAN J EXP BIOL, APRIL 2003
of Azospirillum and Azotobacter were picked up and transferred to respective media slopes. The bacterial isolates were checked for purity by streaking on nutri- ent agar. The selected pure cultures were screened for ARA in semi-solid MM and solid Jensen's media slopes after incubating at 28° ± 1°C for 4 days.
fAA production by selected isolates - Production of indole acetic acid (IAA) like compound was de- termined in liquid MM and Jensen's media supple- mented with 100 mg
r'
tryptophan. After 7 days of growth, the broth was centrifuged at 12,000 rpm for 5 min. To the supernatant, Salkowski's reagent9 was added and the intensity of pink colour developed was graded visually from 1+ to 4+. The Salkowski's re- agent in general reacts with compounds having in- dole-ring with development of pink colour.Characterization of diazotrophs - Isolates selected on the basis of relative ARA and IAA production were identified based on cell shape, spore formation, motility, Gram reaction, capsulation, pigment produc- tion and biochemical characteristics according to standard procedures of bacterial identification'o,,,.
Effect on seedling and plant growth- The effect of the selected isolates on growth of pearl millet seed- lings was examined in Petri plates. The strains were grown for 5 days on respective medium slopes. The growth of the cultures on slopes was suspended in 5 ml sterilized normal saline and surface sterilized pearl millet cv. HSB-64 seeds, surface sterilized with acidic alcohol (H2S04: ethanol 4: I) were inoculated with the suspension. After 30 min of bacterial adsorp- tion on seeds, the seeds were transferred to plain agar (0.8%) plates. The plates were incubated at 28° ± 1°C and length of roots and shoots were recorded at 5 and 9-day stage.
After initial screening, 12 promising isolates were selected for pot culture studies. Seeds inoculated with these isolated were sown in earthen pots, each con- taining 8 kg field soil. Basal dose of NPK (40:20:20) was given at the time of sowing. Uninoculated seeds were sown as control and 6 replications were kept for each treatment. Observations for root ARA, plant dry weight and nitrogen content were taken at 45 and 60 days after sowing (DAS). The data were analysed sta- tistically.
Results
M PN counts of naturally associated diazotrophs- The MPN counts (\OgIO values) of commonly associ- ated diazotrophs in the rhizosphere of pearl millet var- ied from 3.176-4.176 in MM and from 3.176-4.653 in
Jensen's medium. The soil of the selected area was sandy loam in texture. Samples from 6 locations were having similar counts in both the media (around 104 g"), while from 5 locations counts of Azotobacter (Jensen's medium) were slightly higher. The counts were low
«
104 g"') in samples from 3 locations in both the media.ARA and fAA production - A total of 97 colonical isolates were picked up (65 from MM medium plates and 32 from Jensen's medium plates) by dilution plat- ing the cultures from ARA positive tubes. On re- screening the pure cultures for ARA and production of IAA, 15 isolates from MM medium and 12 isolates from Jensen's medium showed ARA activity varying from 1.7 to 47.9 11m C2H2 reduced tube" h(' (Table 1).
The production of IAA like compounds in medium containing tryptophan varied from not detectable to very high level (4+). One of the isolates, which did not show C2H2 reduction but produced high amount of IAA (4+) was also used for further studies. Based on
Table I - Relative ARA and IAA production by selec'ed rhizo- bacteria under cultural conditions
Isolate Codc No. Relative ARA IAA levels (11M C2H2 reduced hr"' tube")
Diazotrophs
MIBI 8.2 4+
MIB2 21.0 4+
JIB 8.2 3+
J2A 4.1 3+
J3B 2S.7
JSB 36.7
JSC 32.S 2+
M6CI 33.9 3+
M6C3 2S.7 1+
J6C 3.6 3+
nc
34.3M8B 1.7 4+
M9D 12.8 1+
J9A 21.0
MIOB 19.9 4+
JIOA IS.2 3+
MIlA 4.7
MIlE 11.1 3+
MIIF 44.4 3+
J1IA 30.4
MI2DI 23.3
MI2D3 47.9 4+
MI2D4 20.6 4+
Jl2A 47.9 2+
MI3A 6.7 4+
MI3C 16.1 3+
Jl3B 24.S 3+
Non-diazotroph
M9C 4+
TIWARI et al.: ASSOCIATIVE DIAZOTROPHS OF PEARL MILLET FROM SEMI ARID REGION 343
relative ARA and IAA production, 12 isolates were finally selected for identification and for determining their effect on seedlings and plant growth.
