436

Journal of Basic Microbiology 2007, 47, 436 439

Short Communication Establishment of Azotobacter on plant roots: chemotactic response, development and analysis of root exudates of cotton (Gossypium hirsutum L.) and wheat (Triticum aestivum L.)
Rakesh Kumar1, Ranjana Bhatia1, K. Kukreja1, Rishi Kumar Behl2, Surjit Singh Dudeja1 1 and Neeru Narula
1 2

Department of Microbiology, CCS Haryana Agricultural University, Hisar, India Department of Plant Breeding, CCS Haryana Agricultural University, Hisar, India

Biofertilizers contribute in N2 fixation, P solubilization, phytohormone production and thus enhance plant growth. Beneficial plant-microbe interactions and the stability and effectiveness of biofertilizer depend upon the establishment of bacterial strains in the rhizosphere of the plant. This interaction depends upon many factors, one of them being plant exudates. Root exudates are composed of small organic molecules like carbonic acids, amino acids or sugars etc., which are released into the soil and bacteria can be attracted towards these exudates due to chemotaxis. The chemotactic behaviour of Azotobacter strains was studied using cotton (Desi HD 123 and American H 1098) and wheat (WH 711) seedlings and the root exudates of these two plants were chemically characterized. Analysis of the root exudates revealed the presence of sugars and simple polysaccharides (glucose), amino acids (glutamate, lysine) and organic acids (citric acid, succinic acid, maleic acid, malonic acid). Differences between cotton cultivars in root exudates were observed which influenced chemotactic response in Azotobacter. These results indicate colonization with rhizobacteria which implies that optimal symbionts, on the sides of both plant cultivar and bioinoculant bacteria can lead to better plant growth under cultivation conditions.
Keywords: Root exudates / Azotobacter chroococcum / Chemotaxis / Triticum aestivum L. / Gossypium hirsutum L. / Symbiosis Received: October 24, 2006; returned for modification: January 12, 2007; accepted: April 04, 2007 DOI 10.1002/jobm.200610285

Introduction*
Root exudates are important factors that structure the rhizosphere bacterial community. It is established that seed/root exudates of plants can be processed as nutrients leading to enhanced growth and higher prevalence of degrading strains of bacteria. On the other hand, numbers of certain bacteria are increasing by chemotactic response to root exudates. The resulting rhizosphere microflora can interact with root agglutinins present in the root exudates to form the rhizoplane community. Among rhizospheric bacteria, strains with
Correspondence: Prof. R. K. Behl, Department of Plant Breeding, CCS Haryana Agricultural University, Hisar-125004, India E-mail: rkbehl@hau.ernet.in or rbehalhsr@sancharnet.in 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

plant growth promoting effects have been isolated. The plant growth promotion can be attributed to better nutrient access, e.g. by phosphorus mobilization from soil particles, or by nitrogen fixation in the bacterial symbionts, or can be stimulated by bacterial production and release of phytohormones. At early stages of plant development it seems obvious that root exudates are responsible for changing the physiologically active micro-flora in the vicinity of the roots. As a result, also amino acids and various growth factors synthesized by the primary microbe population in the rhizosphere may give rise to successive populations with different and more complex requirements for nutrition. The specific rhizosphere community that is finally observed on mature roots may include symbionts which either
www.jbm-journal.com

Journal of Basic Microbiology 2007, 47, 436 439

Establishment of Azotobacter on plant roots

437

can enter the root, like e.g. mycorrhizal fungi or rhizobia, or species that are found regularly on root surfaces but not inside the root. Aside from nutrient mobilization, the rhizoplane bacteria can provide protection from soil-borne pathogens thus also aiding plant growth. All roots have the remarkable ability to secrete both low and high molecular weight molecules into the rhizosphere in response to biotic and abiotic stresses (Bertin et al. 2003). The root exudation of a plant can vary considerably both qualitatively and quantitatively due to different environmental conditions. Free living and associative diazotrophs are more numerous in the rhizosphere than in bulk soils, indicating their dependence on organic compounds exuded by roots in the rhizosphere (Gransee and Wittenmeyer 2000). Various nitrogen fixing microorganisms have been found to be present in the rhizosphere of agricultural plants, but the contribution of fixed nitrogen to plant nutrition is controversial (Lima et al. 1987). Diazotrophs found in the soil or associated with roots include Azotobacter chroococcum, Azospirillum brasilense and Gluconacetobacter diazotrophicus (formerly Acetobacter diazotrophicus) and the positive responses of plants to inoculation with these bacteria are attributed to N2 fixation, but in addition due to several other factors like phytohormone and/or ammonium production, etc. (Okon 1985, Okon et al. 1983). We have screened 14 bioinoculant strains which have been used with cotton and wheat crops under green house and field conditions (Narula et al. 2005). Responses of these cultures have been found to be variable depending on plant species and variety. To study these interactions, the relationships of cotton or wheat plants with selected Azotobacter bioinoculants under controlled conditions were observed by (a) studying the chemotactic response of Azotobacter strains to root exudates, (b) comparing root exudates of two plants (cotton and wheat) and two cultivars of cotton belonging to two different species and (c) characterizing plant development in the interactions of the three plants and of 14 bacterial strains.

