Microbiol. Res. (1995) 150, 173 -177 Microbiological Research

© Gustav Fischer Verlag Jena

Nitrogenase activity and nitrogen-fixing bacteria associated with root base and root tip of rice plants

P. K. Kanungo, G. K. Patnaik, T. K. Adhya, V. Rajaramamohan Rao

Division of Soil Science and Microbiology, Central Rice Research Institute, Cuttack-753006, India

Accepted: November 7, 1994

Abstract

An assessment of nitrogenase activity associated with the basal and terminal portions of rice roots from seven rice cultivars was made. Results indicate a definite partitioning of the nitrogenase activity within the root and a plant growth dependent shift in the activity associated with basal and terminal portion of the roots. Nitrogenase activity associated with the root base steadily declined with plant growth, while root tip associated nitrogenase activity increased sharply. Further, thehasal -portion had higher nitrogenase activity in the prereproductive phase while the root tip had higher activity during reproductive and maturity stages of the plant. Microbiological analysis of plant roots indicated the association of higher populations of Azospirillum sp. and Azotobacter sp. with the root base, while the root tips had higher populations of facultative anaerobic nitrogen-fixing bacteria.

Key words: root base - root tip-associative nitrogenase activity - rice cultivar

Introduction

Rice soils provide unique conditions for the activity of microorganisms in view of the abundant moisture availability and fluctuating redox status of the soil throughout the growth of the rice crop. In fact, the rice plant provides a favourable niche for the nitro­gen-fixing bacteria to form associations in the root zone and certainly enhances nitrogen fixation in rice fields (Yoshida and Ancajas 1971; Kalininskaya et al. 1973; Hirota et al. 1978; Rao et al. 1987). The stimu­latory effects of the rice plants on nitrogen fixation in a paddy soil essentially resulted from the activity of

Corresponding author,' V. Rajaramamohan Rao

nitrogen-fixing microorganisms within or around the rice roots (Rao and Rao 1985; Ladha et al. 1986).

Recently, the occurrence of associative bacteria like Azospirillum sp. in and around the rice roots was reported and the interacting factors were investi­gated (Nayak and Rao 1977; Charyulu and Rao 1980; Ladha et al. 1993). Varietal variation with respect to nitrogen fixation in the rice rhizosphere has also been demonstrated (Hirota et al. 1978; Rao and Rao 1985). Presumably, nitrogen fixation is effected by the bacteria that depend on the plant roots for energy, nutrient supply and other factors. Although few data are available on root associated nitrogen fixation under tropical rice cultivation conditions, the partitioning of nitrogenase activity on the root is not well understood. We investigated the nitrogenase activity associated with the basal and terminal por­tions of roots of several rice cultivars. In addition three different groups of nitrogen-fixing bacteria associated with root base and tip were quantified.

Materials and methods

Sampling o/root base and tip. A field experiment was conducted in the experimental plots of the institute by transplanting 25-day old seedlings! of 7 rice cultivars (Annapurna, Swarnaprava, I~-36, IR-50, Sarasa, Rasi and Ratna) to plots (10 m2 area) maintained under uniform field conditions. Each variety was grown in triplicate plots randomly arran­ged and the sampling of the root was done at periodic intervals to study the nitrogenase activity associated with the root base and tip. The soil used is a sandy-loam (Typic Haplaquept) with pH 6.2, organic

Microbial. Res. 150 (1995) 2 173

matter 1.6%, total N 0.09%, electrical conductivity 0.5 dS/m, clay 25%, silt 22% and sand 53%. Roots of each variety were collected from the experimental field at desired intervals over the growing period of rice. They were throughly washed under running tap water to remove the adhering soil particles followed by rinsing several times in sterile distilled water. The roots were then differentiated into the root base (1- 2 cm portions of the root from the base) and root tip (1-2 cm portions from the tip). The portions were collected seperately and arranged in five replica­tes in B - D vacutainer tubes (Becton-Dikinson, New Jersey, USA) each having 100 mg fresh weight of the root material.

Acetylene reduction analysis. The tubes containing the root material were stoppered with serum caps and filled with 10% (by volume) of high purity acetylene (C2H 2)

gas by a hypodermic syringe; ethylene (C2H4 ) produc­tion was determined after 24 h incubation in the dark at 28 °C. Tubes without C2H 2 were also included for the assay of endogenous C2H4 • At the end of the incubation before the analysis, the tubes were thor­oughly shaken in order to equilibrate the head space with the soil trapped ethylene. The preparation of the samples, their incubation and analysis were done as described previously (Rao et al. 1983). For the determination of C2H 4 , 0.5 ml of the gas phase from each tube (five replications) was injected into a gas chromatograph (AIMIL-NUCON, New Delhi, India) fitted with a hydrogen flame ionization detector (FID) and a 1500 x 3 mm column filled with 100 - 120 mesh Porapak-R at a column temperature of 50 °C, injector temperature of80 °C and detector temperature of80 0C. High purity nitrogen at a flow rate of30 ml/min served as carrier gas. The nitrogenase activity (means of five replicates) was expressed as nMoles ofC2H4 formed/g dry root x h. The results were subjected to statistical analysis following Duncan's multiple range test.