Identification of selected isolates - All the 12 iso- lates were Gram negative, motile rods. Isolate M9C produced green fluorescent pigment in King's B me- diuml2 with oxidase positive test but ARA was not detected. The isolate was catalase +ve, hydrolysed gelatin but did not hydrolyse starch thus resembling Pseudomonas sp. All other 11 isolates were having C2H2 reduction activity. Isolates MIl E, 112A and 113B produced brownish pigment in Jensen's medium with colonies resembling to Azotobacter. Isolates J5B, M12D3 and M12D4 utilized citrate, hydrolysed urea, were fermentative in glucose medium with production of gas and MR +ve, thus resembling Klebsiella. Based on Gram staining, morphology and biochemical char- acteristics, the isolates MIB2, M6C3, M9D, MI0B and 111A were identified as Azospirillum sp., MllE, 112A and 113B as Azotobacter sp., J5B, M 12D3 and M 12D4 as Klebsiella sp. and M9C as Pseudomonas sp.
Inoculation effect on seedlings of cv. HSB64- Effect of seeds inoculation with selected isolates resulted in an increase in root length in most of the isolates, except M6C3 after 5 days of incubation (Table 2). Isolate J 12A showed maximum increase
Table 2 - Effect of rhizobacterial inoculation on root and shoot elongation of pearl millet cv. HSB-64 under in vitro conditions
in plates
Isolate No. Root length (mm) Shoot length (mm) 5 day 9 day 5 day 9 day
Control 74 ± 3 77±2 51±2 102±2
Azospirillum sp.
MIB2 91 ±2 182±3 50± I 85±2
M6C3 74±3 144±3 61 ±2 113±2
M9D 103±3 125±2 54± I 94± I
MIOB 89±3 154± 1 51 ±2 78±3
lilA 133±4 232±6 70± 1 105±2
Azotobacter sp.
MilE 106±3 175±2 64±2 92± I
Jl2A 144±3 223±4 62±2 123±3
113B I08±3 143±3 62± I 90±2
Klebsiella sp.
15B 122±3 162±2 67± I 112± I
M12D3 84±3 165±2 61 ±2 106± 1
MI2D4 I04±3 149±4 61 ±2 114±3
Pseudomonas sp.
M9C 103±3 216±2 61 ± I 112±2
Values are average of 30 plants.
(193%) in root length. At 9 days of incubation, all the isolates induced consistent increase in root length.
Maximum increase was observed with isolates J II A, MIB2, JI2A and M9C. The shoot length increased on 5lh day by inoculation of isolates M6C3, M9D and JlIA (A zospirillum) as well as other isolates of Azotobacter, Klebsiella and Pseudomonas. At 9 days of incubation, the consistency in shoot elongation was not noticed. However, inoculation with M6C3 (Azospirillum), Jl2A (Azotobacter), M12D4 and J58 (Klebsiella) and M9C (Pseudomonas) resulted in in- crease in shoot length by 110-121 %.
Inoculation effect on plant growth-Under pot culture conditions, seed inoculation of pearl millet cv.
HSB64 with isolate JlIA (Azospirillum sp.) which had recorded the highest ARA value in vitro (Table 2) and was a non-producer of IAA, showed significant gains in root dry weight at 45 DAS and total nitrogen at both 45 and 60 DAS (Table 3). The other isolate M9D, which showed the least ARA value and pro- duced very less IAA (l +) under cultural conditions, also showed significant gains in the three parameters (root length, root N content and ARA). Inoculation with isolates MIB2 and MI0B also resulted in sig- nificant increase in weight and N content of roots at 45 DAS but not at 60 DAS.
The Azotobacter isolates had variable effect. While inoculation with Mil E resulted in significant gains in root dry weight and N content at later stage, inocula- tion with 112A was ineffective. However, isolates J 13B showed significant gain in root N content at both 45 and 60 DAS. Among the 3 strains of Kleb- siella, inoculation with M 12D3 and M 12D4 resulted in significant increase in root weight and root nitrogen at 60 DAS. Inoculation with Pseudomonas isolate M9C, which was ARA negative but had high level of IAA production did not result in any significant effect on dry weight and nitrogen content of roots.
The effect of inoculation on dry weight and N con- tent of shoot is depicted in Table 4. Isolates M lOB and MIB2 (Azospirillum), MllE (Azotobacter), M 12D4 (Klebsiella), showed significant gains in dry weight and N content at 60 DAS. Although, other iso- lates also had positive influence, but the results were inconsistent.
Discussion
Cereal and forage crops are known to be highly re- sponsive to inorganic fertilizer nitrogen, but its high cost, limited availability and heavy losses due to leach- ing and denitrification requires alternate strategies for
344 INDIAN J EXP BIOL, APRIL 2003
Table 3 - Effect of inoculation on root biomass and root nitrogen of pearl millet under pot culture conditions
Treatment Root ARA
45 DAS 60 DAS (nMC2H2 reduced prl h(l)
Root dry wt Root N Root dry wt RootN 45 DAS 60 DAS
Control 1.07 3.81 2.96 6.82 95.6 46.7
Azospirillum sp.