Khurana (1988). Root exudates were filter sterilized (0.45 m) and kept at 20 C. Root exudates were concentrated 10 folds in a vacuum oven at a temperature 40 C. Chemotactic behaviour of Azotobacter strains was studied by using a capillary method. Sterilized capillary tubes were first dipped in filter sterilized root exudates and then in the culture broth of inoculant strains, respectively. After half an hour, all liquid from the capillary tube was serially diluted and plated on appropriate media (Jensens nitrogen free medium) and incubated at 30 C for 46 72 h. Cfu ml1 was taken after three to four days of incubation. Analysis of variance was carried out and means were compared using critical differences (CD) values. In order to study the effect of root exudates on growth of Azotobacter strains, 0.5% concentrated exudates were added to Jensens medium (without carbon) alone and also along with 0.5% glucose in 50 ml tubes. 50 l of the cell inoculum (109 cfu ml1) was added to above tubes and incubated at 28 + 2 C for 3 4 days. Growth was measured by optical density at 600 nm. Analysis of root exudates Total sugars were determined by using anthrones method (Dubios et al. 1951). Sugars, amino acids and organic acids present in root exudates were analyzed by ascending paper chromatography (Block et al. 1958). Amino acids were also characterized by TLC.

Results and discussion

Chemotactic response of Azotobacter towards root exudates In the rhizosphere, roots serve as a source of nutrients by means of exudates and thus create gradients in the rhizosphere soil. Chemotaxis may be important in assisting motile bacteria to approach their host. We compared chemotaxis of 14 strains of Azotobacter chroococcum used as bioinoculants in an in vitro assay, using the capillary method, which allows the quantitative measurement of chemotactic response. In order to test for adaptation to the micro-environment created by the host plant, different plant species and varieties were used. Chemotactic studies were performed to find out whether an attractant was present in the root exudates. Maximum bacterial colonies (cfu/ml) were observed with strain 103 (9 103), followed by AVK 51 (5 103) and HT 57 (3 103) in Desi cotton HD 123, whereas in American cotton variety H 1098 maximum colonies were observed with E 12 (16 103) followed by AVK 51 (9 103), HT 57 and AVK 42 (7 103 each), respectively. Likewise,
www.jbm-journal.com

Materials and methods

Bacterial strains and their maintenance Strains of A. chroococcum were maintained on Jensens media slants and stored at 4 C. Seeds of cotton (Desi cotton-Gossypium herbacium L. var. HD 123 and American cotton-Gossypium hirsutum L and var. H 1098) and wheat (Triticum aestivum L. var. WH 711) were grown and root exudates were collected as described by Dudeja and
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

438

R. Kumar et al.

Journal of Basic Microbiology 2007, 47, 436 439

Table 1. Analysis of root exudates obtained from control and inoculated plants. Azotobacter Sugars inoculants Cotton HD 123
Control Mac 27 Ala 27 Xylose, Glucose Glucose Glucose

Amino acids Cotton H 1098

Glucose Glucose Glucose

Organic acids Cotton H 1098 Wheat WH 711 Cotton HD 123

Oxalic acid

Wheat WH 711
Glucose Glucose Fructose

Cotton HD 123
Arginine Threonine

Cotton H 1098
Citric acid Maleic acid Malonic acid

Wheat WH 711
Succinic acid Succinic acid Malonic acid

HT 54 HT 57 E 12

Glucose Glucose Glucose

Xylose Glucose Xylose

Sucrose, Glucose Sucrose, Glucose Glucose, Xylose Glucose

MSX 9

Glucose

Glucose

Maleic acid Threonine Threonine Arginine, Oxalic, Threonine Succinic, Malonic acid Unidentified Unidentified Glutamate Maleic acid Arginine Arginine Methionine Malic, Maleic acid Arginine, Arginine, Unidentified Succinic, Glutamate Glutamate Malonic, Maleic acid Lysine Unidentified Unidentified Malonic acid