Microbiological studies. Nitrogen-fixing Azospirillum sp., Azotobacter sp. and anaerobic nitrogen-fixing bacteria associated with root base and tip were enumerated in 70 d old rice cultivars. The microbio­logical analysis was carried out by conventional serial 10-fold dilution technique on N-free media. The popUlation of nitrogen-fixing Azospirillum sp. was counted following the method suggested by Okon et al. (1977). The population of Azotobacter sp. and anaerobic nitrogen fixers by the methods ofRao et al. (1973). Serial dilutions were plated on N-free agar media for counting Azotobacter sp. in triplicates for each dilution. The anaerobic nitrogen-fixing bacteria were counted following the MPN technique of the tubes containing N-free liquid medium with CaC0 3

and incubated under anaerobic conditions. Tubes

174 Microbio!. Res. 150 (1995) 2

with visible microbial growth, vigorous gas produc­tion and ARA were considered as positive (Rao et al. 1973). Results presented are the means offive replica­tes for Azospirillum sp. and anaerobic nitrogen fixers and three replicates for Azotobacter populations.

Results and discussion

Nitrogenase activity associated with the root base among the two rice varieties, IR-36 and Annapurna (Figs. 1 and 2), steadily declined with the growth of the plant. On the other hand, the activity asso-

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~ \ .... ~ 0 500 , , "0 ...

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45 55 75 85 105 115

Age of Planf

Fig. 1. Alterations in the nitrogenase activity associated with root base and tip (Cultivar IR-36) at different stages of plant growth.

~ ~ OJ

2000

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45 55 75 85 105 115

Age of Plant

Fig. 2. Changes in the nitrogenase activity associated with root base and tip of rice cultivar Annapurna.

ciated with root tip showed a sharp increase after 55 days in IR 36 while in Annapurna the increase was noticed only after 75 days. Thus, there is a clear variation with respect to the nitrogenase activity among the rice cultivars. Moreover, the association of nitrogenase with root base and root tip has shown a reverse trend during the later phases of plant growth.

The total weighted average of root base and root tip associated nitrogenase activity for all the seven cultivars indicates that the root base associated nitrogenase gradually declined with the age of the crop (Fig. 3). On the contrary, the nitrogenase activi­ty associated with the root tip which was low in the initial stages of the plant growth increased gradually and steadily, particularly during the reproductive phase (after panicle initiation) in most of the culti­vars. These results indicate a clear partitioning of the nitrogenase activity associated with the root and a plant growth dependent shift in the nitrogenase activity with basal and terminal portions of the root. Thus, higher nitrogenase is associated with the root base in the pre-reproductive phase of the plant, whereas the root tip had higher activity during the later growth phases extending to the reproductive and maturity stages. Possibly, differences in the nature of organic matter suplied by the root, redox status and the changes in the micro-habitat might be responsible for the differences in the nitrogenase activity with root base and tip.

The average nitrogenase activity associated with root base and tip for individual rice cultivars before panicle initiation (vegetative) and after panicle initia-

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2000

1500

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Age of Plant

Fig. 3. Weighted average nitrogenase activity associated with root base and tip of seven rice cultivars at different growth stages.

tion (reproductive) phases brings out interesting information (Table 1). At the pre-panicle initia­tion stage, almost all the seven cultivars showed a higher nitrogenase activity associated with root base although these varieties differed in the extent and magnitude of activity. Thus, IR-36, Sarasa and Ratna had higher root base associated nitrogenase activity than other cultivars. Cultivar Annapurna had the lowest root tip associated nitrogenase activi­ty. Interestingly, IR-36 had the highest root base associated nitrogenase activity, while its root tip associated nitrogenase was one of the lowest. Com­pared with other cultivars, Sarasa had reasonably high root base as well as root tip associated nitroge­nase activity.

After the panicle initiation stage the nitrogenase associated with the root tip was higher than in the root base. IR-50 and IR-36 showed lower root base associated nitrogenase activity than other cultivars. Though the nitrogenase activity varied among the cultivars, the trends remained consistent with root tips supporting a higher activity. While IR-50 and IR-36 had an almost 3-fold increase in the activity associated with root tips, the other cultivars exhibited less than 2.5 fold increase compared with their basal portions. Apparently, the partition pattern of the root base and tip associated nitrogenase activity is uniform among the rice cultivars though there existed variation in the extent of the activity. Varietal variation with respect to associative nitrogen fixation has been reported (Rao and Rao 1985 ; Ladha et al. 1986).

Table 1. Alterations in the weighted average* of nitro-genase activity associated with the root tip and root base among the rice varieties.

Rice variety Average nitrogenase activity n moles C2H4 formed/g dry root/h

Before panicle initi- After panicle initi-ation (vegetative) ation (reproductive)

Root base Root tip Root base Root tip

Annapurna 820" 33- 353b 698b

Swarnaprabha 887- 508d 341 b 594-IR-50 797- 458<d 298b 748< IR-36 1339c 240b 214" 713c

Rasi 815- 365< 879d 1230e

Sarasa 1087b 764f 481< 917d

Ratna 1018b 663e 345b 746c

* Weighted average of nitrogenase activity was based on the total of observed nitrogenase activity for each variety throughout the growth phase (vegetative/reproductive)/ no. of observations. Means followed by the same letter do not significantly differ (P < 0.05) by Ducan's multiple range test (DMRT)

Microbiol. Res. 150 (1995) 2 175

Table 2. Nitrogen-fixing bacterial populations associated with root base and root tips of different rice cuItivars.

Rice Portion of Populations of N 2-fixing bacteria x 103/g dry root* cultivar the root

Anaerobic Azospirillum sp. Azotobacter sp.

Ratna Root base 9.3 ± 3" 350 ± 33 0.57 ± 0.12 Root tip 17.0 ± 2 130 ± 18 0.42 ± 0.16

Sarasa Root base 4.5 ± 1.2 350 ± 28 0.46 ± 0.18 Root tip 130.0 ± 19 170 ± 20 0.34 ± 0.13

Annapuma Root base 13.0±2 540±42 0.88 ± 0.13 Root tip 49.0 ± 5 220 ± 19 0.43 ± 0.14

* Root base and tip samples were collected from 70-day old plants. a Values of the replicates ± standard deviation of the mean.

We also investigated the alterations in the distribu­tion pattern of the populations of Azospirillum sp., Azotobacter sp. and anaerobic nitrogen-fixing bac­teria associated with root bases and tips in plant samples collected 70 d after planting from three rice cultivars. Root bases harboured higher populations of Azospirillum sp. and Azotobacter sp., while the root tips had higher populations of facultative an­aerobic bacteria (Table 2). The three rice cultivars exhibited almost similar trends with regard to har­bouring the microbial populations at root bases and tips. Thus, lower populations of Azotobacter sp. and Azospirillum sp. were associated with root tips while higher numbers of anaerobic nitrogen-fixing organisms with the basal portion of the root. The composition of the populations also varied among the three cultivars investigated. Thus, the qualitative and quantitative differences in the association of various groups of bacteria with the basal and termi­nal parts of the root might also contribute to the variations in the nitrogenase activity. Alterations in the number and groups of various nitrogen-fixing bacteria among the cultivars of different crop plants has been reported (Boddey and Dobereiner 1984; Ladha et al. 1986, 1993 ; Rao el af. 1987) .

Our results further suggest that the nitrogenase activity associated with the root base steadily de­creased with plant growth, while the root tip showed a steady increase in activity. Almost throughout the growth of the plant this trend seems to be identical among the seven rice cultivars studied. This could be due to maturity, partial deterioration or decline in metabolic activity of roots from the base towards the tip. In addition, the depletion of available organic matter and nutrients around the root base at maturity could also contribute to the lower activity. Further, the trends remained almost identical among the cultivars with respect to root base and tip associated activities. Perhaps, differences in the oxygen status in

176 Microbiol. Res. 150 (1 995) 2

the root region at the base and tip might reflect in the resultant activity. Root tip portions of the rice plants remain more or less at a higher redox potential (Eh) than the basal portion (Flessa and Fischer 1992). Predictably, such alterations in the redox potential at root base and tip portions during the different stages of plant growth might have contributed to the relative differences in the nitrogenase activity at the two sites of the root. Detailed investigations are needed to evaluate the factors affecting the nitroge­nase activity and the microorganisms associated for a better understanding of the plant-bacteria interac­tions.

Acknowledgements

The authors thank Dr. B. Venkateswarlu, Director, for his keen interest and encouragement.

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