MIB2 1.90* 6.45* 3.17 7.32 605.4 266.5
M6C3 1.07 2.68 3.80* 12.14* 105.2 33.1
M9D 2.57* 5.63* 2.79 12.89* 696.3 51.0
MIOB 2.04* 7.05* 3.24 13.55* 132.7 44.1
JlIA 2.42* 7.29* 3.62 9.63* 329.5 141.0
Azotobacter sp.
MilE 1.45 5.15 4.53* 18.04* 613.8 94.7
JI2A 1.32* 5.06 3.37 7.94 141.3 25.3
MI2D4 1.52 7.66* 3.04 10.97* 420.4 82.2
Klebsiella sp.
J5B 1.76* 4.52 3.28 12.88* 96.3 203.3
M12D3 1.68* 3.57 4.04* 16.19* 94.4 313.1
MI2D4 1.52 5.16 4.30* 17.13* 82.4 27.5
Pseudomonas sp.
M9C 1.24 4.37 2.99 8.05 93.6 9.9
CD. 0.52 1.63 0.76 2.29
Root dry weight in g and N in mg planrl; *values differ significantly from control Table 4 - Effect of inoculation on shoot biomass and shoot nitro-
gen of pearl millet under pot culture condition
Treatment 45 DAS 60 DAS
Shoot dry wt Shoot N Shoot dry wt Shoot N
Control 3.78 11.02 10.93 82.31
Azospirillum sp.
MIB2 6.20* 18.23* 14.25* 122.94*
M6C3 4.19 10.51 11.24 104.85
M9D 9.50* 19.63* 11.12 78.69
MIOB 5.19 20.09* 23.49* 146.04*
JIIA 7.36* 21.02* 13.56* 99.35
Azotobacter sp.
MilE 4.43 9.37 17.71 * 151.98*
JI2A 4.62 11.57 12.86 117.71*
JI3B 2.70 8.97 10.78 72.09
Klebsiella sp.
J5B 4.58 14.75 13.20 105.89
MI2D3 5.52 18.07* 14.69* 92.24
MI2D4 5.94* 15.94 16.90* 126.16*
Pseudomonas sp.
M9C 4.39 10.06 15.69* 97.92
CD. 1.96 6.27 2.59 33.47
Shoot dry weight in g and N in mg planrl; * values differ signifi- cantly from control.
better crop production at minimal input costs. One such strategy is to look for microorganisms that asso- ciate with these crops and increase rhizosphere devel- opment. These rhizosphere microbes augment the availability of crop nutrients and enhance the nutrient uptake efficiency of the crop plants. The beneficial effects of root associative diazotrophs on crop plants like pearl millet are well documented. The extensive development of fibrous roots in millets affords good opportunity for the proliferation of root-associated diazotrophs as well as for PGPR.
The present study has shown that the rhizosphere of crop plants harbours native diazotrophs but thcir number rarely exceeds 104_105 gO' of rhizosphere soil at tillering stage of growth (40 DAS). Yadav and Chaudhary'3 reported similar values for diazotrophs in pearl millet rhizosphere. In the present study, about 62.5% of the rhizobacteria isolated in Jensen's and 74.5% in MM media failed to show C2H2 reduction, though these were growing on nitrogen free medium.
Some of these bacteria could be nitrogen scavengers, while the others may be nitrogen fixers requiring spe- cific root exudates as nitrogenase inducers. Several of the isolates were of 'pseudomonad' type, which could be plant growth promoters.
TIWARI et al.: ASSOCIATIVE DIAZOTROPHS OF PEARL MILLET FROM SEMI ARID REGION 345
The rhizobacteria when used as seed inoculants en- hanced root growth under aseptic conditions, mark- edly perceptible at 9 days of seedling growth. The effect on shoot length was more pronounced at 5
thday, although the trend was maintained subsequently due to nutrient limitation. Venkateswarlu and Rao
l4reported similar effect of A.
brasilenseon root-shoot elongation in pearl millet seedlings. Conversely, Dobbelaere
et al.15reported that inoculation of wheat with wild type strains of
A. brasilenseresulted in a significant decrease in root length and increase in root hair formation. Adding tryptophan to the plates fur- ther enhanced this effect.