Lysine, Arginine Threonine Unidentified Methionine

Succinic acid Maleic acid Succinic acid Citric acid, Succinic acid, Maleic acid Succinic acid Succinic acid

Maleic acid Malonic acid Oxalic acid Succinic acid, Maleic acid Succinic acid Succinic acid

AVK 42

Glucose

Glucose

Glucose

Threonine, Lysine Arginine

Unidentified Threonine

AVK 51

Glucose

Glucose

Glucose

Arginine, Glutamate

Arginine

Maleic, Succinic acid Citric, Malonic acid

in case of wheat WH 711, E 12 (9 103) was followed by AVK 51 (7 103 ), IS 16 and Ala 27 (5 103 each). Plant response on Azotobacter inoculation If adaptation towards a specific host plant plays a role it might be argued that specific changes in root exudates of the host plant are induced after contact. In order to test this hypothesis, we performed inoculation assays and analyzed the resulting exudates. First we analyzed total sugar content as prepared C-source of the bacteria. The two varieties of cotton plants exuded 20.8 to 36 g ml1 sugars after inoculation with cultures of diazotrophs. Maximum sugar in exudates was found after inoculation with Ala 27 (36.2 g ml1) followed by AVK 42 (35.4 g ml1) and HT 57 (30.7 g ml1) in Desi cotton HD 123 whereas in American cotton variety H 1098, E 12 (37.1 g ml1) induced maximum excretion of sugars. The greatest effects of inoculation were seen with wheat where the maximum sugars in exudates was observed after inoculation with Ala 27 (106.3 g ml1) followed by HT 57 (102.8 g ml1). The other strains did not show an increase in total sugar excretion as compared to the control. Most of the exudates showed the presence of glucose as identified by paper chromatography except in confrontation with E 12 and HT 54, where xylose was
2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

also observed. Ala 27 induced excretion of fructose and HT 54 and HT 57 confrontation showed the presence of sucrose in wheat root exudates. Organic acids identified were citric acid, succinic acid, malonic acid and maleic acid in case of American cotton H 1098. In Desi cotton HD 123, oxalic acid, succinic acid, maleic, malonic and citric acid were observed while in case of wheat succinic, maleic and malonic acid were observed. Amino acids in the exudates of two cotton varieties were identified by paper chromatography and some common amino acids like glutamate, lysine, threonine and arginine were observed. In case of wheat, in addition to arginine, threonine, and glutamate, methionine was also observed. Few unidentified amino acids were also observed in exudates in the both crops (Table 1). Growth of Azotobacter on root exudates Wittenmeyer et al. (1995) and Merbach et al. (1996) observed sugars, amino acids and carboxylic acid with sugars usually representing the largest part in root exudates of maize plants. Azotobacter cultures were grown in 0.5% exudates alone and in glucose supplemented with exudates. Maximum growth was found with E 12, Ala 27 and Mac 27 in exudates of cotton HD 123. In American cotton H 1098 exudates, E 12 and
www.jbm-journal.com

