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Series G: Ecological Sciences, Vol. 37 Azospirillum VI and Related Microorganisms Genetics - Physiology - Ecology

Edited by Istvan Fendrik Institute of Biophysics,University of Hannover Herrenhauser StraBe 2 0-30419 Hannover, Germany Maddalena del Gallo Department of Food Technology, Environmental and Microbiological Sciences, University of Molise Via Tiberio 21 1-86100 Campobasso, Italy Jos Vanderleyden F. A. Janssens Laboratory of Genetics Department of Applied Plant Sciences, K. U. Leuven Willem de Croylaan 42 B-3001 Heverlee, Belgium Miklos de Zamaroczy Unite de Physiologie Cellulaire, CNRS URA 1300 Departement des Biotechnologies, Institut Pasteur F-75724 Paris Cedex 15, France

Springer Proceedings of the NATO Advanced Research Workshop on Azospirillum VI and Related Microorganisms, held at Sarvar, Hungary, September 4-7,1994

Llbrar-y of Congress Cataloglng-ln-Pu~llcatlon Data

Azosplrillum VI and related microorganisms: genetics, physiology, ecology / edited by Istv~n Fendrlk '" [et al.1. p. cm. -- (NATO ASI series. Series G, Ecological sciences no. 37) Includes bibliographical references and Index. ISBN-13:978-3-642-79908-2 (hard) 1. Azosplrillum. I. Fendrlk, I. II. Series. QR82.A9A9725 1995 589.9'5--dc20 95-33021 CIP

Additional material to this book can be downloaded from http://extra.springer.com.

ISBN-13:978-3-642-79908-2 e-ISBN-13:978-3-642-79906-8 001: 10.1007/978-3-642-79906-8 This work is subject to copyright. All rights are reserved. whether the whole or part of the material is concerned, specifically the rights oftranslation, reprinting. reuse of illustrations, recitation, broadcast• ing, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9. 1965, in its current version. and permission for use must always be obtained from Springer-Verlag. Violations are liable for prosecution under the German Copyright Law. © Springer-Verlag Berlin Heidelberg 1995 Soltcover reprint 01 the hardcover 1st edititon 1995 Typesetting: Camera ready by authors/editors Printed on acid-free paper SPIN 10477097 31/3136 - 5 4 3 210 VI

pathway might be dependent on changes in the proton motive force. Although the role of indole-3-acetic acid (IAA) biosynthesis in Azospirillum is still not clear, significant progress was reported on the genetics and biochemistry of IAA biosynthesis. In the "Ecology session", new and intriguing research fields were introduced: the growth of Azotobacter inside algal cells proves the possibility to produce stable and functional artificial symbiosis; the formation of stable coaggregates of Azospirillum and Rhizobium cells was reported as an interesting model for delivery beneficial with multiple applications. Using new methodologies, such as the use of ribosomal sequences, taxonomic and ecologic studies of diazotrophic bacteria were presented; soil parameters which affect Azospirillum survival were for the first time systematically analysed and lead to the conclusion that this bacterium cannot survive, or at least cannot survive in a high number in the soil, without the associated plant. As a logical consequence, tactic response appears to be one of the most important capability of Azospirillum to occupy its ecological niche. The key lecture on Field Application evaluated the physiological and agronomical aspects of Azospirillum inoculations. A survey of worldwide field experiments over the past 20 years revealed that in 60 to 70% of the trials, an increase in crop yield could be observed. These increases compensated the inoculation costs and replaced generally about 30% N-fertilizer. A better coordination of field experiments among the researchers and more clarification of the basic mechanisms of plant growth promotion will be necessary in the future. The workshop demonstrated how important it is to encourage contacts between scientists of NATO and Central and East European Countries in particular - to evaluate the impact of research on environmental friendly and low energetically demanding agriculture and food prodction. Such meetings are especially useful in eliminating differences between research activities of laboratories and in initiating co-operation in future projects. The exchange of ideas and results are improving efficiency both in basic and applied sciences. The Organizing Committee wants to express its deepest gratitude for the very generous support of the Workshop given by the North Atlantic Treaty Organization. The Organization Committee also wishes to thank the following sponsoring Organizations: Hannoversche Hochschulgemeinschaft, Agricultural University of G5dollo, University of Hannover, ENEA, Deutsche Forschungsgemeinschaft, University of Molise, Hungarian Academy of Sciences. We gratefully acknowledge the Township of Sarvar, especially Dr. M. Radics (Manager of the City) and I. SzabO (Major of Town). Our special thanks go to all members of the Local Organizing Committee (Peter GaaI, Eva Karpati), who were able to overcome all difficulties and are fully responsible for the success of the meeting. Many thanks must go to Roswitha Fendrik for her excellent management of the organization before and during the Workshop.

I. Fendrik Director M. Del Gallo Co-Director T. Sik Co-Director J. Vanderleyden Co-Director Preface

Three years ago the 5th Azospirillum Workshop was held in Hannover. Since 1991, Azospirillum research has made significant progress and the NATO A.R.W. in Sarvar/Hungary was very important to consolidate the present interest and to discuss novel aspects. Among these novel aspects, major emphasis was given to bacteria that live inside the plants - endophytes - and their mode of infection. Fungi are presently considered as good candidates for infection needles. This means that the ecosystem to consider becomes more complex and that the experimental settings to study so-called associative bacteria-plant interactions, might have to be adjusted. The Workshop covered in an interdisciplinary way, genetical aspects, interactions with other microorganisms, physiology and molecular biology, ecology and field applications. Among the large number of isolated from the rhizosphere of cereal crops, Azospirillum appears as a model system to study root colonization during association. However, in this type of association, which occurs in the absence of root differentiated structures, it is difficult to analyse what limits transfer to the host plant. Therefore new tools had to be developed, and are now successfully applied in Azospirillum and other associative diazotrophs. An area which has progressed rapidly in the last few years deals with plant-endophytic nitrogen fixing organisms. Several genera of bacteria are involved and these and their various hosts were reviewed by 1. D5bereiner. Also 1. Sprent developed this theme, discussing first, ways in which endophytic bacteria enter host plants and second, cells/intercellular spaces/vessels as an environment of supporting . This led to the formulation of key questions for further study in the near future. Endophytic bacteria are present in high numbers in all plants analysed (wild and cultivated ). This is known for a long time but was not considered in the right perspective. Now we are aware that endophytes are part of the plant ecosystem and it should therefore be possible to reconstruct a more equilibrated crop-environment through inoculation with selected endophytes and VAM-fungi. Meanwhile the study of Rhizobia as possible endophytes for non-leguminous plants continues. The group of E. Cocking studies the structure of pseudo-nodules induced in roots of wheat and rice by Rhizobium sp. Using the most advanced tools in molecular genetics, several groups reported on genome analysis, gene identification and gene regulation in Azospirillum. The new findings with respect to nif and gin gene regulation are of particular interest, since Azospirillum holds also for other plant root associated diazotrophs. It could also be observed from the various presentations at the Workshop, that statements, concerning the genetics and the physiology of nitrogen fixation and association with plant roots, are slowly merging, as was previously observed in the field of symbiotic nitrogen-fixing bacteria. In the session "Physiology and Molecular Biology" several papers focussed on the Wheat• Azospirillum interactions. Colonization of wheat roots causes alterations in the metabolism of both the plant and bacterial partner. In colonized plant roots, accumulation of hydroxy• proline rich proteins and activation of glycosidases occurs. The role of bacterial polysaccharides and plant lectins in the interaction Azospirillum-plant roots was addressed by several groups. New ideas for the lectin-recognition hypothesis have been forwarded and appear worthwhile to pursue in the future. It should be noticed that the lectin-hypothesis gains also renewed interest in the symbiotic Rhizobium-legumes interaction. With respect to sensing between Azospirillum and plant roots, nice studies on chemo- and aerotaxis of Azospirillum were presented. At least for , the main chemosensing TABLE OF CONTENTS

SECTION I - PLENARY LECTURES Endophytic Occurrence of Diazotrophic bacteria in Non-Leguminous Crops J. D6bereiner, V.L. Baldani and V.M. Reis ...... 3 N2-Fixation by Endophytic Bacteria: Questions of Entry and Operation J. I. Sprent and E. K. James ...... 15 Achievements in the Genetics of Nitrogen Fixation in Hungary T. Sfk ...... 31

SECTION II - GENETICS Azospirillum-Cereals: a Partnership with Future J. Vanderleyden, A. Costacurta, J. Desair, P. De Troch, S. Dobbelaere, V. Keijers, K. Michiels, A. Milcamps, S. Moens, A. My Bekry, D.Petersen, E. Van Bastelaere, A. Vande Broek, A. Van Dommelen...... 47 Phylogenetic Studies of the Genus Azospirillum R. Fani, C. Bandi, M. Bazzicalupo, G. Damiani, F.P. Di Cello, S. Fancelli, E. Gallori, L. Gerace, A.M. Grifoni, P. Lib and E. Mori ...... 59 Genetic Control of Nitrogen Assimilation and Nitrogen Fixation in Free Living Azospirillum brasilense: a Review M. de Zamaroczy ...... 77 Involvement of nitA and glnB Genes in Expression of Nitrogen Fixation Genes in Azospirillum brasilense F. Arsene and C. Elmerich ...... 91 Polysaccharide Synthesis in Azospirillum brasilense P. De Troch, D. J. Petersen and J. Vanderleyden ...... 97 Biodegradation of Polysaccharides by Azospirillum V. Keijers, S. Dobbelaere, M.A. Bekri and J. Vanderleyden ...... 105 Genetical Aspects of Indole Acetate Production in Azospirillum brasilense Sp245 E. Katzy, L. Petrova, I. Borisov and V. Panasenko ...... 113 Auxin Biosynthesis and Denitrification in Plant Growth Promoting Bacteria W. Zimmer, K. Kloss, B. Hundeshagen, E. Niederau and H. Bothe ...... 121 Azospirillum Phylogeny Based on rrs (16S rRNA Gene) Sequences R. Bally, J. Haurat and P. Normand ...... 129 Involvement of Ion Protease of Azospirillum brasilense in Iron Uptake E. Mori, R. Fani, M. Fulchieri, E. Gallori and M. Bazzicalupo ...... 137 Cloning and Analysis of an Azospirillum brasilense Iteron and hsl UV Operon Containing Region P. Lib, M. Bazzicalupo, AM. Grifoni, E. Mori and R. Fani ...... 143 VIII

Cloning of a Streptomyces Endoglucanase Gene in Azospirillum brasilense B. Perito, R Fani and G. Mastromei ...... 149 Initiation of Monocot Plant Development in vitro for Studying Plant-Microbe Interaction G. Gyulai, L. Murenyetz, J. Janovszky, M. Tarczi,1. Racz ...... ;155 Chitinase Expression in Strawberry Root Colonization by Azospirillum brasilense and V.A Mycorrhiza. C. H. Bellone, S. C. de Bellone and R O. Pedraza ...... 161

SECTION III - INTERACTIONS WITH OTHER MICROORGANISMS Endophytic Bacteria: Their Possible Role in the Host Plant S. Di Fiore and M. del Gallo ...... 169 YAM-Fungi and Their Interactions with Other Microorganisms F. Schonbeck ...... 189 Interaction of Rhizobia with Non-Legume Crops for Symbiotic Nitrogen Fixation Nodulation E. D. Cocking, S. L. Kothari, C. A. Batchelor, S. Jain, G. Webster, J. Jones, J. Jotham and M. R. Davey ...... 197 Diazotrophic Mixed Cultures of Azospirillum brasilense and Enterobacter cloacae P. Kaiser ...... 207 Molecular Study of the Interaction of with Wheat Germ Agglutinin E. Karpati, P. Kiss, M. Afsharian, F. Marini, S. Buglioni,1. Fendrik and M. Del Gallo ...... 213 Short-Term Attachment of Bacteria from the Rhizobiaceae Family to the Roots of Cereals G. K. Solovova, V. A. Velikov, Y. V. Krivopalov and M. 1. Chumakov ...... 223 Interactions Between Azospirillum and Phialophora radicicola F. Flouri, K. Sini and C. Balis ...... 231 Establishment of Two Nitrogen-Fixing Bacteria on Roots of Kallar Grass Using Alginate-Coated Seed in Mixed Inoculation T. Stein, J. Ueckert and 1. Fendrik ...... 239 First Results of Individual and Dual Inoculation with Azospirillum• Glomus on Wheat A. Gori and F. Favilli ...... 245 Morphogenesis of Strawberry Roots Infected by Azospirillum brasilense and V.A. Mycorrhiza C. H. Bellone and S. C. de Bellone ...... 251 Specific Interaction Between Chickpea (Cicer arietinum L.) and Three Chickpea-Rhizobium Strains Inoculated Singularly and in Combination with Azospirillum brasilense Cd P. Fabbri and M. del Gallo ...... 257 IX

SECTION IV - PHYSIOLOGY AND MOLECULAR BIOLOGY Interactions Between Partners in the Association "Wheat• Azospirillum brasiiense Sp245" V. Ignatov, G. Stadnik, O. Iosipenko, N. Selivanov, A. Iosipenko and E. Sergeeva ...... 271 Azospirillum brasiiense Exopolysaccharide Complexes, Their Possible Involvement in Bacteria-Wheat Roots Interactions and the Suggested Nature of These Interactions I. Skvortsov, S. A. Konnova, O. Makarov, R. Prokhorova and V. Ignatov ...... 279 Continuous Culture Application in Physiological investigations of Diazotrophic Bacteria J. Ueckert and I. Fendrik ...... 291 Changes in Membrane Potential upon Chemotactic Stimulation of Azospirillum brasilense . I. B. ZhuIin, L. E. Sarmiento and B. L. Taylor ...... 299 Physiological Aspects of Phytohormone Production by Azospirillum brasilense Sp7 A. Iosipenko and V. Ignatov ...... 307 Laccase Activity in Azospirillum lipoferum: Characteristics, melanization and Tn5 Mutants D. Faure, C. Jacoud, M. L. Bouillant and R. Bally ...... 313 Wheat Lectin Possibly Serves as a Signal Molecule in the Azospirillum-Wheat Association L. P. Antonyuk, O. Fomina, A. Kalinina, S. Semenov, M. Nesmeyanova and V. Ignatov ...... 319 Biochemical Characterization of IAA Mutants of Azospirillum brasilense Sp7 Induced by Transposon Tn5 Mutagenesis G. Gastelum-Reynoso, C. Cruz-Rufz, Y. G. Xochihua-Corona, J. Luzuriaga-Galicia and B. E. Baca ...... 325 Influence of Asparagine and Aspartic Acid on Growth of Azospirillum K. Sini, F. Flouri and C. Balis ...... 331 Inoculation with Azospirillum brasilense Cd Affects the Root System Development of Sorghum bicolor P. Barbieri, C. Trambaioli, G. Zanetti and E. Galli ...... 335 Laccase Activity in Azospirillum lipoferum: Substrates and Inhibitors C. Jacoud, D. Faure, A. Effosse, P. Wadoux and M. L. Bouillant ...... 341 Conversion of Tryptophan, Indole-3-Pyruvic Acid, Indole-3-Lactic Acid and Indole to Indole-3-Acetic Acid by Azospirillum brasilense Sp7 T. Bar and Y. Okon ...... 347 Transformation of Aniline and Its Chlorinated Derivatives by Azotobacter agilis S. Russel and 5. Muszynski ...... 361 Reduction of 4-Chloronitrobenzene by Azospirillum lipoferum S. Russel and S. Muszynski ...... 369 x

Immunological Properties of Azospirillum Cell Surface: the Structure of Carbohydrate Antigens and Evaluation of Their Involvement in Bacteria-Plant Contact Interactions L. Matora, G. Solovova, O. Serebrennikova, N. Selivanov, S. Shchyogolev ...... 377 Role of Choline and Glycine Betaine in Azospirillum brasilense Cd under Salinity Stress A. K. Tripathi, B. M. Mishra and M. Schloter ...... , ...... 383 Bioreactor Systems for Research on Rhizosphere Bacteria J. Ueckert, K. Huckfeldt and I. Fendrik ...... 393

SECTION V - ECOLOGY Artificial Plant-Azotobacter Symbiosis for Atmospheric Nitrogen Fixation I. Gyurjan, P. Koranyi, E. Preininger, Sz. S. Varga and G. Paless ...... 401 Taxonomic and Ecologic Studies of Diazotrophic Rhizosphere Bacteria Using Phylogenetic Probes A. Hartmann, J. I. Baldani, G. Kirchhof, B. ABmus, P. Hutzler, N. Springer, W. Ludwig, V. L. D. Baldani and J. D6bereiner ...... 415 Coaggregation of Azospirillum with other Bacteria: Basis for Functional Diversity C. A. Neyra, A. Atkinson and O. Olubayi ...... 429 Soil Parameters wich Affect the Survival of Azospirillum brasilense Y. Bashan, M.E. Puente, M. N. Rodriguez-Mendoza, G. Holguin, G. Toledo, R. Ferrera-Cerrato and S. Pedrin ...... 441 Chemotaxis in Plant-Associated Bacteria: the Search for the Ecological Niche I. B. Zhulin and B. L. Taylor ...... 451 Occurrence, Isolation and Identification of Azospirillum Strains in Greece I. Kefalogianni, F. Flouri and C. Balis ...... 461 Influence of the Carbon Substrate on the Composition of the Exocellular Polysaccharides by Azospirillum brasilense F. Marini, S. Speranza and M. del Gallo ...... 467 Identification of Azospirillum by Oligonucleotide Probes After Isolation from Soil and Sorghum Rhizoplan Contaminated or not by the Parasitic Plant: Striga M. Kabir and D. Faure ...... 479 Ecological Aspects of Azorhizobium caulinodans in the Rhizosphere of Rice Seedlings C. Van Nieuwenhove, L. Van Holm, R. Vanden Bruel and K. Vlassak .... .487 Associative, Symbiotic N2-Fixers and Scavenger Strains, Affected by Cu2+ and Zn2+ in vitro B. Bir6, H. E. A. F. Bayoumi Hamuda and M. Kecskes ...... 495 XI

SECTION VI - FIELD EXPERIMENTS, APPLICATIONS Inoculation Effects of Azospirillum on Sunflowers (Heliantus annus) Under Different Fertilization and Irrigation Regimes R. Itzigsonh, Z. Abbass, S. Sarig and Y. Okon ...... 503 Inoculation of Genetically Modified Strains of Azospirillum: Monitoring of Population Dynamic C. Fogher, M. Delledonne and L. Frigerio ...... 515 Growth Stimulation in Graminees, Legumes, and Crucifers by Pseudomonas-Bacteria G. Hoflich and W. Wiehe ...... 523 Experimental System for Creating New Type of N2-Fixing Plants Sz. S. Varga, E. Preininger, P. Koninyi and I. Gyurjan ...... 529 Survival of Associative Nitrogen Fixers in Rhizoplane as a Criterion for Estimation of Their Effect on Inoculated Plants A. A. Belimov, A. M. Kunakova, E. G. Alekseyeva, O. N. Martsinkevitch and A. P. Kojemyakov ...... 535 Adhesion of Rhizosphere Bacteria to Roots of Maize and Wheat R. Pastorelli, A. Gori and F. Favilli ...... 543 Occurrence of Dinitrogen-Fixing Bacteria and Acetylene Reduction Activity in a Rhizosphere of Five Selected Plants from Tropics S. Russel and C. Chidolue Ifiorah ...... 549 Dominant N2-Fixing Microflora in a Paddy Soil and Effect of Rice Inoculation with Azospirillum Bacteria N. Kaloianova and Ognian Kostov ...... 555 Isolation and Characterization of Azospirillum Strains from Soil and Rice Plants in North Vietnam N. Ngoc Dung, M. Kraut and W. Klingmiiller ...... 559

Subject Index ...... 567

List of Participants from NATO Countries ...... 571

Other Participants ...... 572

List of Participants from Non-NATO Countries ...... 574