Inoculation of cereals with diazotrophs has been reported to stimulate plant dry matter and grain yield by affecting some physiological events like photosyn- thesis, nitrogen fixation and nutrient uptake in crops like wheat
l6,pearl millet
l7,sorghum
l8and maize
'9.In the present study, inoculation with the native isolates under unsterilized soil conditions resulted in signifi- cant increases in root and shoot nitrogen with some of the isolates. Significant increase in shoot dry weight was recorded with MIOB
(Azospirillumsp.) inocula- tion. Root dry weight was also significantly higher in the treatments and maximum effect was seen with Mil E
(Azotobactersp.) inoculation. The N content of shoot and root increased significantly on inoculation with M lOB
(Azospirillumsp.), Mil E
(Azotobactersp.) and M 12D4
(Klebsiellasp.). The plant biomass promoting effect could be the consequence of in- creased root biomass enhancing the nutrient uptake efficiency as well as due to nitrogen fixation by the diazotrophs. These results indicate that the native rhizobacteria could be exploited as better bioinocu- lants for pearl millet.
References
I Astrom B, Gustafsson A & Gerhardson B, Characteristics of a plant deleterious rhizosphere pseudomonad and its inhibitory metabolite(s), J Appl Bacterial, 74 (1993) 20.
2 Bolton J H, Elliot L F, Turco R F & Kennedy A C, Rhizoplane colonization of pea seedlings by Rhizobiul/l leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth, Plant Soil, 123 (1990) 121.
3 Kloepper J W, Lifschilz R & Zablotowiez R M, Free-living bacterial inocula for enhancing crop productivity, Trellds Biotechnol, 7 (1989) 39.
4 Patriquin D G, Dobereiner J & Jain D K, Sites and processes of association between diazotrophs and grasses, Call J Microbial, 29 (1983) 900.
5 Wani S P, Dart P J & Upadhyaya M N, Factors affecting nitrogenase activity (C2H2 reduction) associated with sorghum and millet estimated using the soil core assay, Can J Microbial, 29 (1983) 1063.
6 Dobereiner J, Isolation and identification of root associated diazotrophs. In: Nitrogell fixation with lIoll-legllmes. edited by F A Skinner, R M Boddey & I Fendrick (Kluwer Academic, London) 1989, 103.
7 Jensen H L, The Azotobacteriaceae, Bact Rev, 18 (1954) 195.
8 Kumar S, Paroda Shashi & Dadarwal K R, Root growth stimulating Azospirilla associated with root of sunnower, Helianthus anllus L. Indian J Exp Bioi, 33 (1995) 565. 9 Sharma K S B, Lakshmi Kumari, Apte R & Subba Rao N S,
Physiological characteristics of R. meliloti and R. trifolii in relation to symbiosis with Lucerne (Egyptian clover), Plallt Soil, 38 (1973) 299.
10 Kreig N R & Dobereiner J, The genus Azospirilllllll. in:
Bergey's Mallllal of Systelllatic Bacteriology Vol. I, edited by N R Kreig & J L Holt (The Williams & Wilkins Co.
Baltimore) 1984,94.
II Cappucino J G & Sherman N, V. Biochemical activities of microorganisms, Microbiology: A LaboratDlY Mallllal, (Addison-Wesley Publishing Company Inc.) 1983, 131.
12 King E 0 & Ward M K, Two simple media for the demonstration of pyocyanin and nuorescein, J Lab Ciill Med, 44 (1954) 301.
13 Yadav K S & Chaudhary K, Rhizosphere and rhizoplane bacteria from desert grown pearl millet, Illdiall J Microbiol.
29 (1989) 381.
14 Venkateswarlu B & Rao A V, Response of pearl millet to inoculation with different strains of AzospirillulIl brasilense.
Plant Soil, 74 (1983) 379.
15 Dohbelaere S, Croonenborghs A, Thys A, Broek A V &
Vanderleyden J, phyto stimulatory effect of Azospirilllllll brasilense wild type and mutant strains altered in IAA production on wheat, Plant Soil, 212 (1999) 155.
16 Boddey R M, Baldani V L D, Baldani 1 I & Dobereiner J, Effect of inoculation of AzospirillulIl spp. on nitrogen accumulation by field grown wheat, Plalll Soil, 95 (1986) 709.
17 Wani S P, Chandrapalaiah S & Dart P 1, Response of pearl millet cultivars to inoculation with nitrogen-fixing bacteria, Expl Agric, 21 (1985) 175.
18 Sadhu A C, Patel J R, Patel P C & Patil B G, Effect of biofertilizers and nitrogen levels on cereal fodder crops, Forage Res, 17 (1991) 59.
19 Pandey A, Sharma E & Palni L M S, Innuence of bacterial inoculation on maize in upland farming system of the Sikkim Himalayas, Soil Bioi Biochelll, 30 (1998) 379.