Journal of Basic Microbiology 2007, 47, 436 439

Establishment of Azotobacter on plant roots

439

Ala 27 gave maximum growth in terms of optical density, whereas in few cultures it increased with addition of sugar. In case of wheat, increase in the growth of two cultures (Ala 27 and HT 57) was observed. Some of these compounds might be influencing the growth and development of surrounding plants and soil microorganisms. Our results indicate that Azotobacter chroococcum in the rhizosphere are stimulated by the presence of crop and specifically react to crop variety. The main source of this stimulation is the availability of complex carbohydrates near to or on plant roots for assimilation and growth. Plants, on the other hand, often benefit from the nitrogen compounds, either fixed or released from organic material by bacterial metabolism, e.g. A. vinelandii, a free living nitrogen fixing bacteria, ultimately depends on carbon released by the plants, while the plants growing in soils with low nitrogen colonized by these bacteria benefit from the fixed nitrogen compounds. This is an example for mutually beneficial associations of free living nitrogen fixing bacteria with no direct contact. Preference of Azotobacter strains for root exudates The bacteria clearly showed preference for one exudate over the other. This might well be related to the substances found in each exudate. As our results indicate, two strains, HT 54 and 1S-16, preferred wheat exudates at least 2-fold over cotton exudates. The other strains preferred cotton, but differentiated between the varieties. American cotton variety H1098 was preferred by 9 strains with 2 to 14-fold higher numbers attracted to variety American over Desi cotton HD 123 and Desi was preferred by 3 strains with 2.5 to 6-fold preferences. It therefore, seemed logical to analyze the root exudates of the different plants used. These differences were statistically significant. As can be seen from the control analysates in Table 1, most bacteria seemed to prefer citric acid (from American cotton) over oxalic acid (from Desi cotton) over succinic acid (from wheat). It might be argued that the energy yield from C6-compounds is better, thus resulting in observed preferences.

tant for the initiation and establishment of the association between host and bacterium, can be reached from the analyses of influences excerted by each interaction partner on the other. In addition, there is a need to know how diazotrophs may benefit the plant. Investigations on the production of phytohormones and the action of siderophores produced by Azotobacter strains might help to understand this aspect of interactions. A consideration of the factors that might play a role in interactions of bacteria and hosts has to be extended to the mechanisms likely to be involved in stable colonization of distinct microhabitats of the rhizosphere. Only then, application of the results seems possible in agriculture which may benefit the ecosystem if less mineral fertilizers have to be used in the future.

References
Bertin, C., Yand, X. and Weston, A., 2003. The role of root exudates and allelochemicals in the rhizosphere. Plant Soil, 256, 67 83. Block, R.J., Durrum, E.L. and Zweig, G., 1958. A Manual of Paper Chromatography and Paper Electrophoresis. Academic Press New York. Dubios, M., Gilles, K., Hamilton, J.K., Rebes, P.A. and Smith, F., 1951. Colorimetric method for the determination of sugars. Nature, 168, 167. Dudeja, S.S. and Khurana, A.L., 1988. Effect of high temperature on root exudate of pigeon pea-Rhizobium symbiosis. Ind. J. Microbiol., 28, 193 198. Gransee, A. and Wittenmeyer, L., 2000. Qualitative and quantitative analysis of water soluble root exudates in relation to plant species and development. J. Plant Nutr. Soil Sci., 163, 381 385. Lima, E., Boddey, R.M. and Dbereiner, J., 1987. Quantification of biological nitrogen fixation associated with sugarcane using a 15N aided nitrogen balance. Soil Biol. Biochem., 19, 165 170. Merbach, W., Knof, G., Augustin, J., Jacob, H.J., Jager, R. und Toussaint, V., 1996. kophysiologische Wechselbeziehungen zwischen Pflanze und Boden. In: Reaktionsverhalten von agrarischen kosystemen homogener Areale (Mhle, H., Claus, S., eds.), pp. 195 207. Teubner Verlagsgesellschaft Stuttgart. Narula, N., Saharan, B.S., Kumar, V., Bhatia, R., Bishnoi, L.K., Lather, B.P.S. and Lakshminarayana, K., 2005. Impact of the use of biofertilizers on cotton (Gossypium hirsutum) crop under irrigated agroecosystem. Arch. Agron. Soil Sci., 51, 69 77. Okon, Y., 1985. Azospirillum as a potential inoculant for agriculture. Trends Biotechnol., 3, 223 228. Okon, Y., Heytler, P.G. and Hardy, R.W.F., 1983. Nitrogen fixation by Azospirillum brasiliense and its incorporation into host Setaria italica. Appl. Environ. Microbiol., 46, 694 697. Wittenmeyer, L., Gransee, A. und Schilling, G., 1995. Untersuchungen zur quantitativen and qualitativen Bestimmung von organischen Wurzelausscheidungen bei Mais und Erbsen. Mitteil. Dtsch. Bodenkundl. Gesellsch., 76, 971 974.
www.jbm-journal.com

Conclusions
Our results obtained for the cotton/wheat association with diazotrophs indicate a high degree of adaptation between the host plant and the most abundant diazotrophs. The microhabitat provided by the host plant seems to generate a selection pressure in favor of the micro-organisms, which in turn best benefit the host. An understanding of the mechanisms, which is impor 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim