ISSN 1510-7809

LOTUS NEWSLETTER 2005 Volume 35, Number 1

Editor: M. Rebuffo INSTITUTO NACIONAL DE INVESTIGACION AGROPECUARIA Editor: M. Rebuffo INSTITUTO NACIONAL DE INVESTIGACION AGROPECUARIA

Editorial Office INIA La Estanzuela This Newsletter consists of informal Colonia, Uruguay reports which are presented to further Phone: +598-574-8000 the exchange of ideas and information Email: [email protected] between research workers. Fax No.: +598-574-8012 Consequently the data presented here Web: http://www.inia.org.uy/sitios/lnl/ are not to be used in publications without the consent of the authors. Images are copyright of the authors, and their reproduction is strictly prohibited without their consent.

Front cover: The photographs on the front cover shows the symptoms of crown and root rot in a diseased birdsfoot trefoil plant (left), which was recorded during the epidemiological studies carried out in Uruguay by Altier and Kinkel (pp. 42-58). Middle- up: the most frequently and consistently isolated species from diseased crown and root tissues was Fusarium oxysporum. Middle-down: The lack of complementation among the isolates that composed the F. oxysporum population associated with birdsfoot trefoil indicated a large genetic diversity, as measured by vegetative compatibility. Right: A culture plate method was used by Altier and Groth (pp. 59-74) to characterize F. oxysporum isolates for aggressiveness to seeds and seedlings of birdsfoot trefoil: significant differences were observed among the isolates.

The opinions in this publication are those of the authors and not necessarily those of the Lotus Newsletter. The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the Newsletter concerning the legal status of any country, territory, city, or area, or of its authorities, or concerning the delimitation of its frontiers or boundaries. Where trade names are used this does not constitute endorsement of or discrimination against any product by the Newsletter. Lotus Newsletter (2005) Volume 35, Number 1.

Contents

Contents i

Newsletter Announcements and Instructions iv

Personalia vi

Conference announcement

P.M. GRESSHOFF. Third International Conference on Legume Genomics and Genetics: From Genes to Crops 1

Workshop abstracts

O. RUIZ (Chairperson). Interdisciplinary workshop on genetic, molecular and eco- physiological aspects of Lotus spp. and their symbionts. [Taller interdisciplinario sobre aspectos genéticos, moleculares y fisioecológicos del Lotus spp. y sus simbiontes]. 3

E. CAMADRO. Theoretical bases for the elaboration of a breeding program. Lotus glaber as an example. 7

M. REBUFFO. Plant breeding: Lotus corniculatus and Lotus uliginosus. 9

H. ACUÑA and A. CONCHA. Condensed tannin concentrations in Lotus spp. 11

A. CLÚA, M. BARRAGÁN, M.S. TACALITI, D. GIMÉNEZ and A.M. CASTRO. Assisted and traditional assessment of saline stress tolerance in Lotus glaber. 13

M. SISTERNA and G.A. LORI. Fungal diseases on Lotus spp in Argentina. 15

A. ALIPPI. Bacterial diseases of Lotus spp. 17

F. CASSÁN, V. LUNA and O.A. RUÍZ. Physiological studies of tolerance to saline stress in Lotus glaber and their correlation with the establishment and efficiency of the symbiotic association with Mesorhizobium loti. 19

C. LABANDERA and M. JAURENA. Rhizobiology research in Lotus species. 21

A.I. SANNAZZARO, A.B. MENÉNDEZ, E. ALBERTÓ and O.A. RUIZ. The arbuscular mycorrhizal fungi of Lotus glaber. 22

R. MENDOZA. Seasonal variation of arbuscular mycorrhizal fungi in temperate grasslands along a wide hydrologic gradient. 23

D. COGLIATTI, L. LETT, M. BARUFALDI, P. SEGURA and J. CARDOZO. Growth, Nitrogen and Phosphorus economy in two Lotus glaber Mill. Populations grown under contrasting P-availability. 24

i

G.G. STRIKER, P. INSAUSTI and R.J.C. LEÓN. Comparative responses between Lotus glaber and Paspalum dilatatum to the flooding-grazing interaction. 25

A. ANDRES, B. ROSSO and O. SCHENEITER. Morpho-physiological characterization of Lotus glaber naturalized populations. 27

A.I. SANNAZZARO, E. ALBERTÓ, O.A. RUIZ and A.B. MENÉNDEZ. Influence of the arbuscular mycorrhizal fungus Glomus intraradices on the saline stress physiology of Lotus glaber. 29

R. PAZ, D.H. SANCHEZ, F. PIECKENSTAIN, S. MAIALE, A. SANNAZZARO, J. CRUZ CUEVAS, A. CHIESA, G. BONA and O.A. RUIZ. Molecular and biochemical approximation of polyamine roles in tolerance mechanisms to salt stress in Lotus spp. 31

P.A. SANSBERRO, F.D. ESPASANDIN, C.V. LUNA and L.A. MROGINSKI. Adventitious shoot regeneration in Lotus glaber Mill. 33

A.V. AVILA JACQUES and I. HERINGER. Fire on native pastures - effects on soil and vegetation. 35

M. BAILLERES. Is arriving the Lotus glaber time in the pampa deprimida del salado? 36

M. DALL´AGNOL and S.M. SCHEFFER-BASSO. Perspectives of utilization of native legumes in Rio Grande do Sul. 37

O.R. VIGNOLIO. Lotus glaber productivity changes under different management conditions. 39

Feature articles and reports

N.A. ALTIER and L.L. KINKEL. Epidemiological studies on crown and root rot of birdsfoot trefoil in Uruguay. 42

N.A. ALTIER and J.V. GROTH. Characterization of aggressiveness and vegetative compatibility diversity of Fusarium oxysporum associated with crown and root rot of birdsfoot trefoil. 59

J. REYNAUD and M. LUSSIGNOL. The Flavonoids of Lotus corniculatus 75

P. DÍAZ, O. BORSANI, A. MÁRQUEZ and J. MONZA. Nitrogen metabolism in relation to drought stress responses in cultivated and model Lotus species. 83

V.C. LEPEK and A.L. D´ANTUONO. Bacterial surface polysaccharides and their role in the rhizobia-legume association. 93

S.L. GREENE. U.S. Germplasm Collection of Lotus: Activities over the last decade. 106

C. PACIOS-BRAS. The symbiosis between Lotus japonicus and rhizobia: Function of nod factor structural variation. [Spanish version] 109

ii Lotus Newsletter. 2003. Volume 33.

R. MENDOZA, I. GARCÍA and V. ESCUDERO. Can the symbiosis between arbuscular mycorrhiza and Lotus glaber tolerate waterlogging in a saline-sodic soil? 115

S. HERNÁNDEZ, M. REBUFFO, S. ARRIVILLAGA, M. JAURENA, C. LABANDERA, D. RISSO and J. CILIUTI. Evaluation of the genotype-environment interaction in the establishment of Lotus uliginosus (Schkuhr) with soil-cores. [Spanish version] 120

Research centers and projects

P.M. GRESSHOFF. The Australian Research Council’s Centre of Excellence for Integrative Legume Research 131

J. SANJUÁN and M. REBUFFO. The LOTASSA proposal: the success of enthusiasm and tenacity. 134

D. REAL, G.A. SANDRAL, J. WARDEN, M. REBUFFO, D.F. RISSO, J.F AYRES, W.M KELMAN and S. J. HUGHES. Breeding birdsfoot trefoil for Mediterranean-type environments in Southern Australia 136

Lotus activities: Background and present research 138

Current list of Lotus researchers. Database last updated August 30 2005 143

iii Lotus Newsletter (2005) Volume 35. Number 1.

Newsletter Announcements and Instructions

The Lotus Newsletter has been published annually since 1971 by Dr. W.Grant - Canada, Dr. R.McGraw and Dr. P.Beuselinck - USA, and it has been on the web site of INIA, Uruguay since 2003. It is intended as a worldwide communication link for all those who are interested in the research and development of Lotus species. Persons interested in trefoil improvement, genetics, molecular biology, microbiology, production, marketing, or utilization are invited to contribute to the Lotus Newsletter. Previous issues may be used as a guide. It is expected that the work reported will be developed further and formally published later in refereed journals. It is assumed that contributions in LN will not be cited unless no alternative reference is available.

The life of the Newsletter depends on the contributions. Therefore, many thanks to all contributors and to those that wrote the summary about their activities. In response to several contributors, that requested a more frequent edition of LN, two numbers of Volume 35 will be on the web site this year: Number 1 in August and Number 2 in December.

The web site is under transformation, since it will host two databases. The first database will be available by the end of year 2005 and it will consist of all researchers’ information. The second database for the Literature will be available as a small database in 2005 and it will be built throughout year 2006 with the researchers’ contributions. I would like to encourage everybody to make contributions to the list of bibliography for next year.

Acknowledgement

I would like to express my gratitude to Dr. Paul Beuselinck and Dr. William F. Grant for their permanent support and advice. We can provide electronic copies of all articles that are not available on the web, since Dr. Paul Beuselinck donated all his collection of LN issues. Dr. W. Grant made a great contribution last year with the list of cultivars of Lotus species of agronomic relevance. Although he will not be able to write new articles for LN, we will all share his permanent enthusiasm through the email.

INIA provided funding for my attendance to the XX International Grassland Congress, held in Dublin in June 2005, where I presented a poster about LN in the session “The role of the International Grassland Congress and Grassland Societies in technology interaction and influencing policy: a discussion session”. All papers related to trefoil will be summarized for Volume 35 Number 2, and I invite all who wish to contribute with their view of the Congress.

The priority of the Newsletter was the update of the recipient list, and I am pleased to inform that the registration increased to 185 researchers in August 2005. Thanks to all for helping me with this task.

What to contribute?

Send us the kind of information you would like to see in LN. - Contributions should be current, scholarly, and their inclusion well-justified on the grounds of new information. - Results of recently concluded experiments, recent additions to germplasm collections, information on new or tentative cultivars with descriptions. We want to include an adequate cultivar description, including disease reactions and origin if possible. - Notes on acreage, production, varieties, diseases, etc., especially if they represent changing or

iv Lotus Newsletter (2005) Volume 35. Number 1.

unusual situations. - Genome maps and information on probe-availability and sequences, and populations synthesized for specific traits being mapped. Glossy black and white prints of maps should be included, if possible. Partial maps can also be submitted. - Short reports of workshops, conferences, symposia, field days, meetings, tours, surveys, network activities, and recently launched or concluded project. - Details of recent publications, with full bibliographic information and short reviews. - Personal news (new appointments, awards, promotions, change of address, etc.)

How to format contributions

- Include the full address with telephone, fax, and e-mail numbers of all authors. - Keep the items brief – remember, LN is a newsletter and not a primary journal. - Give the correct Latin name of every crop, pest, or pathogen at the first mention. - If possible, table should fit within the normal typewritten area of a standard upright page (not a ‘landscape’ page). You may include figures and photographs (black and white or color). Please send disk-files (with all the data) whenever you submit line figures and maps. - Supply the essential information: round off the data-values to just one place of decimal whenever appropriate, choose suitable units to keep the values small (e.g. use tons instead of kg). - All lists of references should have been seen in the original by the author and year. Provide all the details such as author/s, year, title of the article, full title of the journal, volume, issue, and page numbers (for journal articles), and place of publication and publishers (for books and conference proceedings) for every reference. Incomplete references will not be accepted. - The language of the Newsletter is English, but we encourage researchers to submit their articles in other languages as well; the translation will be linked within the LN web site. Authors should closely follow the style of the reports in this issue. Contributions that deviate markedly from this style will be returned for revision, and could miss the publication date. If necessary, we will edit communications so as to preserve a uniform style throughout the Newsletter. This may shorten some contributions, but particular care will be taken to ensure that the editing will not change the meaning and scientific content of the article. Wherever we consider that substantial editing is required, we will send a draft copy of the edited version to the contributor for approval before printing. - Contact the Editor for detailed guidelines on how to format text.

Material may be submitted at any time during the year. Deadline for Volume 35 Number 2 will be 30th November 2005. Please send all contributions and request for inclusion in the recipients list to:

Monica Rebuffo, Lotus Newsletter Editor, c/o INIA La Estancuela, 70000 Colonia, Uruguay Email [email protected] Fax +598-574-8012

v Lotus Newsletter. 2005. Volume 35 (1).

Personalia

Welcome to the new subscribers of the Lotus Newsletter: Aïssa Abdelguerfi – Algerie; Adriana Alippi, Analía Sannazzaro, Marina Sisterna and Pedro Alfonso Sansberro – Argentina; Steve Hughes – Australia; Edgar Cárdenas – Colombia; Doublas Martin and Andy Pollard – Falklands Islands; Seishiro Aoki – Japan; Katharina Pawlowski – Sweden; Heathclife Riday and Matt Sanderson – USA; Athole Marshall, Jilliam Perry and Christina Marley – United Kingdom.

Several Lotus researchers have been recently reassigned or no longer work on the subject. Dr Lene Krusell moved from Max Planck Institute of Molecular Plant Physiology – Germany to University of Aarhus – Denmark. Dr. Jiri Stiller – USA, has switched the fields and currently work on sugarcane. Dr. Mark McCaslin – USA, is president of Forage Genetics International. Dr. Andreas Roussis – Denmark, is presently working at the Center for Human and Clinical Genetics, Leiden University Medical Center in The Netherlands. Dr. Ken Vogel – USA, no longer works with Lotus since his research focus on sorghum and forage grass species. Dr. Mónica Tourn – Argentina, works with tropical pastures at the present time.

The following personal information has been updated during the current year: Ana Arambarri, José De Batista and Miguel Cahuépé – Argentina; John Ayres – Australia; William F. Grant – Canada; Lene Heegaard Madsen, Christina Cvitanich, Niels Sandal and Lene Krusell – Denmark; Emanolis Flementakis – Greece; Ariel Asuaga – Uruguay;

vi Lotus Newsletter (2005) Volume 35 (1), 1-2.

Third International Conference on Legume Genomics and Genetics: From Genes to Crops

PETER M. GRESSHOFF* ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia http://www.iclgg3.org/ *Corresponding author

The major aim of research is to discover the connection between structure and function. This biennial conference focuses on the genetics and genomics of one of the major plant groups, namely the legumes. Legumes are major crop plants for the benefit of human food, animal feed, vegetable oil and nutriceutical production. Their benefits are recognized around the world. Legumes also serve as a model plant, providing useful information for the improvement of other crops such as tomato, sunflower, cotton, corn and rice. The biochemistry of legumes is distinct from that of other plant groups and many unique molecules, often used in biomedical applications, are found among legume metabolites. Isoflavones and plant sterols are just two major examples.

The ICLGG-3 will be held in Brisbane, Australia and is hosted by the ARC Centre of Excellence for Integrative Legume Research. The meeting will bring together world experts, reporting their findings in both crops as well as model legumes. The model legume Lotus japonicus and Medicago truncatula have led the recent push towards understanding the genomic structure of legumes. By April 2006 a significant part of their genomes will be available, allowing comparative genomic approaches. Concurrently the rate of gene discovery in complex developmental processes, such as lateral branching, embryogenesis, flower development, and nodulation has increased to a rapid rate, providing first-time insights into the mechanistic events underlying plant development. Many of the discoveries were impossible in the existing model plant rice and Arabidopsis, because of their growth habit and biological inabilities to develop, for example, nodules.

The central theme will be: From genes to crops. Thus reductionist and fundamental science will be reported together with ‘real world’ applications and progress. The format of the meeting will include plenary lectures of broader interest and overview, short research reports, and integrative discussion sessions. Experts from related fields of yeas, Arabidopsis or human genomics will provide possibilities to discuss future directions of the legume genomics field. Experts from the bio-pharmaceutical and nutritional sciences, representing end users will be invited. Posters will provide a venue of large scale data presentation and a forum for discussion.

1 2 PeterM. Gresshoff

Lotus Newsletter (2005) Volume 35 (1), 3-6.

Interdisciplinary workshop on genetic, molecular and eco-physiological aspects of Lotus spp. and their symbionts. [Taller interdisciplinario sobre aspectos genéticos, moleculares y fisioecológicos del Lotus spp. y sus simbiontes].

Chairperson: OSCAR A. RUIZ*

Held at: Instituto de Investigaciones Biotecnológicas - Instituto Tecnológico de Chascomús. IIB-INTECh. (UNSAM-CONICET). Chascomús. Provincia de Buenos Aires. Argentina. 9 –10 September 2004 * Corresponding author

Program

Day 9 September: Plant breeding workshop

Discussion Leader: Ing. Agr. MSc, RAÚL RODRÍGUEZ (Unidad Integrada Balcarce)

Planteo teórico sobre cómo se elabora un plan de mejoramiento genético, tomando al Lotus como base [Theoretical bases for the elaboration of a breeding program, with Lotus as an example.] Dra. ELSA CAMADRO, Unidad Integrada Balcarce, CONICET, Argentina

Mejoramiento genético: Lotus corniculatus y Lotus uliginosus. [Plant breeding: Lotus corniculatus and Lotus uliginosus.] Ing.Agr. M.Phil. MÓNICA REBUFFO, INIA, Estación Experimental “La Estanzuela”, Colonia, Uruguay.

Evaluación de las concentraciones de taninos en una población de Lotus glaber naturalizada. [Evaluation of tannin concentrations in a population of naturalized Lotus glaber.] Ing.Agr. MSc. PhD HERNÁN ACUÑA, INIA, Estación Experimental Quilamapu, Chile.

Evaluación de una población seleccionada de Lotus glaber frente al estrés salino. [Evaluation of a selected population of Lotus glaber under saline stress.] Dra. ANA CASTRO, Facultad de Ciencias Agrarias, Universidad Nacional de La Plata, Argentina.

3 4 Oscar Ruiz

Phytopathology Workshop

Discussion Leader: Ing.Agr. ADRIANA M. ALIPPI (CIC - Centro de Investigaciones de Fitopatología, Fac. de Cs. Agrarias y Ftales, UNLP).

Enfermedades de origen fúngico. [Fungal diseases.] Ing.Agr. GLADYS A. LORI and Ing.Agr. MARINA SISTERNA, CIC - Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, UNLP, Argentina.

Enfermedades de origen bacteriano. [Bacterial diseases.] Ing.Agr. ADRIANA ALIPPI, CIC - Centro de Investigaciones de Fitopatología, Facultad de Ciencias Agrarias y Forestales, UNLP, Argentina.

Microbiology Workshop

Discussion Leader: Dr. JUAN SANJUAN (CSIC – Estación Experimental del Zaidin, Departamento de Microbiología del Suelo y Sistemas Simbióticos, Granada, Spain).

Evaluaciones de biodiversidad y algunas consideraciones tecnológicas acerca de la producción de un inoculante eficiente para el Lotus glaber. [Evaluation of biodiversity and some technical considerations on the production of efficient inoculant for Lotus glaber.] Dr. FABRICIO CASSÁN, Universidad Nacional de Río Cuarto e IIB-INTECh (UNSAM-CONICET), Argentina.

Actividades de Investigación del Departamento de Microbiología de Suelos en Rhizobiología en especies del Género Lotus. [Research activities of the Department of Soil Microbiology on Rhizobiology in species of the Genus Lotus.] Licenciado MARTÍN JAURENA, Laboratorio de Microbiología de Suelos, Ministerio de Agricultura, Ganadería y Pesca, Uruguay.

Biodiversidad de micorrizas asociadas al Lotus glaber en la Pampa Deprimida del Salado. [Biodiversity of mycorrhiza associated to Lotus glaber in the Pampa Deprimida del Salado.] Licenciada ANALÍA SANNAZZARO. IIB-INTECh (UNSAM-CONICET), Argentina.

Asociación Lotus-Micorrizas en pastizales afectados por gradiente hidrológico y salino. [Association Lotus-Mycorrhiza in grasslands affected by hydrologic and saline gradient.] Dr. RODOLFO MENDOZA, CEFYBO-CONICET, Argentina.

Lotus Workshop, Argentina 5

Day 10 September: Physiology Workshop

Respuestas comparativas entre Lotus glaber y Paspalum dilatatum a la interacción pastoreo-inundación. [Comparative responses between Lotus glaber and Paspalum dilatatum to the flooding-grazing interaction.] Dr. GUSTAVO STRIKER, Dr. PEDRO INSAUSTI and Dr. ROLANDO LEÓN, IFEVA, Facultad de Agronomía de la Universidad de Buenos Aires (UBA), Argentina.

Caracterización morfo-fisiológica de poblaciones naturalizadas de Lotus glaber. [Morpho-physiological characterization of Lotus glaber naturalized populations.] Ing.Agr. MSc. PhD. ADRIANA ANDRES, Ing.Agr. MSc. BEATRIZ ROSSO, Ing.Agr. MSc. OMAR SCHENEITER, INTA-Pergamino, Argentina.

Economía del nitrógeno y del fósforo en dos poblaciones de Lotus glaber crecidas bajo condiciones contrastantes de fósforo. [Nitrogen and Phosphorus economy in two populations of Lotus glaber Mill. grown under contrasting conditions of Phosphorus.] Dr. DANIEL COGLIATTI, Ing. LINA LETT, Ing. MÓNICA BARUFALDI, Ing. PAOLA SEGURA and Dr. JORGE CARDOZO. Facultad de Agronomía de Azul, Universidad del Centro de la Provincia de Buenos Aires y Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.

Influencia de las micorrizas sobre la fisiología del estrés salino en Lotus glaber. [Influence of mycorrhiza on the saline stress physiology of Lotus glaber.] Dr ANA MENÉNDEZ, Facultad de Ciencias Exactas de la UBA e IIB-INTECh (UNSAM-CONICET), Argentina.

Biotechnology Workshop

Una aproximación bioquímica y molecular del rol de las poliaminas en la tolerancia al estrés salino del Lotus glaber. [Molecular and biochemical approximation of polyamine roles in tolerance mechanisms to salt stress in Lotus glaber.] Licenciada ROSALÍA PAZ, IIB-INTECh (UNSAM-CONICET), Argentina.

Cultivo in vitro de Lotus glaber. [In vitro culture of Lotus glaber Mill.] Dr PEDRO SANSBERRO, IBONE, Facultad de Ciencias Agrarias de la Universidad Nacional del Nordeste (UNNE), Argentina.

Ecology, Management and Production Workshop

Queima de pastagens naturais - efeitos sobre o solo e a vegetação. [Fire on native pastures - effects on soil and vegetation.] Dr. AINO VICTOR AVILA JACQUES, Professor Titular Aposentado da Universidade Federal do Rio Grande do Sul – UFRGS, Brazil.

6 Oscar Ruiz

Lotus glaber, su nuevo rol en los sistemas ganaderos de la Cuenca del Salado. [Lotus glaber, its new role in the grazing systems of “Cuenca del Salado”.] Ing. Agr. MATÍAS BAILLERES, Estación Experimental de Manantiales, Ministerio de Asuntos Agrarios de la Provincia de Buenos Aires, Argentina.

Perspectivas de Utilização de Leguminosas Nativas en RGS. [Perspectives of utilization of native legumes in Rio Grande do Sul.] Dr MIGUEL DALL´AGNOL Professor da UFRGS, Faculdade de Agronomia, Dep. de Plantas Forrageiras e Agrometeorologia, Brazil.

Cambio de la productividad del Lotus spp. bajo distintos tipos de manejo. [Changes in the productivity of Lotus spp. under different management conditions.] Dr. OSVALDO VIGNOLIO, Universidad Nacional de Mar del Plata, Unidad Integrada Balcarce, Argentina.

Lotus Newsletter (2005) Volume 35(1), 7-8. Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Theoretical bases for the elaboration of a breeding program. Lotus glaber as an example.

ELSA CAMADRO* EEA Balcarce, INTA-FCA, UNMdP. CONICET. C.C. 276, 7620 Balcarce, Bs. As., Argentina * Corresponding author

Before starting a breeding program, problems or aspects that need to be improved in the species of interest have to be identified and listed. Among others, the following are apparently of concern in Lotus glaber: slow seed germination, seedling weakness, lack of persistence, susceptibility to stresses (abiotic: soil salinity and flooding, and biotic: insects) and winter and early spring forage production. Next, it is necessary to consider: which problems can be solved by cultural and/or chemical practices, what is the effectiveness of these practices and their cost in relation to the expected results, which could be solved only by breeding and, if the solution to a problem can be approached in both ways, which of them is the most cost-effective. If the breeding approach seems convenient from the economic or environmental point of view, it is necessary, before making a decision, to know (or determine if not known) the type of genetic control of the trait(s) of interest: (a) nuclear (mono- or oligogenic with none or little environmental influences on the expression, or oligo- or polygenic, with the expression highly influenced by the environment) or (b) cytoplasmic. Nuclear genes have biparental inheritance and follow Mendel’s laws, thus predictions can be made regarding expected genotypes/phenotypes and proportions in controlled crosses, in contrast with cytoplasmic genes that can have either uni- or biparental inheritance and do not follow Mendel’s laws. Also, it has to be considered that a given trait can be under control of nuclear or cytoplasmic genes, or under control of cytoplasmic genes that interact with nuclear genes (i.e. chlorophyll content or male sterility) and that an enzyme can be composed of various polypeptides, some of them codified by nuclear genes and the others by cytoplasmic genes (i.e. the enzyme RUBISCO).

If breeding is deemed appropriate, it has to be decided what type of cultivar will be developed (improved population, synthetic variety, hybrid, etc.). For doing this, it has to be considered: (a) that Lotus glaber is a species herbaceous, perennial, diploid and self-incompatible, that reproduces sexually and has related diploid and polyploid species that can be manipulated in vitro and are amenable to transgenesis, and (b) the complexity of the process, the minimum required time, the likelihood of maintenance of the genetic identity of the cultivar (given the feasibility of natural hybridizations and re-seeding), the available infrastructure and equipments, and costs in relation to benefits. The first step to start a breeding program is to gather a collection of germplasm (commercial cultivars, advanced selections, natural populations), and to study the heritable variation within and between genetic materials. If no heritable variation is found for the trait(s) of interest, the

7 8 Elsa Camadro breeder can recur to: (a) interspecific hybridization (a collection of species and genotypes has to be gathered and controlled crosses performed; if hybridization barriers are detected, strategies have to be applied or developed to circumvent them, i.e., ploidy level manipulations), (b) in vitro manipulations to select pre-existing somaclonal variation or generate it, (c) induced mutagenesis with chemical or physical agents, (d) transgenesis for monogenic traits. It is important to analyze the advantages and disadvantages of each approach taking into account effectiveness and costs. For in vitro manipulations, the correlation with the in vivo behavior has to be established. Breeding has been considered an art and a science. But modern breeding has to be carried out on solid scientific bases, making use of sophisticated tools only if the cost-benefit relations warrant it.

Lotus Newsletter (2005) Volume 35 (1), 9-10.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Plant breeding: Lotus corniculatus and Lotus uliginosus.

MÓNICA REBUFFO* Instituto Nacional de Investigación Agropecuaria (INIA), INIA La Estanzuela, Colonia, Uruguay * Corresponding author

Beef and dairy production are based on grazing systems in Uruguay. Natural grasslands dominated by grasses represent over 70% of the total grazing area (about 13 millions hectares), while agricultural areas are cultivated with legume pastures in rotation with cereal crops (1.2 millions hectares). Lotus corniculatus (birdsfoot trefoil), either pure or in mixtures, is the legume most extensively utilized, whereas the relevance of Lotus uliginosus (big trefoil) to improve forage production and quality of natural grasslands has sharply increased in recent years.

The main restriction for forage productivity in Uruguay is the lack of persistence of the legumes. Birdsfoot trefoil breeding program began in 1988. The objectives are the improvement of persistence and forage production, maintenance of seasonal forage distribution and seed production. San Gabriel is the most widely utilized cultivar in the country. Altier (1997) clearly pointed at the root rot complex (Fusarium oxysporum, F. solani, Colletotrichum, etc) as the main reason for the lack of persistency of this cultivar. The progress in persistence has been sustained throughout 4 cycles of recurrent selection performed under field conditions (Rebuffo and Altier, 1997). Dead plants in the third year dropped from 88% in Cycle 0 to 53% in Cycle 4. Breeding determined the increment in the tolerance to F. oxysporum (Altier et al., 2000), in addition to the increment in crown size and in the proportion of plants with lateral roots. Cultivar INIA Draco, the outcome of Cycle 2, produces 15% more forage than San Gabriel (range from 5% up to 61 % increment in years with high incidence of root rot diseases). Additional strategies to improve persistency have been added to the objectives of the breeding program, such as the introgression of rhizome into the adapted germplasm.

The main restriction of big trefoil late flowering materials is the low seed production, due to the frequent water deficit during summer. The program has been working with diploid and tetraploid materials looking for the improvement of seed production. The breeding program started in 1983, with the introduction of a large collection of diploid accessions. The aim was to identify germplasm suitable for forage and seed production in the country. An early flowering experimental line is under evaluation at the present time. Its high seed production could build up the soil seed bank, and therefore facilitate reseeding in natural grasslands. The recent appearance of severe damages caused by Uromyces (rust) added another objective for this species (Ciliuti et al., 2003). At the present time we are evaluating rust resistant lines for introgression into outstanding forage and seed production lines.

9 10 Mónica Rebuffo

References

ALTIER N. 1997. Enfermedades del Lotus en Uruguay. [Lotus diseases in Uruguay.] INIA, Montevideo, Uruguay. Serie Técnica 93, 16 p. [In Spanish]

ALTIER N., EHLKE N.J. and REBUFFO M. 2000. Divergent selection for resistance to Fusarium root rot in birdsfoot trefoil. Crop Science 40, 670-675.

CILIUTI J., ARRIVILLAGA S., GERMÁN S., STEWART S., REBUFFO M. and HERNÁNDEZ S. 2003. Studies of rust fungi on Lotus subbiflorus and L.uliginosus. Lotus Newsletter, 33, 18-24.

REBUFFO M. and ALTIER N. 1997. Breeding for persistence in Lotus corniculatus L. Proceedings of The XVIII International Grassland Congress, Canada. ID 1881.

Lotus Newsletter (2005) Volume 35 (1), 11-12.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Condensed tannin concentrations in Lotus spp.

HERNÁN ACUÑA* and ALEX CONCHA Instituto de Investigaciones Agropecuarias,INIA, Centro Regional de Investigación Quilamapu, Casilla 426, Chillán, Chile * Corresponding author

The most important species of genus Lotus used in Chile are L. corniculatus (Lc), L. glaber (Lg) and L. uliginosus (Lu). These species, like other forage legumes, contain appreciable amounts of condensed tannins (CT) which are important compounds for reducing incidence of pasture bloat, produced by the formation of the stable foam originated in the soluble plant protein released in the rumen, preventing the normal expulsion of the gases (Tanner et al., 1995). Condensed tannins are also recognized as a factor that affects the palatability and digestibility of feeds, reducing intake and nutrient absorption (Barry, 1984). Low levels of CT in plants may improve utilization of herbage protein by ruminants without impairing feed intake and digestibility (Wang, 1996).

The objective of this study was to determine CT concentrations in Lc cultivar (13) and in Lg (11) and Lu (22) accessions collected in Chile between 32° - 38° S and 36° - 45° S, respectively, by Butanol – HCl method, and to detect variability attributable to genetics.

The experiments were established in autumn 1998 at Cato, volcanic medium textured soil: Cabrero, sandy soil; Chillán, clay soil under irrigated conditions; San Ignacio and Vilcún, both localities of volcanic soil without irrigation. There was one experiment per species at each location in a two random block design. Terril et al. (1992) procedure was used to separate total CT in forage into extractable, protein–bound and fibre–bound fractions. The samples for analysis were taken in December 1999 (2d cut), 40 – 45 days after the first cutting, when plants were completely blossomed.

The results show, in general, significant differences (P<0.05) between Lc cultivar or Lg and Lu accessions, in fractions and in total CT concentrations, in all different environments. Lotus corniculatus range from 1.9 to 5.6, 0.8 to 3.1 and 0.9 to 2.1 % of DM for extractable, protein-bound and fibre-bound CT, respectively. Lotus uliginosus DM % of CT range from 3.2 to 6.5, 1.3 to 3.9 and 0.9 to 2.9 for extractable, protein-bound and fibre-bound fractions, respectively. The above values are in the ranges reported in the literature, but the concentrations of CT in Lg are notoriously high (around 4.5 % of total CT in DM) considering that some authors reported absence of CT in Lg leaves, presence in stems and abundance only in roots, measured with vanillin-HCl reaction (Strittmatter et al.,1994). It is concluded that the studied materials show enough variability to be used in future breeding programs to develop cultivars of these species which contain appropriate amounts of CT to avoid pasture bloat without negative effects on digestibility or herbage intake.

11 12 Hernán Acuña and Alex Concha

Acknowledgements: Financed by Project FONDECYT 1980004.

References

BARRY T.N. and DUNCAN S.J. 1984. The role of condensed tannins in the nutritional value of Lotus pedunculatus for sheep. 1. Voluntary intake. British Journal of Nutrition, 51, 485-491.

STRITTMATTER C.D., RICCO R.A., KADE M., WAGNER M.L. and GURNI A.A. 1994. Condensed tannins in Lotus tenuis Waldst. Et Kit. Lotus Newsletter, 25, 41-44.

TANNER G.J., MOATE P.J., DAVIS L.H., LABY R.H., LI Y., LARKIN P.J. and LI Y. 1995. Proanthocyanidins (condensed tannin) destabilize plant protein foams in a dose dependent manner. Australian Journal of Agricultural Research, 46, 1101-1109.

TERRIL T.H., ROWAN A.M., DOUGLAS G.B. and BARRY T.N. 1992. Determination of extractable and bound condensed tannin concentration in forage plants, protein concentrate meals and cereal grains. Journal of Science and Food Agriculture, 58, 321-329.

WANG Y., WAGHORN G.C., MCNABB W.C., BARRY T.N., HEDLEY M.J. and SHELTON I.D. 1996. Effect of condensed tannins in Lotus corniculatus upon the digestion of methionine and cysteine in the small intestine of sheep. Journal of Agricultural Science, 127, 413 - 421.

Lotus Newsletter (2005) Volume 35 (1), 13-14.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Assisted and traditional assessment of saline stress tolerance in Lotus glaber.

ARIEL CLÚA1, MARIANA BARRAGÁN1, MARÍA SILVIA TACALITI1, DANIEL GIMÉNEZ1 and ANA MARÍA CASTRO 1,2 * 1 INFIVE y Genética, Facultad Ciencias Agrarias y Forestales, UNLP, Argentina 2 CONICET, Argentina. * Corresponding author

Narrowleaf birsdfoot trefoil is a naturalized grass in the Salad River Basin, which is a region with variable type of soils, being the Natralboles and Natracualfs the most frequent (Salazar et al. 1980). These soils induce plant salinity stress. High salinity in soils is a common abiotic distress, producing deprived water supply, affecting plant growth and productivity by osmotic stress and/ or ion toxicity. The most typical plant symptom of salinity injury is a retarded growth due to inhibition of cell elongation (Nieman, 1965). Largely differences have been found between and within species in the degree of adaptation to water stress. It is important to investigate the metabolic changes involved in plant adaptive strategies to water stress and their physiological basis could be useful for breeding and management purposes. Consequently, the aims of this work are to study: (i) the Narrowleaf birdsfoot trefoil intrapopulation variation in osmotic adjustment capability; (ii) the morphological and metabolic changes in response to water stress caused for salinity, and (iii) the assessment of biochemical and molecular markers associated with tolerance to saline stress.

The birdsfoot trefoil plants were collected from different populations of the Salado river basin, living in contrasting environments. Shoot explants of 5 cm long, were sectioned from plants to obtain clones, under natural conditions of light, temperature, and humidity. The explants grew in trays containing Hoagland’s solution (Hoagland & Arnon 1950). After 20 days half plantlets of every genotype were transferred to another tray containing 200mM of ClNa. The rest of the plantlets remained in Hoagland solution as controls. It was recorded the survival, fresh and dry weights in aerial and root parts, root volume, foliar area, non-structural carbohydrates, protein contents, proline and isozymes. Samples were collected after 15 days of treatment. A sample of 0.5 gr of fresh weight was collected in one-week plantlets to analyze DNA by RAPDs in a bulk screening method.

The most tolerant genotypes subjected to saline stress showed the highest survival and their foliar area, fresh and dry weights and root volumes were not significantly different from control plants. On the other hand, susceptible genotypes showed losses of 60-100% in the above mentioned parameters. Similarly, reduced, non-reduced and total carbohydrates were similar in control and stressed plants of tolerant genotypes. The susceptible genotypes showed a significant increase of the carbohydrate contents when subjected to saline stress, comparing with their controls. The protein contents decreased in every genotype under stress. Tolerant genotypes showed a high significant increase of proline under stress

13 14 Ariel Clúa, Mariana Barragán, María Silvia Tacaliti, Daniel Giménez and Ana María Castro. compared to their checks. Peroxidase system discriminated the tolerant genotypes from susceptible ones under stress. Three RAPDs were identified only in tolerant genotypes.

The traditional and assisted selection permitted the identification of tolerance to saline stress in Lotus glaber.

References

HOAGLAND D.R. and ARNON D.I. 1950. The water-culture method for growing plants without soil. California Agricultural Experimental Station, Circular 347, 1-32.

NIEMAN R.H. 1965. Expansion of bean leaves and its supression by salinity. Plant Physiology, 40, 156-161.

SALAZAR J.C., MOSCATELLI G.N., CUENCA M.A., FERRARO R.F., GOGAGNONE R.E., GRIMBERG H.L. and SÁNCHEZ J.M. 1980. Carta de suelos de la Provincia de Buenos Aires, Argentina. 1:500.000. [Soil Map of Buenos Aires Province, Argentina. 1:500.000.] Instituto Nacional de Tecnología Agropecuaria (INTA), Buenos Aires, Argentina. p. 505. [In Spanish]

Lotus Newsletter (2005) Volume 35 (1), 15-16.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Fungal diseases on Lotus spp in Argentina.

MARINA SISTERNA and GLADYS A. LORI* CIC-CIDEFI (Centro de Investigaciones de Fitopatología), Facultad de Ciencias Agrarias y Forestales, 60 y 119, cc. 31, (1900) La Plata, Prov. Buenos Aires, Argentina. * Corresponding author

Diseases can limit persistence of Lotus spp. in production systems. Several pathogens are involved in a “disease complex”. Nevertheless, little is known about diseases and their impacts, both in the world and in Argentina. Fungal pathogens are the most prevalent organisms and according to the plant tissue they can affect, diseases are classified as follows (the references correspond to the diseases cited for Argentina):

Crown and root diseases: They are considered chronic diseases, causing plant death and losses of 60 to 80 %. Typical symptoms are rot and wilt. The main Genus is Fusarium (F. solani, F. oxysporum, F.verticillioides and F. equiseti). (Teyssandier, 1976a; Dal Bello and Sisterna, 1992; Madia de Chaluat, 1994; Monterroso et al., 1998; Juan et al., 2000).

Stem and foliar diseases: In contrast to crown and root diseases, foliar ones do not cause directly the plant death. They contribute to the progressive weakness of the plant through the effects on the basic metabolic processes. The reported pathogens are: Phomopsis loti (blight) (Teyssandier, 1976b; Colletotrichum destructivum (anthracnose) (Wolcan and Dal Bello, 1988; Monterroso et al., 1998); Stemphylium loti and S. spp. (leaf spot) (Dal Bello and Wolcan, 1989; Monterroso et al.,1998; Juan et al., 2000); an unidentified rust (Dal Bello, 1986); Uromyces loti (rust) (Monterroso et al., 1998; Juan et al., 2000) and Botrytis cinerea (Monterroso et al., 1998; Juan et al., 2000).

Seed diseases: They cause decrease in germination and damping-off. Fungi genera present in the seed are: Alternaria, Aspergillus, Bipolaris, Botrytis, Cladosporium, Colletotrichum, Curvularia, Epicoccum, Fusarium, Leptosphaerulina, Penicillium, Phoma ,Phomopsis ,Stemphylium and Verticillum.(Teyssandier, 1976a, b; Wolcan.and Dal Bello, 1988; Dal Bello and Sisterna, 1992; Madia de Chaluat, 1994).

References

CHAO L., DE BATTISTA J.P. and SANTIÑAQUE F.1992. A survey of diseases affecting Lotus corniculatus in west Uruguay and Entre Ríos Province (Argentina). Lotus Newsletter, 22, 61-62.

15 16 Marina Sisterna and Gladys Lori

DAL BELLO G. 1986. A new rust of Lotus spp. Lotus Newsletter, 17, 3-4.

DAL BELLO G. and WOLCAN S. 1989. Stemphylium spp. on plants of Lotus tenuis in Argentina. Lotus Newsletter, 20, 6-8.

DAL BELLO G. and SISTERNA M. 1992. Seed Pathology of Lotus spp. Lotus Newsletter, 23, 57-60.

JUAN V.F., MONTERROSO L., SACIDO M.B. and CAUHÉPÉ M.A. 2000. Postburning legume seeding in the Flooding Pampas, Argentina. Journal of Range Management, 53, 300-304.

MADIA DE CHALUAT M. 1994. Identificación y patogenicidad de hongos hallados en semillas de Lotus spp. en Argentina.[Identification and pathogenicity of fungi found on Lotus spp. seed in Argentina]. Boletín de Sanidad vegetal. Plagas, 20, 827-831. Ministerio de Agricultura, Pesca y Alimentación. [In Spanish]

MONTERROSO L., JUAN V.J., CAUHÉPÉ M.A. and SACIDO M.B. 1998. Incidencia y severidad de Fusarium spp. sobre Lotus tenuis, durante su establecimiento post-quema en pajonales de paja colorada (Paspalum quadrifarium). [Incidence and severity of Fusarium spp. on Lotus tenuis plants, during its postburn stablishment in grassland of Paspalum quadrifarium] Fitopatología, 33 (4), 224-227. [In Spanish]

TEYSSANDIER E. 1976a. Identificación de patógenos en semillas de Lotus corniculatus. [Identification of Lotus corniculatus seed pathogens] Plan N° 136, Enfermedades de forrajeras transmitidas por sus semillas. p.64-66. Cátedra de Fitopatología, Fac. de Agronomía, UBA. [In Spanish]

TEYSSANDIER E. 1976b. Tizón del Lotus corniculatus causado por Phomopsis loti.[Blight of Lotus corniculatus caused by Phomopsis loti] Plan N° 136, Enfermedades de forrajeras transmitidas por sus semillas p. 67-71. Cátedra de Fitopatología. Fac. de Agronomía, UBA. [In Spanish]

WOLCAN S. and DAL BELLO G. 1988.Colletotrichum destructivum O’Gara, causal agent of a new disease on Lotus tenuis Waldst. et Kit. Agronomie, 8, 741-744. Lotus Newsletter (2005) Volume 35 (1), 17-17.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Bacterial diseases of Lotus spp.

ADRIANA M. ALIPPI* CIC - Centro de Investigaciones de Fitopatología (CIDEFI), Facultad de Ciencias Agrarias y Forestales, Universidad Nacional de La Plata, c.c. 31, calles 60 y 118, 1900 La Plata, Argentina * Corresponding author

There are no reports about diseases of bacterial origin affecting species of Lotus in Argentina. On a world-wide basis, only Pseudomonas viridiflava and Clavibacter michiganensis subsp. insidiosus have been reported affecting birdsfoot trefoil (Lotus corniculatus; Bradbury, 1986).

Main symptoms caused by P. viridiflava on trefoil are wilting of affected plants and reduction of root growth. The tops were wilted and the crowns appeared yellowish gray (Lukezic et al., 1983).

P. viridiflava also produces other diseases in several plants. It has been reported to cause basal stem rot of tomato, fruit rot on tomato, discolored pith on chrysanthemum, bacterial wilt of sweet onion, root and crown rot of alfalfa, bacterial canker of poinsettia, blossom blight of kiwifruit, necrosis of melon, blite, eggplant, basil, bean, cabbage, cauliflower, dill, grape, lettuce, lupine, parsnip, passion fruit, pea, poppy, pumpkin, rape, and other hosts (Gitaitis et al., 1998; Alippi et al., 2003). It has also been reported as a secondary invader and epiphyte. In Argentina P. viridiflava has been associated with symptoms of pith necrosis of tomato and pepper (Alippi et al., 2003) and leaf necrosis of basil (Alippi et al., 1999), being a potential pathogen to species of Lotus due to its ubiquitous nature and transmission through contaminated seed.

Bacterial wilt caused by C. m. subsp. insidiosus has been reported affecting Medicago sativa, Lotus spp., Melilotus spp., and Trifolium spp. (Bradbury, 1986). The disease occurs throughout most of the alfalfa growing areas of the world (Graham et al., 1980). Infected plants are scattered throughout the stand and are easily detected by their yellow-green color and stunted growth. Mild symptoms consist of leaf mottling with slight cupping or upward curling of the leaflets and some reduction in plant height. Severely infected plants are stunted and yellow-green, with many spindly stems and small, distorted leaflets. Diseased plants are usually most evident in the regrowth after clipping with appearance of witches’ broom symptoms. Cross sections of taproots show first a yellowish brown discoloration of the outer vascular tissue, and subsequently the entire stele discolors. When the bark is peeled away, the stele is yellowish brown, in contrast with the white of healthy plants. Pockets of infection sometimes appear on the inner surface of the bark (Graham et al, 1980). The pathogen can survive in plant material in the soil, hay and seed for several years. It can be spread plant to plant via rain, irrigation or contaminated implement. Long distance spread is

17 18 Adriana M. Alippi due to contaminated seed and hay. Bacteria usually infect plants through wounds in the root and crown that can be caused by winter injury, nematodes, or mechanical injury.

Dwarfing symptoms of alfalfa, probably caused by Xylella fastidiosa has been reported in USA (Graham et al, 1980), and similar symptoms were described on L. corniculatus in Uruguay (Altier, 1997), but the identity of the causal agent was not confirmed. Due to the wide range of hosts reported for X. fastidiosa there is high probability that the bacterium can also affects Lotus spp. From this limited information about bacterial diseases, it is obvious that much remain to be done to elucidate interactions between bacterial diseases and development of Lotus spp.

References

ALIPPI A.M., WOLCAN S. and DAL BÓ E. 1999. First report of bacterial leaf necrosis of basil caused by Pseudomonas viridiflava in Argentina. Plant Disease, 83 (9), 876.

ALIPPI A.M., DAL BÓ E., RONCO L.B., LÓPEZ M.V., LÓPEZ A.C. and AGUILAR O.M. 2003. Pseudomonas populations causing pith necrosis of tomato and pepper in Argentina are highly diverse. Plant Pathology, 52 (3), 287-302.

ALTIER N. 1997. Enfermedades del Lotus en Uruguay. [Diseases of Lotus in Uruguay]. INIA, Montevideo, Serie Técnica N ° 93, 16 p. [In Spanish]

BRADBURY J.F. 1986. Guide to Plant Pathogenic Bacteria. C.A.B. International Mycological Institute, Kew, Surrey, England, 332 pp.

GRAHAM J.H., FROSHEISER F.I., STUTEVILLE D.L. and ERWIN D.C. 1980. A compendium of alfalfa diseases. APS Press, USA, 65 pp.

GITAITIS R., MAC DONALD G., TORRANCE R., HARTLEY R., SUMNER D.R., GAY J.D. and JOHNSON III W.C. 1998. Bacterial streak and bulb rot of sweet onion: II. Epiphytic survival of Pseudomonas viridiflava in association with multiple weed hosts. Plant Disease, 82, 935-938.

LUKEZIC F.L., LEATH K.L. and LEVINE R.G. 1983. Pseudomonas viridiflava associated with root and crown rot of alfalfa and wilt of birdsfoot trefoil. Plant Disease, 67, 808-811.

Lotus Newsletter (2005) Volume 35 (1), 19-20.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Physiological studies of tolerance to saline stress in Lotus glaber and their correlation with the establishment and efficiency of the symbiotic association with Mesorhizobium loti.

FABRICIO CASSÁN1,2*, VIRGINIA LUNA1 and OSCAR A. RUÍZ2 1Laboratorio de Fisiología Vegetal. Universidad Nacional de Río Cuarto. Río Cuarto. Argentina. 2Unidad de Biotecnología 1. IIB-INTECh. UNSAM. CONICET. Chascomús. Argentina. * Corresponding author

Soil salinity represents the main cause of abiotic stress in cultivable plants around the world. At least 34.000.000 Has. are subjected to excess of water and mineral salts in Argentina, These factors determine extreme ecological conditions causing a drastic reduction in their productivity. Salt stress has two components: osmotic stress, caused by a relative increase of solute concentration and the consequent decreased in water availability in the soil, and ionic stress, caused by the modification of the K+/Na+ relationship and by concentrations of Na+ and Cl- that are harmful for plant tissues. Plants strategies to overcome these conditions are directed to the activation of multiple metabolic pathways in order to: facilitate the acquisition and retention of water, protection of cell functionality, modifications of growth patterns and maintenance of general homeostasis. These biochemical and physiological modifications include changes in mechanisms such as: (a) phytohormones balance, (b) osmoprotectans synthesis, (c) specialized proteins synthesis for active species of oxygen (AOS) scavenging, (d) toxic ions mobilization and compartmentation.

The rapid increase in world population makes it necessary to find alternative ways to improve agricultural productivity and make the available resources be used more efficiently. One of the strategies consists on obtaining plants tolerant to different stresses in order to introduce them in unfavourable lands. The Argentinean region called Pampa Deprimida del Salado concentrates near 70% of the Argentinean livestock activity, and their productivity is intimately linked to its edaphic characteristics: nutritionally poor soils with high concentrations of minerals salts, high pH, and high fluctuations of the useful water determined by seasonal cycles of drought and flood. In this way, the search for tolerant plant populations, as well as the understanding of the physiological basis of salinity tolerance in Pampa Deprimida ecosystem species, can be considered as a high-priority topic to be studied. Preliminary observations in the IIB-INTECh indicate the existence of high salinity tolerant genotypes in different populations of Lotus glaber (Vignolio et al., 1994). The soil conditions in the Pampa Deprimida are a natural obstacle for the efficient installation of pastures based in traditional leguminous as alfalfa or clover. In this sense, the possibility of obtaining improved populations of Lotus glaber represents an important productive alternative for those ecosystems and makes this species an excellent experimental model. In relation to its symbiotic associations, previous work in the IIB-INETCh indicates that for a

19 20 Fabricio Cassán, Virginia Luna and Oscar A. Ruiz significant nodulation in an established population of Lotus glaber it is necessary to use several Mesorhizobium loti strains (Fulchieri et al., 2001). Studies to verify if the physiological conditions of the tolerant plant affect the infection process, colonization and effective biological N fixation have not been addressed yet, nor has the question if the fixation efficiency is due to a minimum number of nodulating strains in the soil.

Our hypothesis suggests that salinity tolerance in Lotus glaber would depend, at least in part, on the plant capacity to associate with different native Mesorhizobium loti strains. The physiological conditions of the plant in this moment would be decisive for the effective bacterial colonization and later biological N fixation. To corroborate this hypothesis we propose the following general objectives: (a) to elucidate physiological and biochemical salinity tolerance mechanisms in Lotus glaber populations and their nodulation through association with Mesorhizobium loti; (b) to identify efficient symbiotic associations among tolerant populations of Lotus glaber and Mesorhizobium loti strains that could facilitate their successful installation under adverse edaphic conditions.

References

FULCHIERI M., ESTRELLA M. and IGLESIAS A. 2001. Characterization of Rhizobium loti strains from the Salado River Basin. Antonie Van Leeuwenhoek. International Journal of General and Molecular Microbiology, 79, 119-125.

VIGNOLIO O., MACEIRA N. and FERNANDEZ O. 1994. Efectos del anegamiento en invierno y verano sobre el crecimiento y la supervivencia de lotus tenuis y Lotus corniculatus. [Flooding effects in winter and summer on the growth and the survival of Lotus tenuis and Lotus corniculatus.] Ecología Austral, 4, 19-28. [In Spanish]

Lotus Newsletter (2005) Volume 35 (1), 21.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Rhizobiology research in Lotus species.

CARLOS LABANDERA and MARTÍN JAURENA* Department of Soil Microbiology, MGAP, Montevideo, Uruguay. * Corresponding author

The Uruguayan natural grasslands are the nutritional base of livestock production and occupy more than 70 % of the surface of the country. These grasslands are very stable, but they present limitations of quantity and quality of forage inside and between years. The improvements of grasslands are been done by sod seeding of forage legumes inoculated with specific strains of Rhizobium sp. and phosphate fertilizers. This technology was validated in commercial farms in the eastern and the center regions of the country. These improvements are realized principally with species of the genera Lotus (L. subbiflorus cv. “Rincón ", L. uliginosus cv. "Makú ", L. corniculatus cv. "San Gabriel "). However the situation is not the same in the basaltic region in the north part of the country, for which new species of forage legumes are been evaluated, since traditional Lotus species are not adapted due to restrictions related to edaphically factors (mix of superficial and deep soils) and climatic conditions (high temperature in summer and freezes in winter). Main lines of rhizobiological research in species of Lotus are: • Rhizobia strain selection for species of agronomic interest. • Evaluation of compatibility between rhizobia strains and Lotus species • Analysis of biodiversity patterns of strains able to nodulate Lotus corniculatus. In the first case the introduction and isolation of new rhizobia strains allows the evaluation in symbiosis of the agronomic potentiality of Lotus species of current and potential use. This activity begins with a preliminary testing of symbiotic efficiency of strains and isolations in growth chambers under controlled conditions. Superior strains pass to greenhouse evaluation in "soil cores" to imitate field conditions. In this stage there is an evaluation of Lotus and grassland dry matter production. The strains of better behavior in greenhouse go to field evaluation in plots in several localities.

Final selection is based in field results. Selected strains are evaluated for genetic stability and are used to study interspecific compatibility, and ability to growth in the industry conditions for inoculant production.

We are also performed biodiversity studies of the native and introduced rhizobia populations in soils and in the roots system associated with the persistence of commercial Lotus species. The principal outcomes of this research relates with: I) the knowledge of the characteristics of the native and introduced rhizobia strains, II) the isolation and conservation of about 150 isolations able to nodulated Lotus species, III) Inoculant development and use in farm conditions.

21 Lotus Newsletter (2005) Volume 35 (1), 22.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

The arbuscular mycorrhizal fungi of Lotus glaber.

ANALÍA I. SANNAZZARO1*, ANA B. MENÉNDEZ12, EDGARDO ALBERTÓ1 and OSCAR A. RUIZ1 1Unidad Biotecnológica 1. IIB-INTECH/UNSAM-CONICET. Ruta Circunvalación Laguna km 6, (7130) Chascomús, Pcia. de Buenos Aires, Argentina. 2DBBE, FCEN, UBA, Buenos Aires, Argentina. * Corresponding author

We intended to study the composition and structure of arbuscular mycorrhizal (AM) fungal community associated with Lotus glaber in sodic soils of the Salado River basin.

Roots were cleared, stained with Trypan blue and the amount of intraradical mycorrhizal structures estimated by the slide method. Spores were identified after isolation by wet sieving and decanting. The chemical analysis of rhizospheric soil was performed.

Spores of eighteen different AM species were detected. Glomus geosporum, the most frequently isolated AM fungus, was also the dominant one. Shannon-Wiener diversity index varied between 0.65 and 1.65.

Morphological types of AM fungi associated with L. glaber were also studied. At least eight colonization patterns (IP) of AM fungi in roots of L. glaber were observed. Arum- and Paris-types of infection were found in the same plant species. This result supports the idea that the morphology of AM is not solely under plant control, but is also influenced by fungal identity. One IP presumably corresponding to G. intraradices and a second one possibly assignable to G. tenue were the most commonly found.

Additionally, DNA from the root samples was isolated and intergenic DNA sequences from the AM fungi were amplified through a nested PCR with taxon-specific primers. Problems in cloning and sequencing caused that only two fragments have been successfully sequenced. Sequencing of these clones and the comparisons with the data in the Genebank revealed that the amplified fragments possess a high homology percentage with sequences from G. intraradices

Our findings reinforce previous suggestions that G. geosporum and G. intraradices are well adapted to sodic-saline conditions and would play a role in the resistance of L. glaber to these soils.

22 Lotus Newsletter (2005) Volume 35 (1), 23.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Seasonal variation of arbuscular mycorrhizal fungi in temperate grasslands along a wide hydrologic gradient.

RODOLFO MENDOZA* Centro de Estudios Farmacológicos y Botánicos (CEFYBO), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Serrano 669, 1414 Buenos Aires, Argentina. * Corresponding author

We studied seasonal variation in population attributes of AM fungi over two years in four sites of temperate grasslands of the Argentinean Flooding Pampas. The sites represent a wide range in soil conditions, hydrologic gradient, and floristic composition. Lotus glaber, a perennial herbaceous legume naturalised in the Flooding Pampas, was dominant at the four plant community sites. Its roots were highly colonised by AM fungi. Temporal variations in spore density, spore type, AM root colonisation, floristic composition and soil chemical characteristics occurred in each site and were different among sites. The duration of flooding had no effect on spore density but depress AM root colonisation. Eleven different types of spores were recognized and four were identified. Two species dominated at the four sites: Glomus fasciculatum and Glomus intraradices. Spore density was highest in summer (dry season) and lowest in winter (wet season) with intermediate values in autumn and spring. Colonisation of L. glaber roots was highest in summer or spring and lowest in winter or autumn. The relative density of G. fasciculatum and G. intraradices versus Glomus sp. and Acaulospora sp. had distinctive seasonal peaks. These seasonal peaks occurred at the four sites, suggesting differences among AM fungus species with respect to the seasonality of sporulation. Spore density and AM root colonisation when measured at one time were poorly related to each other. However, spore density was significantly correlated with root colonisation three months prior suggesting that high colonisation in one season precedes high sporulation in the next season.

23 Lotus Newsletter (2005) Volume 35 (1), 24.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Growth, Nitrogen and Phosphorus economy in two Lotus glaber Mill. Populations grown under contrasting P-availability.

DANIEL COGLIATTI1,2, LINA LETT1*, MÓNICA BARUFALDI1, PAOLA SEGURA1 and JORGE CARDOZO2. 1 Facultad de Agronomía de Azul, Universidad del Centro de la Provincia de Buenos Aires. Av. República de Italia 780, CP 7300 Azul, Provincia de Buenos Aires Argentina. 2 Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina * Corresponding author

With the aim of comparing the growth and economy of phosphorus (P) and nitrogen (N) of two Lotus glaber populations differing in their ploidy level, an experiment was performed under two soil P-availabilities. Plants were grown in the open air from mid January to April 2003 in soil filled pots kept under field capacity. The experimental design was a 2x2 factorial consisting of two Lotus glaber populations, a colchicine-induced autotetraploid (Barufaldi. et al., 2000) and diploid cv. Chaja, provided by KWS Argentina S.A., and two P-fertilization doses (0 and 100 ppm of P as triple super phosphate). The original extractable P concentration was 4 ppm (Bray and Kurtz N°1). At transplanting time, all germinated seeds were inoculated with 108 cells per plant of the commercial inoculant strain LL32 produced by Laboratorios Biagro S.A. Argentina. Twenty plants were harvested at 46, 74 and 102 days after germination and on each occasion growth parameters, P and N concentration were determined. In addition to the greater growth of all fertilized plants, the two populations showed similar growth rate (GR) and relative growth rate (RGR) at each P-availability. Variability of dry biomass was greater for tetraploid than for haploid plants. In spite of similar means at each harvest, the tetraploid population included the plants with the highest individual dry biomass. Leaf area of diploid plants at the end of the experiment was larger than that of tetraploids, due to a higher biomass partitioning to leaves. Phosphorus concentration was similar between populations and greater in P-fertilized plants. Differences were not found either for P-absorption and P-utilization efficiencies or P-partitioning between shoots and roots. As was expected, P-fertilization increased P-uptake and reduced P-utilization efficiencies, but partitioning was not affected in either population. In a similar manner to the results obtained with phosphorus, neither N-concentration nor N-use efficiencies were different between populations. Differences in N-content observed between P-availabilities were partially attributed to the higher number of nodules observed in P-fertilized plants. Under P-fertilization higher nodule number and nodule dry weight were observed in the diploid population.

References

BARUFALDI M., ANDRÉS A., CROSTA H. and ESEIZA M. 2000. Obtención de una población autotetraploide de Lotus glaber Mill. (Lotus tenuis Waldst. & Kit). [Obtaining an autotetraploide population of Lotus glaber Mill. (Lotus tenuis Waldst. & Kit).] Revista de Tecnología Agropecuaria INTA Pergamino, V (15), 45-50. [In Spanish]

24 Lotus Newsletter (2005) Volume 35 (1), 25-26.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Comparative responses between Lotus glaber and Paspalum dilatatum to the flooding-grazing interaction.

GUSTAVO G. STRIKER*, PEDRO INSAUSTI and ROLANDO J.C. LEÓN. IFEVA-CONICET, Facultad de Agronomía, UBA, Av. San Martín 4453, Buenos Aires, Argentina. * Corresponding author

Flooding and grazing are the two most important factors of the disturbance regime that affects the natural grasslands of the Flooding Pampa. There is enough evidence that indicates that two functional groups of these grasslands, native gramineous and exotic dicotyledonous differ in the tolerance of the above-mentioned disturbances (Insausti et al., 1999). Paspalum dilatatum and Lotus glaber, representatives of these two groups, respectively, are frequently subject to the action of cattle trampling and defoliation, as sequelae from grazing during large flooding periods. Our aim was to assess a series of response variables that characterize the water relations, the tissue aeration and the growth of the plants of L. glaber and P. dilatatum when they are simultaneously affected by grazing and flooding. In this hypothesis we propose that L. glaber does not tolerate the simultaneous action of trampling, defoliation and flooding, such as the gramineous P. dilatatum. We predict that L. glaber responds to these disturbances with a lower water potential, conductance, and foliar transpiration than P. dilatatum. Besides, it shows lower porosity in their tissues and it does not place its leaves above water level, as fast as P. dilatatum does.

In order to test this hypothesis, an experiment was carried out in 25 individuals of each species extracted from the grassland in soil blocks. After a two-month acclimatizing period, the treatments were initiated in an experimental garden: 1) flooding (F), 15 days at a 6 cm level, 2) simulated cattle trampling (T), at the onset of the experiment, 3) defoliation (D), at the onset, by cutting above 6 cm, 4) control without alterations (C). The flooding period was followed by a recovery period of 30 days. The design was a factorial arrangement with 5 treatments (C-F-T-D-FTD) and 5 replicates. The following response variables were measured: a) leaf waterpotential (Ψw), b) stomatal conductance (gs), c) transpiration (E), at 3-5 day intervals, d) porosity of roots and shoots/sheaths, by pycnometry at the end of the flooding, e) time record of the height of 10 young shoots/tillers and f) number of green and dry shoots/tillers.

The results showed an increase in the porosity of the roots and sheaths of P. dilatatum in the treatments with flooding or in the FTD interaction. Besides, in this species the greater values of Ψw, gs and E were recorded in the days of high atmospheric demand with respect to the control ones and, as from the 5th day of the flooding, the tillers were above water level. The number of tillers was lower with the trampling (T) and increased with the defoliation (D or FTD), compared to the control ones, but there were no differences with respect to the flooding. The L. glaber plants with FTD treatment died five days after the onset of the

25 26 Gustavo G. Striker, Pedro Insausti and Rolando J.C. León experiment. The ones that were only subject to flooding increased their porosity, in the roots and shoots, however they showed lower values of Ψw, gs and E along the time with respect to the control ones. Once the flooding was discontinued, such plants recovered. The control shoots of the L. glaber plants took three times less time than the flooded ones to reach the height that the water level had in the flooding treatment. In this species, trampling caused a decrease in the radical porosity and the quantity of shoots. This last variable was also reduced with the flooding but increased with the defoliation. It is concluded that flooding and grazing show a differential effect on these species, in their water relations and their growth, and that these responses would be related to the different tolerance that they express with respect to those grassland disturbances.

References

INSAUSTI P., CHANETON E.J. and SORIANO A. 1999. Flooding reverted grazing effects on plant community structure in mesocosms of lowland grassland. Oikos, 84, 266-276.

Lotus Newsletter (2005) Volume 35 (1), 27-28.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Morpho-physiological characterization of Lotus glaber naturalized populations

ADRIANA ANDRÉS*, BEATRIZ ROSSO and OMAR SCHENEITER INTA EEA Pergamino, Argentina * Corresponding author

Lotus glaber Mill (Lotus tenuis Waldst. et Kit) is a legume that has become naturalized in lowlands of Argentinean pampas. The productive cycle of this perennial legume native from Europe is during spring and autumn and summer and is considered a great alternative for forage production in stressed soil conditions including flooding and phosphorous deficiency. In addition, Lotus glaber is well known for its anti-bloating and high nutritional properties. (Mazzanti et al, 1992).

Our principal aim in this work is to evaluate the morpho-physiological genetic variability looking to contribute to future breeding programs in the species.

During 2003 and 2004 were collected several genotypes of Lotus glaber in diverse environments of Buenos Aires Province. The places where the samples were obtained were evaluated for their edaphically properties as well as their historical cow managements and production. Using an experimental block randomized method; at least 50 seeds were colleted for each genotype.

A biodiversity evaluation of vegetal species implanted around the Lotus glaber completed the study.

In May 2004 the seeds collected were sown in pots containing compost and maintained in controlled environmental conditions in a greenhouse. One month later, the plants obtained were transplanted to farm condition in the experimental field of EEA Pergamino. Eighty genotypes for each population were analyzed using the method described elsewhere (Turesson, 1922).

The variables evaluated were: • Plant vigor (low, medium and high) • Growth style (erect, prostrated and intermediate) • Stem and blade numbers per plant • Dry weight per plant • Time flowering after farm transplantation. • Seed production • Existence or not of bacterial and fungal diseases

27 28 Adriana Andrés, Beatriz Rosso and Omar Scheneiter

The results obtained will be presented and discussed.

References

MAZZANTI A., CASTAÑO J., SEVILLA G. and ORBEA J. 1992. Características agronómicas de variedades de gramíneas y leguminosas forrajeras adaptadas al sudeste de la Provincia de Buenos Aires. [Agronomic characteristics of forage grasses and legumes varieties adapted to Buenos Aires Southeast Province.] Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria. INTA (Argentina). Boletín de Divulgación Técnica, Nº 102, 20 p. [In Spanish]

TURESSON G. 1922. The genotipical response of the plant species to the habitat. Hereditas, 3, 211-350. Lotus Newsletter (2005) Volume 35 (1), 29-30.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Influence of the arbuscular mycorrhizal fungus Glomus intrarradices on the saline stress physiology of Lotus glaber.

ANALÍA I. SANNAZZARO1, EDGARDO ALBERTÓ1, OSCAR A. RUIZ1 and ANA B. MENÉNDEZ12* 1Unidad Biotecnológica 1. IIB-INTECH/UNSAM-CONICET. Ruta Circunvalación Laguna km 6, (7130) Chascomús, Pcia. de Buenos Aires, Argentina. 2DBBE, FCEN, UBA, Buenos Aires, Argentina. * Corresponding author

Lotus glaber is a glycophytic, perennial legume from Europe that occurs widely in saline habitats. Previous observations indicate the occurrence of genotypes highly tolerant to salinity in different populations of L. glaber (Mujica and Rumi, 1997). Several workers have shown that AM fungi protect the plant against salinity (Al-Karaki et al., 2001; Feng et al., 2002). In turn, a high diversity of AM fungal colonization patterns in roots of L. glaber was found in fields characterized by their high salinity (Sannazzaro et al., 2004).

Our aim was to evaluate the effect of mycorrhizal colonization on growth response to salt stress in two genotypes of L. glaber differing in their tolerance to salinity. We also hypothesized that polyamines, small organic cations that are thought to play a role in the plant responses to salt stress (Bouchereau et al., 1999; Simon-Sarkadi et al., 2002) are involved in such a process.

The experiment consisted of a randomized block design with two factors: (1) mycorrhizal treatments (with or without AM fungus) and (2) two salinity levels of 0 and 200 mM NaCl. L. glaber plants colonized by G. intrarradices grew better than non-AM ones, particularly under saline condition, where they showed higher values of net growth, shoot/root ratio, K/Na rate, and protein and chlorophyll contents. An increase in total free polyamine content of mycorrhized L. glaber plants compared to non-mycorrhized ones, suggests that these amines may be involved in the salt stress alleviation of this species. The increment in spermine levels in sensitive L. glaber plants grown under salt stress condition could be due to de novo synthesis from its metabolic precursors. In addition, high proline levels were observed under salt stress conditions in both genotypes. Our results interestingly indicate that G. intrarradices established a more efficient symbiosis with the tolerant than with the sensitive genotype. Results suggest that the fungal symbiont could play an important role in the adaptation of L. glaber plants to salt stress under field conditions.

References

AL-KARAKI G.N. and HAMMAD R. 2001. Mycorrhizal influence on fruit yield and mineral content of tomato grown under salt stress. Journal of Plant Nutrition, 24, 1311-1323.

29 30 Analía I. Sannazzaro, Edgardo Albertó, Oscar A. Ruiz and Ana B. Menéndez

BOUCHEREAU A., AZIZ A., LARHER F. and MARTIN-TANGUY J. 1999. Polyamines and environmental challenges: recent development. Plant Science, 140, 103-125

FENG G., ZHANG F.S., LI X.L., TIAN C.Y., TANG C. and RENGEL Z. 2002. Osmotic adjustment in mycorrhizal plants: a symbiotic-association determined higher tolerance to salt stress. Mycorrhiza, 12, 185-190

MUJICA M.M. and RUMI C.P. 1997. Alterations observed in Lotus glaber (syn. L. tenuis) seedlings apparently induced by ethephon. Lotus Newsletter, 28 http://www.psu.missouri.edu/lnl/v28/Lotus_NL.htm#mujica

SANNAZZARO A.I., RUIZ O.A., ALBERTÓ E. and MENÉNDEZ A.B. 2004. Presence of different arbuscular mycorrhizal infection patterns in roots of Lotus glaber plants growing in the Salado River basin. Mycorrhiza, 14, 139-142

SIMON-SARKADI L., KOCSY G. and SEBESTYÉN Z. 2002. Effect of salt stress on free amino acid and polyamine content in cereals. Acta Biol Szeged, 46, 73-75.

Lotus Newsletter (2005) Volume 35 (1), 31-32.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Molecular and biochemical approximation of polyamine roles in tolerance mechanisms to salt stress in Lotus spp.

ROSALÍA PAZ, DIEGO H. SANCHEZ, FERNANDO PIECKENSTAIN, SANTIAGO MAIALE, ANALÍA SANNAZZARO, JUAN CRUZ CUEVAS, AMALIA CHIESA, GONZALO BONA and OSCAR A. RUIZ*. Unidad Biotecnológica 1. IIB-INTECH/UNSAM-CONICET. Ruta Circunvalación Laguna km 6, (7130) Chascomús, Pcia. de Buenos Aires, Argentina.

* Corresponding author

Lotus glaber is the most important legume in the saline-alkaline lowlands of the Salado River basin. This region (approximately 9,000,000 ha), located in Buenos Aires Province (Argentina) is devoted to the breeding of beef cattle. In order to increase forage yield and improve the quality of their pastures, regional farmers utilize L. glaber, whose adaptability to saline soils is well-known. The economic importance of this legume has led to an increasing number of studies regarding the physiological basis of its salt tolerance. Polyamines are aliphatic amines of low molecular weight charged positively at physiological pH. The distribution of these positive charges permits their interaction with proteins, membrane lipids and DNA. It’s well known that the activity of the plant enzymes of polyamine biosynthesis is induced under abiotic stress, including salinization. With this idea in mind, we evaluated the effect of salt stress in polyamine pathway in Lotus glaber. As proline is a traditional stress marker in plants, we evaluate their levels under similar stress conditions. To understand the response of L. glaber to salt stress, we evaluated 15 day-old seedlings germinated and grown under 0, 25, 50 and 75 mM NaCl. We observed a progressive accumulation of Na+ and loosening of K+. Simultaneously we observed an increasing in proline levels and accumulation in Spm, coincidently with a diminishing in Spd levels.

In addition, we analyzed a natural population of L. glaber collected from lowlands of Salado River Basin. We isolated 103 genotypes of L. glaber and cloned them by nodal cutting. Thirty days old genotypes were exposed to 300 mM NaCl (n=5), and death days average was determined. A Gaussian distribution was obtained. The time survival varied from 12 till 30 days. Five genotypes of each extreme were selected and cloned and sub irrigated with 0 and 150 mM NaCl. Based on their differential relative growth rates the genotypes were classified in sensitive and tolerant. We concluded that the preliminary classification based on average days of “survival-time” under strong saline conditions are not representative of their tolerance physiological conditions because some genotypes identified firstly as sensible were tolerant and vice versa.

Analysis in polyamine content demonstrated that spermine accumulates less in tolerant genotypes than in sensitive. Simultaneously, proline presents a progressive accumulation in

31 32 Rosalía Paz, Diego H. Sánchez, Fernando Pieckenstain, Santiago Maiale, Analía Sannazzaro, Juan Cruz Cuevas, Amalia Chiesa, Gonzalo Bona and Oscar A. Ruiz both genotypes but was smaller in tolerant ones. Complementary and taking in to account the results obtained, we generate Lotus corniculatus transgenic plants that over express under control of the constitutive promoter CaMV35S, a putative gene of spermidine synthase previously cloned from tobacco. The lines obtained presented constitutive high levels of Spd and Spm than controls non-transformed. Moreover, we observed that these transgenic plants under saline stress showed a decrease in the spermidine concentrations and an increment in the spermine levels suggesting activation in the spermine synthase activity. Under similar conditions the proline levels diminished.

Actually, we are working in the generation of Lotus spp transgenic plants that potentially over express regulatory enzymes in the polyamine biosynthesis under control of a promoter inducible by stress. This promoter denominated RD29A was cloned from Arabidopsis thaliana and is under evaluation in our lab. Binary vectors harboring the sequence of arginine decarboxylase (ADC) under control of this promoter was assayed successfully in hairy roots of Lotus corniculatus, suggesting a conservative stress signaling pathway between species.

Lotus Newsletter (2005) Volume 35 (1), 33-34.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Adventitious shoot regeneration in Lotus glaber Mill.

PEDRO A. SANSBERRO*, FABIANA D. ESPASANDIN, CLAUDIA V. LUNA and LUIS A. MROGINSKI Instituto de Botánica del Nordeste (IBONE-CONICET), Facultad de Ciencias Agrarias (UNNE). Sgto. Cabral 2131. CC: 209; CP: 3400. Corrientes, Argentina. Fax: 54 3783 427131. * Corresponding author

In order to get a suitable protocol of regeneration for the genetic transformation of Lotus glaber Mill., several experiments from different explants were conduced. Roots, cotyledons and leaves from seedlings grown in vitro were cut into pieces and used as source of explants. Subsequently, they were cultured on 11 cc glass tubes containing 3 ml of Murashige and Skoog (1962) (plus sucrose 3%) semisolid medium (agar 0.65%), supplemented with different combinations of auxins (either naphtalenacetic acid or indoleacetic acid; NAA and IAA, respectively) and cytokinins (benciladenine, kinetin or thidiazuron; BA, KIN and TDZ, in that order). The cultures were incubated under 116 μmol·m-2·s-1 PPFD (photoperiod 14 h) and 27±2ºC.

After 45 days of culture, de novo shoot organogenesis was noticed from all explants tested. The type of explants markedly influenced organogenesis and the growth of the regenerated shoots. The regeneration frequencies were higher with leaf and cotyledons explants while the number of shoots formed per responsive explant was greater with leaf and roots.

The number of shoots produced per responsive leaf explant increased from 4 to 23, as the percentage of leaf explants producing shoots increased from 10 to more than 40%. NAA in combination with BA induced the highest regeneration rate (40.1±18.3%) bringing 19.7±3.3 shoots per responsive leaf explant. Histological examination confirmed the direct process of organogenesis. The regenerated shoots from the best induction treatment were transferred to a fresh medium of similar chemical composition and without plant growth regulators for 30 days; in which, the in vitro rooting was stimulated. In all cases, the morphogenetic process was characterized by a direct pattern of root formation without callus proliferation. Plantlets with fully expanded leaves and well-developed roots were acclimatized in pots with transparent covers that were subsequently lifted to reduce humidity. The acclimatized plantlets were successfully established in soil. All plantlets were phenotypically normal.

In conclusion, our study provides a practical technique for efficient plantlets production of Lotus glaber. The procedure described here for the direct shoot organogenesis from various explants, and subsequently plantlets regeneration, facilitates the rapid propagation of this species. It will also be of use in cryoconservation and genetic breeding aimed at improving the abiotic stress tolerance.

33 34 Pedro A. Sansberro, Fabiana D. Espasandin, Claudia V. Luna, Luis A. Mroginski.

References

MURASHIGE T. and SKOOG F.A. 1962. A revised medium for rapid growth and bioassays with tobacco culture. Phisiologia Plantarum, 15, 473-497.

Lotus Newsletter (2005) Volume 35 (1), 35.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Fire on native pastures - effects on soil and vegetation.

AINO VICTOR AVILA JACQUES1* and INGRID HERINGER2 1 Ph.D. Retired Full Professor, Invited Colaborator of Federal University of Rio Grande do Sul, Porto Alegre, RS, Brazil. 2 Doctor in Animal Science (UFRGS). Professor of the University of Xanxerê, Xanxerê, SC, Brazil

* Corresponding author

The effects of fire and alternative managements on soil and vegetation of native pastures were revised from several research works, jointing personal observations developed during almost 40 years in range areas of Rio Grande do Sul, Brazil. Soil parameters such as potential acidity; aluminum concentration; basis saturation; and others were considered with vegetation parameters such as production and quality of green forage dry matter during the year; botanical composition; volumetric amount of water in soil; nutrients cycling through the forage and dead material; etc. Some results: Burning results in higher contents and saturation of aluminum, and higher potential soil acidity; Mowing reduces potential soil acidity and increases soil basis saturation; Lime, fertilization and mowing favor native species of higher forage value such as Paspalum notatum, Paspalum plicatulum and Desmodium incanum; Burning favor the andropogonea species in detrimental of prostrate grasses and legumes, and also ciperaceae, reducing the floristic diversity; Burning reduces the forage green dry matter and dead material, and the volumetric amount of water in soil, resulting in considerable proportion of uncovered soil surface; Higher nutrient cycling through forage and dead material in areas not burned; The regrowth of native species during the spring period is delayed in areas burned every two years for more than 100 years as compared with other alternative managements. Eryngium horridum, undesirable species, increases its participation in burned areas as compared with other alternative managements. The general conclusion, based on the results of the revised works, is that the burning of natural pastures, in the high altitude region of southern Brazil, must be avoided as a routine practice, because it is detrimental to the environment, reduces forage yield and quality, and it is not a sustainable practice.

35 Lotus Newsletter (2005) Volume 35 (1), 36.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Is arriving the Lotus glaber time in the Pampa Deprimida del Salado?

MATIAS A. BAILLERES Chacra Experimental Manantiales, Ministerio de Asuntos Agrarios, Provincia de Buenos Aires, Argentina. * Corresponding author

The National farming situation showed a clear tendency to intensify agriculture to the detriment of cow production. This situation is induced by the clear differences that exist between the economic prices actually obtained by farmer’s through agriculture and livestock. This occurs in part by the high price of the grains and the low livestock productive efficiencies in result to the absence of innovations.

In addition, there is an increasing demand of soils for agricultural activities producing a substantial reduction in the quality and the extension of lands devoted to cow feeding.

One alternative to increase forage capacity and productive efficiency is the implantation of pastures in soils that edaphically stressing, traditionally not utilized for forage production. Lotus glaber is ideal to grow in typical unfavorable conditions that exist in lowlands frequently associated with marginal soils in the region.

The Lotus glaber in normally sowed associate ie: a mix pasture with Festuca arundinacea. In our experimental field, the Chacra Experimental Manantiales, we analyzed this pasture that was sowed in 2002. This assay was development in typical “mosaic soils” using capacities class IV to VII.

The whole forage production was 12672 Kg dry weigh /ha; estimating a cow consumption of 4738 Kg we estimated a mean meat production of 600,5 Kg /ha. The results obtained and the economic equations in productivity will be shown and discussed.

In our opinion, this information is demonstrative that we still can "make something" for a more rentable livestock in the region in using Lotus glaber and modifying a farm characterized by only cattle breeding exploitation to cattle breeding and fattening.

36 Lotus Newsletter (2005) Volume 35 (1), 37-38.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Perspectives of utilization of native legumes in Rio Grande do Sul.

MIGUEL DALL´AGNOL1* and SIMONE M. SCHEFFER-BASSO2 1Universidade Federal do Rio Grande do Sul, Faculdade de Agronomia, Dep. Plantas Forrageiras e Agrometeorologia, Bolsista CNPq, Brazil. 2Universidade de Passo Fundo,Instituto de Ciências Biológicas, Brazil. * Corresponding author

The importance of legumes for animal production has been shown since a long time ago by many authors (Blaser, 1982; Petritz et al., 1980). The inclusion of legumes on pastures promotes several advantages, such as an increase on animal production, as a result of better forage quality, a better forage distribution of forage yield along the year and an increase on soil fertility and microbiological activity, due to incorporation of N to the soil. Recently Dall’Agnol and Scheffer-Basso (2004) have related the general benefits of temperate and tropical legumes, as well as the situation of temperate and native legumes in the “Zona Campos” of South America (Dall´Agnol et al., 2002).

Although temperate legumes are species considered important for many grazing systems, their lack of persistence has been pointed as the major limitation to their utilization, as well as bad management practices by the farmers (Beuselinck et al., 1994). In Southern Brazil, the grazing systems used are typically extensive, with the native pasture being the base of most pastures. On those systems, legumes are not extensively used, but some of them have a relative importance. Among those, the most important are white clover (Trifolium repens), red clover (T. pratense), birdsfoot trefoil (Lotus corniculatus). These species are used as cultivated pastures or are over-sown on the native pastures, aiming an improvement on forage quality and yield distribution, but on both cases with poor persistence as earlier related.

Regardless of the importance that the maintenance and persistence of legumes on pastures have, very little attention has been given to their study and for the understanding of their relationships on the different grazing systems. This point is particularly true in relation to our native species, which demonstrates the lack of collaborative work among the different researchers and research institutes.

An increase in legume utilization is possible and one of the possibilities is through their inclusion on areas integrated with cultures, such as soybean-pastures, rice-pastures, etc. However, an increase on legume utilization, especially the natives, must be made with special care on conserving this valuable natural resource. This germplasm is unique, with species of excellent value of forage and it is our obligation to study and to preserve it for the next generations. Among the several genera and species with potential, it is worth mentioning: Adesmia (A. bicolor and A. bicolor); Trifolium (T. polymorphum, T.

37 38 Miguel Dall’Agnol and Simone M. Scheffer-Basso riograndense and T. argentinense); Desmodium (D. incanum, D. uncinatum) and Macroptlium, Vigna and Vicia.

Finally, an increase on legumes utilization, natives or not, only will be possible if we can make a real integration of different areas of knowledge, such as plant pathology, microbiology and physiology, among others. Besides that we should make an effort to decrease the increasing number of burocratic barriers for germplasm exchange and collaborative work, including those existing in our home countries.

References

BEUSELINCK P.R., BOUTON J.H., LAMP W.O., MATCHES A.G., MCCASLIN M.H., NELSON C.J., RHODES L.H., SHEAFFER C.C. and VOLENEE J.J. 1994. Improving legume persistence in forage crop systems. Journal of Production Agriculture, 7, 311-322.

BLASER R.E. 1982. Integrated pasture and animal management. Tropical Grassslands, Melbourne, 16 (1), 9-24.

DALL´AGNOL M., NABINGER C., MONTARDO D.P., SAIBRO J.C. DE, FRANKE L.B., SCHIFINO-WITTMANN M.T. and SCHEFFER-BASSO S.M. 2002. Estado atual e futuro da produção e utilização de leguminosas forrageiras na zona campos: RS. [Actual knowledge and future of the production and utilization of forage legumes on the grasslands zone: RS.] Reunião do Grupo Técnico em Forrageiras do Cone Sul, XIX. Anales. Mercedes, 2002. p. 83-90. [In Portuguese]

DALL´AGNOL M. and SCHEFFER-BASSO S.M. 2004. Utilização de Recursos Genéticos de Leguminosas para Ruminantes. [Legume genetic resources utilization by ruminants.] Reunião Anual da Sociedade Brasileira de Zootecnia, Campo Grande, 2004. [In Portuguese]

PETRITZ D.C., LECHTENBERG V.L. and SMITH W.H. 1980. Peformance and economics return of beef cows and calves grazing grass-legume herbage. Agronomy Journal, 72, 581-584. Lotus Newsletter (2005) Volume 35 (1), 39-41.

Abstract, Workshop held at Chascomús, Argentina, 9-10 September 2004

Lotus glaber productivity changes under different management conditions.

OSVALDO RAMÓN VIGNOLIO* Facultad de Ciencias Agrarias (UNMdP)-EEA INTA Balcarce C.C. 276 (7620) Balcarce. Argentina. * Corresponding author

Lotus glaber productivity changes pure and in mixtures with other species were analyzed. L. glaber productivity was high in the first year, but it declined in time (Colabelli and Miñón 1994; Quadrelli et al., 1997). The changes in the productivity and quality of pasture sown in the Flooding Pampas (Buenos Aires, Argentina) have been studied with a succession viewpoint. The pasture was gradually replaced by native species, present in the seed bank (León and Oesterheld, 1982; Oesterheld and León, 1987). Pasture changes were caused by soil compaction (Oesterheld and León, 1993), plant mortality and nutrition deficiency (Guaita et al., 1996), among other factors. Lotus glaber productivity declined in three years’ time while the productivity of native and exotic species increased, some of them of poor quality (Colabelli and Miñón, 1994; Quadrelli et al., 1997). Lotus glaber productivity changes may be due to reduction of stem density (Miñón and Refi, 1993; Acuña and Cuevas, 1999) and seedling mortality caused by Fusarium spp. (Monterroso et al., 1998). Lotus glaber plant mortality caused by flooding (Vignolio et al., 1994) and soil compaction (Striker et al., 2005) was also reported. Plant mortality by mechanical shoot cut carried out at the beginning of the reproductive season also was recorded. Lotus glaber spreads by seed; therefore, if seed production is affected by grazing (Miñón and Refi, 1993; Acuña and Cuevas, 1999) or mechanical cut (Colabelli and Miñón, 1993; Quadrelli et al., 1997), besides the aspects previously mentioned,. it is possible to understand the reduction of its productivity in time Bovine can spread L. glaber seeds, but, the number of seedlings dying in dung during the establishment phase is very high (Sevilla et al., 1996). Furthermore, if the animal eats immature pods and seeds, the seed bank could exhaust and delay L. glaber population recovery. In order to recover pasture productivity is recommendable: (a) to control weeds, although some studies have reported that the weeds biomass was increased with fertilization (Quadrelli et al., 1997.); (b) to fertilize with P (Guaita et al., 1996); (c) to sow the species that are in low quantity and (d) to maintain the seed bank for natural reseeding (Taylor et al., 1973). The persistence of L. glaber populations in pasture and in grassland could be possible by means of seeds bank. Lotus glaber has an important seedling emergence pulse at the end of winter (Sevilla et al., 1996); therefore, if the farmers generate favorable conditions for its establishment, L. glaber population could be maintained.

39 40 Osvaldo R. Vignolio

References

ACUÑA H. and CUEVAS G. 1999. Efecto de la altura y frecuencia de la defoliación, bajo corte y pastoreo, en el crecimiento y productividad de tres especies de género Lotus en suelos arcillosos. [Effect of defoliation height and frequency, under cutting and grazing, on growth and productivity of three species of the genus Lotus in loamy soils.] Agricultura Técnica (Chile), 59, 296-308. [In Spanish]

COLABELLI M. and MIÑON D. 1994. Rendimiento y cambios botánicos de pasturas de Lotus tenuis puro y en mezcla bajo régimen de corte. [Yield and botanical changes in pure and mixed swards of Lotus tenuis under cutting conditions.] Agricultura Técnica (Chile), 54, 39-45. [In Spanish]

GUAITA M.S., ESCUDER C.J. and ECHEVERRIA H.E. 1996. Fertilización de una pastura de raigrass perenne y trébol rojo: 1. Nivel de nitrógeno y frecuencia de corte. [Fertilization of ryegrass and red clover pasture: 1. Nitrogen label and cutting frequency.] Revista Argentina de Producción Animal, 16, 253-260. [In Spanish]

LEÓN R.J.C and OESTERHELD M. 1982. Envejecimiento de pasturas implantadas en el norte de la Depresión del Salado. Un enfoque sucesional. [Decay of cultivated pastures in the Salado River Basin: A successional viewpoint evolutionary.] Revista de la Facultad de Agronomía (UBA, Argentina), 3, 41-49. [In Spanish]

MIÑON D. and REFI R.O. 1993. Persistencia de pasturas de Festuca arundinacea, Trifolium repens y Lotus tenuis bajo pastoreo continuo. [Persistence of pastures of Festuca arundinacea, Trifolium repens and Lotus tenuis under grazing conditions.] Dialogo (PROCISUR, Montevideo, Uruguay), 38, 95-102. [In Spanish]

MONTERROSO L., JUAN V.J., CAUHÉPÉ M. and SACIDO M.B. 1998. Incidencia y severidad de Fusarium spp. sobre Lotus tenuis durante su establecimiento post-quema en pajonales de paja colorada (Paspalum quadrifarium). [Incidence and severity of Fusarium spp. on Lotus tenuis during its postburn establishment in grassland of Paspalum quadrifarium.] Fitopatología (Argentina), 33, 224-227. [In Spanish]

OESTERHELD M. and LEÓN R.J.C. 1987. El envejecimiento de pasturas implantadas: su efecto sobre la productividad primaria. [Aging of sown pastures: their effect on the primary productivity.] Turrialba, 37, 29-35. [In Spanish]

OESTERHELD M. and LEÓN R.J.C. 1993. Cambios en la compactación del suelo durante el envejecimiento de pasturas implantadas. [Changes in soil during the aging of sown pastures.] Revista Argentina de Producción Animal, 13, 149-153. [In Spanish]

QUADRELLI A.M., LAICH F.S., ANDREOLI E. and ECHEVERRIA H.E. 1997. Respuesta de Lotus tenuis Waldst a la inoculación con Rhizobium loti y a la fertilización fosfatada. [Response of Lotus tenuis Waldst to inoculation with Rhizobium loti and phosphate fertilization.] Ciencia del Suelo (Argentina), 15, 22-27. [In Spanish] Lotus Workshop, Argentina 41

SEVILLA G.H., FERNANDEZ O.N., MIÑON D.P. and MONTES L. 1996. Emergence and seedling survival of Lotus tenuis in Festuca arundinacea pastures. Journal of Range Management, 49, 509-511.

STRIKER, G.G., INSAUSTI, P. and LEÓN R.J.C. 2005. Comparative responses between Lotus glaber and Paspalum dilatatum to the flooding-grazing interaction. Lotus Newsletter, 35, 23-24.

TAYLOR T.H., TEMPLETON W.C.Jr. and WYLES J.W. 1973. Management effects on persistence and productivity of birdsfoot trefoil (Lotus corniculatus L.). Agronomy Journal, 65, 646-648.

VIGNOLIO O.R., MACEIRA O.N. and FERNANDEZ N.O. 1994. Efectos del anegamiento en invierno y verano sobre el crecimiento y la supervivencia de Lotus tenuis y Lotus corniculatus. [Effects of waterlogging in winter and summer on the growth and survival of Lotus tenuis and Lotus corniculatus.] Ecología Austral, 4, 19-28. [In Spanish]

Lotus Newsletter (2005) Volume 35 (1), 42-58.

Epidemiological studies on crown and root rot of birdsfoot trefoil in Uruguay

NORA A. ALTIER1* and LINDA L. KINKEL2 1Department of Plant Protection, National Institute for Agricultural Research, INIA Las Brujas, 90200 Canelones, Uruguay 2 Department of Plant Pathology, University of Minnesota, St. Paul, MN, USA *Corresponding author

Abstract

Birdsfoot trefoil fields in 3 locations in Uruguay were surveyed to determine incidence and severity of crown and root diseases in 1- to 3-yr-old stands. Twenty-five plants in each of 12 permanent quadrats were evaluated at each site (n = 300 plants per site, 3 sites per location). Plants were scored for disease severity following a 5-class scale: 0 = no disease, 4 = dead plant. Crown and root rot occurred in every site, with average incidences (percent infected plants) of 43, 96, and 100% and average severities of 0.51, 1.51, and 1.86 in 1-, 2-, and 3-yr-old stands, respectively. Fusarium oxysporum was the primary pathogen associated with diseased plants. Variance to mean ratios for disease severity among quadrats within sites were consistently less than 1, suggesting that disease was not aggregated among quadrats in individual sites. Stand counts decreased with age, from 200 plants/m2 in 1-yr-old to less than 50 plants/m2 in 3-yr-old stands. Stand counts also decreased with increasing disease intensities among stands of the same age, suggesting a relationship between crown and root rot and plant persistence. Resistant cultivars and proper utilization practices should be considered as potential means for disease management. Keywords: birdsfoot trefoil, crown and root rot, Fusarium oxysporum, Lotus corniculatus, persistence

Introduction

Birdsfoot trefoil (Lotus corniculatus L.) is the most important forage legume in Uruguay, where it is used for pasture, hay or silage. In addition, seed production is an important export enterprise. Birdsfoot trefoil is usually undersown with a cereal crop during the fall, and plays an important role in the sustainability of crop-pasture rotations. The major constraint for the use of birdsfoot trefoil is its relatively poor persistence. In Uruguay, significant plant losses are observed in pastures in stands two-years-old and older, especially following periods of environmental stress (i.e. summer drought) or under continuous grazing systems (Formoso, 1993). These stands become unprofitable and the farmer may decide to start a new crop, resulting in a short legume-phase in the rotation. A similar situation is reported by several authors in North America (Beuselinck et al., 1984; Hoveland, 1989; Miller et al., 1964;

42 Crown and root rot in Lotus corniculatus 43

Taylor et al., 1973). The limited persistence is generally attributed to the interaction of several abiotic and biotic factors such as climatic and edaphic stresses, diseases and pests, and management practices that produce a cumulative stress load (Grau, 1996; Leath, 1989). The use of birdsfoot trefoil could be increased if highly productive stands could be maintained under intensive management for several years.

Diseases are a major cause of premature stand decline and reduced productivity in most temperate forage legumes (Leath, 1989; Leath et al., 1996). Crown and root diseases have been identified as the most important limitations to birdsfoot trefoil production and persistence (Berkenkamp et al., 1972; Beuselinck, 1988; Drake, 1958; Grant and Marten, 1985; Henson, 1962; Hill and Zeiders, 1987; Hoveland et al., 1982; Hoveland et al., 1987; Kainski, 1960; Miller et al., 1964; Pettit et al., 1966; Taylor et al., 1973). Severe losses from these diseases are usually associated with warm weather and high humidity, and thus these are of greater importance in the South than in the Northeast or Northcentral U.S. (Beuselinck, 1988; Grant and Marten, 1985). However, these diseases have been recently reported in most regions where birdsfoot trefoil is grown (Altier, 1994; Bergstrom et al., 1995; Viands et al., 1994). In Uruguay, Altier (1994, 1997) found that 93% of birdsfoot trefoil plants from a space plant nursery died by the end of the second year, and 82% of plant losses were due to crown and root diseases.

The first symptom of crown and root infection is the failure of the plants to resume growth after harvest (Berkenkamp et al., 1972; Grau, 1996; Henson, 1962; Kainski, 1960). Infected plants have a low tolerance to water stress during summer months and reduced vigor; if the invasion continues, plants become wilted and die. Diseased plants show necrosis and rotting of crown and root cortical tissues, but discoloration may be restricted to the central core and follow the vascular system (Figure 1). As the disease develops, both the cortex and central core may be invaded by the fungus. Necrotic areas are often associated with wounds in the crown or root surface (Altier, 1994; Leath et al., 1971). Insect feeding injury by root curculio probably enhances infection by soil pathogens (Kalb et al., 1994; Leath and Hower, 1993). Figure 1. Symptoms of crown and root rot in a diseased birdsfoot trefoil plant.

44 Nora A. Altier and Linda L. Kinkel

Crown and root diseases are caused by a complex of soil organisms. Although several genera of fungi including Rhizoctonia, Mycoleptodiscus, Macrophomina, Phoma, and others have been isolated from diseased plants, Fusarium species make up the largest number of pathogens causing crown and root diseases of birdsfoot trefoil (Berkenkamp et al., 1972; Beuselinck, 1988; Drake, 1961; Henson, 1962; Kainski, 1960; Ostazeski, 1967). The species of Fusarium most frequently associated with crown and root rots of forage legumes is F. oxysporum, followed by F. avenaceum, F. solani, F. acuminatum, F. tricinctum, and F. moniliforme (Grau, 1996; Leath, 1989). In addition, F. oxysporum has been reported as the causal organism of Fusarium wilt of birdsfoot trefoil (Gotlieb and Dorisky, 1983; Zeiders and Hill, 1988). More recently, Bergstrom and Kalb (1995) described a wilt organism of birdsfoot trefoil as a specific pathogen of this species, for which they proposed a new taxon, F. oxysporum f.sp. loti. In Uruguay, Altier (1994, 1997) studied the fungi associated with diseased birdsfoot trefoil plants in a nursery and found that the majority of fungi isolated from crown and root tissues were Fusarium spp. (72%), with the two most frequently isolated species being F. oxysporum (54% of total) and F. solani (9% of total). Similar results were found by Chao et al. (1994), who reported Fusarium as the main genus (>80%) associated with infected crowns and roots during a survey of diseases affecting birdsfoot trefoil in western Uruguay and the Entre Rios Province, Argentina.

While the information published on crown and root diseases of Lotus focuses on descriptions of pathogens, etiology, or yield impacts, studies on disease ecology and epidemiology are limited (English, 1999). Information on the ecological aspects of Fusarium crown and root disease comes from studies done with other plant hosts. Forage legume roots are most likely colonized by Fusarium species shortly after planting. However, disease symptoms may not appear for some time. This delay has been generally attributed to the weak pathogenicity of root rotting Fusaria, which cause more severe rot when plants are under stress (Grau, 1996; Leath, 1989). Fusarium rot then progresses gradually, increasing in severity with the age of the plant (Kalb et al., 1994; Leath, 1989).

Knowledge of disease development on individual plants must be coupled with epidemiological studies to understand host population responses to pathogen population pressures in the field. Although gross estimates on the impact of crown and root rots on forage legumes are available, there is much less research concerning the quantitative measurement of disease incidence and severity and the dynamics of these diseases in time and space (Nutter and Gaunt, 1996). The understanding of root disease development in time and space is of critical importance for the management of a perennial forage crop. The longevity of each plant, and therefore the productivity and persistence of the crop, will be directly dependent on healthy root systems. Until epidemiological data are available, disease management in forages will be imprecise (Leath, 1989).

Quantitative characterization of root disease epidemics has been difficult because of the relative inaccessibility of the roots (Campbell, 1986). The progress of root disease epidemics is most commonly monitored as an increase in incidence and severity of root symptoms. Obtaining these measurements requires destructive sampling which does not allow repeated assessments on the same plant. Sampling strategies must be carefully designed in order to study the temporal development and spatial pattern of disease (Campbell and Madden, Crown and root rot in Lotus corniculatus 45

1990).

The current study was aimed to assess the importance of crown and root rot complex on birdsfoot trefoil production in Uruguay and characterize disease epidemiology. This knowledge should provide insight into potential means of disease management to aid farmers in decision-making. Specifically, the major objectives of this research were to determine (1) the incidence and severity of crown and root diseases of birdsfoot trefoil as affected by stand age in diverse ecological regions of Uruguay, and (2) the main pathogens associated with diseased plants.

Materials and Methods

Field survey

Between September 1994 and March 1996, 12 birdsfoot trefoil fields were surveyed in three areas representing distinct physiographic regions of Uruguay, distinguished by soil type, topography, and prevalent production systems. These fields where located in areas near INIA La Estanzuela, Colonia, INIA Tacuarembó, Tacuarembó, and INIA Treinta y Tres, Treinta y Tres (INIA, National Institute for Agricultural Research, Uruguay) (Figure 2).

Figure 2. Location of three regions in Uruguay, in which birdsfoot trefoil was surveyed for crown and root diseases from 1994 to 1996.

Characteristics at each area are as follows; Colonia: clay-loamy, horizon B textural soils (O.M.=2.1-4.3%; pH=5.8-7.0; Al<0.1meq/100g; P>5ppm), sown pasture for very intensive production systems (livestock-crop and dairy farms); Tacuarembó: sandy/sand-loamy, deep soils (O.M.=1.1-3.3%; pH=4.5-5.5; Al=0.4-0.8meq/100g; P<5ppm), sown pasture for intensive to more extensive production systems (livestock, small area under crop rotation and dairy farms); Treinta y Tres: loamy/loam-sandy soils (O.M.=1.5-4.7%; pH=5.1-5.6; Al=0.1-0.5meq/100g; P<5ppm; high erosion risk), sown pasture for diversified production systems (livestock, hay-seed production of forage crop). 46 Nora A. Altier and Linda L. Kinkel

Each September (spring 1994 and 1995), birdsfoot trefoil fields were selected to compose a matrix of nine sites: three locations and three ages of stand (1-, 2-, and 3-yr-old). One-yr-old stands represented pastures sown during the fall months (April-May-June) of the current year. In September 1995 at each location, the 3-yr-old field was dropped while a new 1-yr-old field was included, in order to keep the nine site matrix. This new matrix was sampled at September 1995 and at March 1996.

A stratified sampling design was employed using twelve permanent 5x5 m quadrats per site. In each site, stand counts and plant samplings were performed twice a year, at the end of winter (September) and at the end of summer (March). Stand counts (no. of plants/m2) were performed in the central square meter of the quadrat, using a 1.0x0.1 m transect. Sample size in each quadrat was 25 plants, with one plant randomly sampled from each 1x1 m cell of the quadrat. Plants were dug and removed with the entire root system, placed in ice chests and taken to the laboratory. At the laboratory, roots and crowns were washed and split longitudinally. Each plant was scored for disease severity (crown rot and root rot separately) following a visual five-class scale: 0 = no disease, 1 = slight rot or discoloration (less than 30% affected tissue), 2 = moderate crown/root rot or discoloration (30-70% affected tissue), 3 = severe crown/root rot or discoloration (more than 70% affected tissue), 4 = plant dead. Disease incidence (crown rot and root rot separately) was calculated as percentage of diseased plants per quadrat. The assessed unit was the whole crown or root: a scale value of 1 or higher constituted disease. Variance-to-mean ratios were calculated for the crown and root severity data for each quadrat at each sampling time to provide insight into the spatial pattern of the disease among quadrats (Campbell and Madden, 1990).

Descriptive statistics and analyses of variance (general linear model procedure, SAS Institute) were performed on crown and root rot incidence (CRI, RRI), crown and root rot severity (CRS, RRS), stand counts (NP), and variance-to-mean ratios for crown and root rot severity (VMC, VMR). Mean separations were performed using Fisher's protected LSD test (P<0.05). For the seven variables analyzed (CRI, RRI, CRS, RRS, NP, VMC, VMR), the variances were not homogeneous among different years (1994-1995 vs. 1995-1996) and different sampling seasons (September vs. March), therefore data were analyzed separately.

Fungal isolations

Subsamples of diseased roots from each quadrat of each site, sampled in September of 1994 and September of 1995, were used for fungal isolation. The roots were randomly selected from those representing the median severity class in the given quadrat, most commonly roots in classes 1 and 2. Pieces of 0.5-1.0 cm2 from the interface of infected and non-symptomatic tissue were washed under flowing tap water overnight, surface-sterilized by soaking in 95% ethanol for 1 min, then soaking in 1% sodium hypochlorite for 3 min, followed by a rinse in sterile distilled water, and finally plated on PDA. Two and five pieces were plated per quadrat, for roots sampled in September of 1994 and September of 1995, respectively. The intention was to obtain at least one fungal isolate per quadrat per site (12 quadrats x 3 locations x 3 stand ages = 108 isolates). Hyphal tip growth of each different fungal colony (except for easily identified genera) was transferred to PDA plates and tubes for further Crown and root rot in Lotus corniculatus 47 identification and storage. Each year (1994 and 1995) a collection of Fusarium spp. isolates was maintained on PDA slants at 4 C during the identification process (four months). Subsequently, selected isolates were stored on silica gel crystals at 5 C until needed (Windels, 1992). Identification of F. oxysporum was done using the procedures outlined by Nelson et al. (1983). Three randomly selected isolates were sent to the International Mycological Institute (IMI-CAB International, UK) for confirmation of identification (IMI No. 368015, 368016, 368017, report from Dr. D. Brayford). Two collections of F. oxysporum isolates were finally composed.

Results

Field survey

Crown and root rot occurred in every field surveyed, independent of location and stand age. Locations followed no consistent trend as a source of variation for crown and root rot incidence. Stand age had a large and significant effect on disease incidence (Table 1). At both sampling times (September and March), crown rot incidence and root rot incidence were significantly lower in 1-yr-old stands than in 2- or 3-yr-old stands. The largest increase in crown rot incidence was observed between September and March of 1-yr-old stands, i.e. after the first summer of the crop, when incidence reached levels close to 80% (Figure 3A). Root rot incidence increased more slowly than crown rot incidence, but by September, in two-yr-old stands, levels were very close to 100% (Figure 3A). From that time on, levels of crown and root rot incidence were always higher than 90% (Figure 3B) but differences in disease incidence between 2-yr-old vs. 3-yr-old stands were still usually significant (Table 1).

Disease severity data showed similar trends as disease incidence (Table 2, Figure 4). Location did not always have a significant or consistent effect on crown rot and root rot severity. Crown rot severity as well as root rot severity increased significantly with the age of the stand. As compared with disease incidence, disease severity continued to increase gradually from 2-yr-old stands to 3-yr-old stands, when there were no or few nondiseased plants left (disease incidence was close to 100%). The largest increase in disease severity occurred during the winter for both years. Crown rot severity was always higher than root rot severity, except in September in 1-yr-old stands, where crown rot severity was equal to or lower than root rot severity.

Stand counts were significantly affected by location and stand age (Table 3). Two and 3-yr-old stands at Tacuarembó generally had lower counts than the other two locations. The average number of plants per square meter declined as stands aged. The largest reduction was observed from 2- to 3-yr-old stands. Within each field, the stand counts indicated that most plants died during the summer, as determined by large differences between September counts and March counts (Figure 5). Data on stand counts showed the reverse trend from data on disease level, i.e. the older the stand, the higher the disease level and the lower the number of plants per square meter. 48 Nora A. Altier and Linda L. Kinkel

Table 1. Incidence1 of Fusarium crown and root rot in birdsfoot trefoil, for each sampling time2 as affected by stand age.

Crown Rot Incidence (%) Root Rot Incidence (%) Stand Age S942 M95 S95 M96 S94 M95 S95 M96 1-yr-old 163 78 2 80 17 43 26 79 2-yr-old 96 95 100 99 91 92 96 99 3-yr-old 99 99 100 100 99 98 99 100

LSD (0.05) 2 3 1 4 3 4 4 4 CV (%) 7 8 4 8 9 12 11 8

1 Disease incidence: No. of diseased plants/total No. of assessed plants. 2 September 1994 (S94), March 1995 (M95), September 1995 (S95), March 1996 (M96). Data were from the same 9 site matrix for Sept. 94 vs. March 95, and for Sept. 95 vs. March 96, so these two pairs of columns of values can be compared. However, variances were not homogeneous, therefore LSDs were not calculated. 3 Average of three locations.

100 A 80 60 40 20 0 Sep-94 Mar-95 Sep-95 Mar-96 Disease Incidence (%)

Crow n Rot C Crow n Rot T Crow n Rot TT Root Rot C Root Rot T Root Rot TT

100 B

80

60

40 Sep-94 Mar-95 Sep-95 Mar-96 Disease Incidence (%)

Crow n Rot C Crow n Rot T Crow n Rot TT Root Rot C Root Rot T Root Rot TT

Figure 3. Progress of disease incidence of crown and root rot of birdsfoot trefoil in 1- and 2-yr-old stands (A) and 2- and 3-yr-old stands (B), surveyed in Colonia (C), Tacuarembó (T) and Treinta y Tres (TT). Disease incidence was calculated as No.diseased plants/total No. assessed plants.

Crown and root rot in Lotus corniculatus 49

Table 2. Severity1 of Fusarium crown and root rot in birdsfoot trefoil, for each sampling time2 as affected by stand age.

Crown Rot Severity Root Rot Severity Stand Age S942 M95 S95 M96 S94 M95 S95 M96 1-yr-old 0.173 0.90 0.02 1.06 0.18 0.48 0.28 0.97 2-yr-old 1.33 1.49 1.82 1.91 1.20 1.26 1.43 1.65 3-yr-old 1.67 1.75 2.22 2.39 1.52 1.49 1.80 2.00

LSD (0.05) 0.06 0.07 0.08 0.14 0.07 0.09 0.09 0.13 CV (%) 12.0 11.4 11.9 16.0 15.5 16.8 16.4 17.4 1 Disease severity: 0 = no disease, 1 = slight crown/root rot (<30% affected tissue), 2 = moderate crown/root rot (30-70% affected tissue), 3 = severe crown/root rot (>70% affected tissue), 4 = plant dead. Since the scale included a class 0, crown and root rot severity are expressed as a disease index (DSI). 2 September 1994 (S94), March 1995 (M95), September 1995 (S95), March 1996 (M96). Data were from the same 9 site matrix for Sept. 94 vs. March 95, and for Sept. 95 vs. March 96, so these two pairs of columns of values can be compared. However, variances were not homogeneous, therefore LSDs were not calculated. 3 Average of three locations.

3 A 2.5 2 1.5 1 0.5 0 Sep-94 Mar-95 Sep-95 Mar-96 Disease SeverityIndex

Crow n Rot C Crow n Rot T Crow n Rot TT Root Rot C Root Rot T Root Rot TT

3 B 2.5 2 1.5 1 0.5 0 Sep-94 Mar-95 Sep-95 Mar-96 Disease SeverityIndex

Crow n Rot C Crow n Rot T Crow n Rot TT Root Rot C Root Rot T Root Rot TT

Figure 4. Progress of disease severity of crown and root rot of birdsfoot trefoil in 1-and 2-yr-old stands (A) and 2- and 3-yr-old stands (B), surveyed in Colonia (C), Tacuarembó (T) and Treinta y Tres (TT). Disease severity was rated using a visual 5-class scale: 0 = no disease, 1 = slight crown/root rot (<30% affected tissue), 2 = moderate crown/root rot (30-70% affected tissue), 3 = severe crown/root rot (>70% affected tissue), 4 = plant dead. Since the scale included a class 0, crown and root rot severity are expressed as a disease index (DSI). 50 Nora A. Altier and Linda L. Kinkel

Table 3. Number of plants of birdsfoot trefoil per square meter, for each sampling time1 as affected by stand age.

No. of plants/m2 Stand Age S941 M95 S95 M96 1-yr-old 3142 168 188 106 2-yr-old 214 116 146 63 3-yr-old 89 46 86 1

LSD (0.05) 25 16 21 15 CV (%) 26 31 32 47

1 September 1994 (S94), March 1995 (M95), September 1995 (S95), March 1996 (M96). Data were from the same 9 site matrix for Sept. 94 vs. March 95, and for Sept. 95 vs. March 96, so these two pairs of columns of values can be compared. However, variances were not homogeneous, therefore LSDs were not calculated. 2 Average of three locations.

A 500

2 400 300 200

No.plants/m 100 0 Sep-94 Mar-95 Sep-95 Mar-96

Colonia Tacuarembó Treinta y Tres

B 500

2 400 300 200

No.plants/m 100 0 Sep-94 Mar-95 Sep-95 Mar-96

Colonia Tacuarembó Treinta y Tres

Figure 5. Reduction in the number of plants of birdsfoot trefoil in 1- and 2-yr-old (A) and 2- and 3-yr-old (B) stands surveyed in Colonia, Tacuarembó and Treinta y Tres.

Crown and root rot in Lotus corniculatus 51

The average variance-to-mean (VM) ratios were always less than 1, which suggests a rather uniform distribution of the disease independent of sampling time, stand age, and location (Table 4). Location did not have a significant effect on VM ratios, except for March 96. However, VM ratios were significantly affected by stand age, the younger the stand the higher the VM ratio. The highest average VM ratios recorded in September of 1-yr-old stands were closest to 1, which is indicative of a nearly random pattern of the disease in new stands. VM ratios for crown rot severity tended to be slightly lower than VM ratios for root rot severity, suggesting a marginally more uniform distribution of crown rot than root rot.

Table 4. Variance to mean ratios (V/M) of Fusarium crown and root rot severity in birdsfoot trefoil, for each sampling time1 as affected by stand age.

V/M Crown Rot Severity V/M Root Rot Severity Stand Age S941 M95 S95 M96 S94 M95 S95 M96 1-yr-old 0.892 0.34 0.26 0.48 0.80 0.67 0.77 0.45 2-yr-old 0.24 0.28 0.23 0.28 0.31 0.32 0.26 0.28 3-yr-old 0.20 0.19 0.16 0.17 0.21 0.21 0.23 0.23

LSD (0.05) 0.08 0.04 NS 0.07 0.09 0.06 0.07 0.07 CV (%) 40.9 33.8 119.0 42.8 41.9 31.8 37.6 41.8

1 September 1994 (S94), March 1995 (M95), September 1995 (S95), March 1996 (M96). Data were from the same 9 site matrix for Sept. 94 vs. March 95, and for Sept. 95 vs. March 96, so these two pairs of columns of values can be compared. However, variances were not homogeneous, therefore LSDs were not calculated. 2 Average of three locations.

Fungal isolations

Fungal colonies were recovered from root and crown pieces of plants sampled in all three locations. Independent of the location, root and crown pieces from 1-yr-old plants yielded few fungal colonies (11.9% and 14.9% of the total, for 1994 and 1995, respectively), while fungi were readily isolated from root and crown pieces from 2- and 3-yr-old plants.

The majority of fungi isolated from diseased crown and root tissues of birdsfoot trefoil were Fusarium spp., with the most frequently and consistently isolated species being F. oxysporum (Table 5). The second most frequently isolated fungi included presumed saprophytic genera, Penicillium, Aspergillus, Gliocladium, Epicoccum, Cladosporium, Rhizopus, and Mucor. Unknown fungi included sterile hyphomycetes and coenocytic, nonsporulating species, and were recovered in relatively low frequencies (Table 5). One isolate recovered in September 1994, identified tentatively as Mycoleptodiscus spp., and two isolates recovered in September 1994, identified presumptively as Rhizoctonia solani, were counted as unknown fungi.

52 Nora A. Altier and Linda L. Kinkel

Table 5. Percent frequency of fungi isolated from diseased crowns and roots of birdsfoot trefoil plants from three locations (Colonia, Tacuarembó and Treinta y Tres) in Uruguay, for two sampling dates (September 1994 and September 1995).

Percent frequency Fungi isolated 1994 1995 Fusarium 53 57 F. oxysporum 35 40 Other Fusarium spp. 18 17 Presumed saprophytic genera1 35 38 Unknown2 12 5

Total number of isolates3 120 365 Number of pieces examined 216 540

1 Species of Penicillium, Aspergillus, Epicoccum, Cladosporium, Rhizopus, Mucor, Gliocladium, and others. 2 Sterile hyphomycetes, coenocytic, nonsporulating fungi and others. 3 Total number of yielding colonies on PDA from 0.5-1.0 cm 2 pieces cut from surface-sterilized diseased birdsfoot trefoil crowns and roots.

Sixty four Fusarium spp. isolates were recovered from roots sampled in 1994, and 208 isolates from roots sampled in 1995, and composed the two Fusarium spp. collections. Forty two F. oxysporum isolates from 1994, and 146 isolates from 1995 composed the two F. oxysporum collections.

Discussion

Deterioration of roots and crowns of birdsfoot trefoil was demonstrated to occur in diverse ecological areas of the country, representing distinct physiographic regions distinguished by soil type, topography, and prevalent production systems. The range of edaphic conditions surveyed does not limit the development of crown and root diseases in birdsfoot trefoil. We confirm the hypothesis that the occurrence of the crown and root rot complex is a widespread phenomenon in Uruguay and has a negative impact on birdsfoot trefoil production and persistence (Altier, 1994; 1997). Our results also agree with preliminary information obtained by Chao et al. (1994) during a survey of diseases affecting 12 birdsfoot trefoil pastures in western Uruguay and the Entre Rios Province, Argentina, which reported that crown and root rot were the most prevalent diseases.

The repeated isolation of Fusarium oxysporum from symptomatic plants of birdsfoot trefoil suggests this species is frequently responsible for crown and root rot and stand decline. No other known pathogen that is alone capable of causing these disease symptoms was isolated Crown and root rot in Lotus corniculatus 53 from diseased crown and root tissues. These results are consistent with previous findings when studying the fungi associated with crown and root rot complex of birdsfoot trefoil and other forage legumes (Altier, 1994; 1997; Beuselinck, 1988; Chao et al., 1994; Grau, 1996; Kainski, 1960; Leath, 1989; Zeiders and Hill, 1988).

The random initial spatial pattern of crown and root rot (variance to mean ratios slightly less than 1) may result from the ubiquitous nature of F. oxysporum and indicates that the potential for disease development is independent of location and site. The fact that VM ratios decreased with the age of the stand suggests a gradual saturation of the system, where every plant sampled was diseased as a consequence of the dispersal over space and time. The observed lower VM ratios for crown rot severity as compared with VM ratios for root rot severity indicates that infection of crown tissue progresses faster than infection of root tissue and saturation of the system occurs early.

The large and significant effect of stand age on disease incidence and severity was expected and previously reported (Berkenkamp et al., 1972; Beuselinck, 1988; Drake, 1958; Grant and Marten, 1985; Grau, 1996; Henson, 1962; Hill and Zeiders, 1987; Hoveland et al., 1982; Hoveland et al., 1987; Kainski, 1960; Leath, 1989; Leath et al., 1971; Miller et al., 1964; Pettit et al., 1966; Taylor et al., 1973). However, we did not expect the high disease incidence levels as early as March, when the plants had not completed one year in the field. Root rot incidence increased slower than crown rot incidence, but by September in the second production year most of the plants were symptomatic. Large areas of necrosis limit the amount of healthy tissue available to maintain the essential physiological functions of water and nutrient absorption, nitrogen fixation, carbohydrate storage and translocation to the growing points (Grau, 1996).

In the conditions of Uruguay, summer appears to be the critical season for plant survival. While the largest increases in crown and root rot severity occurred during the winter, stand count results indicate that most plants died during the summer. The high soil temperatures registered during that season, interacting with periods of drought, most likely accentuate the stress on plants already weakened by disease. Since data on stand counts supported data on disease level, i.e. the older the stand, the higher the disease level and the lower the number of plants per square meter, a relationship between disease level and persistence in the field is clearly established.

Fusarium crown and root rot is affected by environmental, biological and management factors that stress the plant and is a chronic rather than an acute disease (Grau, 1996; Leath, 1989). The utilization of perennial forage legumes is in itself the most serious stress repetitively imposed on the plants (Beuselinck et al., 1984; Hoveland, 1989; Leath, 1989; Miller et al., 1964; Taylor et al., 1973). In the production systems of Uruguay, pastures are utilized during most of the year, therefore grazing animals are imposing a severe stress on the legume. Animals influence legume performance by selective grazing, trampling and excretion (Hoveland, 1989). Animal trampling causes direct injuries to the crown of the plants producing entry points for pathogens. Despite the fact that Fusarium species may directly penetrate unwounded tissues, wounding alters the host-pathogen interaction and favors fungal development in the tissues (Chi et al., 1964; Stutz et al., 1985). Wounding of 54 Nora A. Altier and Linda L. Kinkel roots and crowns is a common phenomenon and F. oxysporum is a wound parasite that can readily invade tissues (Kalb et al., 1994; Leath, 1989; Leath and Hower, 1993). Fusarium spp. are primarily cortical invaders which can survive and increase in the cortex until conditions favor pathogenicity (Kommedahl and Windels, 1979). Once the infection has taken place, the plant remains diseased. The impact of the disease and the rate at which it develops are functions of the environment and management. Because climate cannot be impacted, proper management becomes the prime strategy. Correct and timely application of crop management practices during the winter and summer months must contribute to reduce the stresses imposed on the plants and therefore, to reduce the rate of disease development and stand decline. Crop management practices, such as frequency and intensity of utilization, play a role in the development of Fusarium crown and root rot of red clover (Fezer, 1961; Fulton and Hanson, 1960; Rufelt, 1986; Siddiqui et al., 1968) and alfalfa (Lukezic et al., 1969). The effect of utilization management on the rate of disease development and consequently the impact on birdsfoot trefoil productivity need to be investigated.

Additionally, phenotypic selection has proved to be effective in increasing the level of resistance to F. oxysporum, when developing birdsfoot trefoil populations (Altier et al., 2000; Rebuffo and Altier, 1997; Zeiders and Hill, 1988). The release of cultivars with enhanced resistance need to be coupled with improved management practices to provide an integrated management scheme for Fusarium crown and root diseases.

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GRANT W.F. and MARTEN G.C. 1985. Birdsfoot trefoil. In HEATH M.E., BARNES R.F. and METCALFE D.S. (Eds.). Forages: the science of grassland agriculture. 4th. ed. Ames, Iowa State University Press. pp. 98-108.

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HOVELAND C.S. 1989. Legume persistence under grazing in stressful environments of the United States. In MARTEN G.C., MATCHES A.G., BARNES R.F., BROUGHAM R.W., CLEMENTS R.J. and SHEATH G.W. (Eds.) Persistence of forage legumes. Madison, ASA-CSSA-SSSA, Madison, WI, USA. pp. 375-383.

HOVELAND C.S., ALISON M.W.Jr. and DOBSON J.W.Jr. 1987. Performance of birdsfoot trefoil varieties in North Georgia. Athens, Agricultural Experiment Stations, University of Georgia. Research Report, 528. 5 p.

HOVELAND C.S., HAALAND R.L., HARRIS R.R. and MCGUIRE J.A. 1982. Birdsfoot trefoil in Alabama. Auburn, Alabama Agricultural Experiment Station, Auburn University. Bulletin 537.

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KALB D.W., BERGSTROM G.C. and SHIELDS E.J. 1994. Prevalence, severity, and association of fungal crown and root rots with injury by clover root curculio in New York alfalfa. Plant Disease, 78, 491-495.

KOMMEDAHL T. and WINDELS C.E. 1979. Fungi: pathogen or host dominance in disease. In KRUPA S.V. and DOMMERGUES Y.R. (Eds.). Ecology of root pathogens. Elsevier Scientific Publishing Company: Amsterdam, The Neatherlands. pp. 1-103.

LEATH K.T. 1989. Diseases and forage stand persistence in the United States. In MARTEN G.C., MATCHES A.G., BARNES R.F., BROUGHAM R.W., CLEMENTS R.J. and SHEATH G.W. (Eds.) Persistence of forage legumes. ASA-CSSA-SSSA: Madison, WI, USA. pp. 465-479.

LEATH K.T.and HOWER A.A. 1993. Interaction of Fusarium oxysporum f.sp. medicaginis with feeding activity of clover root curculio larvae in alfalfa. Plant Disease, 77, 799-802. Crown and root rot in Lotus corniculatus 57

LEATH K.T., LUKEZIC F.L., CRITTENDEN H.W., ELLIOT E.S., HALISKY P.M., HOWARD F.L. and OSTAZESKY S.A. 1971. The Fusarium root rot complex of selected forage legumes in the Northeast. Pennsylvania State University, PA, USA. Bulletin 777.

LEATH K.T., WELTY R.E., PRATT R.G. and SONODA R.M. 1996. Pasture/forage crops and diseases in the United States. In CHAKRABORTY S., LEATH K.T., SKIPP R.A., PEDERSON G.A., BRAY R.A., LATCH G.C.M. and NUTTER F.W.Jr. (Eds.) Pasture and forage crop pathology. ASA-CSSA-SSSA: Madison, WI, USA. pp. 33-58.

LUKEZIC F.L., BLOOM J.R. and CARROLL R.B. 1969. Development of Fusarium root rot and its effect on root and crown carbohydrate levels of clipped alfalfa plants grown in a gnotobiotic environment. Phytopathology, 59, 1575-1579.

MILLER J.D., KREITLOW K.W., DRAKE C.R. and HENSON P.R. 1964. Stand longevity studies with birdsfoot trefoil. Agronomy Journal, 56, 137-139.

NELSON P.E., TOUSSOUN T.A. and MARASAS W.F.O. 1983. Fusarium species: an illustrated manual for identification. Pennsylvania State University Press: University Park, PA, USA.

NUTTER F.W.Jr. and GAUNT R.E. 1996. Recent developments in methods for assessing disease losses in forage/pasture crops. In CHAKRABORTY S., LEATH K.T., SKIPP R.A., PEDERSON G.A., BRAY R.A., LATCH G.C.M. and NUTTER F.W.Jr. (Eds.) Pasture and forage crop pathology. ASA-CSSA-SSSA, Madison, WI, USA. pp. 93-118.

OSTAZESKI S.A. 1967. An undescribed fungus associated with a root and crown rot of birdsfoot trefoil (Lotus corniculatus). Mycologia, 59, 970-975.

PETTIT R.E., CALVERT O.H. and BALRIDGE J.D. 1966. Leptodiscus terrestris colonization of birdsfoot trefoil roots in Missouri. Plant Disease Reporter, 50, 753-755.

REBUFFO M. and ALTIER N. 1997. Breeding for persistence in Lotus corniculatus. In Proceedings of the XVIII International Grassland Congress. Winnipeg and Saskatoon, Canada. pp. 4/73-74.

RUFELT S. 1986. A threshold value for cutting intensity in relation to Fusarium root rot in red clover. In Studies on Fusarium root rot of red clover (Trifolium pratense L.) and the potential for its control. Ph.D. Thesis. Uppsala, Swedish University of Agricultural Sciences. Plant Protection Reports, Dissertation, 10.

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SIDDIQUI W.M., HALISKY P.M. and LUND S. 1968. Relationship of clipping frequency to root and crown deterioration in red clover. Phytopathology, 58, 486-488.

STUTZ J.C., LEATH K.T. and KENDALL W.A. 1985. Wound-related modifications of penetration, development, and root rot by Fusarium roseum in forage legumes. Phytopathology, 75, 920-924.

TAYLOR T.H., TEMPLETON W.C.Jr. and WYLES J.W. 1973. Management effects on persistence and productivity of birdsfoot trefoil (Lotus corniculatus L.). Agronomy Journal, 65, 646-648.

VIANDS D.R., EHLKE N.J., PAPADOPOULOS Y.A. and SMITH R.R. 1994. Cooperative project to develop birdsfoot trefoil with multiple disease resistance. Lotus Newsletter, 25, 45-46.

WINDELS C.E. 1992. Fusarium. In SINGLETON L.L., MIHAIL J.D. and RUSH C.M. (Eds). Methods for research on soilborne phytopathogenic fungi. APS Press: St. Paul, MN, USA. pp. 115-128.

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Lotus Newsletter (2005) Volume 35 (1), 59-74.

Characterization of aggressiveness and vegetative compatibility diversity of Fusarium oxysporum associated with crown and root rot of birdsfoot trefoil

NORA A. ALTIER1* and JAMES V. GROTH2 1Department of Plant Protection, National Institute for Agricultural Research, INIA Las Brujas, 90200 Canelones, Uruguay 2Department of Plant Pathology, University of Minnesota, St. Paul, MN, United States of America *Corresponding author

Abstract

Birdsfoot trefoil (Lotus corniculatus) fields were selected in a 9-site-matrix of three locations and three stand ages, and surveyed twice a year during two successive years. Twenty-five plants in each of 12 permanent quadrats were sampled at each site and date. Samples of infected crown and root tissues were used for fungal isolation. Fusarium oxysporum was the primary pathogen associated with diseased plants (40% of isolations). Vegetative compatibility assessed using nitrate non-utilizing mutants was used as a measure of genetic relatedness of F. oxysporum isolates. No complementation was found among 18 isolates (630 pairings of 36 nit complementary mutants), indicating a high degree of genetic diversity in the pathogen population. A culture plate method was used to characterize isolate aggressiveness to birdsfoot trefoil seeds and seedlings, based on a 5-class scale: 1 = healthy seedling, 5 = dead seed. Most F. oxysporum isolates (36 out of 44) were pathogenic to birdsfoot trefoil, and were highly variable in aggressiveness (range: 1.44-3.85). The variability observed in the pathogen population needs to be considered when selecting isolates for resistance screening. Additional keywords: crown and root diseases, forage legumes.

Introduction

Crown and root diseases are a major cause of premature stand decline and reduced productivity in birdsfoot trefoil pastures (Altier, 1997; Altier et al., 2000; Bergstrom et al., 1995; Berkenkamp et al., 1972; Beuselinck, 1988; Henson, 1962; Hill and Zeiders, 1987; Kainski, 1960; Miller et al., 1964; Pettit et al., 1966; Taylor et al., 1973). Fusarium species make up the largest number of pathogens causing crown and root diseases of birdsfoot trefoil (Berkenkamp et al., 1972; Beuselinck, 1988; Drake, 1961; Henson, 1962; Kainski, 1960). The species of Fusarium most frequently associated with crown and root rots of forage legumes is F. oxysporum, followed by F. avenaceum, F. solani, F. acuminatum, F. tricinctum, and F. moniliforme (Grau, 1996; Leath, 1989; Leath et al., 1971). More recently, Bergstrom and Kalb (1995) described a wilt organism of birdsfoot trefoil as a specific pathogen of this species, for which they proposed a new taxon, F. oxysporum f.sp. loti. In

59 60 N.A. Altier and J.V. Groth

Uruguay, Altier (1994, 1997) studied the fungi associated with diseased birdsfoot trefoil plants in a space-planted nursery and found that the majority of fungi isolated from crown and root tissues were Fusarium spp. (72%), with the two most frequently isolated species being F. oxysporum (54% of total fungi) and F. solani (9% of total fungi).

Authors disagree on the role that Fusarium spp. have in the development of the root and crown disease complex of forage legumes (Grau, 1996). Some contend that pathogenic forms only invade root tissues damaged or killed by other causes; others assert that the fungus plays a major role in root and crown disease development (Leath et al., 1971). The pathogenicity of Fusarium species varies considerably among and within species depending on their ability to penetrate roots directly, their degree of host specificity, and their interaction with plant stress factors (Fezer, 1961; Leath, 1989; Leath and Kendall, 1978; Pederson et al., 1980; Stutz and Leath, 1983; Venuto et al., 1995). A number of environmental factors including soil moisture, drainage, air and soil temperature, nutrients, stand density, plant age, frequency and height of cutting, crop rotation, insect injury, and previous invasion by viruses and nematodes, have an effect on the expression of the disease and on plant susceptibility (Leath et al., 1971).

Root rotting Fusaria may penetrate unwounded roots directly, but most species have limited ability to initiate root rot on their own (Chi et al., 1964; Stutz et al., 1985). The wounding of roots increases the frequency of penetration by disrupting the mechanical barrier imposed by the epidermis. Furthermore, Stutz et al. (1985) asserted that wounding alters the host-pathogen interaction to favor fungal development in the root. Wounding can be the result of insect damage or mechanical injuries by soil heaving, harvesting machinery, and trampling and soil compaction caused by animals.

Kommedahl and Windels (1979) asserted that F. oxysporum is mainly a wilt-inducing pathogen. However, it is so frequently isolated from necrotic roots that it is regarded as a root rot, wound-associated, pathogen (Kalb et al., 1994; Leath, 1989; Leath and Hower, 1993). F. oxysporum has been described as an aggressive, pioneer colonizer of moribund tissues and it can readily invade roots (Leath, 1989).

An understanding of the evolutionary basis for the pathogenicity of F. oxysporum and the genetic diversity of the fungal population is critical and would help to develop, or improve, the effectiveness of strategies for disease management (Gordon and Martyn, 1997; Kistler, 2001). Classical genetics based on segregation and recombination is not possible with this anamorphic fungus, since it lacks a known perithecial state (Kistler, 1997). Among diverse approaches in genetic techniques, heterokaryosis or vegetative compatibility analysis provides the opportunity to study the genetics of F. oxysporum with greater precision than before (Bosland, 1988; Klein and Correll, 2001). The mechanisms available for genetic exchange in this species are still largely unknown, but numerous possibilities exist beyond simple sexual or clonal reproduction. Genetic evidence consistent with horizontal genetic transfer (transposable elements) and past genetic hybridization between lineages indicate that means for recombination and production of novel genotypes are effectively available (Kistler, 1997). Heterokaryosis and parasexual recombination have been postulated as mechanisms that play a role in explaining the diverse pathogenic potential of F. oxysporum Fusarium oxysporum diversity in Lotus corniculatus 61

(Bosland, 1988). Heterokaryon formation is favored by vegetative compatibility, which is mediated by nuclear loci, called het or vic genes (Leslie, 1993). Two strains are vegetatively compatible if they have the same allele at each incompatibility locus. In an asexual population (as in F. oxysporum), differences at the vic loci are assumed to effectively limit the exchange of genetic information to those individuals that belong to the same vegetative compatibility group (VCG). Since sexual recombination does not occur, members of each VCG will form a genetically isolated subpopulation that will be subjected to standard population genetic forces such as selection, mutation, migration, and drift (Leslie, 1993). Vegetative compatibility can serve as a natural means to further subdivide closely related fungal populations and has been used to estimate genetic diversity within and among these same populations (Correll, 1991; Klein and Correll, 2001; Leslie, 1993).

Puhalla (1985) initiated the idea of grouping isolates of F. oxysporum into VCGs based on the use of nitrate nonutilizing (nit) mutants generated on medium containing potassium chlorate. Mutants resistant to the salt are usually also nit mutants. On a minimal medium with nitrate as the sole nitrogen source, mutants have a radial growth comparable to wild type, but their colonies are very thin. If mutants produce a dense, wild-type growth when paired, they are vegetatively compatible (Puhalla, 1985). This author reported evidence for a correlation between VCG and forma specialis, and proposed an evolutionary model to explain the origin of formae speciales, races and VCGs. He asserted that vegetative compatibility may be a fast and easy way to distinguish pathotypes of F. oxysporum with unique virulence capabilities (Puhalla, 1985). Recently, Klein and Correll (2001) asserted that molecular markers and VCG are usually not independently associated, which may mean that VCGs in F. oxysporum represent clones, or closely related strains descended from a common ancestor (lineage). However, when pathotypes have been considered, there often has been no clear-cut association between these and molecular genotypes (Kistler, 1997).

Soil populations of some phytopathogenic fungi are extremely diverse with respect to VCG (Correll et al., 1986; Gordon and Martyn, 1997; Gordon and Okamoto, 1991; 1992). Studies that have used nit mutants to differentiate strains in the nonpathogenic portion of a F. oxysporum population revealed a large number of distinct VCGs (Correll et al., 1986; Gordon and Okamoto, 1991; Steinberg et al., 1997). Moreover, most of the strains associated with a given host within the same geographic area, or even isolated from a single field in two consecutive years, belonged to different VCGs (Correll et al., 1986; Gordon and Okamoto, 1991). Thus, the frequency with which anastomosis occurs within such populations is likely to be low.

Despite many references concerning VCGs in diverse formae speciales, few references are found concerning F. oxysporum that cause crown and root diseases of forage legumes. Venuto et al. (1995) studied the virulence, legume host specificity and genetic relatedness of F. oxysporum isolates from red clover, and reported that VCGs were not useful in predicting host reaction because isolates from distinct groupings elicited similar host reactions. Their results indicate that the number of genes controlling compatibility seems to be higher than the number of virulence genes (Venuto et al., 1995).

Information on the pathogenicity and genetic diversity of F. oxysporum isolates from 62 N.A. Altier and J.V. Groth birdsfoot trefoil is required if breeding for resistance is to be explored as a means of managing the Fusarium crown and root disease complex (Altier et al., 2000). The major objective of our study was to characterize the F. oxysporum population associated with diseased plants of birdsfoot trefoil, in terms of aggressiveness and genetic relatedness.

Materials and methods

Fungal isolations

Birdsfoot trefoil fields were selected in a 9-site-matrix of three locations (Colonia, Tacuarembó and Treinta y Tres, Uruguay) and three stand ages (1-, 2-, and 3-yr-old), and surveyed during September of 1994 and 1995. A stratified sampling design was employed using 12 permanent 5x5 m quadrats per site and sample size was 25 plants per quadrat. At the laboratory, subsamples of five diseased roots from each quadrat of each site were used for fungal isolation. Pieces of 0.5-1.0 cm2 from different areas of the root and crown (primarily from the interface of infected and non-symptomatic tissues) were washed under flowing tap water overnight, surface-disinfested by soaking in 95% ethanol for 1 min, then soaking in 1% sodium hypochlorite for 3 min, followed by a rinse in sterile distilled water, and finally plated on PDA. Two and five pieces were plated per quadrat, for roots sampled in September of 1994 and September of 1995, respectively. The intention was to obtain at least one fungal isolate per quadrat per site (12 quadrats x 3 locations x 3 stand ages = 108 isolates). Hyphal tips from each fungal colony (except for easily identified genera) were transferred to PDA plates and tubes for further identification and storage. Each year (1994 and 1995) a collection of Fusarium spp. isolates was maintained on PDA slants at 4 C during the identification process (four months). Subsequently, selected isolates were stored on silica gel crystals at 5 C until needed (Windels, 1992). Isolates were identified as F. oxysporum using the procedures outlined by Nelson et al. (1983). Three randomly selected isolates were sent to the International Mycological Institute (IMI-CAB International, UK) for confirmation of identification (IMI No. 368015, 368016, 368017, report from Dr. D. Brayford).

Two core collections of F. oxysporum isolates (composed of 15 out of 64 isolates of the 1994 Fusarium spp. collection, and 36 out of 208 isolates of the 1995 Fusarium spp. collection) were used to perform aggressiveness and vegetative compatibility tests. Isolates to compose the core collections were selected as follows: 1. the three geographical locations were represented; 2. different stand ages within locations were represented; 3. different quadrats within sites were represented, and if there were more than one isolate per quadrat within a site, one isolate was randomly selected.

Aggressiveness of isolates

A culture plate method was used to characterize F. oxysporum isolates for aggressiveness to seeds and seedlings of birdsfoot trefoil. The seedling test was not aimed to parallel the development of the Fusarium crown and root rot in the field, which primarily occurs in mature plants, but rather to compare the behavior of isolates on a potential host plant as Fusarium oxysporum diversity in Lotus corniculatus 63 reported by Fulton and Hanson (1960), Kainski (1960), and Kilpatrick et al. (1954). The method is similar to one used to select alfalfa germplasm for resistance to Pythium seedling diseases (Altier and Thies, 1995).

Fungal inoculum, consisting of mycelia and conidia, was produced in a 9 cm-diameter petri plate containing PDA. A 3 mm-diameter disc of inoculum was removed from the periphery of the resulting 4 to 5-day-old colony, placed in the center of a 9 cm-diameter petri plate containing 1.5% water agar (WA) and incubated at 22 C for 7 days. Using a vacuum template, 25 surface-disinfested birdsfoot trefoil seeds were placed equidistantly to the inoculum disc in a radiate pattern on the agar surface. The plates were incubated in growth chambers at 22 C for 7 days under cool-white light (12-h photoperiod; 330 FT. candles). Noninoculated plates of WA containing 25 surface-disinfested seeds were used as controls to determine seed germination, and expected percentage of dead and hard seed.

Disease severity was used as a measurement of isolate aggressiveness and was rated using a five-class scale, in which 1 = healthy seedling, primary root free of necrosis or with slight discoloration; 2 = infected seedling, primary root tip necrotic but firm, cotyledons free of disease; 3 = severely infected seedling, primary root tip and/or cotyledons rotted and soft, seedling will die as infection progresses; 4 = dead seedling, germinated seed with emerged radicle rotted; 5 = dead seed, nongerminated seed rotted. Aggressiveness was expressed as disease severity index (DSI), calculated as the numerical value of each class times the number of individuals in the class, divided by the number of seeds expected to germinate as determined in the noninoculated control, and percentage of surviving plants (PSP), calculated as the total of classes 1 and 2 divided by the number of seeds expected to germinate as determined in the noninoculated control.

Eight F. oxysporum isolates from the 1994 core collection were tested against San Gabriel and Estanzuela Ganador birdsfoot trefoil cultivars in June 1995, and 36 F. oxysporum isolates from the 1995 core collection plus one isolate from 1994 (used as a control) were tested against San Gabriel birdsfoot trefoil in June 1996. The experimental design was a randomized complete block with four replications over time totaling 100 seeds per treatment, with a factorial arrangement of treatments (isolates x birdsfoot trefoil cultivars). For both experiments, data on DSI and PSP were subjected to analysis of variance (general linear model procedure, SAS Institute) and means were separated using Fisher's protected LSD test (P<0.05).

Genetic relatedness of isolates

Vegetative compatibility was used as a measure of genetic relatedness using the methodology developed by Puhalla (1985). Nitrate-nonutilizing (nit) mutants were recovered by plating the F. oxysporum isolates on a chlorate-containing medium (KPS), and complementation tests were performed on a minimal agar medium (MM) that contained sodium nitrate as the sole source of nitrogen (Puhalla, 1985).

For the recovery of nit mutants, each isolate of F. oxysporum was grown on MM at 22 C for 3-4 days. Four 3 mm-diameter mycelial plugs were taken from each colony and spaced well 64 N.A. Altier and J.V. Groth apart on each plate of KPS. The KPS plates were incubated at 22 C for 14 days, during which time they were inspected for fast-growing, chlorate-resistant sectors. Different sectors from the same isolate were then transferred to MM. Very thin, but normally expansive growth on MM indicated that the sectors were also unable to reduce nitrate (nit mutants). Different nit mutants of a given isolate were plated on MM as follows: 1 mm3 mycelial block of one of them, arbitrarily designated nitA, was placed at the center of a plate of MM, and five of the other nit mutants of that isolate were spaced in a circle of radius 15 mm around nitA. Plates were incubated at 22 C for 7 days and then examined. Any outer nit mutant that developed a line of dense growth where it contacted the central nitA colony was designated nitB. Based on Puhalla's results concerning efficiency of recovery of nit mutants (number of nit mutants per inoculum plug = 0.58, number of nitB mutants per inoculum plug = 0.13), we estimated that for each isolate, at least 16 wild type inoculum plugs should be plated on KPS (four KPS plates per isolate), to obtain nine nit sectors and two nitB mutants (Puhalla, 1985). The number of nit mutants tested for each isolate ranged from 12 to 24.

The number of isolates from which nit mutants were tested, the number of isolates from which nitA and nitB complementary mutants were obtained, and the number of isolates from which no complementary mutants were obtained, were recorded. Complementary nitA and nitB mutants from each of 18 F. oxysporum isolates were then paired on MM in all possible combinations to perform complementation tests among isolates (Puhalla, 1985). Nine isolates from the 1994 core collection and eight isolates from the 1995 core collection, plus the isolate 067NY-94 of F.o. f.sp. loti provided by Dr. G.C. Bergstrom (Dept. of Plant Pathology, Cornell University, Ithaca, NY 14853) were characterized for vegetative compatibility. Complementation tests for the 18 isolates (630 pairings of 36 nit complementary mutants) were repeated once during 1995 and 1996.

Results

Fungal isolations

Fungal colonies were recovered from root and crown pieces of plants sampled at the three locations. Independent of the location, root and crown pieces of 1-yr-old plants yielded few fungal colonies, but they were isolated readily from root and crown pieces of 2- and 3-yr-old plants.

The majority of fungi isolated from diseased crown and root tissues of birdsfoot trefoil were Fusarium spp., with the most frequently and consistently isolated species being F. oxysporum (66% and 70% of isolates, n=42 and n=146, in 1994 and 1995, respectively). Taxonomic identification was not done for the other Fusarium spp. Additional frequently isolated fungi included presumed saprophytic genera, Penicillium, Aspergillus, Gliocladium, Epicoccum, Cladosporium, Rhizopus, and Mucor. Unknown fungi (that were recovered in relatively low frequencies) included sterile hyphomycetes and coenocytic, nonsporulating species. One fungal isolate recovered in September 1994, identified tentatively as Mycoleptodiscus spp., and two isolates recovered in September 1994, identified tentatively as Rhizoctonia solani, were counted as unknown fungi. Fusarium oxysporum diversity in Lotus corniculatus 65

Sixty-four isolates of Fusarium spp. were recovered from roots sampled in 1994, and 208 isolates from roots sampled in 1995, and composed the two Fusarium spp. collections. Fifteen F. oxysporum isolates from 1994, and 36 isolates from 1995, were selected to compose the two F. oxysporum core collections.

Aggressiveness of isolates

The eight F. oxysporum isolates tested from the 1994 core collection were pathogenic to seed and seedlings of birdsfoot trefoil, but significant differences in aggressiveness were observed among the isolates (Table 1). The effect of host cultivar was significant, with Estanzuela Ganador being more susceptible than San Gabriel (data not shown). Results could be due to differences in the germplasm reaction and/or to differences in the seed vigor of the seed lots. However, there was no interaction between isolates and cultivars and therefore, data on DSI and PSP were averaged over the two cultivars. The ranges for average DSI and PSP among all isolates were 1.44-2.25 and 58.7-85.7, respectively (Table 1). One isolate from Treinta y Tres (TT1C8) was significantly more aggressive than all the rest, as determined by the highest DSI and the lowest PSP. The other isolates showed a continuous range in variation for aggressiveness.

Table 1. Aggressiveness on birdsfoot trefoil of Fusarium oxysporum isolates from the 1994 core collection, as determined by disease severity and percentage of surviving plants.

Isolate1 Disease severity2 Percentage of surviving plants3 (DSI) (PSP) TT1C8 2.254 58.64 C2C1 1.73 76.9 T2C1 1.68 79.5 C2C2 1.64 80.9 T3C1 1.61 80.1 T3C8 1.57 83.8 TT3C8 1.46 85.7 T2C6 1.44 85.6 LSD (0.05) 0.30 9.8 CV (%) 17.28 12.1 1 The first letter(s) refer(s) to the geographic location of an isolate; TT= Treinta y Tres, C= Colonia, T= Tacuarembó. The first number refers to the age in years of the diseased plant from which an isolate was derived; the second number refers to the quadrat in the field from which the diseased plant was sampled (1-12). 2 DSI, disease severity index based on a 5-class scoring system of individual seedlings: 1= healthy seedling, 2= primary root tip necrotic and firm, 3= primary root tip and cotyledons rotted and soft, 4= dead seedling, 5= dead seed. 3 PSP, percentage of surviving plants: percentage of seedlings in classes 1 and 2. 4 Values were averaged over birdsfoot trefoil cultivars San Gabriel and Estanzuela Ganador.

66 N.A. Altier and J.V. Groth

Since the results from the first aggressiveness test (1994 isolates) indicated that the interaction between isolates and cultivars was not significant, and the number of isolates to be tested from the 1995 core collection was large, the second aggressiveness test (1995 isolates) included only birdsfoot trefoil cv. San Gabriel.

All of the 36 F. oxysporum isolates tested from the 1995 core collection were pathogenic to seed and seedlings of birdsfoot trefoil, but significant differences in aggressiveness were observed among the isolates (Table 2). The range for average DSI was 1.69-3.85 and for average PSP, it was 4.8-78.9. With few exceptions, isolates from Colonia were more aggressive than isolates from the other two locations. One isolate from Colonia (C2C7) was significantly less aggressive than all other isolates, as determined by the lowest DSI and the highest PSP. Isolates from Tacuarembó and Treinta y Tres showed a large variability in aggressiveness (DSI = 2.44-3.76). There was a tendency for isolates from different quadrats from the same site to have similar DSI and PSP values. The control isolate TT1C8, from the 1994 core collection, had low aggressiveness as compared to isolates from the 1995 core collection (DSI = 2.61, PSP = 48.9). However, these values are similar to those obtained from the previous test (DSI = 2.25, PSP = 58.7) (Table 2).

Genetic relatedness of isolates

Nit mutants were obtained for 45 out of 52 isolates: 15 of 15 isolates that composed the 1994 core collection, 29 of 36 isolates of the 1995 core collection, and for the isolate of F.o. f.sp. loti provided by Dr. Bergstrom. The number of nit mutants tested for each of the 45 isolates ranged from 12 to 24. Complementary nitA and nitB mutants were obtained for only 17 out of 45 isolates: 9 of 15 isolates of the 1994 core collection, 8 of 29 isolates of the 1995 core collection and for the F.o. f.sp. loti isolate (Figure 1). No complementary mutants were obtained from the rest of the isolates.

Figure 1. Complementary nitA and nitB mutants derived from Fusarium oxysporum isolates on minimal medium. The plate in the upper left corner is used as an example: one mutant, arbitrarily designated nitA, was placed in the center of the plate; then five other nit mutants obtained from the same isolate were spaced in a circle of radius 15mm around the nitA block. In this case the five outer nit mutants were complementary with the nitA mutant and were designated nitB mutants. Fusarium oxysporum diversity in Lotus corniculatus 67

Table 2. Aggressiveness on birdsfoot trefoil cv. San Gabriel of Fusarium oxysporum isolates from the 1995 core collection, as determined by disease severity and percentage of surviving plants.

Isolate1 Disease Exp. Surv. Isolate1 Disease Perc. surv. severity2 Plants3 severity2 plants3 (DSI) (%) (DSI) (PSP) C2C11 3.85 4.8 C2C10 3.53 14.7 C2C2 3.82 7.6 TT1C9 3.48 15.6 C3C1 3.77 7.4 TT3C1 3.48 16.1 TT3C3 3.76 7.5 T2C9 3.44 14.5 C3C5 3.75 11.4 T3C5 3.44 18.4 TT3C10 3.73 8.8 T3C4 3.43 16.6

C3C4 3.73 9.9 T3C7 3.35 19.3 C3C10 3.73 10.5 TT1C10 3.29 21.3 C3C11 3.73 12.3 T3C9 3.25 24.2 C2C9 3.71 8.6 T3C12 3.25 25.4 T3C10 3.70 14.3 TT3C4 3.17 25.8 TT3C12 3.67 11.5 TT3C5 3.15 19.0

TT2C9 3.67 12.5 C3C2 3.14 26.4 T3C8 3.64 12.2 TT3C8 3.11 25.5 C3C8 3.64 12.7 T1C2 3.10 35.8 TT1C4 3.63 14.2 TT2C3 3.03 30.2 TT2C6 3.58 12.1 TT2C11 2.44 53.4 T3C2 3.58 12.3 C2C7 1.69 78.9

TT1C8/944 2.61 48.9 LSD (0.05) 0.42 16.1 LSD (0.05) 0.42 16.1 CV (%) 8.77 58.9 CV (%) 8.77 58.9 1 The first letter(s) refer(s) to the geographic location of an isolate; C= Colonia, TT= Treinta y Tres, T= Tacuarembó. The first number refers to the age in years of the diseased plant from which an isolate was derived; the second number refers to the quadrat in the field from which the diseased plant was sampled (1-12). 2 DSI, disease severity index based on a 5-class scoring system of individual seedlings: 1= healthy seedling, 2= primary root tip necrotic and firm, 3= primary root tip and cotyledons rotted and soft, 4= dead seedling, 5= dead seed. 3 PSP, percentage of surviving plants: percentage of seedlings in classes 1 and 2. 4 Control isolate from the 1994 core collection.

68 N.A. Altier and J.V. Groth

No complementation was found either among the nine F. oxysporum isolates from the 1994 core collection or among the eight isolates from the 1995 core collection. Pairings among the nine 1994 isolates and the eight 1995 isolates also resulted in no complementation (Fig. 2). When the 17 isolates from Uruguay were tested with the F.o. f.sp. loti isolate, no complementation was obtained. Based on these results, the 17 isolates should be assigned to different VCGs. Since each plate contained two complementary nit mutants for at least one isolate, we were assured that the methodology was adequate to detect compatibility among isolates if it did exist (Fig. 2). In these tests, complementation always and only occurred between complementary nitA and nitB mutants from the same isolate, used as controls.

Figure 2. Complementation tests among Fusarium oxysporum isolates from which complementary nitA and nitB mutants were obtained, on minimal medium. No complementation occurred among different isolates. The observed complementation only and always occurred between complementary nitA and nitB mutants of the same isolate.

Discussion

The repeated isolation of F. oxysporum from diseased roots of birdsfoot trefoil suggests this species is primarily responsible for crown and root rot and stand decline, consistent with previous findings (Altier, 1994; 1997; Berkenkamp et al., 1972; Beuselinck, 1988; Henson, 1962; Kainski, 1960). In addition, F. oxysporum has been reported as the causal organism of Fusarium wilt on birdsfoot trefoil (Bergstrom and Kalb, 1995). No other known pathogen that is alone capable of causing these disease symptoms was isolated from diseased crown and root tissues.

All the examined isolates of F. oxysporum incited a host reaction in birdsfoot trefoil. This means they may have similar genetic factors that determine pathogenicity and host disease reaction. However, isolates showed a continuous range in aggressiveness to birdsfoot trefoil seeds and seedlings. The observed variability was expected, and was consistent with previous reports (Leath and Kendall, 1978; Venuto et al., 1995). Aggressiveness is defined as a property of the pathogen reflecting the relative amount of damage caused to the host without regard to resistance genes (Shanner et al., 1992). With few exceptions, the highest aggressiveness was expressed by isolates from sites with long legume pasture history (e.g., Colonia as compared with the other two locations). There was a tendency for isolates from Fusarium oxysporum diversity in Lotus corniculatus 69 the same site to have similar values for DSI and PSP; however, the reason for this is unknown. Results in our tests were similar to those of Fulton and Hanson (1960), Kainski (1960), and Kilpatrick et al. (1954), who used seedling tests to compare aggressiveness of Fusarium spp. isolates causing crown and root rot on forage legume hosts, and reported variability in the pathogen. Kainski (1960), while studying the fungi involved in root rots and seedling diseases of birdsfoot trefoil, showed that most of the fungi that were pathogenic to seeds and seedlings were also pathogenic to established plants but differed in their relative aggressiveness. Kilpatrick et al. (1954) studied the pathogenicity of 72 isolates of F. oxysporum associated with root rots of red clover and observed a wide variation in disease severity as measured by percentage dead plants (range among isolates: 26-95%). This variable is the complement of the one we used, percentage of surviving plants (PSP).

Despite obtaining nit mutants for 45 out of 52 isolates, complementary nitB mutants were obtained for only 17 out of 45 isolates. There are two plausible explanations for these results. First, the efficiency of recovering nitB complementary mutants is low and isolate dependent (average 0.13 per inoculum plug, range 0-0.25; Puhalla, 1985). Therefore, a large number of inoculum plugs per isolate should have been plated on KPS to recover more nitB mutants. Secondly, some isolates could have been heterokaryon self-incompatible (HSI), as defined by Correll et al. (1987), though HSI strains usually occur at low frequency in F. oxysporum populations (1-2%, J.F. Leslie, Kansas State University, U.S.A., 1996, pers. comm.; 4%, Steinberg et al., 1997).

The lack of complementation among the isolates that compose the F. oxysporum population associated with birdsfoot trefoil indicates a large genetic diversity, as measured by vegetative compatibility. Isolates do not share genes for complementation and thus, they belong to different VCGs. However, they may share genetic factors that induce host disease reaction, since all of them have the ability to cause disease symptoms in birdsfoot trefoil seeds and seedlings. We may conclude that similarity or dissimilarity in vic genes does not reflect pathogenicity and aggressiveness capabilities. Our results agreed with those of Venuto et al. (1995) who showed that VCGs were not useful in predicting host reaction of red clover to isolates of F. oxysporum, because isolates from distinct groupings elicited similar host reactions. This indicates that the number of genes controlling compatibility seems to be greater than the number of virulence genes. Correll (1991) reported that over 46 distinct VCGs have been identified among a collection of F. o. f.sp. asparagi isolates pathogenic to asparagus in greenhouse pathogenicity tests, and that race 1 isolates of F. o. f.sp. lycopersici were found to belong to at least 41 VCGs.

Some genetic diversity studies have been consistent with Puhalla's initial generalization and have stated a correlation between pathogenic phenotype and genotype (Puhalla, 1985). However, the examination of numerous formae speciales of F. oxysporum has revealed that the relationship between host specialization (formae speciales), virulence capabilities (races) and VCGs can vary from simple to complex (Correll, 1991; Gordon and Martyn, 1997; Kistler, 1997; 2001; Klein and Correll, 2001). Based on pieces of evidence, authors assume that virulence and VCG phenotypes change independently of one another and at different rates (Correll, 1991; Klein and Correll, 2001). Kistler (2001) asserted that horizontal gene transfer could explain the partitioning of host specificity into genetically distant lineages. 70 N.A. Altier and J.V. Groth

From this hypothesis, the prediction is that genes for host specificity (as determined by pathogenic phenotype) may be more closely related than can be accounted for by the underlying phylogeny (as determined by molecular markers or VCGs). He concluded that strains pathogenic to a given host may emerge rapidly in a genetic background preadapted for fitness on any plant species currently colonized by F. oxysporum (Kistler, 2001). Several working models have been proposed to help explain the degree of VCG diversity thus far observed in F. oxysporum (Correll, 1991; Gordon and Martyn, 1997; Klein and Correll, 2001). It has been suggested that the parasitic, but nonpathogenic portion of the population may represent some primitive or basal population structure of this species and a largely unexplored reservoir of genetic diversity. From this primitive population, which has a high degree of VCG diversity, mutations to virulence may occur among isolates of the various VCGs. If selection of existing variants or a mutation occurred in isolates that are brought into proximity with a susceptible host (e.g., the roots), then they may proliferate and lead to an epidemic. This will likely result as a consequence of the intense selection pressure imposed by agricultural practices.

Soils in Uruguay support the growth of a wide range of leguminous species, either introduced crops, native species (including woody trees and shrubs) or weeds. In areas of intensive livestock production, forage legumes are used in short rotations with cereals and grasses. Under this situation, in most agricultural soils, F. oxysporum populations do find conditions conducive to development of host specialization, and survive, at least in part, by colonizing leguminous-host plants. Furthermore, results from a study to characterize soil populations of F. oxysporum under different rotation systems have demonstrated that even soil under continuous agriculture without any legume crop supports high population densities of this fungus (Altier, 2003). We could speculate that this population may represent what Correll (1991) designates the primitive or basal population structure of this species. Given the global distribution of F. oxysporum and its pervasive association with plants, this gives reason for concern. Future work should focus especially on the role of alternate hosts in maintaining pathogen populations in soil and in the study of factors that influence the dynamics of isolate competition within a heterogeneous F. oxysporum population.

Characterization of the pathogen population has practical implications for the success of breeding for resistance to Fusarium crown and root rot in birdsfoot trefoil. The high degree of variability for aggressiveness among F. oxysporum isolates supports using isolates from different locations in the inoculation and selection procedures of a breeding program for cultivar development for the region. If the primary variation in the reaction of the host is due to variation in the pathogen, selection for resistance to one or a few isolates would not result in resistance to other isolates (Pederson et al., 1980; Venuto et al., 1995). Developing germplasm with increased resistance to F. oxysporum should involve the screening of birdsfoot trefoil against several genetically divergent isolates of the pathogen, if the resistant cultivars are to be widely used.

Acknowledgements

The authors gratefully acknowledge Dr. Gary C. Bergstrom for providing the isolate 067NY-94 of Fusarium oxysporum f.sp. loti. Fusarium oxysporum diversity in Lotus corniculatus 71

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STEINBERG C., EDEL V., GAUTHERON N., ABADIE C., VALLAEYS T. and ALABOUVETTE C. 1997. Phenotypic characterization of natural populations of Fusarium oxysporum in relation to genotypic characterization. FEMS Microbiology Ecology, 24, 73-85.

STUTZ J.C. and LEATH K.L. 1983. Virulence differences between Fusarium roseum "Acuminatum" and F. roseum "Avenaceum" in red clover. Phytopathology, 73, 1648-1651.

STUTZ J.C., LEATH K.T. and KENDALL W.A. 1985. Wound-related modifications of penetration, development, and root rot by Fusarium roseum in forage legumes. Phytopathology, 75, 920-924.

TAYLOR T.H., TEMPLETON W.C.Jr. and WYLES J.W. 1973. Management effects on persistence and productivity of birdsfoot trefoil (Lotus corniculatus L.). Agronomy Journal, 65, 646-648.

VENUTO B.C., SMITH R.R. and GRAU C.R. 1995. Virulence, legume host specificity, and genetic relatedness of isolates of Fusarium oxysporum from red clover. Plant Disease, 79, 406-410.

WINDELS C.E. 1992. Fusarium. In SINGLETON L.L., MIHAIL J.D. and RUSH C.M. (Eds). Methods for research on soilborne phytopathogenic fungi. APS Press: St. Paul, MN, USA. pp. 115-128. Lotus Newsletter (2005) Volume 35 (1), 75-82.

The Flavonoids of Lotus corniculatus

JOËL REYNAUD* and MONIQUE LUSSIGNOL Université Claude Bernard Lyon 1, ISPB Faculté de Pharmacie, Laboratoire de Botanique, 8 avenue Rockefeller, 69373 Lyon Cedex 08, France. *Corresponding author

Introduction

Since the first studies published in the fifties and sixties (Nakaoki et al., 1956; Harney and Grant, 1964; Bate-Smith, 1965), many authors have investigated the flavonoid chemistry of Lotus corniculatus (Table 1 and Table 2) and demonstrated the richness and diversity of flavonoid compounds in this species. Some authors have examined the variation with altitude of the flavonoid content of Lotus corniculatus. Others have used flavonoids as speciation markers within the Lotus corniculatus complex.

Flavonoids

Flavonoids are a large class of secondary plant metabolites of widespread occurrence in higher plants (more than 6000 known structures; Harborne and Baxter, 1999). Of the two most frequent subclasses, flavones and flavonols (Figure 1), only derivatives of flavonols have been identified in Lotus corniculatus (3-OH free or substituted by a sugar).

A recent study (Sarelli et al., 2003) has revealed that Lotus corniculatus also contained insignificant amounts of two isoflavonoids at budding and flowering stages: formononetin and biochanin A (Figure 2). These two phytoestrogens are in too small a quantity to have adverse effects on reproductive functions.

Figure 1. Structure of flavones and flavonols Figure 2 Structure of isoflavonoids

R O B 2 2 O 7 3 A C 3 5 R 4' R1 O R O 3

Flavones: R3=H Formononetin: R1=H, R2=OH, R3=Ome Flavonols: R3=OH Biochanin A: R1=R2=OH, R3=OMe

75 76 Jöel Reunaud and Monique Lussignol

Aglycones (Table 1)

As in most plants, flavonoids of Lotus corniculatus are not present as free aglycones: in the different studies reported in Table 1, the aglycones were obtained after acid hydrolysis of the plant material (leaves or flowers). The 10 compounds mentioned in the different studies are all derivatives of kaempferol and quercetin. The species is particularly rich in 5-desoxyflavonols (structures characteristic of the polyphenoic profile of Fabaceae) and, in flower material only, in 8-hydroxy or 8-methoxy flavonols. A LC-MS (Liquid Chromatography-Mass Spectrometry) study with different detection modes recently published by de Rijke et al. (2004) did not reveal the presence of these methoxy and desoxy derivatives as free aglycones.

Table 1. Flavonoid aglycones identified in Lotus corniculatus (after acid hydrolysis of the plant material).

3'

2' 4' 8 O 5' 7 6' 6 3 5 O Plant organs and references Aerial Seeds Leaves Flowers parts Kaempferol (3,5,7,4'-tetrahydroxyflavone) 1,2,3,4 3,4 Quercetin (3,5,7,3',4'-pentahydroxyflavone) 1,2,3,4 3,4 Isorhamnetin 3,4 3,4 (3,5,7,3'-tetrahydroxy-4'-methoxyflavone) Desoxy-5-Kaempferol (3,7,4'-trihydroxyflavone) 3,4 3,4 Desoxy-5-Quercetin (Fisetin) 3,4 (3,7,3',4'-tetrahydroxyflavone) Desoxy-5-Isorhamnetin (Geraldol) 3,4 3,4 (3,7,3'-trihydroxy-4'-methoxyflavone) Methoxy-8-Kaempferol (Sexangularetin) 3,4 (3,5,7,4'-tetrahydroxy-8-methoxyflavone Methoxy-8-Quercetin (Corniculatusin) 3,4 (3,5,7,3',4'-pentahydroxy-8-methoxyflavone) Methoxy-8-Isorhamnetin (Limocitrin) 3,4 (3,5,7,3'-tetrahydroxy-8,4'-dimethoxyflavone) Hydroxy-8-Quercetin (Gossypetin) 3,4 (3,5,7,8,3',4'-hexahydroxyflavone)

Flavonoids of Lotus corniculatus 77

Monosides and Diosides (Table 2)

Lotus corniculatus is particularly characterized by the great diversity of its flavonol glycoside content (12 monosides and 10 diosides have been reported to date). In 1969, Harborne reported on the presence of 7-O-methyl-gossypetin, but the information was inaccurate and was further rectified; in 1978, the same author corrected the identification to 8-O-methylgossypetin (or 8-methoxyquercetin). Although the presence of isorhamnetin has been reported in the literature (Hasan, 1976; Jay et al., 1978), no glycoside based on this molecule has been evidenced to date. The plant seeds are particularly rich in flavonol glycosides (5 monosides and 6 diosides). The recent study performed by de Rijke et al. (2004) has shown that the 2 major compounds present in Lotus corniculatus are two isomers of 3-O-rhamnoglusosyl-kaempferol.

Flavonoids and flower color

For Jay and Ibrahim (1986), the predominant flavonoids (present as glycosides) in the flower buds of Lotus corniculatus are kaempferol and quercetin. Small amounts of gossypetin are also present. The yellow coloration of flower petals is concomitant with the accumulation of large amounts of gossypetin and corniculatusin and much smaller amounts of sexangularetin. For these authors, gossypetin and corniculatusin are mostly responsible for the intensity of the yellow color during flower development.

In some individuals, the flowers have entirely yellow keel petals ("light-keeled Lotus"). In other, less common individuals, the keel petals are red-brown ("dark-keeled Lotus"). Several authors, like Jones and Crawford (1977), have shown a cline in keel color frequencies in different parts of Western Europe (England and Wales, Denmark, West Germany, the Netherlands, Austria, France, Spain and Sweden). These authors have also shown the lack of relationship between the color of keel petals and cyanogenesis.

A study by Jones et al. (1986) of the relation between keel color, insect visits and reproductive output has indicated that "keel color does not influence pollinator foraging behavior nor colonization by flower insects". Their data show that the phenotypes do not differ in pod and seed production.

Relation between flavonoids and altitude

An article published in 1972 by Ceruti et al. investigated the total flavonoids of Lotus corniculatus flowers collected at various altitudes in Northern Italy. After extraction, they quantified their flavonoid content by measuring the Optical Density (OD) at 350 nm (the wavelength corresponding to maximum absorption of kaempferol and quercetin glycosides). Their measures revealed that variations of the flavonoid content of the plant (OD, maximum value = 1) correspond to 3 areas: * from 230 to 600m, OD increased from 0.2 to 0.4 * from 600 to 1600m, OD remained stable at approximately 0.4 * from 1600 to 2600m, OD increased from 0.4 to 0.7 78 Jöel Reunaud and Monique Lussignol

Table 2. Flavonoid glycosides identified in Lotus corniculatus

Plant organs and references Aerial Seeds Leaves Flowers parts Monosides Glucosyl-3-Kaempferol 5 Rhamnosyl-3-Kaempferol 5 Glucosyl-7-Kaempferol 6 5 Rhamnosyl-7-Kaempferol 5 Arabinosyl-3-Quercetin 7 Galactosyl-3-Quercetin 7 5 Rhamnosyl-3-Quercetin (Quercitrin) 7 4, 5 Rhamnosyl-7-Quercetin 6 5 Galactosyl-3-Gossypetin 8 Galactosyl-3-Corniculatusin 5 9 Glucosyl-3-Corniculatusin 5 Glucosyl-3-Sexangularetin 5

Diosides Diglucosyl-7-Kaempferol 7 Diglucosyl-3,7-Kaempferol 7 5 Dirhamnosyl-3,7-Kaempferol 6,7 6,10 Glucosyl-3-Rhamnosyl-7-Kaempferol 6,7 6 Rhamnosyl-3-Glucosyl-7-Kaempferol 5 (*) Dirhamnosyl-3,7-Quercetin 6 5 Glucosyl-3-Rhamnosyl-7-Quercetin 6 (*) Rhamnosyl-3-Glucosyl-7-Quercetin 5 (*) Rhamnosyl-3-Glucosyl-7-Sexangularetin 5 Rhamnoglucosyl-3 or 7-Quercetin (*) (*) for these diosides, the aglycone and the sugars have been identified but the exact positions of the sugars on the aglycone skeleton remain to be determined (3 or 7).

References for Table 1 and Table 2. 1 Harney and Grant, 1964 6 Waleska and Strzelecka, 1984 2 Bate-Smith, 1965 7 Gorski et al., 1975 3 Jay et al., 1978 8 Harborne, 1969 4 Hasan, 1976 9 Nielsen, 1970 5 Reynaud et al., 1982 10 Nakaoki et al., 1956 Flavonoids of Lotus corniculatus 79

From these findings, they concluded that the upregulation of the flavonoid content is related with the quantity and the quality of sun radiations received by Lotus corniculatus individuals as a function of altitude. For these authors, the stability observed between 600 and 1600m would be due to the fact that individuals were collected in forest habitats.

Several years ago, in my thesis work, I assessed the ratio of flavonoid diosides to monosides (D/M) in Lotus corniculatus samples collected at various altitudes in two French regions (Massif Central and Alps). Variations of the D/M ratio were not similar in the two areas. In plants collected between 600 and 1400m in the Massif Central (ancient hercynian massif), the D/M ratio varied from 2.3 at 600m to 14.8 at 1600m, whereas in the Alps (a recent mountain range) the ratio varied from 5.5 at 1200m to 0.6 at 1800m. My conclusion was that Lotus corniculatus populations of the Massif Central correspond to early plant settlements, probably all tetraploids, with more evolved flavonoid chemistry and a strong capacity to synthesize diosides. In the Alps, the plant populations are more recent (recolonization after the last ice age), with tetraploid individuals at lower altitudes and diploid individuals (sometimes named Lotus alpinus) at higher altitudes. The capacity of these high altitude diploid populations to synthesize diosides is reduced.

Flavonoids as speciation markers

We have studied 412 individuals collected from diploid and tetraploid populations of Lotus corniculatus growing in the Southern French Alps (Mercantour, Ventoux and Lure Mountain). After extraction and HPLC analysis of their polyphenolic content, a polyphenolic "fingerprint" of each individual was obtained. A statistical analysis of the 412 HPLC profiles led us to the following conclusions:

● in this geographic area, at low altitudes, there are tetraploid plants with a rich and diversified polyphenolic content.

● at higher altitudes, where conditions are more unstable, we find two poor and homogeneous polyphenolic profiles corresponding to two types of diploid Lotus corniculatus: one type is characteristic of the inner Alps and the other one of the western Alps (Mont Ventoux, for instance).

Results of the different studies described above have been published in this journal and elsewhere (Reynaud and Jay, 1989; 1990; 1991; Jay et al., 1991; Reynaud et al., 1991).

Conclusion

Lotus corniculatus, a plant with high agronomic value in some regions of the world, is also of particular interest for more theoretical research due to its rich flavonoid content. Though only based on kaempferol and quercetin flavonols, the rich and numerous flavonoid compounds synthesized by the plant can be used to study speciation in the Lotus corniculatus complex or variations of flavonoid chemistry as a function of altitude. The increasing sensitivity of isolation and identification methods should make it possible to identify occurrences of new, yet undisclosed flavonoids in this species. 80 Jöel Reunaud and Monique Lussignol

Acknowledgements Special thanks to Marie-Dominique Reynaud for her assistance in the English translation of this paper.

References

BATE-SMITH E.C. 1965. The phenolic constituents of plants and their taxonomic significance. 1. Dicotyledons. Journal of the Linnean Society of Botany, 58, 95-173.

CERUTI A., FIUSSELLO N. and LUPPI MOSCA A.M. 1972. Flavonoids in Lotus corniculatus petals in relation to altitude. Atti Cl. di Scienze fisiche, 106, 333-350.

GORSKI P.M., JURZYSTA M. and RZADKOWSKA-BODALSKA H. 1975. Flavonoids from the bird's foot trefoil seeds (Lotus corniculatus L.). Acta Soc. Bot. Pol., 44, 289-295.

HARBORNE J.B. and BAXTER H. 1999. The handbook of natural flavonoids. John Wiley and sons, New York, vol.2, pages??

HARBORNE J.B. 1969. Gossypetin and herbacetin as taxonomic markers in higher plants, Phytochemistry, 8, 177-183.

HARBORNE J.B., SALEH N.A. and SMITH D.M. 1978. On the natural occurrence of gossypetin-7- and 8-monomethyl ethers. Phytochemistry, 17, 589-591.

HARNEY P.M. and GRANT W.F. 1964. Chromatographic study of the phenolics of species of Lotus closely related to L. corniculatus and their taxonomic significance. American Journal of Botany, 51, 621-627.

HARNEY P.M. and GRANT W.F. 1965. A polygonal representation of chromatographic investigations on the phenolic content of certain species of Lotus. Canadian Journal of Genetic and Cytology, 7, 40-51.

HASAN A. 1976. Contribution à l'étude biochimique de trois représentants de la tribu des Lotées : Dorycnium Vill., Bonjeania Reichenb., Lotus L. (Légumineuses). [Contribution to the biochemical studie of three representant of the tribe of Loteae : Dorycnium Vill., Bonjeania Reichenb., Lotus L. (Leguminosae).]. These 3°C., Lyon. [in French]

JAY M., HASAN A., VOIRIN B. and VIRICEL M.R. 1978. Les flavonoides du Lotus corniculatus. [The flavonoides of Lotus corniculatus]. Phytochemistry, 17, 827-829. [In French]

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JAY M. and IBRAHIM R.K. 1986. Biosynthesis of 8-substituted flavonols in relation to ontogeny of flower color in Lotus corniculatus. Biochemie und Physiologie der Pflanzen, 181, 199-206.

JAY M., REYNAUD J., BLAISE S. and CARTIER D. 1991. Evolution and differentiation of Lotus corniculatus/Lotus alpinus populations from French south-western Alps. III. Conclusions. Evolutionary Trends in Plants, 5, 157-160.

JONES D.A., COMPTON S.G., CRAWFORD T.J., ELLIS W.M. and TAYLOR I.M. 1986. Variation of the colour of the keel petals in Lotus corniculatus L. 3 Pollination, herbivory and seed production. Heredity, 57, 101-112.

JONES D.A. and CRAWFORD T.J. 1977. Variation of the colour of the keel petals in Lotus corniculatus L. 1. The polymorphism in Western Europe. Heredity, 39, 313-325.

NAKAOKI T., MORITA N., HIRAKI A. and KUROKAWA Y. 1956. Medicinal resources. V. Components of the leaves of Lotus corniculatus var. japonicus, Microlespedeza striata, Magnolia obovata, and Abutilon avicennae. Yakugaku Zasshi-Journal of the Pharmaceutical Siciety of Japan, 76, 347-349.

NIELSEN J.G. 1970. Flavonoids of Lotus: I. Isolation and structure of the 3-galactoside of a new flavonol 5,7,3',4'-tetrahydroxy-8-methoxyflavonol (corniculatusin) from Lotus corniculatus. Tetrahedron Letters, 11, 803-804.

REYNAUD J. and JAY M. 1989. Evolution of populations of Lotus corniculatus s.l. in the South-western area of the French Alps. Lotus Newsletter, 20, 20-22.

REYNAUD J. and JAY M. 1990. Evolution of Lotus corniculatus s.l. populations in the Mercantour (French Prealps). Lotus Newsletter, 21, 24-28

REYNAUD J. and JAY M. 1991. Evolution and differentiation of Lotus corniculatus/Lotus alpinus populations from French south-western Alps. II. Contribution of phenolic metabolism markers. Evolutionary Trends in Plants, 5, 149-155.

REYNAUD J., JAY M. and BLAISE S. 1991. Evolution and differentiation of populations of Lotus corniculatus s.l. (FABACEAE) from Southern French Alps (Massif du Ventoux and Montagne de Lure). Canadian Journal of Botany, 69, 2286-2290.

REYNAUD J., JAY M., and RAYNAUD J. 1982. Flavonoid glycosides of Lotus corniculatus (Leguminosae). Phytochemistry, 21, 2604.

RIJKE E.DE, ZAPPEY H., ARIESE F., GOOIJER C. and BRINKMAN U.A.T. 2004. Flavonoids in Leguminosae: analysis of extracts of T. pratense L., T. dubium L., T. repens L., and L. corniculatus L. leaves using liquid chromatography with UV, mass spectrometry and fluorescence detection. Analytical and Bioanalytical Chemistry, 378, 995-1006. 82 Jöel Reunaud and Monique Lussignol

SARELLI L. TUORI M., SAASTAMOINEN I., SYRJÄLÄ-QVIST L. and SALONIEMI H. 2003. Phytoestrogen content of birdsfoot trefoil and red clover: effects of growth stage and ensiling method. Acta Agriculturae Scandinavica, Section A- Animal Science, 53, 58-63.

WALEWSKA E. and STRZELECKA H. 1984. Flavonoid compounds in aerial parts of Lotus corniculatus L. Herba Polonica, 30, 151-157.

Lotus Newsletter (2005) Volume 35 (1), 83-92.

Nitrogen metabolism in relation to drought stress responses in cultivated and model Lotus species

PEDRO DÍAZ1*, OMAR BORSANI1, ANTONIO MÁRQUEZ2 and JORGE MONZA1. 1Laboratorio de Bioquímica, Departamento de Biología Vegetal, Facultad de Agronomía, Av. E. Garzón 780, CP 12900 Montevideo, Uruguay. 2Departamento de Bioquímica Vegetal y Biología Molecular, Facultad de Química, Sevilla España. *Corresponding author

Abstract

Amino acid profiles of Lotus corniculatus, L. glaber, L. japonicus, L. subbiflorus and L. uliginosus in response to drought stress were studied. All Lotus species accumulate proline, derived from de novo synthesis, in response to drought stress. Asparagine analized by HPLC showed the highest abundance accounting for 20 to 25 % of total amino acids, without revealing any change in response to drought stress. Additionally, an increase in the amount of photorespiratory pathway intermediates, serine and glycine, was observed in some species as a consequence of drought stress, but intracellular ammonium non change in response to drought stress. The differential effect of drought stress on amino acid profiles and proline accumulation in Lotus species plants is discussed.

Introduction

There are four species of Lotus that have been domesticated and improved by selection and plant breeding: birdsfoot trefoil (Lotus corniculatus), greater lotus (L. uliginosus), narrow-leaf trefoil (L. glaber) and hairy birdsfoot trefoil (L. subbiflorus) (Blumenthal and McGraw, 1999; Díaz et al., 2005a). Besides its agronomical attributes, there is a great deal of interest in Lotus because the species are extremely amenable to tissue culture, in particular L. corniculatus and L. japonicus (Webb et al., 1990; Handberg and Stougaard, 1992). In spite of the fact that L. japonicus is not used as forage legume, this species could be a good model for a wealth of genetic, biochemical, molecular biological (Orea et al., 2002) and symbiotic studies which cannot be carried out in other model species as Arabidopsis thaliana.

Drought is the major limitation on crop productivity worldwide. In broad terms, drought can be permanent, seasonal or random. Another type of drought to be considered is named non-apparent drought and is observed in hot summer days when high temperature or wind, induce an increase on transpiration rates that exceeds water root absorption rates (Sánchez-Díaz y Aguirreolea, 1993).

Lotus species are sowed in geographical zones where the plants can be exposed to random or

83 84 Pedro Díaz, Omar Borsani, Antonio Márquez and Jorge Monza

non-apparent drought. They are adapted to temperate and humid environments, wherein these types of drought are frequent, and therefore, they could have acquired mechanisms to tolerate the drought.

For several years it has been known that plants respond to drought stress by undergoing biochemical adaptative processes such as ion transport and by accumulating different compounds, named compatible osmolytes. These osmolytes, which are known to increased there synthesis under osmotic stress, include proline, they accumulate to high concentrations without interfering with cell metabolism (Bray, 1993). As well as proline, other nitrogen compounds could be accumulating in plants in response to drought stress (Good and Zaplachinski, 1994; Chiang and Dandekar, 1995). The amino acid metabolism may play an important role in plant stress tolerance, by osmotic adjustment through to accumulation of compatible osmolytes; by detoxification of active oxygen species, xenobiotics and heavy metals; and by intracellular pH regulation (Rhodes et al., 1999; Alia et al., 2001).

During drought stress, protein residues may be altered by chemical processes; some proteins are irreversibly damaged by the effects of drought stress and are degraded by proteases. It has been suggested that proteases mobilize amino acids from proteins to the synthesis into compatible osmolytes (Campalans et al., 1999). In our study the analyses were focused in amino acid profiles and in drought-induced proline accumulation in Lotus agronomical species and in the model species Lotus japonicus.

Material and methods

Plant material, growth conditions and drought treatment

L. corniculatus cv San Gabriel (AGROSAN S.A.), L. uliginosus cv Grassland Maku (Ing. Agr. D. Formoso), L. subbiflorus cv El Rincón (AGROSAN S.A.), L. glaber cv Herminia (PAS S.A. Montevideo Uruguay) and L. japonicus (Regel) Larsen ecotype Gifu (Prof. A. Márquez) were treated according to Orea et al. (2002) and germinated at 28 ºC for 2 days. The plants were grown under controlled conditions: 16/8 h light/dark with photosynthetic photon flux density of 250 µmol. m-2. s-1, 22/18 ºC and relative humidity 70/80%. The plants were grown during 28 days in hydroponic assemblies (Borsani et al., 1999) with a modified Hornum nutrient solution described by Handberg and Stougaard (1992) with 8 mM of KNO3. The drought stress was induced as described by Borsani et al. (1999) the measurements of leaf tissues were performed 0 and 12 h after and this is know as fast drought stress (Díaz et al., 2005b).

Analytical determinations

The relative water content (RWC) was calculated according Antolín et al. (1995). Total protein was quantified according to Bradford (1972), chlorophyll concentration according to Wellburn (1994) and proline concentration according to Borsani et al. (1999). Nitrate, ammonium and total free amino acids were extracted as proposed by Izaguirre-Mayoral et al. (1992) with potassium phosphate buffer 10.0 mmol L-1 – ethanol (1 – 1). Nitrate was Biochemical responses to drought stress in Lotus 85 analysed according to Cataldo et al. (1975), ammonium according Solorzano (1969), and total amino acids were quantified with ninhydrin reagent according to Moore and Stein (1948). The identification of amino acids was achieved by an extraction derivatised with o-pthaldialdehyde and the samples were separated-analysed by HPLC (Díaz et al., 2005b in press). Proteolytic activity was measured according to Roy-Macauley et al. (1994).

Results

Biochemical responses to drought stress in plants are usually evaluated through the osmolyte accumulation and detoxification enzymes of reactive oxygen species, among others. Our work was focussed in nitrogen osmolytes as proline, GABA, amino acids, nitrate and ammonium.

Leaf RWC in different Lotus species averaged as follows: control 84 % and 12 h drought stress 63 % (Table 1). L. subbiflorus showed the lowest difference in RWC between the control and the drought stressed plant in our assay conditions. This fact could be explained since this is the most pubescent plant species and this feature could diminish the water loss by the leaf.

Photosynthetic pigments were used to determine the physiological status of the plants. No changes were observed in chlorophyll a and b concentration in response to 12 h of drought stress in the Lotus species studied, and also chlorophyll a/b ratio was largely unaffected by drought stress (Table 1).

Table 1. RWC, clorophyll (clo), proline, total free amino acid (tot aa), protein, nitrate and ammonium in one month old plants control (c) and subjected to drought stress for 12 h (ds).

L.corniculatus L. glaber L. japonicus L. subbiflorus L. uliginosus c ds c ds c ds c ds c ds RWCa 83.5 63.4* 81.0 58.3* 87.5 66.8* 82.5 70.2* 85.9 58.5* clo ab 7.1 6.7 8.8 7.3 10.6 10.7 7.8 8.7 5.3 5.9 clo bb 4.7 3.9 5.6 5.3 5.9 5.7 4.1 4.8 3.5 3.6 a/b 1.8 1.9 1.9 1.8 1.9 1.9 1.8 2.0 1.8 1.9 Prolineb 2.5 12.2* 1.3 10.2* 2.6 6.2* 1.3 2.9 1.2 9.7* Tot aab 164.8 173.5 100.9 93.9 131.0 169.3 103.8 107.2 113.7 145.8 Proteinc 119.2 104.0 110.4 129.7 135.3 148.5 89.7 92.3 118.4 95.3 nitrateb 330.5 360.2 174.4 94.1 365.5 310.9 123.4 51.9 419.5 230.3 Ammonium 21.5 14.1 0 0 0 0 0 0 24.4 30.8 The * represent a significant difference between drought stressed and control plants at 5 % of Duncan´s method. a, %; b, µmol g-1DW and c, mg g-1 DW.

L. corniculatus, L. japonicus and L. uliginosus plants showed higher nitrate levels than L. glaber and L. subbiflorus plants and we did not found changes in response to drought stress. 86 Pedro Díaz, Omar Borsani, Antonio Márquez and Jorge Monza

On the other hand, ammonium was detected in L. corniculatus and L. uliginosus and no changes were observed in response to drought stress. Ammonium was not detected in the other three species (Table 1).

Proline content increased in drought stressed plants; these increases were between 3 and 7 fold (Table 1). Based on the Van´t Hoff equation, this proline increase could be enough to account for only -0.1 to -0.5 MPa of osmotic adjustment in leaf tissue. However, results obtained by our group showed that in L. corniculatus subjected to 9 days of slow drought stress, the proline content accounts for -2.5 MPa of osmotic adjustment (Díaz et al., 2005b; P. Díaz and M. Sainz, unpublished data).

Total free amino acids and protein content did not change significantly when the plants were subjected to drought stress (Table 1); so, the increase in proline content was also with respect to total amino acids. Additionally, protein content did not change in response to drought stress conditions; this could suggest that there is no protease increase. This point was verified assaying proteolytic activity with azocasein as substrate at different pH. The proteolytic activity was found to be similar in control and drought stressed plants (Figure 1).

Free amino acid composition of control and drought stress Lotus species was analyzed by HPLC and is showed in Table 2. The amino acids analyzed represent the 80% of total free amino acids in leaf tissues. Asparagine was present in high amounts, and accounted for 20 to 25 % in the different Lotus species, and arginine was prevalent in L. uliginosus (Table 2).

Table 2. Major free amino acid composition (expressed as µmol g-1 DW) of one month old plants control (c) and subjected to drought stress for 12 h (ds).

L.corniculatus L. glaber L. japonicus L. subbiflorus L. uliginosus c ds c ds c ds c ds c ds ala 14.9 10.4* 10.3 8.6 7.6 9.7 4.3 11.5 4.3 15.5* asp 17.3 6.1* 9.3 2.8* 6.2 3.1* 1.3 2.7 15.5 9.8* asn 38.6 46.0 31.5 34.6 40.9 42.6 31.0 32.1 29.5 53.0 glu 23.8 18.6* 11.6 14.5 17.2 15.6 9.5 12.1 20.2 19.9 gln 1.8 5.4* 0.4 2.7* 3.2 3.3 1.3 2.1 2.1 5.4 gly 6.3 16.1* 4.6 7.0* 5.7 8.6 4.3 7.9 10.4 17.5 ser 12.5 20.5* 8.9 12.4* 17.1 15.0 12.5 14.8 7.4 13.3* arg 6.6 4.9 5.7 5.4 2.7 3.2 3.4 4.1 9.5 34.6* GABA 13.0 11.1 45.5 46.5 32.4 27.3 25.2 31.9 6.6 30.3* The * represent a significant difference between drought stressed and control plants at 5 % of Duncan´s method.

Biochemical responses to drought stress in Lotus 87

Figure 1. Proteolytic activity in response to drought stress in different Lotus species. Close point, control and open point, drought stress treatment. No difference between drought stressed and control plants at 5 % of Duncan´s method were observed.

88 Pedro Díaz, Omar Borsani, Antonio Márquez and Jorge Monza

No changes in asparagine concentration were observed in Lotus species subjected to drought stress. L. corniculatus plants showed the most significant changes in the amino acid profile as a consequence of drought stress. For instance, a decrease in aspartate, alanine and glutamate, and an increase in glutamine, serine and glycine were observed in this species. Some similar changes in the amino acid profiles found in L. corniculatus could be observed in L. glaber, L. japonicus and L. uliginosus. The latter showed a decrease in aspartate concentration and L. glaber also exhibited an increase in serine and glycine concentration. A significant increase in GABA concentration was observed only in L. uliginosus as a consequence of 12 h of drought stress.

Discussion

The present study outlines the changes in proline and free amino acid concentration in several Lotus species during the onset of drought stress in plants. Proline was negatively correlated with RWC in Lotus species (Tables 1 and 2). Previous works showed that L. corniculatus and L. japonicus under drought stress conditions decreased RWC of leaves and this was accomplished by an increase in proline concentration (Borsani et al., 1999; Díaz et al., 2002). The amount of accumulated proline is too low to account for osmotic adjustment according to the Van´t Hoff equation, so proline may be regarded as a scavenger of hydroxyl and singlet oxygen radicals (Smirnoff and Cumbes, 1989; Alia et al., 2001).

Our work shows that proline accumulation in all Lotus species results from de novo synthesis and not from protein hydrolysis, since total amino acids and protein content remained unaltered (Table 1). Additionally, no changes in proteases activity were detected (Figure 1). As well, no differences in protein content were found in Lotus species subjected to fast drought stress. The decreases in protein content are associated with slow drought stress which has been found to occur in tomato (Bauer et al., 1997), Phaseolus vulgaris and Vigna unguiculata (Roy-Macauley et al., 1992).

Lotus species showed different nitrate concentrations, which may probably accumulate in the vacuoles, and so nitrate might be regarded as having a role as an osmoregulator (Blom-Zandstra and Lampe, 1985; Márquez et al., 2005). A decrease in nitrate concentration was observed in tobacco during dehydration. This decrease was accompanied by a general decreased in total free amino acids content (Ferrario-Méry et al., 1998), but this metabolic event was not found in the Lotus species studied.

Different amino acids were prevalent in the different Lotus species studied: for instance, asparagine and glutamate were found to be the most abundant in L. corniculatus and L. uliginosus control treatment, and asparagine and GABA were abundant in the others species (Table 2). Lotus genus is similar to Medicago sativa in that asparagine is the principal amide (Girousse et al., 1996). In temperate legumes, asparagine is the main molecule used to transport reduced nitrogen within the plant, and in that case can account high content. No changes in asparagine content were observed in response to drought stress in Lotus species. In A. thaliana and Brassica napus leaves an increase in asparagine content has been reported in response to osmotic stress (Chiang and Dandekar, 1995; Good and Zaplachinski, 1994). Biochemical responses to drought stress in Lotus 89

High glutamate concentration is necessary for proline synthesis through the pyrroline 5-carboxylate synthetase and pyrroline 5-carboxylate reductase (Rhodes et al., 1999). An increase in Fd-GOGAT responsible of glutamate supply for proline accumulation has been reported in L. corniculatus leaves under stress conditions (Borsani et al., 1999; Díaz et al., 2005b in press).

A decline in alanine content and an increase in serine and glycine content were observed in Lotus species, and may be due to higher rates of photorespiration in the drought stress condition, since alanine is a major donor of amino groups in photorespiratory metabolism.

A special feature show L. uliginosus which increased GABA, and this fact could lead to the reduction in the cytoplasmatic concentration of glutamate (Cayley et al., 1992). This reduction could be carried out through a transport into the vacuole, or by GABA synthesis. Similarly, elevated GABA levels are observed under conditions when glutamine synthesis is limited, protein synthesis is inhibited and protein degradation is induced, all these metabolic alterations occur under drought stress conditions in plants (Bray. 1993; Shelp et al.,1999). Also significant increases of arginine were observed in L. uliginosus, which may be associated to polyamines metabolism (Rhodes et al., 1999).

Similar results obtained on proline accumulation and amino acid profiles in the L. corniculatus, L. glaber and L. japonicus showed that the latter species could be used as an excellent model to understand and therefore improve drought stress tolerance in cultivated Lotus species through osmolyte synthesis.

Acknowledgements This research was supported by PEDECIBA, CSIC Uruguay and AECI España. The authors wish to thank to Cecilia MacDonald for review of manuscript.

References

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BAUER D., BIEHLER K., FOCK H., CARRAYOL E., HIREL B., MIGGE A. and BECKER T. 1997. A role for cytosolic glutamine synthetase in the remobilisation of leaf nitrogen during water stress tomato. Physiologia Plantarum, 99, 241-248.

BLOM-ZANDSTRA M. and LAMPE J. 1985. The role of nitrate in the osmoregulation of lettuce (Lactuca sativa L.) Grown at different light intensities. Journal of Experimental Botany, 36, 1043-1052.

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BLUMENTHAL M. and MCGRAW R. 1999. Lotus adaptation, use and management. In: BEUSELINCK P. (Ed.) Trefoil, the science and technology of Lotus. American Society of Agronomy, Inc. and Crop Science Society of America, Inc. Madison Wisconsin, USA. p 97-120

BORSANI O., DÍAZ P. and MONZA J. 1999. Proline is involved in water stress responses of Lotus corniculatus nitrogen fixing and nitrate fed plants. Journal of Plant Physiology, 155, 269-273.

BRADFORD M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 72, 248-254.

BRAY E. 1993. Molecular responses to water deficit. Plant Physiology, 103, 1035-1040.

CAMPALANS A., MESSEGUEER R., GODAY A. and PAGÈS M. 1999. Plant responses to drought, from ABA signal transduction events to the action of the induced proteins. Plant Physiology and Biochemistry, 37, 327-340.

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CAYLEY S., LEWIS B. and RECORD M. 1992. Origins of osmoporotective properties of betaine and proline in Escherichia coli K-122. Journal of Bacteriology, 174, 1586-1595.

CHIANG H. and DANDEKAR M. 1995. Regulation of proline accumulation in Arabidopsis thaliana (L.) Heynh during development and in response to desiccation. Plant, Cell and Environmental, 18, 1280-1290.

DÍAZ P., BORSANI O. and MONZA J. 2005a. Lotus related species and their agronomic importance. In MÁRQUEZ A. (Ed.). Lotus japonicus Hanbook. [In press]

DÍAZ P. MONZA J. and MÁRQUEZ A. 2005b. Drought and saline stress. In: MÁRQUEZ A. (Ed.) Lotus japonicus Hanbook. [In press].

DÍAZ P., OREA A., ARCONDÉGUY T., PAJUELO E., PAJUELO P., MONZA J. and MÁRQUEZ A. 2002. Análisis molecular y respuesta a estrés hídrico en mutantes de Lotus japonicus deficientes en glutamina sintetasa cloroplástica. [Molecular analysis and drought stress response in Lotus japonicus mutants without chloroplastic glutamine synthetase.] In: APARICIO-TEJO P. (Ed.) Avances en el Metabolismo del Nitrógeno: Desde la Biología Molecular a la Agronomía. [Advances in Nitrogen Metabolism: From Molecular Biology to Agronomy.] Universidad Pública de Navarra. Pamplona. España. p. 207-217. [In Spanish]

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MÁRQUEZ A., BETTI M., GARCÍA-CALDERÓN M., PAL´OVE-BALANG P., DÍAZ P. and MONZA J. 2005. Nitrate assimilation in Lotus japonicus. Journal of Experimental Botany, 56, 1741-1749.

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OREA A., PAJUELO P., PAJUELO E., QUIDIELLO C., ROMERO J. and MÁRQUEZ A. 2002. Isolation of photorespiratory mutants from Lotus japonicus deficient in glutamine synthetase. Physiologia Plantarum, 115, 352-361.

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WELLBURN A. 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of Plant Physiology, 144, 307-313.

Lotus Newsletter (2005) Volume 35 (1), 93-105.

Bacterial surface polysaccharides and their role in the rhizobia-legume association.

VIVIANA C. LEPEK* and ALEJANDRA L. D´ANTUONO IIB-INTECh-UNSAM, Colectora Av. General Paz 5445 entre Constituyentes y Albarellos, INTI. Edificio 24 (1650) General San Martín, Provincia de Buenos Aires, Argentina. *Corresponding author

Introduction

The establishment of a nitrogen-fixing symbiosis is an economically important plant phenomenon. Biological reduction of dinitrogen to ammonia is among the most effective fixation systems facilitating the plant growth in nitrogen starved soils without the requirement of massive inputs of fertilizers. This process occurs in legumes roots in structures called nodules. Nodule development is induced when legumes enters into association with gram-negative soil bacteria that belong to the Rhizobiaceae family, such as Bradhyrhizobium, Mesorhizobium, Sinorhizobium, Rhizobium and Azorhizobium, all denoted with the general term, rhizobia. Rhizobia invade the nodule and differentiate into a state called bacteroid able to fix the atmospheric nitrogen under the appropriate conditions generated inside the nodule. The nodulation process is the result of a selective interaction between rhizobia and the legume family (Leguminosae or Fabaceae), which means that each legume is nodulated only by specific bacterial species. For example, Medicago sativa by S. meliloti, Glycine max by B. japonicum and Lotus spp. by M. loti. Some bacteria display broad host range such as the Rhizobium NGR234 that can nodulate more than 112 legume genera. On the other hand M. loti has a reduced host range allowing nodulation of different Lotus species (L. corniculatus, L. japonicus, L. glaber and L. uliginosus) and Leucaena leucocephala. Nodules are classified as determinate or indeterminate, depending on the legume that is nodulated. Indeterminate nodules are elongated and present a persistent meristem at their tip, which is infected by rhizobia residing in the nodule. This characteristic results in a gradient of development stages from the older tissue near the root to the growing meristem at the nodule tip. Determinate nodules lack a persistent meristem, are usually round, and don’t show a gradient of development stages as is the case of indeterminate nodules. Examples of indeterminate nodule morphology are the ones formed on roots of Medicago sativa (alfalfa), Medicago truncatula, Pisum sativum (pea), Trifolium species (clovers) and L. leucocephala, meanwhile those formed on roots of Lotus species, Glycine max (soybean) and Vicia faba (bean) are determined.

Due to the importance of nodulation for agriculture, intensive researching is being carried on in this area in order to understand the molecular bases of this process. The new knowledge could be used to obtain more efficient nitrogen fixation process, modification of the host range or increased competitiveness that may influence its capacity to compete in the rhizosphere with other bacteria.

93 94 Viviana C. Lepek and Alejandra L. D’Antuono

Rhizobium-legume associations take place trough a sequence of events, starting with the exchange of signal molecules between the two symbiotic partners. Rhizobia are chemoattracted toward root legume proximity by some of the nutrients present in the root exudates: sugars, amino acids, some dicarboxilic acids and various aromatic compounds such as some flavonoids (Brencic and Winans, 2005). Flavonoids also are necessary to induce the bacterial production of the so called Nod factors (NF) which are lipochitooligosaccharides that have the capacity to induce the transcription of genes that control the morphological changes observed in the plant roots: deformation, branching and curling of root hairs, and cortical cells division which in turn gives rise to nodule primordium. Moreover in some legumes NF favors mature nodule development and pre-infection threads formation (Geurts and Bisseling, 2002). Simultaneously to these changes, bacterial adhesion to the hair root tip, colonization and entrance into the hair root through tubular structures called infection threads, occurs. The infection threads filled with bacteria grow toward the base of the root hair, branch and penetrate into the nodule cells. Upon released from the infection threads, bacteria became internalized in legume nodules cells through an endocytosis-like process (Gage, 2004). Rhizobia remain surrounded by the plant cell plasma membrane and differentiate into nitrogen-fixing bacteoids. Microsymbiont and host derived plasmatic membrane (or peribacteroid membrane) form the symbiosome. In indeterminate nodules each symbiosome contains one bacteroid (Brewin, 1998). In determinate nodules, one symbiosome contains several bacteroids (Cremola et al., 2000).

Together with Nod factors, other bacterial components have been involved in bacterial adhesion, formation and extension of the infection thread, releasing of bacteria into the nodule cells and differentiation into bacteroids. Between these components, polysaccharides have a relevant role. The present review will described the importance of rhizobial polysaccharides in the establishing of symbiosis in general, and we will focus on the results obtained in our laboratory in the Lotus glaber-M. loti system in particular.

Bacterial polysaccharides

Structure, characteristics and function in the free-living bacteria

Rhizobia synthesize different classes of polysaccharides: exopolysaccharide (EPS), capsular polysaccharides (KPS), lipopolysaccharides (LPS) and the cyclic glucan. Some of them are secreted to the media, others are exposed on the surface or present in the periplasmic space (Figure 1). EPS is a heteropolysaccharide formed by a repetitive unit constituted by hexose residues such as glucose, galactose, mannose rhamnose, glucuronic acid and galacturonic acid with piruvyl, acetyl, succynil and hydroxybutanoil substitutions. EPSs are abundant extracellular products secreted to the sorrouding media and accumulated on the cell surface. In free-living bacteria it acts as a physical barrier against external agents and it is also involved in the attachment to surfaces (Spaink, 2000). The KPS is a class of exopolysaccharide that remains attached to the bacterial surface. It has a role in the protection against desiccation and it confers resistance to bacteriophages (Fraysse et al., 2003). Its structure is strain specific meanwhile the EPS is generally common to all the Polysaccharides role in rhizobia-legume associations 95

Figure 1. Schematic representation of bacterial surface polysaccharides. OM: outer membrane, IM: internal membrane. strains of the same specie. KPS also differentiate from the EPS in that it contains 3-deoxy-d-manno-2-octulosonic acid (KDO) (Forsberg and Reuhs, 1997). LPS is a polysaccharide anchored in the bacterial outer membrane and is formed by three different regions, the lipid A, the core and the O-antigen. Lipid A is the part of the molecule involved in the membrane anchoring. The core moiety is a polysaccharide region attached to the lipid A by KDO. The third region consists of a chain of repeating units called O-antigen formed principally by deoxy and/or dideoxy-sugar residues. LPS, in free-living bacteria, is involved in membrane stabilization and it also acts as a barrier against antibacterial compounds such as the cationic peptides. Composition and structure analysis revealed that LPSs are also strain specific (Lerouge and Vanderleyden, 2001). For M. loti only the LPS composition of two strains were reported, that of the strain NZP2213 which O-antigen is formed principally by 2-O-acetyl-6-deoxy-L-talose (Russa et al., 1995) and that of M. loti Ayac 1 BII, an autochthonous strain from Argentina, which O-antigen is composed mainly by rhamnose (D’Antuono et al., 2005). Finally, cyclic glucans are cyclic molecules formed by glucose that could be or not substituted with anionic groups. They are accumulated principally in the periplasmic space and they were found in bacteria belonging to the group of the α-proteobacteria. M. loti, S. meliloti and the pathogenics Agrobacterium tumefaciens and Brucella spp. present a β (1-2) glucan type (Lepek et al., 1990; Breedvald and Miller, 1998; Briones et al., 2001), meanwhile B. japonicum presents a β (1-3) β (1-6) glucan type (Gore and Miller, 1992). Many observations are consistent with the hypothesis that cyclic glucans are involved in protection against hypoosmotic conditions (Spaink, 2000). More details about the different rhizobia polysaccharide structures could be found in the reviews of Spaink (2000) and Fraysse et al. (2003).

Polysaccharides function during the nodulation process

Exopolysaccharides EPS appears to be essential for the successful invasion of indeterminate nodules (Pellock et al., 2000). Two different EPS were described for S. meliloti, the succinoglycan (EPS I) and the galactoglycan (EPS II). Each of them can be found in the form of high or low molecular mass molecule depending on the number of repetitive units. The low molecular mass EPS I, 96 Viviana C. Lepek and Alejandra L. D’Antuono

EPS II and KPS of S. meliloti were described to be required for the extension of the infection thread although the three does not have the same efficiency in promoting this process (Fraysse et al., 2003). Addition of the low molecular mass EPS to an exo- mutant reverts the non-infective phenotype. This may indicate that this polysaccharide acts as a signaling molecule (Gonzalez et al., 1996). Interestingly EPS does not have the same relevance in the formation of determinate nodules (Borthakur et al., 1986). This was elegantly demonstrated studying the nodulation induced by M. loti exo- mutants on Lotus uliginosus (determinate nodules) and on Leucaena leucocephala (indeterminate nodules). Meanwhile M. loti EPS-altered mutants were ineffective on the indeterminate nodulating host, they maintain their fully effectiveness on a determinate one (Hotter and Scott, 1991). However, since the structure and classes of M. loti EPS were not studied in detail, it is not possible to rule out the presence of other bacterial structures able to fulfill the EPS function in the exo- studied mutants. Moreover, there are results that suggest that EPS have also some role in the formation of determinate nodules. Nodules of plants inoculated with a mixture of exo- mutant and wild type strains of B. japonicum were principally occupied with the wild type strain (Parniske et al., 1993).

One of the functions attributed to the EPS is the organization of root hair cytoskeleton influencing in this way the infection thread extension (Pellock et al., 2000). Other authors suggested a function as negative modulator of the plant defense response (Fraysse et al., 2003). This conclusion was obtained from experiments made both with determinate and indeterminate systems (Niehaus et al., 1993; Niehaus et al., 1997; Parniske et al., 1994).

Lipopolysaccharides LPS was involved in several developmental stages of the nodulation process. In root adhesion of R. leguminosarum-clover, R. leguminosarum bv. trifoii LPS was found to bind specifically to a lectin, accumulated in the tips of the root hair, called Trifoliin A (Dazzo and Brill, 1979). However, this effect cannot be generalized to other legume-rhizobia associations. In the case of M. loti selective binding of LPS molecules does not appears to be the determinant of the specificity of the interaction since a R. leguminosarum strain with the information necessary to express the M. loti Nod factors can nodulate Lotus in spite of the differences between the LPS of both bacteria (Pacios Bras et al., 2000). Contrary to what happens with EPS, LPS molecules are involved principally in the formation of determinate nodules, especially during the initiation and elongation of the infection thread (Lerouge and Vanderleyden, 2001; Noel et al., 2000). O-antigen-deficient and core-altered LPS mutants of R. leguminosarum, R. etli and B. japonicum exhibit defective infection thread formation and induce aberrant nodules (Carlson et al., 1995; Noel and Duelli, 2000; Noel et al., 2000; Perotto et al., 1994). In the development of indeterminate nodules it was found for M. truncatula, but not for M. sativa, that S. meliloti LPS is involved in the stage of colonization of plant nodule cells and in the differentiation into bacteroid (Niehaus et al., 1998). However, a different role of LPS in determinate and indeterminate nodule development is not clear. For M. sativa (indeterminate nodules) it was described that S. meliloti lps- mutant induce the formation of normal nodules but this mutant is less competitive that the wild type strain (Lagares et al., 1992). A similar result was obtained in our laboratory in the case of the association M. loti-Lotus glaber (determinate nodules) (D’Antuono et al., 2005). M. loti lpsβ2 and lpsβ1 mutant strains, affected in the synthesis of the O-antigen, presented a Polysaccharides role in rhizobia-legume associations 97 normal nodulation development but were out-competed when were co-inoculated on Lotus plants with the wild-type strain (Table 1). As other bacterial strains affected in the LPS synthesis, M. loti lps- shows an increased sensitivity to cationic peptides such as Polimixyn B and Melitin than the wild type strain. One of the suggested functions for LPS during the nodulation process is the protection against possible cationic peptides generated by the plant as part of its defense response. When the three strains studied are comparing, a correlation between lower competitiveness and higher sensitivity to the antimicrobial compounds is observed. Contrary to the results observed for other interactions that lead to the formation of determinate nodules, in the case of the M. loti-Lotus glaber system, normal infection threads are formed. Moreover, a significant higher number of infection threads were formed in plants inoculated with the lpsβ2 mutant (Figure 2). Taken together all these data suggest that the event that affect competitiveness in the case of the M. loti lpsβ2 mutant is down-stream the infection thread formation and may be the consequence of the absence of a protection barrier against the plant antibacterial compounds. Data obtained by different groups suggest that among the LPS functions several result of its relation with the plant defense response. LPS could act either as protection barrier or as a molecule that masks bacterial compounds that induce the host defense response and it may also act as suppressor of this response. Accordingly, not only the determinate or indeterminate legume phenotype should be relevant for the influence of the LPS in the nodulation process but also the class and intensity of the defense response generated in a particular legume.

Table 1. Comparison of competitiveness index and sensitivity to cationic peptides of the different M. loti LPS-affected mutants. Bacteria cells were incubated for one hour at room temperature with 20 mg/ml of PmB (polimixin B). Bacterial survivance was tittered by plating a series of dilutions on Minimal medium-Agar plates. For Competitiveness index determination, plants were inoculated with a mixture of an equal number of wild-type and mutant strains. After 3 weeks, bacteria were recovered form the nodules and plated. Cl= colony forming units (CFU) of mutant recovered from the nodule/CFU of total bacteria recovered from the nodule. Data were extracted from D’Antuono et al. (2005).

98 Viviana C. Lepek and Alejandra L. D’Antuono

Figure 2. Comparison between the number of infection threads formed by the wild-type (″ ) and by the lpsβ2 mutant (□ ) at different postinoculation times. Infection threads were visualized using for inoculation bacteria expressing green fluorescent protein. Mean number of infection threads is marked with a horizontal line. Statistically significant differences (P< 0.05) are marked with asterisk.

An important characteristic of the LPS is its variation in response to external signals. There were found changes in the O-antigen structure when rhizobia were subjected to seed or root compounds (Noel et al., 2004). Also, during the differentiation of the bacterium into bacteroid, LPS undergoes changes in its structure detected using LPS-specific monoclonal antibodies (Kannenberg et al., 1998). A general observation is that the LPS changes during symbiosis lead to an increasing of surface hydrophobicity that influence the interaction between bacterial and plant cell membranes and could be relevant for the sincronic division bacteoid / symbiosome (Vedam et al., 2004;; Kannenberg and Carlson, 2001). Rhizobia as several other bacteria belonging to the α-2 subclass of proteobacteria group, have as a distinctive feature of its lipid A, the presence of a long fatty acid chain, the 27-hydroxyoctacosanoic acid (27-OH-C28:0) (Bhat et al., 1991). It was hypothesized that this molecule increases the stability of the bacterial membrane (Vedam et al., 2003). During symbiosis a doubling of 27-OH-C28:0 amounts in the lipid-A molecule was reported (Kanneberg and Carlson, 2001) and this could be responsible in part of the increased surface hydrophobicity. The fact that inside the nodules a different LPS is present could explain why, in occasions, mutation in genes involved in the free-living bacterial LPS synthesis does not affect the nodulation process. Analyzing M. loti LPS by polyacrilamide gels electrophoresis, we observed that the pattern of bands of the LPS obtained from bacteria isolated from nodules was different to that extracted from free-living bacteria (unpublished results). Also we found that the mutation in the lpsβ2 gene that has only effect on the bacterial competitiveness does not alter the pattern of bands of the bacteroid LPS (unpublished results).

Polysaccharides role in rhizobia-legume associations 99

Cyclic glucans Both in determinate and indeterminate nodule formation the absence of cyclic glucan synthesis affect the invasion capacity of the bacteria. As was mentioned above, S. meliloti and M. loti synthesize a β (1-2) cyclic glucan. Mutants affected in the synthesis of this polysaccharide induce the formation of empty nodules and does not induce the formation of the infection threads (Geremía et al., 1987; D’Antuono et al., 2005; Figure 3). It was described in M. loti, in Agrobacterium tumefaciens and in Brucella abortus that the absence of this polysaccharide in the periplamic space causes a pleiotropic phenotype at the level of the bacterial envelope. Mutants are no motile and more sensitive to detergents (D’Antuono et al., 2005; Briones et al., 2001). In the absence of the cyclic glucan, problems in the flagellum ensemble (Swart et al., 1994) and reduction in the accumulation of proteins, which are components of a transport system (Banta et al., 1998), occurs. The mechanism by which the glucan stabilizes the membrane and its components could be related with its osmoprotective function (Miller et al., 1986) and with its capacity of protecting the membrane against the disruptive action of calcium (D’Antuono et al., 2005). The pleiotropic effect on the bacterial envelope could be the cause of the early interruption of the symbiotic process at the initiation of infection thread formation and bacteria invasion. However it could not be ruled out that the β (1-2) cyclic glucan has itself some other function in the process. β (1-3) β (1-6) cyclic glucan of B. japonicum was involved in the suppression of the host defense response (Bhagwat et al., 1999; Mithofer et al., 2002). Recently it was hypothesized that β (1-2) cyclic glucan might be a virulence factor in Brucella abortus because it was demonstrated that this molecule interacts with the cholesterol of the host membrane lipid rafts, perturbing then the intracellular trafficking to the advantage of the pathogen (Arellano-Reynoso et al., 2005).

Figure 3. Flourescence light microscopy of cross section of nodules induced on Lotus roots inoculated with A, M. loti wild-type strain and B, M. loti cgs mutant strain. Bacteria expressing green fluorescent protein were used. Modified from D’Antuono et al. (2005) Molecular Plant-Microbe Interactions, volume 18, with permission.

100 Viviana C. Lepek and Alejandra L. D’Antuono

Conclusions and perspectives

Different bacterial polysaccharides have been found as relevant in the process that leads to the formation of an effective nodule. The results obtained by several laboratories suggest that they have principally influence on two aspects of the process: the bacterial relation with the defense response that could be generated in the plant host and the intimate interaction that occur between bacterial and plant cell membranes. The molecular mechanisms, by which each polysaccharide acts, remain to be determined.

In the last years, a great advance in the knowledge of the legumes genomic organization and gene expression was achieves. The use of bacterial mutants affected in different steps of the nodulation process helps to the identification of those plant genes which expression characterizes each step (Mitra and Long, 2004). Some polysaccharide-affected mutants were used with this purpose. Recently, Lotus japonicus became a model legume for the study the molecular process that leads to the formation of determinate nodules. About 112,000 Lotus EST sequences are now deposited in the public GenBank database. Technologies for high-throughput measurement of Lotus gene transcript levels during symbiosis began to be applied (Colebatch et al., 2002; Colebatch et al., 2004; Kouchi et al., 2004). Inoculation assays with M. loti cgs and lpsβ2 mutant strains, affected in two different steps of the nodule development, are been carried on in order to study the transcriptional Lotus gene response during the symbiosis event.

References

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BANTA L.M., BOHNE S.D., LOVEJOY D. and DOSTAL K. 1998. Stability of the Agrobacterium tumefaciens VirB10 protein is modulated by growth temperature and periplasmic osmoadaption. Journal of Bacteriology, 180, 6597-6606.

BHAGWAT A.A., MITHOFER A., PFEFFER P.E., KRAUS C., SPICKERS N., HOTCHKISS A., EBEL J. and KEISTER D. 1999. Further studies of the role of cyclic β-glucans in symbiosis. An ndvC mutant of Bradyrhizobium japonicum synthesizes cyclodecakis-(1-3)-β-glucosyl. Plant Physiology, 119, 1057-1064.

BHAT U.R., CARLSON R.W., BUSCH M. and MAYER H. 1991. Distribution and phylogenetic significance of 27-hydroxyoctacosanoicacid in lipopolysaccharides from bacteria belonfing to the alpha-2 subgroup of Proteobacteria. International Journal of Systematic Bacteriology, 41, 213-217.

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BORTHAKUR D., BARBER C.E., LAMB J.E., DANIELS M.J., DOWNIE J.A. and JOHNSTON W.B. 1986. A mutation that blocks exopolysaccharide synthesis prevents nodulation of peas by Rhizobium leguminosarum but not beans by R. phaseoli and corrected by cloned DNA from Rhizobium of the phytopathogen Xanthomonas. Molecular and General Genetics, 203, 320-323.

BREEDVELD M.B. and MILLER K. 1998. Cyclic glucan of members of family Rhizobiaceae. Microbiological Reviews, 58, 145-161.

BRENCIC A. and WINANS S.C. 2005. Detection of and response to signals involved in host-microbe interactions by plant-associated bacteria. Microbiology and Molecular Biology Reviews, 69, 155-194.

BREWIN N.J. 1998. Tissue and cell invasion by Rhizobium: The structure and development of infection threads and symbiosomes. In KONDOROSI A., HOOYKAAS P.J.J. and SPAINK H.P. (Eds.) The Rhizobiaceae. Kluwer Academic Publishers. Dordrecht. The Netherlands. pp 417-429.

BRIONES G., IÑON DE IANNINO N., ROSET M., VIGLIOCCO A., SILVA PAULO P. and UGALDE R. 2001. Brucella abortus cyclic β-1,2-glucan mutants have reduced virulence in mice and are defective in intracellular replication in HeLa cells. Infection and Immunity, 69, 4528-4535.

CARLSON R.W., REUHS B., CHEN T.B., BHAT U.R. and NOEL K.D. 1995. Lipopolysaccharide core structures in Rhizobium etli and mutants deficient in O-antigen. Journal of Biological Chemistry, 270, 11783-11788.

COLEBATCH G., KLOSKA S., TRVASKIS B., FREUND S., ALTMANN T. and UDVARDI M.K. 2002. Novel aspects of symbiotic nitrogen fixation uncovered by transcript profiling with cDNA arrays. Molecular Plant-Microbe Interactions, 15, 411-420.

COLEBATCH G., DEBROSSES G., OTT T., KRUSELL L., MONTANARI O., KLOSKA S., KOPKA J. and UDVARDI M.K. 2004. Global changes in transcription orchstrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus. Plant Journal, 39, 487-512.

CREMOLA M., FEDEROVA E., TATÉ R., RICCIO A., FAVRE R. and PATRIARCA E.J. 2000. Nodule invasion and symbiosome differentiation during Rhizobium etli-Phaseolus vulgaris symbiosis. Molecular Plant-Microbe Interactions, 13, 733-741.

D’ANTUONO A.L., CASABUONO A., COUTO A., UGLADE R.A. and LEPEK V.L. 2005. Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis. Molecular Plant-Microbe Interactions, 18, 446-457.

DAZZO F.B. and BRILL W.J. 1979. Bacterial polysaccharide which binds Rhizobium trifolii to clover root hairs. Journal of Bacteriology, 137, 1362-1373. 102 Viviana C. Lepek and Alejandra L. D’Antuono

FORSBERG L.S. and REUHS B. 1997. Structural characterization of the K antigens from Rhizobium fredii USDA257: evidence for a common structural motif, with strain-specific variation, in the capsular polysaccharides of Rhizobium spp. Journal of Bacteriology, 179, 5366-5371.

FRAYSSE N., COUDERC F. and POINSOT V. 2003. Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis. European Journal of Biochemistry, 270, 1365-1380.

GAGE D.J. 2004. Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiology and Molecular Biology Reviews, 68, 280-300.

GEREMÍA R., CAVAIGNAC S., ZORREGUIETA A., TORO N., OLIVARES J. and UGALDE R. 1987. A Rhizobium meliloti mutant that forms ineffective pseudonodules in alfalfa produces exopolysaccharides but fails to form β (1-2)glucan. Journal of Bacteriology, 169, 880-884.

GEURTS R. and BISSELING T. 2002. Rhizobium Nod factor perception and signaling. The Plant Cell (Supplement), 14, S239-S249.

GONZALEZ J.E., REUHS B.L. and WALKER G.C. 1996. Low molecular weight EPS II of Rhizobium meliloti allows nodule invasion in Medicago sativa. Proceedings of the National Academy of Sciences Of the United States of America, 93, 8636-8641.

GORE R.S. and MILLER K.J. 1992. Cell surface carbohydrates of micro aerobic, nitrogenase-active, continuous cultures of Bradyrhizobium sp. Strain 32H1. Journal of Bacteriology, 174, 7838-7840.

HOTTER G.S. and SCOTT D.B. 1991. Exopolysaccharide mutants of Rhizobium loti are fully effective on a determinate nodulating host but are ineffective on an indeterminate nodulating host. Journal of Bacteriology, 173, 851-859.

KANNENBERG E.L., REUHS B.L., FORSBERG L.S. and CARLSON R.W. 1998. Lipopolysaccharides and K-antigens: their structures, biosynthesis, and functions. In SPAINK H.P., KONDOROSI A. and HOOYKAAS P.J.J. (Eds.) The Rhizobiaceae: Molecular Biology of Model Plant-Associated Bacteria. Kluwer Academic Publishers, Dordrecht/Boston/London. pp. 120-154.

KANNENBERG E.L. and CARLSON R.W. 2001. Lipid A and O-chain modifications cause rhizobium lipopolysaccharides to bexome hydrophobic during bacteoid development. Molecular Microbiology, 39, 379-391.

Polysaccharides role in rhizobia-legume associations 103

KOUCHI H., SHIMOMURA K., HATA S., HIROTA A., WU G., KUMAGAI H., TAJIMA S., SUGANUMA N., SUZUKI A., AOKI T., HAYASHI M., YOKOYAMA T., OHYAMA T., ASAMIZU E., KUWATA C., SHIBATA D. and TABATA S. 2004. Large-scale analysis of gene expression profiles during early stages of root nodule formation in a model legume, Lotus japonicus. DNA Research, 11, 263-274.

LAGARES A., HOZBOR D.F., NIEHAUS K., PICH OTERO A.J.L., LORENZEN J., ARNOLD W. and PUHLER A. 1992. Genetic characterization of a Sinorhizobium meliloti chromosomal region involved in lipopolysaccharide biosynthesis. Journal of Bacteriology, 183, 1248-1258.

LEPEK V.C., NAVARRO DE NAVARRO Y. and UGALDE R.A. 1990. Synthesis of β(1-2) glucan in Rhizobium loti. Archives of Microbiology, 155, 35-41.

LEROUGE I. and VANDERLEYDEN J. 2001. O-antigen structural variation: mechanisms and possible roles in animal/plant-microbe interactions. FEMS Microbiology Reviews, 26, 17-47.

MILLER K.J., KENNEDY E.P. and REINHOLD V.N. 1986. Osmotic adaptation by gram-negative bacteria: possible role for periplasmic oligosaccharides. Science, 231, 48-51.

MITHOFER A. 2002. Suppression of plant defence in rhizobia-legume symbiosis TRENDS in Plant Science, 7, 440-444.

MITRA R.M. and LONG S.R. 2004. Plant and bacterial symbiotic mutants define three transcriptionally distinct stages in the development of the Medicago truncatula/Sinorhizobium meliloti symbiosis. Plant Physiology, 134, 595-604.

NIEHAUS K., KAPP D. and PHULER A. 1993. Plant defense and delayed infection of alfalfa pseudonodules induced by an exoolysaccharide (EPS)-deficient Rhizobium meliloti mutant. Planta, 190, 415-425.

NIEHAUS K., BAIER R., KOHRING B., FLASHL E. and PUHLER A. 1997. Symbiotic suppression of the Medicago sativa plant defence system by Rhizobium meliloti oligosaccharides. In LEGOKI A., BOTHE H. and PUHLER A. (Eds.) Biological Fixation of Nitrogen for Ecology and Sustanaible Agriculture Springe Verlag, Heidelberg, Germany. pp. 110-114.

NIEHAUS K., LAGARES A. and PUHLER A. 1998. A Sinorhizobium meliloti lipopolysaccharide mutant induces effective nodules on the host plant Medicago sativa (alfalfa) but fails to establish a symbiosis with Medicago trunculata. Molecular Plant-Microbe Interactions, 11, 906-914.

104 Viviana C. Lepek and Alejandra L. D’Antuono

NOEL K.D. and DUELLI D.M. 2000. Rhizobium lipopolysaccharide and its role in symbiosis. In TRILETT E.W. (Ed.) Prokaryotic nitrogen fixation: a model system for analysis of a biological process. Horizon Scientific Press, Wymondham, United Kingdom. pp. 415-431.

NOEL K.D., FORSBERG L.S. and CARLSON R.W. 2000. Varying the abundance of O antigen in Rhizobium etli and its effect on symbiosis with Phaseolus vulgaris. Journal of Bacteriology, 182, 5317-5324.

NOEL K.D., BOX J.M. and BONNE V.J. 2004. 2-O-methylation of fucosyl residues of a rhizobial lipopolysaccharide is increased in response to host exudates and is eliminated in a symbiotically defective mutant. Applied and Environmental Microbiology, 70, 1537-1544.

PARNISKE M., KOSCH K., WERNER D. and MULLER P. 1993. ExoB mutants of Bradyrhizobium japonicum with reduced competitivity on Glycine max. Molecular Plant-Microbe Interactions, 6, 99-106.

PARNISKE M., SCHMIDT P.E., KOSCH K. and MULLER P. 1994. Plant defense response of host plants with determinate nodules induced by EPS defective exoB mutants of Bradyrhizobium japonicum. Molecular Plant-Microbe Interactions, 7, 631-638.

PACIOS BRAS C., ALBERICH JORDÁ M., WIJFJES A.H.M., HARTEVELD M., STUURMEAN N., THOMAS-OATES J.E. and SPAINK H.P. 2000. A Lotus japonicus nodulation system based on heterologous expression of the fucosyl transferase NodZ and the acetyl transferase NolL in Rhizobium leguminosarum. Molecular Plant-Microbe Interactions, 13, 475-479.

PELLOCK B.J., CHENG H. and WALKER G.C. 2000. Alfalfa root nodule invasion efficiency is dependent on Sinorhizobium meliloti polysaccharides. Journal of Bacteriology, 182, 4310-4318.

PEROTTO S., BREWIN N.J. and KANNENBERG E.L. 1994. Cytological evidence for a host defense response that reduces cell and tissue invasion in pea nodules by lipopolysaccharide-defective mutants of Rhizobium leguminosarum strain 3841. Molecular Plant-Microbe Interactions, 7, 99-112.

RUSSA R., URBANIK-SYPNIEWSKA T., LINDSTROM K. and MAYER H. 1995. Chemical characterization of two lipopolysaccharides species isolated from Rhizobium loti NZP2213. Archives of Microbiology, 163, 345-351.

SPAINK H.P. 2000. Root nodulation and infection factors produced by rhizobial bacteria. Annual Review of Microbiology, 54, 257-288.

Polysaccharides role in rhizobia-legume associations 105

SPAINK H.P., OKKER R.J.H., WIJFFELMAN C.A., PEES E. and LUGTENBERG B.J.J. 1987. Promoters in the nodulation region of the Rhizobium leguminosarum Sym plasmid pRL1JI. Plant Molecular Biology, 9, 27-39.

SWART S., LOGMAN T.J.J., LUGTENBERG B.J.J., SMIT G. and KIJNE J.W. 1994. Several phenotypic changes in the cell envelope of Agrobacterium tumefaciens chvB mutants are prevented by calcium limitation. Archives of Microbiology, 161, 310-315.

VEDAM V., KANNENBERG E.L., HAYNES J.G., SHERRIER D.J., DATTA A. and CARLSON R.W. 2003. A Rhizobium leguminosarum AcpXL mutant produces lipopolysaccharide lacking 27-hydroxyoctacosanoic acid. Journal of Bacteriology, 185, 1841-1850.

VEDAM V., HAYNES J.G., KANNENBERG E.L., CARLSON R.W. and SHERRIER D.J. 2004. A Rhizobium leguminosarum lipopolysaccharide lipid-A mutant induces nitrogen-fixing nodules with delayed and defective bacteroid formation. Molecular Plant-Microbe Interactions, 17, 283-291.

Lotus Newsletter (2005) Volume 35 (1), 106-108.

U.S. Germplasm Collection of Lotus: Activities over the last decade

STEPHANIE L. GREENE, Curator USDA, ARS National Temperate Forage Legume Germplasm Resources Unit, 24106 North Bunn Road, Prosser, WA 99350. * Corresponding author

Although the germplasm collections of the USDA, ARS National Plant Germplasm System (NPGS) trace back to 1898, the oldest Lotus accession still available today was received in 1947. It is PI 157531, a local strain of Lotus corniculatus, part of a donation of Italian forage legumes presented by the University of Bologna, Italy. In the last 10 years we have received 181 accessions mainly from Russia, Armenia, Turkmenistan and Tajikistan. Our most recent accession, W6 24978, a wild-collected L. corniculatus, was collected in 2003 in Tajikistan, growing in saline conditions along a roadside. Currently the U.S. collection has 900 accessions representing 60 species from 65 countries. A total of 734 are available for distribution. Small quantities of seed are freely available for research purposes and can be requested using the Germplasm Resources Information Network (GRIN) (www.ars-grin.gov/npgs), or by contacting me ([email protected]).

Reflecting an earlier mandate of Plant Introduction, the NPGS Lotus collection has a major emphasis on Old World species. Over 73% of the collection is represented by cultivated species. The expansive geographic distribution of L. corniculatus is well represented although we have no accessions from India, Mongolia or Taiwan. Countries most represented include the United States, Turkey and Italy. A core subset of L. corniculatus has been developed and is available for distribution. Steiner et al. (2001) discusses how the core subset was developed. An interesting test array of L. corniculatus is also available. This set contains 16 wild accessions collected in the Caucasus Mountains, Russia, in 1995 (Greene et al., 1999). The accessions were collected from sites that represent the climatic range of the region, as defined by classifying minimum winter temperature into 7 different zones, and total annual precipitation into 5 different zones (Table 1). Representation of New World species is limited in the U.S. Collection, although efforts are underway to obtain wild species endemic to the U.S. Users interested in New World species may be interested to know that they can request accessions from the University of Arizona Desert Legume Program (DELEP), by specifying DLEG as the repository in the Search GRIN, Accession Search module of GRIN. DELEP has about 20 accessions collected from the Southwest United States and Mexico.

106 U.S. Germplasm Collection of Lotus 107

Table 1. Climatic test array of L. corniculatus collected in the Caucasus Mountains, Russia in 1995, and available for study. Detailed information can be found on GRIN (http://www.ars-grin.gov/npgs), by clicking on Search GRIN; Search Accession, and specifying identifier number.

Acc. No. Elev. Winter Min. Temp Ann. Precip. (W6) (M) (°C) (mm) 18313 59 -24 591 18327 563 -32 653 18347 525 -31 720 18380 469 -23 725 18558 39 -29 726 18464 659 -32 860 18389 675 -24 923 18599 190 -29 929 18523 1153 -33 1042 18513 408 -30 1119 18530 1471 -32 1311 18471 1207 -27 1415 18442 1871 -33 1493 18546 1366 -28 1728 18486 2296 -28 1798 18495 2113 -25 1833

Since 1995, the collection has been regenerated at the USDA, ARS National Temperate Forage Legume Germplasm Resources Unit, in Southeast Washington. We are systematically regenerating the collection using original or most original seed lot, following the germplasm increase guidelines established by Sackville-Hamilton (1997). All Lotus species are started in the greenhouse and transplanted into the field six weeks later. Our goal is to produce seed off of 100 individual plants, enclosing each accession in a pollination cage (Figure 1). As flowering begins, leaf cutter bees (Megachile pacifica Panzer.) are added to the cages as needed. Since many of our accessions are being regenerated from original seed, some of the seed lots we work with are very old, and of poor quality. We have developed a sterile germination technique, which involves disinfecting and imbibing scarified seed using an 18 hours running water rinse. Seeds are then placed in perlite and allowed to germinate 108 Stephanie Greene

Figure 1. Cross-pollinated Lotus species are increased in isolation using cages, and pollinated using leaf cutter bees in Prosser, WA. under grow lights. Seedlings are hardened off, moved to the greenhouse, and depending on the number of seedlings and vigor, transplanted to the field and treated using routine regeneration protocols. Southeastern Washington is an excellent seed production area. We commonly harvest 200-400 grams of seed from a single plot. Since 1999, we have increased 257 Lotus accessions. In the next five years we are hoping to have the entire collection regenerated. Other directions we will be exploring are the establishment of in situ collecting sites for wild Lotus species native to North America. Because many of the wild species are difficult to regenerate in Washington, this provides a cost effective approach for providing seed of wild species to the Lotus user community.

References

GREENE S.L., HART T.C. and AFONIN A. 1999. Using geographic information to acquire wild crop germplasm for ex situ collections: I. Map development and Use. Crop Science, 39, 836-842.

SACKVILLE-HAMILTON N.R. 1997. Standards for regeneration. In MAGGIONI L., MARUM P., SACKVILLE HAMILTON R., THOMAS I., GASS T. and LIPMAN E. (Eds) Report of a Working Group on Forages. 6th meeting, 6-8 March, Beitostoen, Norway. Pp. 103-108.

STEINER J.J., BEUSELINCK P.R, GREENE S.L., KAMM J.A., KIRKBRIDE J.H., ROBERTS C.A. 2001. A description and interpretation of the NPGS birdsfoot trefoil core subset collection. Crop Science, 41, 1968-1980.

Lotus Newsletter (2005) Volume 35 (1), 109-114.

The symbiosis between Lotus japonicus and rhizobia: Function of nod factor structural variation 1

CRISTINA PACIOS-BRAS* Laboratoire des Proteines du Cytosquelette, Leiden University,41, Rue Jules Horowitz, F-38027 Grenobl, Cedex , France 1 Ph.D. Thesis, 132 p. click here for Spanish version of this article *Corresponding author

Chapter 7. Summary and Discussion

Legumes, together with cereals, represent the largest and richest food sources for human and cattle. Additionally, legumes have the ability to enrich the soil with “ready to use” nitrogen for plants by transforming atmospheric nitrogen into ammonia. Other crops unable to fix nitrogen can also use this nutrient. During the classic Roman Empire this property of legumes to increase the concentration of nitrogen in soil was already used by the application of a rotational crop growth. In this way the better growth of other plants was promoted. This system is still used in our days, being an important alternative to the use of artificial nitrogen enrichment of the soil. Hellriegel in 1888 was the first to assign the legume nitrogen fixation property to the existence of bacteria inside the legume root nodules and Bréal in the same year showed that not all legumes could be nodulated by all rhizobia, defining the so called cross-inoculation groups. In 1990 Lerouge, was the first to describe the structure of the specific bacterial signal molecules (Lipochitin oligosaccharides or nod factors), which induces the process of nodulation of a legume by a rhizobial species. Since then the structures of the majority of the nod factors of the Rhizobiaceae family have been elucidated and most of the studies are currently focusing on the analysis of the specific plant perception system for these bacterial molecules. Nevertheless, by the research described in this thesis we show that there still are many open questions about the specific signal molecules produced by rhizobia.

For our nodulation experiments we mostly used the model legume Lotus japonicus and its natural rhizobial microsymbiont, Mesorhizobium loti. This plant belongs to the determinate nodulating legumes group. This group is characterized by the forming of determinate nodules which are round shaped and its formation initiates by cell divisions at the outer cortex of the root. This thesis demonstrates that L. japonicus is a suitable system for the study of determinate nodulation. Using this system we analyzed the structure and function relationship of nod factors recognized by L. japonicus. We also have developed new assays for analysis of functions of nod factors.

For a better detection of the plant responses to rhizobial nod factors, we constructed and characterized transformed L. japonicus lines containing fluorescent and enzymatic reporter proteins, namely green fluorescent protein (GFP) and ß-glucuronidase (X-Gluc), fused to

109 110 Cristina Pacios-Bras plant promoters. An example of a studied plant promoter is the auxin responsive promoter GH3, implied in plant gene regulation during nodulation.

To facilitate the detection of one or more bacterial populations during the plant invasion, we developed a new method with which we can follow the bacteria as fluorescent entities. By this method two or more different fluorescing rhizobial populations can simultaneously been detected in the root.

For the study of the significance of single rhizobial nodulation genes (nod genes) during symbiosis with L. japonicus, we expressed M. loti nod genes in a Rhizobium strain (R. leguminosarum bv. vicia) that naturally does not posses these genes and does not nodulate L. japonicus. When modified to heterologously express the introduced M. loti nod genes, this strain nodulates L. japonicus. This gain of function system allows the study of single nodulation genes without any influence of the genetic background of the naturally infecting bacteria, M. loti. We concentrated our effort in the study of the significance of nolL. Together with the gain of function system, we studied the effect of the mutation of M. loti nolL during symbiosis with L. japonicus.

For this thesis we have also isolated and characterized new nod factor structures, synthesized by a R. etli that, contrary to other R. etli strains, is unable to nodulate L. japonicus. We also show that the change of the structure of the nod factors produced by this strain after mutation of a single gene, allows it to nodulate L. japonicus.

Visualization of rhizobia during interaction with legumes (Chapter 2)

In Chapter 2 we describe a tool developed to allow following and visualization of living bacteria when interacting with plants. To this effect, different stable broad host range vectors were developed coding for different GFP (Green fluorescent protein) and GFP-derived fluorescent proteins. These vectors are efficiently expressed in different bacterial species and showed to be very suitable for the analysis of the infecting bacteria in the nodulation process. Coinoculation of different rhizobial populations, marked each with a different protein fluorescing with a different color [ECFP (enhanced cyan fluorescent protein) or EYFP (enhanced yellow fluorescent protein)] were followed by CLSM (confocal laser scanning microscopy). By applying this system we were able to demonstrate that two differently labeled bacterial populations can form part of the same infection thread and, in later stadia of symbiosis, both can be located in the same nodule. We were also able to describe movements of the bacteroids inside the nodule.

Auxin distribution during nodulation in L. japonicus (Chapter 3)

It is known that one of the first detectable changes in legumes during the onset and further development of nodulation, in response to the rhizobial infection, are changes in the auxin distribution of the plant. With the purpose of analyzing the auxin distribution pattern in L. japonicus during the development of the nodules we made stable transformed lines of L. japonicus expressing the soybean auxin responsive promoter GH3 fused to the GFP and Nod-factor structural variations in Lotus japonicus 111

GUS (ß-glucuronidase) reporter proteins. This promoter is expressed at the positions in the plant were auxin concentration is enhanced.

The GH3 expression was detected as a green fluorescent signal when analyzed for GFP or as blue staining when analyzed for GUS activity. The GH3GUS expression pattern during nodulation of the indeterminate nodulating legume white clover was described before. Diverging from determinate nodules, indeterminate nodules present the first cell divisions at the root pericycle and have a continuous growing meristem. We found divergences in auxin distribution in L. japonicus, when compared with white clover, after root inoculation with bacteria or its purified nod factors. Where cell divisions leading to nodule formation were found a higher GUS and GFP expression was detected at the outer root cortical cells. This cortical expression showed to be separated from the root vascular bundle expression. In nodule primordia and young nodules the outer cortex upregulation was sustained and an upregulated vascular GUS or GFP activity was detected. When nodules, induced by bacteria, reached maturity the basal main root and cortical expression disappeared. We were able to quantify the difference in GH3 expression between primordia and mature nodules by measurement of the GFP expressed. Analysis of the auxin transport of L. japonicus roots inoculated with purified nod factors showed an increased transport in the segment 4 mm distant from the root tip two days after inoculation. At this time point the first nodular cell divisions were also detected. Although further work is required, the upregulation detected by histochemical GUS staining or GFP expression analysis may be connected to the increase in auxin transport.

Function of NolL during symbiosis with L. japonicus (Chapters 4 and 5)

Rhizobium leguminosarum RBL5560 nodulates Vicia, but cannot nodulate Lotus. When modified to heterologously express the flavonoid independent transcription activator, FITANodD and the fucosyl transferase NodZ, this strain (5560DZ) produces fucosylated nod factors and nodulates L. japonicus. The addition of the nolL gene from M. loti to 5560DZ (to give 5560DZL) improves the nodulation on L. japonicus to levels comparable to those obtained after inoculation with the natural microsymbiont, M. loti. This demonstrates the importance of the presence of this group on the bacterial nod factors for a proper symbiotic interaction between L. japonicus and rhizobia. Isolation and analysis of the structures of the nod factors produced by 5560DZL show that after addition of nolL the nod factors produced are acetyl fucosylated, demonstrating that the NolL protein is an acetyl transferase that supplies an acetyl group to the fucose residue added by NodZ. Curiously, 5560DZL produces a much higher amount of nod factors than 5560DZ, the reason of which is still unclear. When 5560DZ and 5560DZL, each expressing a different fluorescent protein, are coinoculated on a single L. japonicus root, the NolL-containing strain (5560DZL) is always more successful during the plant invasion, as visualized by CLSM microscopy.

Microscope studies of the development of nodulation induced by both 5560DZ and 5560DZL during infection of L. japonicus indicate that the presence of NolL is important for infection thread development and in later stadia for development of nodule structure. We also show that when NolL is present in strain 5560DZL the early nodulin plant promoter ENOD40 and the auxin responsive promoter GH3 are regulated in the same temporal way as 112 Cristina Pacios-Bras after inoculation with M. loti. Induction of these promoters in the absence of NolL (5560DZ) is clearly retarded and rarely reaches the expression pattern of wild type bacteria -induced root nodules. Inoculation of these same strains on L. filicaulis does not induce nodulation, indicating a different nod factor perception by this legume and showing once again the distinctions between the specific perceptions of nod factors by each plant species.

The inoculation of L. japonicus and other Lotus species with M. loti nolL- mutants (R7A::nol-) showed that in the natural background the absence of NolL abolishes nodulation. We also describe for the first time to our knowledge, the phenotype of a M. loti mutant (PN184), being capable of hypernodulation on L. japonicus and L. filicaulis. When the nolL gene is inactive in the hypernodulating strain, normal nodulation is restored. The combination of our results show that in all the cases of loss of NolL function (when absent in the heterologous system, when mutated in M. loti and when mutated in PN184) a negative effect is observed in the amount of nodules produced by L. japonicus. However, the stringency of this effect is completely dependent on the strain background. Because the lack of NolL in PN184 and 5560DZ does not abolish nodulation on L. japonicus and does abolish nodulation by M. loti R7A we believe that NolL may have an additional function related to nodulation on L. japonicus next to acetylation of nod factors. This additional function may be taken over by other genes in the heterologous system and also PN184, making nodulation on L. japonicus possible.

The different recognition of RBL5560DZL by L. japonicus and L. filicaulis suggests specificity for other bacterial factors different from nod factors such as EPS and LPS.

Structures of the nod factors produced by KIM5s and its mutant strain KIM::NodZ and the relation with symbiotic capacity

R. etli usually have pentameric chitin structures with and acetyl fucose substituent at the reducing terminus and a carboamyl group at the non-reducing residue of the chitin backbone..These nod factor structures are identical to those synthesized by M. loti.

The R. etli strain KIM5s is one of the most successful bean-nodulating R. etli strains (due to high competitiveness and high nitrogen fixation rate). We show that KIM5s has a restricted host range when compared with other strains. While R. etli CE3 is able to nodulate Phaseolus vulgaris (common bean) and L. japonicus, R. etli KIM5s cannot induce nodulation on L. japonicus. Knock-out of a gene highly similar to nodZ in R. etli KIM5s results in a strain (KIM::nodZ) which induces nodules on L. japonicus but is not able to nodulate siratro.

We isolated and analyzed the nod factor structures produced by Rhizobium etli strain KIM5S and its mutant strain KIM::NodZ. For the analysis of the nod factors produced we made use of reverse phase capillary column directly coupled to an ion trap spectrometer. This system showed to be an accurate and fast system to elucidate nod factor structures.

Remarkably, R. etli KIM5S synthesizes mainly hexameric nod factors, a structure rarely Nod-factor structural variations in Lotus japonicus 113 detected in the Rhizobiaceae family. This is completely different to the nod factor structures described for all the other described R. etli strains, which are identical to the M. loti nod factors. In addition, no substituents are present at the reducing terminus. Furthermore, these nod factors of KIM5S often contain a non-acylated glucosamine as one or two of the oligosaccharide residues. This kind of nod factor molecules had never been found in any other rhizobial species.

Analysis of the nod factors produced by KIM::nodZ shows that hexameric nod factor structures are absent in this strain, but instead the majority of the produced nod factors are pentameric structures with a chitin backbone. By in vitro Nod factor synthesis and its subsequently inoculation on soybean (Glycine max) roots, a relation between the substituents and length of the nod factor molecule and the plant responses induced was described. Pentameric nod factors with a chitin backbone of five GlucNAc units and a methyl fucose substituent at the reducing unit induced nodule primordia on this legume. The same effect was induced on soybean roots when the LCOs had a four-unit chitin oligomer and the methyl fucose group was not present. Pentameric LCOs without the methyl fucose or tetrameric nod factors presenting this substituent did not induce any biological activity on the same legume. The variation in legume responses induced by different nod factor structures has been proposed to be due to a differential recognition of these molecules by the plant chitinases. This may be the case during recognition of R. etli KIM5S and its mutant strain KIM::nodZ by L. japonicus. The structural differences of the nod factors produced by KIM5s and KIM::nodZ may lead to a different L. japonicus chitinase cleavage in each case that results in lack of nodulation by KIM5s and in successful nodulation by KIM::nodZ.

Future prospects

The intention of this study was to examine the symbiotic behavior of L. japonicus and the importance of Nod-factor structural modifications during symbiosis with this legume. Our results show that although there is a broad knowledge about the structure and biosynthesis of the rhizobial nod factors, still many questions about the bacterial signaling in relation to plant perception during symbiosis remain unanswered.

More experimentation on legumes transformed with genes related to plant responses during nodulation, fused to reporter genes, will help us to see the rhizobia-legume symbiosis as a single whole interacting system. For example the L. japonicus transformed lines used for some experiments in this thesis: ENOD40gusA:intron/gfp and GH3 gusA:intron/gfp. These tools will provide us with more knowledge about the exact bacterial structures required for the symbiotic responses of the plant. For these studies, microscope tools like CLSM and two-photon microscopy together with the availability of model legume systems will play an important role. The rapid advances in proteomics are bringing more insights of plant symbiotic responses in time.

The ultimate goal, a better knowledge of symbiosis, will definitively help us to improve the quality of the crops grown for cattle and human alimentation in sustainable agriculture. For instance our knowledge can be used for the selection of natural strains more optimal in 114 Cristina Pacios-Bras competitiveness during infection of some legumes, like in the case of the heterologous system described during this thesis. This could be applied for favoring the development of symbiosis between the selected rhizobia and the legume of interest. In this way the endogenous rhizobia that often display poor nitrogen fixation capacity can be decreased.

Lotus Newsletter (2005) Volume 35 (1), 115-119.

Can the symbiosis between arbuscular mycorrhiza and Lotus glaber tolerate waterlogging in a saline-sodic soil?

RODOLFO MENDOZA*, ILEANA GARCÍA and VIVIANA ESCUDERO Museo Argentino de Ciencias Naturales “Bernardino Rivadavia”, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Av. Angel Gallardo 470 (C 1405). Buenos Aires. Argentina. E-mail: [email protected] * Corresponding author

Introduction

Lotus glaber Mill. is an important naturalised legume on lowland grasslands in the Flooding Pampa of Buenos Aires Province, Argentina. During the rainy season (autumn, winter and part of the spring) flooding is a common phenomenon in these lowlands. The duration of flooding is variable, ranging from a few days to seven months depending on topography and season (Escudero and Mendoza, 2005). The soils of these lowlands are characterised by high pH, salinity and sodicity (Mendoza, 1980).

Mendoza and Pagani (1997) and Mendoza et al. (2000) have reported high levels of root length colonised and high dependency of L. glaber on arbuscular mycorrhizas (AM) to grow in soils of low P availability. AM symbiosis enables plants to cope with a variety of stressful conditions (Entry et al., 2002). Waterlogging may increase or decrease P availability in soils, and an increase in P availability may decrease AM colonisation in L. glaber roots (Mendoza and Pagani, 1997). Few studies of the effect of soil waterlogging on mycorrhizal colonisation and on nutrient uptake in forage flooding tolerant plants have been reported in extreme saline-sodic conditions (Juniper and Abbot, 1993; Escudero and Mendoza, 2005; Mendoza et al., 2005). The halomorphic condition of the soil adds an extra factor, which increases the stress upon plants.

Escudero and Mendoza (2005) reported high levels of spore density in soil and AM colonisation of L. glaber roots in a soil site waterlogged six months before sampling. Since mycorrhizas colonization is one of the potential strategies for both plant and fungi to tolerate the adverse conditions imposed by waterlogging, we performed a greenhouse pot experience to investigate how the latter affects the level of AM colonisation in L. glaber plants growing in a saline-sodic soil. More details of the experiment can be found in Mendoza et al. (2005). Lotus glaber plants were grown in pots as follow: a) 50 days at field capacity + 40 days at field capacity. b) 50 days at field capacity + 40 days of waterlogging (1 cm above soil surface). Root length (m) and root length colonised (AM%); arbuscular (AC), vesicular (VC) and hyphae-only (HO) root length colonised (m); entry points and nodules in roots, and spore

115 116 Rodolfo Mendoza, Viviana Escudero, Ileana García density in soil, were measured after the initial period of growth (50 days) and at the end of the experiment after an additional 40 more days. The relative rate of change (RRC) of these variables was calculated for waterlogged and non-waterlogged plants: RRC = (ln Wtf – ln Wto)/ tf – to) where W represents the variable tested, tf is the total period of growth from germination (90 days), to is the initial period of growth at near field capacity from germination (50 days). We hypothesised that soil waterlogging would reduce the growth of L. glaber and colonisation of roots by AM fungi and Rhizobium compared to non- waterlogging soil.

Can AM fungi improve Lotus glaber tolerance to waterlogging?

After 90 days of growth, AM fungi colonised more than 49% of the total L. glaber root length in both, waterlogged and non-waterlogged plants (Table 1). However, a 40 days period of soil waterlogging provoked a decrease in the length of the colonised roots by 46 % with respect to non-waterlogged plants.

Table 1. Effect of soil waterlogging on AM fungi colonisation variables and Rhizobium nodules in Lotus glaber roots, and spore density in soil measured at the initial period of growth before (50 days) and after waterlogging (40 days), and the relative rate of change (RRC)(a) of these variables in waterlogged and non-waterlogged plants.

-1 (a) -1 (a) Variable Initial Non-waterlogged Waterlogged RRC (day ) RRC (day ) period plants plants non-waterlogged waterlogged

AM (%) 51.60 a 80.31 b 49.17 a 0.0111 -0.0012 AM root length 0.96 a 26.76 c 14.15 b kw 0.0832 0.0673 (m) AC root length 0.94 a 14.33 b 5.76 ab 0.0681 0.0453 (m) VC root length 0.01 a 0.27 a 1.27 b 0.0082 0.1211 (m) HO root length 0.26 a 15.66 c 7.48 b kw 0.1024 0.0839 (m) Entry points (per 3.87 a 7.09 b 4.36 a kw 0.0151 0.0030 mm root) Spore density 174.00 a 99.00 a 169.10 b kw -0.0141 -0.0007 (per g soil) Nodules 18.99 b 10.20 a 62.62 c ln -0.0156 0.0298 (per g fresh root) Root length (m) 1.85 a 33.32 b 28.78 b 0.0723 0.0686 kw: Kruskal Wallis non-parametric test. ln: log transformation data.. (a): RRC = (ln Wtf – ln Wto)/ tf – to. Waterlogging tolerance of Lotus glaber and mycorrhiza 117

The rate of spread of colonisation by the AM fungi (RRC AM%) within the root during the 40 days of growth decreased in waterlogged plants (Table 1). Root length (AM root length) and the rate of root length colonisation (RRC AM root length) between the initial and the second harvest after waterlogging were significantly lower for the roots of waterlogged plants.

The morphology and phenology of root colonisation was also affected by waterlogging. The arbuscular colonisation root length (AC) and hyphae-only (HO) colonisation root length were higher in the roots of non-waterlogged plants (Table 1). In addition, waterlogging increased the vesicle colonisation root length (VC). These results reflect a depressive effect of soil waterlogging on the AM-Lotus symbiosis. Arbuscules are the sites of nutrient transfer from the fungus to the host and transfer of carbon through the intercellular interfaces (Smith, 1993). Although a reduction of arbuscules does not entail a complete loss of P-transfer because P can also be exchanged from hyphae (Ryan et al., 2003), a decrease in arbuscules in colonised roots of waterlogged plants may represent a tendency of a benefit for the fungus and a C cost for the plant.

The number of entry points per unit of colonised root length was reduced by 38 % in waterlogged soil (Table 1). The relative increase of the number of entry points (RRC entry points) formed per day in roots of waterlogged plants was approximately 0.003, compared to 0.014 entry points formed per day in non-waterlogged roots (Table 1).

The spore density in waterlogged soil after the total period of growth (90 days) was the same as at the initial 50 days (169 per g of dry soil), whereas the density in non-waterlogged soil dropped significantly to 99 per g of dry soil (P < 0.05) (Table 1). Flooding factors appear to be significantly affecting total spore density, but there have been conflicting reports in the literature because spore density has been reported to increase (Rickerl et al., 1994), decrease (Aziz et al., 1995; Muthukumar et al., 1997), or remain invariant with soil moisture or flooding condition (Carvalho et al., 2001, Entry et al., 2002). We propose that differences in spore density between waterlogged and non-waterlogged soil in the present study may be ascribed to differences in spore germination and spore disappearance rather than in sporulation. As a result of the adverse soil condition, spores in waterlogged soil did not germinate. In contrast, spore density decreased after 40 days of growing in non-waterlogged soil as a result of soil conditions appropriate for spore germination. The higher number of entry points per mm of colonised root in non-waterlogged plants (62%) compared to the roots supports that explanation.

After 90 days at the end of the experiment, the number of nodules of Rhizobium per unit of fresh weight was higher in the roots of waterlogged plants (Table 1), with a substantial proportion of the nodules clustered near the soil surface, at the base of the main root. Nitrogen-fixing legumes are usually adversely affected by flooding (Arrese-Igor et al., 1993). However, James and Crawford (1998) reported increases in the number, fresh and dry weights of nodules in two Lotus spp. (L. uliginosus and L. corniculatus) after 60 days of flooding.

In a previous work, Mendoza et al. (2005) found that aerenchyma tissue was developed in 118 Rodolfo Mendoza, Viviana Escudero, Ileana García the cortex of waterlogged and non-waterlogged roots, but was higher in waterlogged roots. AM colonisation exceeded 49% in this roots, suggesting that aerenchyma facilitates root aeration thus may permit AM fungi to survive in low oxygen concentration in soil.

Conclusion

The results of the present work are consistent with our previous field experiment and may explain why we found both high values of spore density and a reduction of AM colonisation in L. glaber roots after six months of flooding on the same soil type (Escudero and Mendoza, 2005). These studies indicate that AM fungi can survive in waterlogged soil, and can colonise roots in the next season by a strategy consisting of colonisation reduction, production of resistance structures such as vesicles with preference to transfer structures such as arbuscules, and inhibition of spore germination. This strategy has a significant implication for the understanding of the biology of AM fungi in flooding condition. Lotus glaber can be classified as a highly tolerant plant to flooding and would make an important contribution to sustain AM infectivity after a long period of flooding at field conditions. However, we need to direct future works to establish if AM fungi can improve the Lotus glaber tolerance to waterlogging.

References

ARRESE-IGOR C., ROYUELA M., DE LORENZO C., DE FELIPE M.R. and APARICIO-TEJO P.M. 1993. Effect of low rhizosphere oxygen on growth, nitrogen fixation and nodule morphology. Physiologia Plantarum, 89, 55-63.

AZIZ T., SYLVIA D. and DOREN R. 1995. Activity and species composition of arbuscular mycorrhizal fungi following soil removal. Ecological Applications, 5, 776-784.

CARVALHO L.M., CAÇADOR I. and MARTINS-LOUÇÂO M.A. 2001. Temporal and spatial variation of arbuscular mycorrhizas in salt marsh plants of the Tagus estuary (Portugal). Mycorrhiza, 11, 303-309.

ENTRY J.A., RYGIEWICZ P.T., WATRUD L.S. and DONNELLY P.K. 2002. Influence of adverse soil conditions on the formation and function of Arbuscular mycorrhizas. Advances in Envionmental Research, 7, 123-138.

ESCUDERO V.G. and MENDOZA R.E. 2005. Seasonal variation of arbuscular mycorrhizal fungi in temperate grasslands along a wide hydrologic gradient. Mycorrhiza. (in press).

JAMES E.K. and CRAWFORD R.M.M. 1998. Effect of oxygen availability on nitrogen fixation by two Lotus species under flooded conditions. Journal of Experimental Botany, 49, 599-609.

Waterlogging tolerance of Lotus glaber and mycorrhiza 119

JUNIPER S. and ABBOTT. 1993. Vesicular-arbuscular mycorrhizas and soil salinity. Mycorrhiza, 4, 45-57.

MENDOZA R.E. 1980 Efecto del agregado de yeso a un suelo sódico de la depresión del Río Salado (Buenos Aires, R. Argentina) y su repercusión sobre algunas características físico-químicas, microbiológicas y de producción de forraje. Revista de Investigaciones Agropecuaria, 15, 573-592.

MENDOZA R.E. and PAGANI E. 1997. Influence of phosphorous nutrition on mycorrhizal growth response and morphology of mycorrhizae in Lotus tenuis. Journal of Plant Nutrition, 20, 625-639.

MENDOZA R., PAGANI E. and POMAR M.C. 2000. Variabilidad poblacional de Lotus glaber en relación con la absorción de fósforo en suelo. Ecología Austral, 10, 3-14.

MENDOZA R. E., ESCUDERO V. and GARCÍA I.V. 2005. Plant growth, nutrient acquisiton and mycorrhizal symbioses of a waterlogging tolerant legume (Lotus glaber Mill.) in a saline-sodic soil. Plant and Soil. (in press).

MUTHUKUMAR T., UDAIYAN K., KARTHIKEYAN A. and MANIAN S. 1997. Influence of native endomycorrhiza, soil flooding and nurse plant on mycorrhizal status and growth of puple nutsedge (Cyperus rotundus L.). Agriculture, Ecosystems and Environment, 61, 51-58.

RICKERL D.H., SANCHO F.O. and ANANTH S. 1994. Vesicular arbuscular endomycorrhizal colonisation of wetland plants. Journal of Environmental Quality, 23, 913-916.

RYAN M.H., MCCULLY M.E. and HUANG C.X. 2003. Location and quantification of phosphorus and other elements in fully hydrated, soil-grown arbuscular mycorrhizas: a cryo-analytical scanning electron microscopy study. New Phytologist, 160, 429-441.

SMITH S.E. 1993. Transport at the mycorrhizal interface. Mycorrhiza News, 5, 1-4.

Lotus Newsletter (2005) Volume 35 (1), 120-130.

Evaluation of the genotype-environment interaction in the establishment of Lotus uliginosus (Schkuhr) with soil-cores

SEBASTIÁN HERNÁNDEZ1, MÓNICA REBUFFO1*, SEBASTIÁN ARRIVILLAGA1, MARTÍN JAURENA2, CARLOS LABANDERA2, DIEGO RISSO3 and JAVIER CILIUTI1 1 Instituto Nacional de Investigación Agropecuaria, INIA La Estanzuela, Colonia, Uruguay. 2 Laboratorio de Microbiología de Suelos (MGAP), Montevideo, Uruguay. 3 Instituto Nacional de Investigación Agropecuaria, INIA Tacuarembó, Tacuarembó, Uruguay. *Corresponding author click here for Spanish version of this article

Introduction

Lotus uliginosus Schkuhr (big trefoil) is a perennial legume highly adapted to different Uruguayan soils. Its extensive underground system formed by the crown and central taproot gives origin to a network of rhizomes, stolons and fibrous roots that successfully colonizes the native swards (Carámbula et al., 1994). The tetraploid cultivar Grasslands Maku (Maku) is the most utilized in the country, even when other diploid cultivars have been evaluated (Castaño and Menéndez, 1998). Maku, released in 1975 in New Zealand, was bred from local ecotypes and introductions from Portugal with outstanding winter growth (Charlton, 1983). Chromosome duplication increased its seed size and seedling vigour. The persistence and high forage production are the main characteristics for the improvement of natural grasslands in extensive cattle raising areas, particularly in the East of the country (Risso et al., 1990; Carámbula et al., 1994; Carámbula et al., 1996; Castaño and Menéndez, 1998). In these conditions, Maku shows an improved performance compared to other legumes of well-known productive capacity, such as Lotus corniculatus (Risso and Berretta, 1996; Castaño and Menéndez, 1998).

The vegetative growth of Maku is opposed to the low seed production under Uruguayan environmental conditions. For this reason INIA's breeding program was aimed to obtain an adapted big trefoil with high seed production. The experimental line LE 627 is an early type diploid material, with good initial growth and high seed potential. Regional evaluation of LE 627 produced inconsistent results; its slow establishment and short persistence has been verified in the basaltic soils, in opposition to the results in the Eastern region, where the experiments have been successfully established (Risso D. Com. Pers; Iglesias and Ramos, 2003; INASE, 2005; Castaño and Menéndez, 1998). This differential performance is probably due to a genotype-environment interaction, either by a specific adaptation of this genotype to different soils, to environmental conditions or an interaction in the symbiotic relationship.

A clear example of symbiotic interactions happens with the introduction of species of the genus Lotus and rhizobia strains that can be effective or parasitic according with host

120 Genotype-environment interaction in Lotus uliginosus 121 combination (Pérez and Labandera, 1998; Irrisarri et al., 1996). Rhizobia strains able to produce nodules in Lotus spp. belong to Rhizobium loti and Bradyrhizobium sp., with a relative specificity between the species and its symbionts. L. corniculatus and L. glaber form a symbiotic effective group with fast growing strains (R. loti) whereas Lotus subbiflorus and L. uliginosus form another effective group with slow growing strains (Bradyrhizobium sp.; Brockwell et al., 1994; Baraibar et al., 1999). These symbiotic groups have incompatible relationships to each other: the bacteria of one symbiotic group produces nodules in the other group host but the relationships are ineffective or parasitic. Under these conditions there is no nitrogen fixation because functional symbiosis does not occur, and so establishment difficulties might arise. Lieven-Antoniou and Whittam (1997) reported mechanisms that lead to differential recognition of host genotypes (L. corniculatus) and their symbionts (R. loti). However, there are fast growing strains (NZP2037) that form effective nodules in both symbiotic groups, although their effectiveness is not as high as the specific ones (Labandera C. Com.Pers.; Pankhurst, 1981; Scott et al., 1987; Barrientos et al, 2002). The rhizobia strain selection programs for L. subbiflorus and L. uliginosus has showed a much better efficiency with slow growing srains (Mayans 2003). The rhizobia strain collection of the Department of Soil Microbiology (RENARE-MGAP, Uruguay) holds several strains and isolations for species of the genus Lotus. Strain U526 (NZP 2309, New Zealand) is recommended for L. uliginosus in Uruguay, whereas strain 531 (NC3, Uruguay) is recommended for L. subbiflorus, although both strains are effective in either host (http://fp.chasque.apc.org:8081/microlab/LMSCI/catalogo/marco.htm).

Native or naturalized rhizobia that effectively nodulate the four Lotus species of agronomic value are generally present in Uruguayan soils. L. corniculatus has been utilized since the 60’s in rotations with crops in the arable area. Therefore, these soils have an important concentration of effective strains for this host. However, without this previous history of cultivation, natural grasslands usually have native Mesorhizobium loti, with high variability in symbiotic efficiency (Baraibar et al., 1999). Effective native rhizobia that nodulate L. uliginosus and L. subbiflorus are in very low concentrations and so, they do not compete with introduced strains. Nevertheless, with the introduction of inoculated legumes of the same genus, the rhizobia remain in the soil in important concentrations. If the Lotus species to be introduced are not of the same symbiotic group of the predominant rhizobia population in the soil, then defective establishment or yield losses might happen (Gwynne et al., 1980). The establishment difficulties in these cases can be attributed to host-strain interactions with less efficiency in nitrogen fixation, which might restrict the symbiotic potential of the host. In conclusion, the successful establishment of species of the genus Lotus depends on the effectiveness of the rhizobia populations present in the soil and the appropriate management of the inoculation technique (Skerman et al., 1991; Labandera et al., 2005).

LE 627 has had a variable performance in the regional trials, with poor establishment in basaltic soils, generating the question about the role of soil types and the native rhizobia population on the establishment of this genotype. The objective of this research was to analyze host-rhizobia-soil interaction in the establishment of the symbiosis and initial development of L. uliginosus, using soil-cores under standardized environmental conditions.

122 Sebastián Hernández, Mónica Rebuffo, Sebastián Arrivillaga, Martín Jaurena, Carlos Labandera, Diego Risso, Mónica Rebuffo and Javier Ciliuti.

Materials and Methods

The treatments consisted of 2 genotypes, with and without inoculation and 4 soil types in a complete factorial design with 6 replicates. Genotypes were cultivar G. Maku and the experimental line LE 627. Each genotype was sown with (WI) and without (NI) the rhizobia strain U-526. Inoculation was done by watering twice the recommended commercial dose.

The four soils are representative of the main areas of extensive production and have different physical-chemical characteristics (Table 1). These correspond to: (1) Eastern lowlands (Lowl) with rice stubble at Paso La Laguna (Treinta y Tres), representative of the lowest topographical levels in the Eastern plains. These humid, heavy soils have a shallow phreatic nape during most of the year. (2) Basaltic soil (Bas) with natural grassland at Glencoe (Paysandu), representative of medium soils of the basaltic region, where the landscape corresponds to hills and sharp valleys. These soils have frequently abundant thick fractions as gravels and stones and the dominant silt is montmorillonite. (3) Rolling hills (Rol) with natural grassland at Palo a Pique (Treinta y Tres), representative of the highest levels in the Eastern landscape known as “Lomadas del Este”. The soil presents alternating higher and depressed areas formed by two profiles: superficial phase with horizon A thickness of 10 - 30 cm and deep phase where horizon A thickness is of 30 - 90 cm. (4) Medium granitic soil (Gra) with natural grassland at La Carolina (Flores), representative of the Central granitic region. This area has shallow soils associated with deep and more fertile soils developed from slime-loamy silts covering the granitic basement.

Table 1. Chemical characteristic of the soil modal profiles (Source: Ministerio de Agricultura y Pesca, 1979)

Location Soil Unit Predominant Horizon pH (water) CEC pH 7* V(%) pH 7 Soil cm 1:2.5 La Solod Lowl A , 0-17 5.5 13.0 52 Charqueda Melanic 1 Queguay Litosol Bas Chico Eutric A1, 0-12 5.9 42.6 79.1 Melanic José Pedro Argisol Rol Várela Subeutric A1, 0-21 5.5 18.5 55 Luvico La Brunosol Gra A , 0-26 6.1 27.1 80.8 Carolina Eutric 1 CEC pH 7 = cationic exchange capacity at pH 7 V(%) pH 7 = (total base / CEC pH 7)100

The soil samples were taken with a soil-corer of 8 cm diameter and 15 cm depth, maintaining intact the soil structure and the natural sward. The soil-cores were placed in plastic bags for watering and sown July 15th 2003 with approximately 20 viable seeds per soil-core. The Genotype-environment interaction in Lotus uliginosus 123 experiment was repeated in two contrasting environments in order to study the temperature effect on the variables under study during the establishment period. The experiments were located in greenhouses: one at 18-25ºC and another at 8-20ºC.

Vigour and initial development were monitored weekly. In September 2003, 10 weeks after sowing, a destructive evaluation of soil-cores was carried out by water immersion. The fresh aerial and root mass were separately weighed. Nodules size, quantity and localization were evaluated by visual estimation with an adapted scale of 10 values Master Class (2000; Table 2).

Tabla 2. Nodulation scale

Scale Description Scale Description 1 Without effective nodules 6 Few effective nodules in main root and many nodules in secondary roots 2 Very few effective nodules in 7 Few effective nodules only in main root secondary roots 3 Few effective nodules in secondary 8 Crowns with few effective nodules roots 4 Many effective nodules in secondary 9 Crowns half-covered with effective roots nodules 5 Few effective nodules in main root 10 Crowns completely covered with and few nodulesI in secondary roots effective nodules

Results and discussion

The temperature had a great influence on big trefoil establishment, with greater growth and larger differences between treatments in the warm environment (18-25°C; Table 3 and 4). There was a triple interaction between genotypes, soils and inoculation at both temperatures on the aerial seedling growth (Table 3), indicating the differential symbiotic response of the genotypes in different soils. Maku responded to the inoculation with U-526 in Lowl in both temperatures (Figure 1a) and in Rol at 18-25°C (Figure 2a), while there was no effect of inoculation in Gra and Bas (Figures 3a and 4a) under both temperatures and in Rol at 8-20°C. In opposition, LE 627 only responded to the inoculation in Bas at 18-25°C (Figure 4b), although seedling aerial weight was smaller than the best growth obtained in Rol with or without inoculation (Figure 2b). Best seedling growth for both genotypes was always achieved in Rol, possibly indicating a favourable soil for L. uliginosus establishment and/or the presence of effective strains (Figure 5c). There was no response to rhizobia inoculation in Gra, probably as an indication of naturalized strains from L. subbiflorus history hindering the impact of inoculation.

Aerial mass data demonstrated the large influence of soil native or naturalized rhizobia populations in the seedlings development of both genotypes. In addition, they could indicate certain strain-host specificity, since only Maku responded to inoculation in Lowl, while LE 627 had a low response to inoculation in Bas not reaching the values obtained in Rol. These 124 Sebastián Hernández, Mónica Rebuffo, Sebastián Arrivillaga, Martín Jaurena, Carlos Labandera, Diego Risso, Mónica Rebuffo and Javier Ciliuti. results corroborated the good performance of the strain U-526 for Maku and the need for further strain selection under field conditions for the diploid LE 627, since genotype-strain interaction for diploid and tetraploid L. uliginosus has already been reported (Barrientos et al., 2001).

Table 3. Fresh weight of aerial part (g/soil-core) for the different temperatures, genotypes, soils and inoculation treatments.

Temperature 18-25ºC 8-20ºC L. uliginosus genotypes LE 627 Maku LE 627 Maku Soil Inoculation Basaltic (Bas) NI 0.54 1.17 0.47 0.79 WI 1.61 1.32 0.30 0.47 Granitic (Gran) NI 1.96 1.94 1.00 0.89 WI 1.57 2.26 0.99 0.52 Rolling Hills (Rol) NI 2.51 2.47 1.88 1.77 WI 2.74 3.27 1.42 1.44 Lowlands (Lowl) NI 1.34 1.09 1.00 0.52 WI 1.74 3.35 1.07 2.59 LSD (5%) 0.78 0.71 Significance 0.008 0.024

Table 4. Root fresh weigh (g/soil-core) and nodulation scale for the different soils and inoculation treatments.

Root weight Nodutation Scale Temperatures 18-25ºC 8-20ºC 18-25ºC 8-20ºC Soils Inoculation Basaltic (Bas) NI 0.25 0.18 3.29 3.38 WI 0.34 0.15 8.21 6.10 Granitic (Gra) NI 0.56 0.31 7.44 6.59 WI 0.47 0.39 7.70 6.69 Rolling Hills (Rol) NI 0.60 0.73 7.93 6.81 WI 0.71 0.53 8.47 7.14 Lowlands (Lowl) NI 0.34 0.24 5.56 5.65 WI 0.62 0.39 9.06 8.72 LSD (5%) 0.15 0.15 1.17 1.50 Significance 0.015 0.01 <0.001 0.008

Genotype-environment interaction in Lotus uliginosus 125

Figure 1. Lowland soil-cores (Lowl) grown at 18-25°C sown with Maku (a) and LE 627 (b). WI inoculated with U-526 and NI without inoculation.

Figure 2. Rolling hills soil-cores (Rol) grown at 18-25°C sown with Maku (a) and LE 627 (b). WI inoculated with U-526 and NI without inoculation. 126 Sebastián Hernández, Mónica Rebuffo, Sebastián Arrivillaga, Martín Jaurena, Carlos Labandera, Diego Risso, Mónica Rebuffo and Javier Ciliuti.

Figure 3. Granitic soil-cores (Gra) grown at 18-25°C sown with Maku (a) and LE 627 (b). WI inoculated with U-526 and NI without inoculation.

Figure 4. Basaltic soil-cores (Bas) grown at 18-25°C sown with Maku (a) and LE 627 (b). WI inoculated with U-526 and NI without inoculation.

Genotype-environment interaction in Lotus uliginosus 127

Soil types had a great effect on big trefoil establishment (Figure 5). The soil main effect demonstrated that both genotypes had better performance at Rol than at Bas, in agreement with field observations (Risso, D. Com. Pers; Iglesias and Ramos, 2003; Carámbula et al., 1996; Castaño and Menéndez, 1998), while the growth was intermediate in Lowl and Gra soils. Soil incidence in L. uliginosus growth was also observed in the root development (Table 4). The root growth was lower in Bas at 18-25°C in comparison with Rol and Gra, while there were only significant differences between Bas and Rol at 8-20°C. This data confirmed previous field experiment results (Iglesias and Ramos, 2003; Castaño and Menéndez, 1998), and discarded temperature as factor of the differential performance between big trefoil genotypes between regions.

Figure 5. Inoculated soil cores grown at 18-25°C: (a) Eastern lowlands with rice stubble (Lowl); (b) basaltic soil with natural grassland (Bas); (3) rolling hills with natural grassland (Rol); (d) medium granitic soil with natural grassland (Gra).

Rhizobia inoculation had a strong interaction with soils on root development at both temperatures, since WI only had bigger roots in Lowl (Table 4). On the other hand, nodulation scale values in both Lowl and Bas were significantly higher in WI than in NI. Inoculation response for Bas was only recorded in the nodulation scale, since there was no significant (P<0.05) aerial or root growth improvement (Tables 3 and 4). These results could indicate the presence of other growth restrictions associated to the soils of the basaltic 128 Sebastián Hernández, Mónica Rebuffo, Sebastián Arrivillaga, Martín Jaurena, Carlos Labandera, Diego Risso, Mónica Rebuffo and Javier Ciliuti. region.

The high response to inoculation in Bas and Lowl could be explained by the absence or low concentration of effective rhizobia strains in these soils, confirming the impact of inoculation for these soils in the establishment and initial growth of L. uliginosus (Vance et al, 1987). In spite of field observations showing difficulties to achieve good and persistent stands of LE 627 in basaltic soils (D.Risso, Com.Pers.; Iglesias and Ramos, 2003), its seedling weight was similar (P<0.05) to Maku in this experiment, providing evidence for restrictions on establishment for both genotypes

The evaluation of soil-cores with natural grasslands allowed a first approach to the study of the complex of L. uliginosus establishment and its response to inoculation in different soils of Uruguay, by discarding the climatic differences among areas through the standardization of the environment. This research study has shown an interaction between host genotypes and U-526 inoculation related to the presence / absence of effective rhizobia strains in different soils. Similarly, difficulties of big trefoil establishment in basaltic soils are probably associated to soil physical characteristics. The understanding of factors involved in these genotype-soil interactions requires more precise research of longer duration, in order to recognize the soil characteristics that restrict establishment and to know the relationships between host and rhizobia genotypes, as well as native populations of rhizobia. Finally, it would be necessary to carry out more research and put greater effort in strain selection of specific rhizobia for the diploid genotype LE 627.

Acknowlegments

Thanks to our colleagues Raul Bermúdez, Robin Cuadro and Rafael Reyno for the collection of soil samples. Thanks are also extended to the permanent staff of the department. We would particularly like to thanks Dinorah Rey, Digno Mirabal and Omar Barolin for the maintenance experiment, and José Rivoir for the evaluation of the experiment.

References

ALLEN O.N. 1962. The inoculation of legumes. In HUGHES H.D., HEATH M.E. and METCALFE D.S. (eds). Forages: the science of grassland agriculture. 2. ed. Ames, Iowa State University Press. p. 119-126.

BARAIBAR A., FRIONI L., GUEDES M. E and LJUNGGREN H. 1999. Symbiotic effectiveness and ecological characterization of indigenous Rhizobium loti population in Uruguay. Pesquisa Agropecuaria Brasilera, 34, 1001-1017.

BARRIENTOS L., HIGUERA M., ACUÑA H., GUERRERO J., ORTEGA F. and SEGUEL I. 2002. Efectividad simbiótica de cepas naturalizadas de Mesorhizobium loti y Bradyrhizobium sp. (Lotus) en plantas de tres especies del género Lotus. [Symbiotic effectiveness of indigenous strains of Mesorhizobium loti and Bradyrhizobium sp. (Lotus) in plants of three Lotus plant species.] Agricultura Técnica (Chile), 62, Genotype-environment interaction in Lotus uliginosus 129

226-236. [In Spanish]

BROCKWELL J., HEBB D.M. and KELMAN W.M. 1994. Symbiotaxonomy of Lotus species and symbiotically related plants and of their root-nodule bacteria. The First International Lotus symposium, 22-24 March 1994 Missouri USA.

CARÁMBULA M., AYALA W. and CARRIQUIRRY E. 1994. Lotus pedunculatus. Adelanto sobre una forrajera que promete. [Lotus pedunculatus. Advances about a forage species that promise]. INIA Treinta y Tres, Uruguay. Serie Técnica, 45, 13 p. [In Spanish]

CARÁMBULA M., BERMUDEZ R. and CARRIQUIRRY E. 1996. Características relevantes de Lotus Maku. [Relevant characteristics of Lotus Maku.] In Producción animal. Unidad experimental Palo a Pique. INIA Treinta y Tres, Uruguay. Serie de Actividad y Difusión, 110, 7-16 p. [In Spanish]

CASTAÑO J. and MENÉNDEZ F. 1998. Caracterización vegetativa y producción de semillas de Lotus. [Vegetative characterization and seed production of Lotus]. Tesis Ing. Agr. Montevideo, Uruguay, Facultad de Agronomía. 67p. [In Spanish]

CHARLTON J.F.L. 1983. Lotus and other legumes. In WRATT G.S. and SMITH H.C. (eds). Plant Breeding in New Zealand. Butterworths of New Zealand, pp.253-262.

GWYNNE D.C. and BECKETT R.E. 1980. The response of Lotus uliginosus L. grown on hill soils to inoculation with Rhizobium. Grass and Forage, 35, 213-217.

IGLESIAS M.P. and RAMOS N. 2003. Efecto de los taninos condensados y la carga sobre la producción y calidad de carne y lana de corderos pesados Corriedale en cuatro especies de leguminosas (Lotus corniculatus, Lotus pedunculatus, Lotus subbiflorus y Trifolium repens). [The effect of condensed tannins and the stocking rates on the meet and wool production and quality of Corriedale heavy lambs in four species of legumes (Lotus corniculatus, Lotus pedunculatus, Lotus subbiflorus and Trifolium repens)]. Tesis de grado, Montevideo, Facultad de Agronomía, Uruguay. 213 p. [In Spanish]

INASE, 2005. Resultados experimentales de evaluación de especies forrajeras para el registro nacional de cultivares: Anuales, bianuales y perennes. Periodo 2004. [Experimental results of evaluation of forage species for the nacional list os cultivars: Annuals, biannuals and perennials. Period 2004]. INIA La Estanzuela, Uruguay. 73 p. [In Spanish]

IRISARRI P., MILNITSKY F., MONZA J. and BEDMAR E.J. 1996. Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils. Plant and Soil, 180, 39-47.

LABANDERA C., JAURENA M., BIASSINI G. and DE MAIO V. 2005. Inoculación de Lotus Makú. [Inoculation of Lotus Maku]. Revista Plan Agropecuario Nº 113, Montevideo, 130 Sebastián Hernández, Mónica Rebuffo, Sebastián Arrivillaga, Martín Jaurena, Carlos Labandera, Diego Risso, Mónica Rebuffo and Javier Ciliuti.

Uruguay, 44-46. [In Spanish]

LIEVEN-ANTONIOU C.A. and WHITTAM T.S. 1997. Specificity in the symbiotic association of Lotus corniculatus and Rhizobium loti from natural populations. Molecular Ecology, 6, 629-639.

MAYANS M.C. 2003. Técnicas utilizadas en la identificación y caracterización de cepas de Rhizobium sp. y Bradyrhizobium sp. [Methods used in the identification and characterization of strains of Rhizobium sp. and Bradyrhizobium sp.] Tesis de grado, Facultad de Ciencias, Uruguay. [In Spanish]

PANKHRUST C.E. 1981. Effect of plant nutrient supply on nodule effectiveness and Rhizobium strain competition for nodulation of Lotus pedunculatus. Plant and Soil, 60, 325-339.

PÉREZ E. and LABANDERA C. 1998. Especificidad simbiótica dentro del genero Lotus. [Symbiotic specificity in the genus Lotus]. [In Spanish] http://fp.chasque.apc.org:8081/microlab/LMSCI/TraTe/espsim.htm

RENGEL Z. 2002. Breeding for better symbiosis. Plant and Soil, 245, 147–162

RISSO D.F. and BERRETA E.J. 1996. Mejoramiento de campos en suelos sobre Cristalino. [Natural grasslands improvement in Granytic soils] In RISSO D.F., BERRETTA E.J. and MORÓN A. (Eds.) Producción y manejo de pasturas. INIA. Montevideo (Uruguay). Serie Técnica, 80, 193-211. [In Spanish]

RISSO D.F., COLL J. and ZARZA A. 1990. Evaluación de leguminosas para mejoramientos extensivos en suelos sobre Cristalino (I). [Legume evaluation for extensive pasture improvement in cristaline soils (I)]. In: INIA, SUP, facultad de Agronomía, CHPA. (Ed.) II Seminario Nacional de Campo Natural. Hemisferio Sur, Uruguay, pp. 231-241. [In Spanish]

SCOTT D.B., WILSON R., SHAW G.J., PETIT A. and TEMPE J. 1987. Biosynthesis and degradation of nodule-specific Rhizobium loti compounds in Lotus nodules. Journal of Bacteriology, 169, 278–282.

SKERMAN P.J., CAMERON D.G. and RIVEROS F. 1991. Relaciones entre los rizobios y las leguminosas. [Relationships between the rhizobia and the legumes.] Roma, FAO. P. 125-141. (Colección FAO Producción y Protección Vegetal No. 2). [In Spanish]

VANCE P.C., REIBACH P.H. and PANKHRUST C.E. 1987. Symbiotic properties of Lotus Pedunculatus root nodules induced by Rhizobium loti and Bradyrhizobium spp. Phisiologia Plantarum, 69, 3, 435-442.

Lotus Newsletter (2005) Volume 35 (1), 131-133.

The Australian Research Council’s Centre of Excellence for Integrative Legume Research

PETER M. GRESSHOFF* ARC Centre of Excellence for Integrative Legume Research, The University of Queensland, St. Lucia, Brisbane, QLD 4072, Australia http://www.cilr.edu.au/ *Corresponding author

The ARC Centre of Excellence for Integrative Legume Research (CILR; www.cilr.uq.edu.au) is a partnership that brings together leading plant research scientists located at the University of Queensland, the Australian National University, the University of Melbourne and the University of Newcastle. The director of the Centre is Professor Peter Gresshoff (who is also Professor of Botany in the School of Integrative Biology at UQ). The Centre aims to drive further development of the genomics and phenomics of legumes, providing the critical mass of human, intellectual and infrastructure resources to function as a world-class research centre.

The Centre was established with an A$10 million Australian Research Council grant over five years starting in 2003. Cash contributions from partner universities and state governments matching the ARC funding, combined with in-kind contributions of staff and facilities has generated a major A$29 million five-year biological science research effort.

Research in the Centre provides critical insights into mechanisms of meristem and organ differentiation and intercellular communication, utilizing comparative genomics on the internationally recognized model legumes Lotus japonicus (Lj) and Medicago truncatula (Mt; see Figure 1). Studies also focus on two major crop legumes – namely garden pea and soybean. New knowledge of plant growth processes through mechanistic analysis of organ induction provides the tools to optimise productivity, quality, and environmental adaptation of legumes. This in turn will have direct impact on agricultural sustainability, environmental quality and potential value-added products for human health.

Major research in the CILR relating to Lotus japonicus deals with the isolation of ethylene and ABA insensitive mutants and transgenics and their interaction with autoregulation control, single seed SNP analysis, generation of multiple mutants, isolation of putative autoregulation of nodulation related genes such as kinase associated protein phosphate (KAPP), the use of Lotus as a transgene testing organism for promoter fusion constructs, promoter-trapping to produce marker lines, RNAi testing of candidate gene function, and analysis of fast neutron deletion mutants. Representative papers from the Centre referring to Lotus japonicus are listed below. The Centre has available about 3,000 EMS M2 families

131 132 PeterM. Gresshoff with large seed supply and welcomes interactions with other scientists for possible selection trials. Additionally promoter-less GUS insertion lines are available for screening (limited number). This is most easily achieved through visitation and selection within the Centre. Please contact the author for availability of genetic material.

The CILR Integrative Project Research program description:

Program 1 Program 2 Program 3 Program 4 Program 5 Program 6

Objective: Objective: Objective: Objective: Objective: Objective: To define the genetic To understand the To discover novel To analyse the To develop genomic To integrate roadmap of components of meristem peptide, RNA structure and and post- genetic and embryo induction and signalling and other signalling function of root genomic tools for chemical development molecules and nodule the control of processes in meristem . meristems flower and microspore control of development plant development

Identification of Identification Control of Identification of genes new Development of Use of of new development of involved in the receptors markers for ‘Systems Biology’ long distance legume microspore molecular and root and nodule to signals and identity of meristems signals meristem integrate meristem floral initiation development controls

Area 1 Area 4 Area 7 Area 11 Area 14 Area 17 Cell biology/ Nodulation / MicroRNA Gene discovery/ Expression Bioinformatics genetics Branching Gene interaction profiling Area 8 Peptide/ Area 2 Area 5 Area 12 Area 15 Area 18 proteins Somatic vs. Xylem and phloem Mutagenesis Microspore Computational sexual embryos signals And TILLING formation Biology Area 9 Biomedical WP1.1 Area 3 Area 6 Area 13 Area 16 Area 19 Classical Area 10 Meristem Developmental Epigenetic Flavone/ Expression signalling transition Modelling changes stress signals Profiles

Embryogenesis Communication Peptide Root and Floral Gene and between and Nodule Meristems Networks: Meristem Meristems Signalling Meristems and Integration Formation Molecules Microspores

Figure 1. Research programs of the CILR. Overlaps between programs generate the integrative, Systems Biology approach.

The Centre also conducts an education and outreach program to bring science findings to students, teachers and the general public. “INFO sheets” and electronic newsletters help provide text material, supported by provision of biological material for class room exercises. The CILR also developed a Natural Science-Social Science Linkage program dealing with a diversity of “legume” issues such as the acceptance of GMO technology, aboriginal use of legumes and nutritional effects of legume enriched diet.

The Centre’s research initiatives have significant intellectual property commercialisation potential, and this will augment Australia’s international standing in scientific discovery and directly benefit the Australian economy. Integrative Legume Research in Australia 133

References

BEVERIDGE C.A., GRESSHOFF P.M., RAMEAU C. and TURNBULL C.G.N. 2003. Many more signals needed to orchestrate development. Journal of Plant Growth Regulators, 22, 15-24.

BUZAS D.M., LOHAR D., SATO S., NAKAMURA Y., TABATA S., VICKERS C.E., STILLER J. and GRESSHOFF P.M. 2005. Promoter trapping in Lotus japonicus reveals novel root and nodule gene expression domains. Plant Cell Physiology (in press).

GRESSHOFF P.M. 2003. Post-genomic insights into nodulation. Genome Biology, 4, 201.

GRESSHOFF P.M. 2005. Positional cloning of plant developmental genes. In MEKSEM K. and KAHL G. (Eds.) Handbook of Genomic Mapping . Wiley-VCH. pp 233-256.

JIANG Q. and GRESSHOFF P.M. 2002. Shoot-control and genetic mapping of the har1-1 (hypernodulation and aberrant root formation) mutant of Lotus japonicus. Functional Plant Biology, 29, 1371-1376.

LOHAR D., SCHULLER K.A., BUZAS D., GRESSHOFF P.M. and STILLER J. 2001. Efficient transformation of Lotus japonicus using selection with the herbicide BASTA. Journal of Experimental Botany, 43, 857-863.

MATAMOROS M., CLEMENTE M.R., SATO S., ASAMIZU E., TABATA S., RAMOS J., MORAN J.F., STILLER J., GRESSHOFF P.M. and BECANA M. 2003. Molecular analysis of the pathway for the synthesis of thiol tripeptides in the model legume Lotus japonicus. Molecular Plant Microbe Interaction, 16, 1039-1046.

MEN A.E., MEKSEM K., KASSEM M. A., LOHAR D., STILLER J., LIGHTFOOT D. and GRESSHOFF P.M. 2001. A Bacterial Artificial Chromosome (BAC) library of Lotus japonicus constructed in an A. tumefaciens - transformable vector. Molecular Plant-Microbe Interaction, 14, 422-425.

SEARLE I.R., MEN A.M., LANIYA T.S., BUZAS D.M., ITURBE-ORMAETXE I., CARROLL B.J. and GRESSHOFF P.M. 2003. Long distance signalling for nodulation requires a CLAVATA1-like receptor kinase. Science, 299, 108-112.

Lotus Newsletter (2005) Volume 35 (1), 134-135.

The LOTASSA proposal: the success of enthusiasm and tenacity.

JUAN SANJUÁN1 and MÓNICA REBUFFO2 1 Estación Experimental del Zaidín, CSIC. Granada, Spain. 2 Instituto Nacional de Investigación Agropecuaria, INIA La Estanzuela, Colonia, Uruguay. *Corresponding author

LOTASSA (LOTus Adaptation and Sustainability in South-America) is the acronym of a project entitled “Bridging Genomics and Agrosystem Management: Resources for Adaptation and Sustainable Production of forage Lotus species in Environmentally Constrained South-American Soils”, which has been recently selected for funding by the EU International Cooperation (INCO) Program. The global objective of LOTASSA is to develop superior biological and genetic resources to 1) assist and speed up selection of Lotus genotypes more tolerant to abiotic stresses and 2) to improve productivity, sustainability and quality of Lotus pastures in environmentally constrained areas of South America. For this, LOTASSA will exploit the close genetic relatedness between the model L. japonicus and cultivated Lotus spp. In this respect, LOTASSA represents a pioneer project where basic and applied plant research are combined for a common goal.

The project is participated by 16 groups belonging to 14 different institutions from 8 countries: Argentina (Oscar Ruiz, IIB-INTECh; Roberto Racca, IFFIVE-INTA), Brazil (Miguel Dall’Agnol and Enilson Saccol de Sá, both from UFRGS), Chile (Hernán Acuña, INIA), Uruguay (Jorge Monza, UDELAR; Carlos Labandera, MGAP; Mónica Rebuffo, INIA), Regional Organization (Emilio Ruz, PROCISUR), Denmark (Jens Stougaard, UAAR), Germany (Michael Udvardi, MPI-GOLM; Martin Parniske, LMU), Slovakia (Igor Mistrik, BU-SAV) and Spain (Antonio Marquez, USEV; Manuel Becana and Juan Sanjuán, both from CSIC), which will share the almost 2 million Euro budget granted by the EU Commission to this project. All partners congratulate for this success that will allow an important international effort directed towards the understanding of Lotus spp. responses to abiotic stresses and the development of resources to improve their adaptation to environmentally-constrained soils in South America. All partners are really excited and expectant about the opportunities that LOTASSA offers to them. Once the proposal has been granted and before the official launching of the project, expected by the end of 2005, it is time to recollect memories that may help to understand that this final success was preceded by lots of difficulties that were overcome by the tenacity and enthusiasm of all people involved.

In fact, the roots of LOTASSA can be tracked back a few years ago, in July 2000, when O. Ruiz and J. Sanjuán, together with M. Udvardi and C. Labandera initiated the preparation of a modest proposal with the acronym LOTAUS (LOTus Adaptation and sustainability in Argentinian and Uruguayan Soils), which could not be finally submitted due to bureaucracy obstacles. The idea of a proposal was kept frozen until 2003, when the thematic priorities of a new INCO call would provide the possibility for a new submission. Between 2000 and

134 The LOTASSA INCO-DEV proposal 135

2003, workshops organized by O. Ruiz in Chascomús (Argentina) on the ecophysiology of Lotus and its symbionts were extremely important in keeping our interest for the topic and especially to facilitate that some of the future LOTASSA partners met for the first time (Ruiz, 2004). It was by the middle of 2003 when additional partners were invited to participate, including J. Stougaard, M. Dall’Agnol, E. Saccol de Sá, H. Acuña, M. Rebuffo, etc., which obviously implied much more complexity for the future proposal. At the same time, another consortium interested in a rather similar topic was building up, headed by A. Márquez (Sevilla, Spain). It was a lucky coincidence that some researchers had been invited to participate in both consortia. Aware of this, O. Ruiz, J. Sanjuán and A. Márquez met in Sevilla, under the heat of July, to evaluate the overlap between the two consortia, concluding that it would be very easy to combine both consortia into a single proposal that would be stronger than the individual ones and would thus avoid unnecessary competition for funding. It was also agreed that J. Sanjuán, who had been the coordinator of the initial LOTAUS, would be the coordinator of the joint consortia and proposal that would end up as LOTASSA. Unfortunately, the proposal, with 13 participants at that time, could not be submitted that year, due to some loose sections that still required further work and time, and to personal problems of the coordinator in the dates previous to the deadline, early September 2003. However, the preparation of the proposal had reached a very advanced state by then and it was decided to submit it to the next INCO call in 2004. At this point, the enthusiastic effort of a partner, M. Rebuffo must be recognized. Not only she was an invaluable support to the coordinator during the proposal preparation in 2003 (and beyond), she also convinced PROCISUR to provide funds to organize a meeting in INIA-La Estanzuela (Colonia, Uruguay) among most South American LOTASSA partners and the project coordinator in March 2004. This meeting was particularly important to evaluate the weaknesses of the 2003 draft, including the needs for additional partners, in order to strengthen the proposal. It was then that PROCISUR, represented by E. Ruz, decided to join the consortium, something that was of great importance to the final success of the proposal. Other partners that joined the consortium after this meeting were M. Becana (Spain) and R. Racca (Argentina).

With all this background, the stage was set up for a strong proposal that was at last submitted in September 2004 and was indeed selected after a tough evaluation, as only 20 proposals were selected for funding among a total of 138 submissions. Lots of bureaucratic steps have been solved after then, and yet a few more formularies will need to be compiled before actual starting of the project this year 2005. We are confident that once we put hands on the bench, the enthusiasm of the partners will make of this international experience a great advance in the genetics, physiology and microbiology of Lotus, towards more productive and sustainable pastures to feed up animal livestocks in the Southern Cone.

On behalf of all partners, we invite Lotus researchers to keep updated on the progress of this project, through the Lotus Newsletter and the LOTASSA web page that will be accessible after project launching.

References RUIZ O.A. 2004. Interdisciplinary workshop on genetic, molecular and ecophysiological aspects of Lotus spp. and their symbionts. Lotus Newsletter, 34, 60-65. Lotus Newsletter (2005) Volume 35 (1), 136-137.

Breeding birdsfoot trefoil for Mediterranean-type environments in southern Australia

DANIEL REAL1*, GRAEME A. SANDRAL1,2, JONATHAN WARDEN1, MÓNICA REBUFFO3, DIEGO F. RISSO4, JOHN F. AYRES5, WALTER M. KELMAN6 and STEVE J. HUGHES7 1Cooperative Research Centre for Plant-Based Management of Dryland Salinity, The University of Western Australia, University Field Station, 1 Underwood Avenue, Shenton Park, WA 6009, Australia Email:[email protected]. 2NSW Department of Primary Industries, Wagga Wagga Agricultural Institute, PMB, Pine Gully Road, Wagga Wagga, NSW 2605, Australia 3National Institute of Agricultural Research, INIA La Estanzuela, Ruta 50 km 11, Colonia, Uruguay. 4National Institute of Agricultural Research, INIA Tacuarembó, Ruta 5 km 386,CP 45000, Tacuarembó, Uruguay. 5Department of Primary Industries, Centre for Perennial Grazing Systems, Glen Innes, NSW 2370, Australia. 6CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601, Australia 7SARDI Genetic Resource Centre, LG02, Main Waite Building, Waite Campus, Adelaide, SA 5001, Australia. * Corresponding author

Australia has a very short agricultural history which commenced with European settlement in 1788. During settlement native perennial vegetation was cleared from large parts of the landscape and replaced with annual crops and annual pastures, such as Triticum aestivum L. (wheat – annual crop) and Trifolium subterraneum L. (subclover – annual pasture). These annual species only use water significantly during the rainy season (autumn – spring), and moreover do not extract significant amounts of water below 1.2 m. In contrast, the native vegetation they have replaced use water all year round and to a much greater depth. The result is that this annual farming system has caused an imbalance in the water cycle and an excess of water leaches through the soil profile into groundwater aquifers. During the leaching process, salts in the soil are dissolved and in many regions the water table becomes saline and salt is deposited in the lower parts of the landscape causing the visual scalding of the land that is associated with salinity. In 2005, 18% of the cleared agricultural land in Western Australia was declared saline. This is predicted to increase to 33% by 2050 unless major changes to the current farming systems occur.

The problem of salinity can be dealt with in a number of ways and one of them is a plant-based solution using perennial species that mimic the water usage of the original native systems. Any new plant-based farming system needs to be profitable and the best example of

136 Lotus corniculatus breeding in southern Australia 137 a plant for this Mediterranean-type system is Medicago sativa L. (lucerne). However, lucerne has several disadvantages: (a) it is not tolerant of acidic soils, (b) it is not waterlogging tolerant, (c) it is susceptible to Redlegged Earth Mite (RLEM: Halotydeus destructor), (d) it predisposes bloat in cattle, and (e) it drops its leaves during dry conditions experienced over summer, which restricts its contribution to the summer/autumn feed-gap. southern Australian (south of latitude 28oS) therefore, requires new plants that have the water usage and drought tolerance capabilities of lucerne but have improved adaptation to its weaknesses identified above.

The search for perennial pasture legumes that can survive in Mediterranean environments in southern Australia and thus have the ability to cope with dry seasons, acidic soils, waterlogging or salinity is a challenge for plant breeders working for the CRC for Plant-based Management of Dryland Salinity (Salinity CRC) based in Perth, Western Australia. To address this problem, the funding body, Australian Wool Innovation has joined the Salinity CRC to fund a Lotus breeding project which will invest over a 5 year period (2003-2008) to develop new Lotus cultivars adapted to these Australian conditions.

One of the species being examined is Lotus corniculatus L. (birdsfoot trefoil) as it is more tolerant of acid soils and waterlogging than lucerne, hence, it will be developed for places in which lucerne is not well adapted. However, birdsfoot trefoil is not as drought tolerant as lucerne and with 5 to 7 months of dry conditions each summer, drought-tolerance is critical and hence this attribute will be targeted in a new breeding program, being managed by Dr. D. Real. As part of this program, Australian researchers by way of formal agreement will have access to breeding material selected for persistence of birdsfoot trefoil from Uruguay’s well-established breeding program conducted by Mrs. M. Rebuffo since 1988 at the National Institute of Agricultural Research (INIA). Birdsfoot trefoil is a long-day species requiring a minimum daylength of 14.0 to 14.5 hours for flowering. Uruguay and Mediterranean Australia are located at similar latitudes; therefore the well adapted germplasm from Uruguay will flower profusely in Mediterranean Australia. This germplasm will be examined along with breeding lines developed by Dr. J. Ayres and Dr. W. Kelman for low latitude permanent pasture applications in Eastern Australia. In addition, the project leader Mr G. Sandral has selected lines which were characterised at the Genetic Resource Centre in south Australia with the assistance of the Curator Mr S. Hughes. The advanced status of these breeding lines from these sources provides an opportunity for this new Uruguay-Australia collaborative breeding program to reduce the normally extended breeding cycle required to produce new cultivars.

Lotus Newsletter (2005) Volume 35 (1), 138-142.

Lotus activities: Background and present research

Aïssa Abdelguerfi Nora Altier Seishiro Aoki standardized tests to characterize Manuel Becana germplasm and to assist in the Edgar Cárdenas Rocha identification of disease resistance in Francesca Cardinale plants. Projects involve interdisciplinary Federico Condón approaches to understanding the ecology of Cristina Cvitanich forage legume microbes and disease Mariana Melchiorre epidemiology as influenced by cultural Martin Parniske practices. Current research also explores Andy Pollard the biological control of Pythium seedling Mark Robbins diseases using native fluorescent Simone Scheffer-Basso Pseudomonas. Norio Suganuma Nancy Terryn Seishiro Aoki JAPAN

Aïssa Abdelguerfi I studied the horizontal gene transfer from ALGERIA Agrobacterium to Nicotiana species in their evolution. I am interested in the evolution I am working on Fabaceae (forage and and phylogeny of legume and rhizobia. I pastoral plants) since 1976; the aspects am collecting Lotus species in Japan and all study are: Ecology, Agronomy, Variability, over the world and isolate symbiotic Taxonomy, Biology, Physiology (salinity, bacteria from their nodules. drought…)

Manuel Becana Nora Altier SPAIN URUGUAY The research of our group is focused on My research program is focused on free radicals, antioxidants and oxidative developing management strategies for stress in legumes, and particulary in minimizing the impact of diseases on nodules. For our studies we use the model forage legume production and persistence. legume Lotus japonicus, both under I work in close collaboration with the nodulating (nitrogen-fixing) and non- breeding programs in the development of nodulating (nitrate-fed) conditions. We use new varieties with improved disease a multidisciplinary approach, by combining resistance. My research also facilitates the physiology and biochemistry with cell and development of new techniques and molecular biology. Currently, we have

138 Lotus activities. 139 concentrated our efforts in three types of Francesca Cardinale antioxidant enzymes (glutathione ITALY synthetases, glutathione peroxidases and superoxide dismutases) and are My background is that of a plant biologist investigating the role(s) of those enzymes who got interested in plant-microbe in the protection of nitrogen fixation as interactions during her studies. After well as their involvement in the plant's working briefly on VA mycorrhizas, during response to several types of abiotic stress my PhD I switched to plant-pathogen (salinity, heavy metals, oxidative stress). interactions focussing at first on the To this end, we are identifying the whole recognition events. During the post-doc set of isoforms, studying their expression years, I worked on the role of new (mRNA, protein, activity) and localization oxylipins in plant defense and on the signal (tissue, cell, organelle), and producing transduction events following biotic and recombinant proteins for detailed abiotic stress perception. Since my arrival biochemical characterization. in Turin in the year 2001 as a staff researcher in Plant Physiopathology, I started working on post-transcriptional Edgar Cárdenas Rocha regulation of ethylene biosynthesis and on COLOMBIA cross-protection mechanisms between different stresses, besides joining the I have been working from 1985 in the existing group on ongoing plant pathology tropical forages research, with emphasis in projects. I’ve been working on Medicago adaptive evaluation of grass, legumes and sativa, Arabidopsis, tobacco and tomato so other forages species germplasm, in the far. I got interested in Lotus japonicus tropical low lands in the Centro because it’s becoming a model plant but Internacional de Agricultura Tropical it’s not so exploited yet as far as molecular (CIAT). Last 7 years I am dedicated to pathology. teaching and I continue with the investigation but in cold climate forages, in the Colombian high altitude Andean Federico Condón region, with the purpose to obtain URUGUAY multipurpose species (cover crops, forages, wood and others), and to make the bovine Phd candidate at the University of production in high altitudes (between 2000 Minnesota, Agronomy and Plant Genetics to 3400 m, 18 to 8 °C). I have interest to Department. Special interest in allelic evaluate subtropical plant species that have diversity, population structure and genetic well develop in our altitudes and latitudes, resources utilization. with the objetive to obtain alternative forages to kikuyo and ryegrass, which are the only base of the feeding in our milk Cristina Cvitanich region. DENMARK

Project Title: Isolation and characterization of Lotus japonicus genes involved in iron and zinc homeostasis. Researchers: Cristina Cvitanich, Winnie Jensen, and 140 Lotus activities.

Erik Østergaard Jensen. The goal of this Martin Parniske project is to find ways to improve the GERMANY nutritional value of legumes by identifying genes and proteins important for iron and My background is the molecular and zinc regulation in the model legume Lotus genetic analysis of plant microbe japonicus. Legumes are important staples interactions. The focus of my laboratory is in the developing world and are a major the genetics of plant root symbiosis and we source of nutrients in many areas. are using Lotus japonicus as genetic model Legumes are frequently grown in soil with organism. We have developed large limited nutrient availability. Plants use populations of EMS mutagenised seed and finely tuned mechanisms to keep have isolated several hundred mutants appropriated levels of iron and zinc in each affected in root symbiosis. We also have of their organs. Several genes involved in established a TILLING reverse genetics iron and zinc homeostasis have been tool for Lotus japonicus, which is described in yeast, and a few orthologs accessible for other laboratories through have been studied in plants. We have used collaboration. these sequences to search for L. japonicus ESTs and genomic loci that are likely to be involved in iron and zinc metabolism. We Andy Pollard have identified sequences corresponding to FALKLAND ISLANDS three ferritins, two putative ferric reductases, seven metal transport proteins Three years out of university, studied at of the ZIP family, and six cation Seale-Hayne Agricultural College in transporters of the NRAMP family. The Devon UK. I have worked in the area of expression patterns of these genes are agronomy for the last 2 years. Research at being studied. Novel plant genes regulated present in regards to Lotus (particularly by iron or zinc availability in the soil will Maku cultivar) has been focused on also be identified and analyzed in details. establishing, only now are we able to study production and quality of the plant. Falkland Island soils are of a low pH and Mariana Melchiorre lime at present is not an economic option. ARGENTINA Lotus uliginosus seems to establish with less difficulty in FI conditions than many At the present, the group is transforming L other legume/grasses. Recent set up of an japonicus in order to overexpress the EU approved abattoir offers it potential in enzymes Mn-Superoxide dismutase and the production of lamb for export. Growth Glutathione reductase. Both enzymes are rates need to be determined for the various involved in the antioxidant system in FI breeds. plants. Our goal is improve the plant response under salt stress and correlate this behavior with the control of oxygen active Mark Robbins species generation. UNITED KINGDOM

Work in the laboratory focuses on polyphenolic metabolism in Lotus spp. Proanthocyanidins (syn. condensed tannins) protect plant proteins from Lotus activities. 141 breakdown when grazed by ruminant nitrogen fixation in nodules. However, animals. Additionally, condensed tannins little is known about the host plant genes appear to have a protein protectant effect that are involved in the establishment of during the ensilage of tanniferous forages. symbiotic nitrogen fixation. Plant Fix- Our current studies include: (1) Animal mutants that form ineffective nodules are feeding experiments using Lotus spp. with useful for studying the regulatory varying CT contents. (2) Analysis of CT mechanism of symbiotic nitrogen fixation pathway regulation by the expression of by host plant genes. Numerous Fix- transgenes in clonal genotypes of Lotus mutants have been isolated from major corniculatus. (3) Molecular analyses of leguminous crops. However, they have a genes that regulate the tissue-specific large genome and are difficult to be accumulation of CT-containing cells in transformed. In contrast, Lotus japonicus Lotus japonicus and Lotus corniculatus. has several characteristics suited to the We have ongoing projects on genes of the model for legume research, and thus its basic-helix-loop-helix class and also on genetic information has been accumulating. R2R3MYB transcription factors (in This allows us to study host plant genes collaboration with John Innes Centre, responsible for symbiosis by map-based Norwich). cloning or gene manipulation. We focus on L. japonicus Fix- mutants. Mutants are generated by chemical mutagenesis and are Simone Scheffer-Basso characterized by phenotypes and gene BRAZIL expression profiles. Furthermore, each

defective gene is cloned by map-based I have worked with morphophysiology and cloning. Our goal is to reveal how host evaluation of forage production of Lotus plants control rhizobial symbiotic nitrogen corniculatus (cutting/grazing tolerance, fixation in nodules. growth habit , nutritive value, competition, mixtures with grasses). Nancy Terryn BELGIUM Norio Suganuma JAPAN Together with my colleagues at the Institute for Plant Biotechnology we have a Biological nitrogen fixation is an important research unit on leguminous plants. Our process that provides nitrogen to all living main focus crops are Phaseolus, cowpea matter on the earth. Rhizobia form root and Lathyrus. Our goal is to exploit nodules on legumes. The bacteria are modern biotechnology for the identification endocytosed into nodule cells, and then and use of novel genes to broaden the have the ability to fix atmospheric nitrogen. genetic base of these crops. This includes Nitrogen fixed by rhizobia is supplied to the development of genetic transformation host plants for their growth and further is protocols, and the introduction of useful utilized for the growth of other organisms. (foreign) genes to address key problems In most cases, rhizobia alone do not fix such as nutritional aspects and insect nitrogen in the soil. They are able to fix tolerance. We have developed and nitrogen only by building a symbiotic improved a P. acutifolius agrobacterium relationship with legumes, indicating that based transformation protocol (De clercq et host plant genes are required for rhizobial 142 Lotus activities. al., 2002; Zambre et al., 2005). With this protocol P. acutifolius or the tepary bean can now routinely been transformed. As P. acutifolius can be hybridised (through embryo-rescue to P. vulgaris) this is an indirect way to genetically improve the common bean. We are also continuing to improve the regeneration and transformation protocols for Phaseolus species in general and particularly P.vulgaris, as well as for cowpea and Lathyrus. Our studies have focused on the seed storage proteins known as arcelins. These are very abundant seed storage proteins found in some wild P. vulgaris genotypes. Seeds of A. thaliana and P. acutifolius plants transformed with arcelin- 5 gene constructs, synthesise arcelin-5 to levels of 15 and 25% of the total protein content, respectively. This high expression level of arcelin-5 is being exploited in two projects, one aiming at improving insect resistance, another aimed at expressing arcelin-5 genes modified to contain extra methionine codons. DE CLERCQ J., ZAMBRE M., VAN MONTAGU M., DILLEN W. and ANGENON G. 2002. An optimized Agrobacterium- mediated transformation procedure for Phaseolus acutifolius A. Gray. Plant Cell Reporter, 21, 333-340. ZAMBRE M., CARDONA C., VAN MONTAGU M., TERRYN N. and ANGENON G. 2005. A reproducible genetic trans- formation system for cultivated Phaseolus acutifolius (tepary bean) and its use to assess the role of arcelins in resistance to the Mexican bean weevil. Theoretical and Applied Genetics, 110, 914-924.

Lotus Newsletter (2005) Volume 35 (1), 143 – 173.

Current list of Lotus researchers

Database last updated Aug 30 2005

Aïssa Abdelguerfi [email protected] Doctor Phone: 56-42-211177 Lab. RGB, INA Fax No.: 56-42-217852 Department of Phytotechnie Lotus corniculatus, Lotus glaber, Lotus El Harrach 16200 uliginosus. Breeding, utilization, germplasm, Alger seed, reclamation, physiology, forage. Algeria Germination, emergence, establishment and Phone: +213 21 29 40 36 vegetative growth of Lotus sp. in clay soils. Fax No.: +213 21 52 58 18 entry last revised Nov 7 2003 [email protected] http://www.ina.dz Shinji Akada Lotus corniculatus, Lotus creticus, Lotus Associate Professor palustris, Lotus uligionosus. Genetics, Hirosaki University Breeding, Germplasm, Taxonomy, Ecology, Gene Research Center Biology, Physiology, Molecular Biology, 3 Bunkyo-cho Microbiology, Microbiology. entry last revised Aug 9 2005 Hirosaki 036-8561 Japan [email protected] Juan Ramón Acebes Ginovés http://nature.cc.hirosaki-u.ac.jp/gene/ Professor Phone: +81-172-3892 Universidad De La Laguna Fax No.: +81-172-3894 Biología Vegetal (Botánica) Lotus corniculatus, Lotus japonicus. Genetics, 38071 La Laguna- Tenerife Breeding, Physiology, Tissue Culture, Islas Canarias Molecular Biology Spain entry last revised Aug 26 2004 [email protected]

Phone: +34-922-318606 Kenneth A. Albrecht Fax No.: +34-922-318447 Professor Lotus sect. Pedrosia. Taxonomy, Ecology, Univ. of Wisconsin-Madison Molecular Biology. Department of Agronomy entry last revised Jun 2 2004 1575 Linden Dr. Madison WI 53706 Hernan Acuña U.S.A. Director [email protected] Centro Regional de Investigación Quilamapu, Phone: +1-608-262-2314 INIA Fax No.: +1-608-262-5217 Casilla 426 Lotus corniculatus, Lotus uliginosus. Ecology; Chillan forage production; utilization. Chile entry last revised Oct 23 2003

143

144 Lotus researchers.

Said Amrani Adriana M. Alippi Project Manager Research Scientist, Comisión de Laboratorie de Biologie du Sol Investigaciones Científicas de la Prov. de Bs. Faculté des Sciences Biologiques As. (CIC) USTHB - BP 32 El Alia - Bab Ezzouar CIDEFI – Unidad de Bacteriología 16111 – Alger Calle 60 y 118 s/n cc 31 Algeria La Plata, Provincia Buenos Aires [email protected] Argentina Phone: +213-21-24-79-50 to 64 ext 936 Fax No.: 54 221 4252346 Fax No.: +213-21-24-72-17 [email protected] All species present in Algeria (Lotus Lotus corniculatus, Lotus glaber, Lotus conimbricensis, Lotus creticus, Lotus uliginosus, Lotus japonicus. Microbiology. roudairei, Lotus jolyi, Lotus glinoides, Lotus Phytobacteriology – Diagnostic and palustris) Microbiology, symbiosis, biological characterization of Bacterial Plant Pathogens. Nitrogen fixation. Taxonomy, seed, utilization. entry last revised Apr 12 2005 entry last revised Mar 5 2004

Nora Altier Seishiro Aoki Researcher Assistant Professor INIA, Nacional Institute for Agricultural University of Tokyo Research Graduate School of Arts and Sciences Department of Plant Pathology Department of Life Sciences INIA Las Brujas 3-8-1 Komaba Ruta 48 km.10 Meguro-ku Canelones CP 90200 Tokyo, 153-8902 Uruguay Japan [email protected] [email protected] http://www.inia.org.uy Phone: +81-3-5454-6638 Phone: +598-2-3677641 Fax No.: +81-3-5454-6638 Fax No.: +598-2-3677609 Lotus japonicus. Evolution of legume-rhizobia Lotus corniculatus, Lotus subbiflorus, Lotus interaction uliginosus. Pathology. entry last revised Apr 21 2005 entry last revised Nov 7 2003

Toshio Aoki Rosario Alzugaray Assistant Professor Researcher Nihom University INIA Uruguay College of Bioresource Sciences Plant Protection Department of Applied Biological Sciences CC 39173 Colonia Kameino 1866 Uruguay Fujisawa [email protected] Kanagawa 252-8510 http://www.inia.org.uy Japan Phone: +598-574-8000 ext 1464 [email protected] Fax No.: +598-574-8012 Phone: +81-466-843703 Lotus corniculatus. Entomology. Fax No.: +81-466-843353 entry last revised Oct 29 2003 Lotus japonicus. Genetics, Physiology, Tissue Culture, Molecular Biology entry last revised Oct 13 2003

Lotus researchers. 145

Ana Arambarri Profesora Walter Ayala Facultad de Ciencias Agrarias y Forestales Researcher Universidad Nacional de La Plata INIA Uruguay Calles 60 y 118 - C.C. 31 Forage Department C. P. 1900 La Plata Casilla de Correo 42 Prov. Buenos Aires CP 33000 Treinta y Tres Argentina Uruguay [email protected] [email protected] [email protected] Phone: +598-452-2023 [email protected] Fax No.: +598-452-5701 Phone: +54-221-423-6618 Lotus corniculatus, Lotus uliginosus, Lotus Fax No.: +54-221-425-2346 glaber, Lotus angustissimus. Ecology, Lotus species, Acmispon, Hosackia and Physiology, Forage Production, Utilization, Syrmatium. Formerly researcher on plant Seed Production. entry last revised Mar 25 2004 taxonomy, seeds. Old and New World Lotus species, epidermal characteristics. Taxonomy of the New World species, known as: John F. Ayres Acmispon, Hosackia and Syrmatium. Presently Principal Research Scientist & Research researcher on medicinal species. Leader entry last revised Feb 14 2005 Department of Primary Industries 'Centre for Perennial Grazing Systems' Alberto Artola Agricultural Research & Advisory Station Breeder NSW Agriculture IPB Semillas PMB Glen Innes Crop Department New South Wales 2370 Enrique Hurtado Nº 11 Australia Colonia del Sacramento. CP 70.000 [email protected] Uruguay Phone: +61-2-67301900 [email protected] Fax No.: +61-2-67301999 Phone: +598-52-23742 Lotus uliginosus, Lotus corniculatus. Lotus corniculatus. Seed Production, Crop Breeding, Ecology, Forage Production, Establishment. Development of seed vigor tests Utilization. entry last revised Aug 12 2005 and seed enhancement methods. entry last revised Nov 24 2003

Andreas Bachmair Ariel Asuaga Group leader Ingeniero Agrónomo Max Planck Institute for Plant Breeding Lancasteriana 2284 Research Montevideo Plant Developmental Biology Uruguay Carl-von-Linné-Weg 10 [email protected] D-50829 Cologne Phone: +598-2-6007821 Germany Fax No.: +598-5325200 [email protected] Lotus corniculatus, Lotus glaber, Lotus http://www.mpiz-koeln.mpg.de/ uliginosus, Lotus subbiflorus. Seed production Phone: +49-221-5062265/266 and trade. Fax No.: +49-221-5062207 entry last revised May 5 2004

146 Lotus researchers.

Lotus japonicus (currently no research activity Lotus corniculatus, Lotus glaber. Remediation with Lotus). Genome organization, of trace element-laden soils with plants. retrotransposons. entry last revised Oct 13 2003 entry last revised Oct 30 2003

Søren Bak Mónica Susana Barufaldi Associate Professor Ayudante graduado The Royal Veterinary and Agricultural Universidad Nacional del Centro de la University, Department Plant Biology Provincia de Buenos Aires (UNCPBA) DK-1871Thorvaldsensvej 40 Facultad de Agronomía Frederiksberg C, Copenhagen Ciencias Básicas Agronómicas y Biológicas Denmark Av. República Italia 780, C.C. 47 [email protected] (7300) Azul, Provincia de Buenos Aires http://www.biobase.dk/P450 Argentina http://plbio.kvl.dk http://www.place.kvl.dk [email protected] Phone: +45-35283346 http://www.faa.unicen.edu.ar/ Fax No.: +45-38283333 Phone: +54-2281-433291/92/93 Lotus japonicus. Metabolite pathways. Fax No.: +54-2281-43329/92/93 Metabolite profiling, transcriptome. Lotus glaber, Lotus corniculatus. Genetics, entry last revised Oct 16 2003 breeding, forage production, seed production.

Generate, verify and evaluate autotetraploid

(via colchicine) populations of L.glaber. Georgget Banchero entry last revised Oct 31 2003 Senior researcher INIA Department of Animal Production Manuel Becana INIA La Estanzuela Professor Ruta 50 Km 12 Estacion Experimental de Aula Dei, CSIC 70000 Colonia Department Plant Nutrition Uruguay Avda Montañana 1005, Apdo 202 [email protected] 50080 Zaragoza Phone: ++598 574 8000 Spain Fax No.: ++ 598 574 8012 [email protected] Lotus uliginosus. Forage production and Phone: +34-976-716055 utilization for strategic nutrition to improve Fax No.: +34-976-716145 sheep reproduction (increase of ovulation Lotus japonicus. Biochemistry, Molecular rate/improve on lactogenesis). Biology. Antioxidants –Abiotic stress (drought, entry last revised Mar 25 2004 salinity, heavy metals), Free radicals, Thiol metabolism, Nodule senescence, Oxidative

stress. Gary S. Bañuelos entry last revised Oct 16 2003 USDA-ARS Water Management Research Laboratory 9611 S. Riverbend Ave. David P. Belesky Parlier CA 93648 Research Agronomist/Research Leader U.S.A. USDA-ARS [email protected] Appalachian Farming Systems Research Phone: +1-559-596-2880 Center Fax No.: +1-559-596-2851 1224 Airport Road Beaver

Lotus researchers. 147

West Virginia 25813-9423 Paul R. Beuselinck U.S.A. USDA-ARS Plant Genetics Research Unit [email protected] University of Missouri http://www.arserrc.gov/Beckley/ 207 Waters Hall Phone: +1-304-256-2841 Columbia, MO 65211 Fax No.: +1-304-256-2921 U.S.A. Lotus corniculatus. Forage production, [email protected] ecology. Phone: +1-314-268-3114 entry last revised Aug 23 2004 Fax No.: +1-314-882-1467 Lotus corniculatus, Lotus glaber, Lotus

uliginosus, Lotus japonicus. Formerly Lotus María Bemhaja spp. genetics; breeding; germplasm. Current Forage Researcher research emphasis is on soybean seed INIA Uruguay composition and seed physiology. Former Forage Department, Animal Production research was on Lotus breeding and selection R. 5 Km 386 for improved persistence and evaluation of Tacuarembó 45000 exotic germplasm. Uruguay entry last revised Nov 7 2003 [email protected] http://www.inia.org.uy Phone: +598-632-4560 Arvid A. Boe Fax No.: +598-632-3969 Professor of Plant Science Lotus corniculatus, Lotus uliginosus. Plant Science Department Physiology, Forage and seed production, South Dakota State University Utilization. NPB 244A, Box 2140C entry last revised Nov 5 2003 Brookings, SD 57007 U.S.A. [email protected] David John Bertioli Phone: +1-605-688-4759 Lecturer/Researcher Fax No.: +1-605-688-4452 Universidade Catolica de Brasilia Lotus corniculatus, Lotus purshianus. Departamento de Biotecnologia e Ciencias Genetics; breeding; seed production; Genomicas entomology. EMBRAPA-CENARGEN entry last revised Nov 6 2003 Parque Estação Biológica-pqEB Final Av. W5 Norte Brasília-DF Omar Borsani CEP: 70770-900 Assistant Professor Brazil Facultad de Agronomía [email protected] Departamento de Biología Vegetal, Bioquímica http://www.bioinformatica.ucb.br/arachisne Av. Garzón 780, Montevideo t/Arachis_Net.htm Uruguay Phone: +55-61-4484735 [email protected] Lotus japonicus. Genome synteny of peanut http://www.fagro.edu.uy/bioquimica with Lotus. Phone: +598-2-3540229 entry last revised Jan 2 2004 Fax No.: ++ 598-2-3543004 Lotus corniculatus, Lotus uliginosus, Lotus japonicus. Biology, Physiology, Tissue Culture, Molecular Biology. entry last revised Nov 18 2003

148 Lotus researchers.

Postbus 9505 Nick Brewin 2300 RA Leiden Project Leader The Netherlands John Innes Centre [email protected] Department of Symbiosis Research Phone: +31-71-5275068 Norwich Research Park Fax No.: +31-71-5275088 Colney Lotus japonicus, Lotus pressli. Physiology, Norwich NR4 7UH Microbiology, Molecular Biology (currently no United Kingdom research activity with Lotus). Autoregulation of [email protected] nodulation of Vetch (Vicia) and Rhizobium Phone: +44-1603-450273 leguminosarum. Fax No. : +44-1603-450045 entry last revised Oct 30 2003 Lotus japonicus. Symbiosis. entry last revised Aug 10 2004 Gustavo Caetano-Anolles Associate Professor of Bioinformatics E. Charlie Brummer University of Illinois Associate Professor Department of Crop Sciences Iowa State University 332 NSRC, 1101 West Peabody Drive 1204 Agronomy Hall Urbana, IL 61801 Ames Iowa 50011 U.S.A. U.S.A. [email protected] [email protected] http://www.cropsci.uiuc.edu/faculty/gca/ http://www.public.iastate.edu/~brummer Phone: +1-217-333-8172 Phone: +1-515-294-1415 Fax No.: +1-217-333-8046 Fax No. : +1-515-294-6505 entry last revised Mar 23 2004

Lotus corniculatus. Genetics; breeding; forage; utilization; ecology. entry last revised Oct 23 2003 Edgar Alberto Cárdenas Rocha Associate Professor Universidad Nacional de Colombia Joe Brummer Department of Sciences for Animal Production Colorado State University Fac. Medicina Veterinaria y de Zootecnia Western Colorado Research Center C ra. 30 No. 45 – 08 P.O. Box 598 Bogotá D.C. Gunnison, CO 81230 Colombia U.S.A. Phone: +57-1-3165148 [email protected] Fax No.: +57-1-3165401 Phone: +1-970-641-2515 [email protected] Fax No.: +1-970-641-0653 http://www.unal.edu.co Lotus corniculatus, Lotus wrightii. Genetics, All Lotus spp. for agronomic and animal breeding; forage, utilization, ecology. evaluation. Germplasm, breeding, forage Improvement of forage production and quality production, seed production in mountain meadows. entry last revised Aug 16 2005 entry last revised Mar 11 2004

Francesca Cardinale Anton van Brussel Staff Scientist Assistant professor University of Turin Leiden University Di.Va.P.R.A. – Plant Pathology Institute of Molecular Plant Sciences via Leonardo da Vinci

Lotus researchers. 149

44 Grugliasco (TO)10095 Italy [email protected] Miguel Cauhépé http://www.divapra.unito.it/personale/dbas Private Researcher and Consultant on Range e/visualizza_singolo.php?ing=yes&singolo and Pasture Management =Cardinale%20Francesca Cereijo 979 Phone: ++39-011-670 8701 or 8875 7620 Balcarce (Prov. de Buenos Aires) Fax No.: ++39-011-236 8875 Argentina Lotus japonicus. Physiology, Pathology, [email protected] Molecular Biology Phone: +54-266-430668 entry last revised June 23 2004 Fax No.: +54-266-430908 Lotus glaber. Ecology, Forage Production, Utilization Bernie Carroll entry last revised Jul 31 2005 Biochemistry and Molecular Biology The University of Queensland Brisbane, 4072 Maurizio Chiurazzi Queensland Institute of Genetics and Biophysics “A. Australia Buzzati Traverso” [email protected] Via Marconi 12 Phone: +61 7 3365 2131 80125 Naples Fax No.: +61 7 3365 4699 Italy entry last revised Mar 15 2004 [email protected] http://www.iigb.na.cnr.it/ Phone: +39-081-7257256 Fabricio Dario Cassán Fax No.: +39-081-5936123 Posdoctoral, CONICET Lotus japonicus. Laboratorio de Fisiología Vegetal-UNRC entry last revised Oct 13 2003 Laboratorio de Biotecnología 1-IIB-INTECh.

Universidad Nacional de Rio Cuarto Daniel H. Cogliatti Campus Universitario, Ruta 36, km 601 Professor of Plant Physiology (5800) Rio Cuarto Facultad de Agronomía, Universidad Nacional Argentina del Centro de la Provincia de Buenos Aires [email protected] Departamento de Ciencias Básicas Phone: +54-358-4676103 Agronómicas y Biológicas Fax No.: +54-358-4676230 Av. República Italia 780 C.C. 47, (7300) Azul Lotus glaber. Microbiology. entry last revised Nov 12 2003 Provincia de Buenos Aires. Argentina [email protected] Ana María Castro http://www.faa.unicen.edu.ar Genetics Proffesor Phone: +54-2281-433292 Faculty of Agricultural Sciences Fax No.: +54-2281-433292 Department of Biological Sciences Lotus glaber. Genetics, Physiology, Mineral CC31, 1900-La Plata nutrition. Argentina entry last revised Nov 17 2003 Phone: ++54-221-423-6758, ext. 421

Fax No: ++54-221-423-3698 Federico Condon [email protected] Researcher Lotus glaber. Genetics, Breeding, Physiology, INIA Uruguay Molecular Biology entry last revised Aug 4 2004 Department of Genetic Resources

150 Lotus researchers.

INIA La Estanzuela Cristina Cvitanich CC 39173, 70000 Colonia University of Aarhus Uruguay Science Park [email protected] Department of Molecular Biology Phone: +598-574-8000 Gustav Wieds Vej 10 C Fax No.: +598-574-8012 DK-8000 Aarhus C Lotus corniculatus, Lotus glaber, Lotus Denmark uliginosus, Lotus japonicus. Genetic Diversity, [email protected] Genetics. Allelic diversity, population structure Fax.no.:+45 86 12 31 78 and genetic resources utilization. Lotus japonicus. Plant molecular genetics and entry last revised Apr 12 2004 general molecular biology.

entry last revised Apr 7 2005

Quentin Cronk Miguel Dall'Agnol Professor of Plant Science and Director Faculdade de Agronomia University of British Columbia Univ. Federal of Rio Grande do Sul UBC Botanical Garden and Centre for Plant Caixa Postal 776 Research 91501-970 Porto Alegre-RS 6804 SW Marine Drive Brazil Vancouver B.C., V6T 1Z4 [email protected] Canada Phone: +51-3316-7405 [email protected] Fax No.: +51-3316-6045 http://www.ubcbotanicalgarden.org Lotus corniculatus, Lotus uliginosus. Plant Fax No.: +1-604-822-2016 Breeding and Genetics. Lotus japonicus, Lotus bertholletii, Lotus entry last revised Oct 8 2003 uliginosus, etc (including N. American Lotus). Gene phylogenies, evolution of florally expressed genes. Francesco Damiani entry last revised Oct 23 2003 Researcher Consiglio Nazionale delle Ricerche Istituto Genetica Vegetale sez Perugia Hilda Nélida Crosta via della Madonna Alta 130 Profesor Adjunto 06128 Perugia Facultad de Agronomía Italy Universidad Nacional del Centro de la [email protected] Provincia de Buenos Aires (UNCPBA). http://www.irmgpf.pg.cnr.it Ciencias Básicas Agronómicas y Biológicas. Phone: +39-075-5014862 Av. República Italia 780, C.C. 47. Fax No.: +39-075-5014869 (7300) Azul, Provincia de Buenos Aires. Lotus corniculatus, Lotus glaber, Lotus Argentina uliginosus, Lotus japonicus. Genetics, [email protected] Breeding, Molecular Biology, Physiology, http://www.faa.unicen.edu.ar Tissue Culture. Phone: +54-2281-433291/92/93 entry last revised Oct 10 2003 Fax No.: +54-2281-433291/92/93 Lotus glaber, Lotus corniculatus. Biology, Taxonomy, Genetics, Tissue Culture, seed David K. Davis production, Molecular Biology. Superintendent entry last revised Nov 25 2003 University of Missouri Agricultural Experiment Station Forage Systems Research Center and Thompson Farm

Lotus researchers. 151

21262 Genoa Rd Pedro Díaz Gadea Linneus, MO 64653 Assistant Professor U.S.A. Facultad de Agronomía [email protected] Departamento de Biología Vegetal, Bioquímica http://www.aes.missouri.edu/fsrc Av. Garzón 780, Montevideo http://www.aes.missouri.edu/thompson Uruguay Phone: +1-660-895-5121 [email protected] Fax No.: +1-660-895-5122 http://www.fagro.edu.uy/bioquimica Lotus corniculatus. Physiology, forage, seed. Phone: +598-2-3540229 Commercial production and sale of Lotus. Fax No.: +598-2-3543004 entry last revised Nov 30 2003 Lotus corniculatus, Lotus glaber, Lotus

uliginosus, Lotus japonicus, Lotus filicaulis.

Biology, Physiology, Tissue Culture, José Pedro De Battista Molecular Biology. Forage researcher entry last revised Nov 18 2003 INTA EEA Concepción del Uruguay Area de Investigación en Producción Animal C.C. Nº 6, Concepción del Uruguay Allan Downie (3260) Entre Ríos Professor Argentina John Innes Centre [email protected] Colney Lane http://www.inta.gov.ar Norwich NR4 7UH Phone: +54-3442-425561 UK Fax No.: +54-3442-425578 [email protected] http://www.jic.ac.uk/staff/allan-downie/ Lotus corniculatus, Lotus glaber, Lotus Phone: +44-1603-450207 subbiflorus. Breeding, Forage production Fax No.: +44-1603-450045 and utilization. Lotus japonicus. Genetics, Molecular Biology, entry last revised May 12 2005 Microbiology, Nodulation signalling. entry last revised Oct 21 2003

Guilhem Desbrosses Jeffrey S. Dukes Lecturer Assistant Professor University of Montpellier II Department of Biology UMR113 / Laboratoire des Symbioses University of Massachusetts Tropicales et Méditerranéennes / Réponse des 100 Morrissey Blvd. plantes aux micro-organismes Boston, MA 02125 UMR113 / CC002 U.S.A. Place Eugène Bataillon [email protected] F-34095 http://globalecology.stanford.edu/DGE/Duk Montpellier Cedex 05 es/Dukes.html France Phone: +1-617-287-6614 [email protected] General interest in Lotus. Plant ecology, Phone: +33-4-67149353 community ecology, ecosystem ecology. Fax No.: +33-4-67143637 entry last revised Aug 24 2004 Lotus japonicus. Genetics, Molecular Biology, Microbiology. entry last revised Oct 28 2003

152 Lotus researchers.

Nancy J. Ehlke Av. República Italia 780, C.C. 47 Department of Agronomy & Plant Genetics (7300) Azul, Provincia de Buenos Aires University of Minnesota Argentina 1991 Buford Circle [email protected] St. Paul MN 55108 http://www.faa.unicen.edu.ar U.S.A. Phone: +54-2281-433291 to 93 [email protected] Fax No.: +54-2281-433291 to 93 Phone: +1-612-625-1791 Lotus glaber, Lotus corniculatus. Biology, Fax No.: +1-612-625-1268 Genetics, Tissue Culture, seed. Lotus corniculatus. Genetics, breeding. entry last revised Nov 26 2003 entry last revised Nov. 30 2003

Osvaldo Néstor Fernández James T. English Associate Professor University of Missouri Universidad Nacional de Mar del Plata 116 Waters Facultad de Ciencias Agrarias – Group Columbia, MO 65211 Agroecology U.S.A. C.C. 276 [email protected] (CP7620) Balcarce Phone: +1-573-882-1472 Argentina Fax No.: +1-573-882-0588 [email protected] Pathology. Soilborne pathogen Phytophthora. Phone: +54-2266-439100/05 No current research with Lotus. Fax No.: +54-2266-439105 entry last revised Oct 14 2003 Lotus glaber. Population ecology and forage production. entry last revised Nov 10 2003 Mike Ewing Associate Professor, Deputy CEO University of Western Australia Emmanouil Flemetakis Cooperative Research Centre for Plant–based Lecturer Management of Dryland Salinity Agricultural University of Athens 35 Stirling Hwy Department of Agricultural Biotechnology Crawley Iera Odos 75 Western Australia 6009 118 55 Botanikos Australia Athens [email protected] Greece http://www.crcsalinity.com Phone: +30-210-5294347 Phone: +61-9-64881876 [email protected] Fax No.: +61-9-648811400 Lotus japonicus, Lotus corniculatus. Lotus corniculatus. Diversity in the Genus. Biochemistry, Molecular Biology, Symbiotic Ecology, Germplasm, Breeding, Rhizobiology, Nitrogen fixation, Carbon Metabolism. Seed Production. entry last revised Sep 2 2004 entry last revised Jan 13 2004

Henk Franssen Marcelo Francisco Eseiza Assistant Professor Jefe de Trabajos Prácticos, Botánica Agrícola I Wageningen University Facultad de Agronomía, Universidad Nacional Molecular Biology del Centro de la Provincia de Buenos Aires Dreijenlaan 3 (UNCPBA) 6703HA Wageningen Ciencias Básicas Agronómicas y Biológicas The Netherlands

Lotus researchers. 153 [email protected] Phone: +31-317-483264 Fax No.: +31-317-483264 William F. Grant Lotus japonicus. Genetics, Molecular Biology, Emeritus Professor developmental program of nodule formation, McGill University nodule evolution. Plant Science entry last revised Oct 24 2003 MacDonald Campus, McGill University P.O. Box 4000 Ste. Anne de Bellevue Gabino Garcia de Los Santos 21,111 Lakeshore Rd. Head of Seed Production Department Quebec H9X 3V9 Instituto de Recursos Genéticos y Canada Productividad [email protected] Colegio de Postgraduados. http://www.mcgill.ca/plant/faculty/grant/ Carretera Mexico-Texcoco Km. 36.5 Web page for publications: Texcoco http://eqb- EDO. de Mexico 56230 dqe.cciw.ca/eman/ecotools/botanists/Grant Mexico WF.html [email protected] Phone: +1-514-3987863 Phone: 595 952 02 62 Fax No.: +1-514-398-7897 Fax No.: 34-85-381511 Lotus corniculatus, Lotus glaber, Lotus spp. Lotus corniculatus. Physiology, ecology, Genetics; taxonomy; germplasm. Genetics of breeding, taxonomy. Germplasm management, Lotus, especially those involved with the adaptation, germplasm screening for forage evolution of Lotus corniculatus. and seed production and breeding. entry last revised Sep 13 2004 entry last revised Nov 21 2003

Alicia Grassano Kirsten Gausing Prof. Titular, Química Analítica. IMSB-University of Aarhus Universidad Nacional de La Pampa C.F. Møllers Alle Facultad de Ciencias Exactas y Naturales Building 130 Departamento de Química DK-8000 Aarhus C Uruguay 151 Denmark (6300) Santa Rosa, La Pampa [email protected] Argentina Fax No.: +45 86 19 65 00 [email protected] Molecular biology. Mainly barley. Phone: +54-2954-436787 entry last revised June 18 2004 Fax No.: +54-2954-432535 Lotus glaber. Microbiology, Ecology. entry last revised Oct 21 2003 José Manuel González García Ing.Agrónomo – Mejoramiento genético INTA- EEA Balcarce Stephanie Greene Departamento de Agronomia Geneticist/Curator C.C. 276- 7620 Balcarce, Bs. As. USDA-ARS Argentina National Temperate Forage Legume mggarcí[email protected] Germplasm Resources Unit Phone: +54 2266-439103 24106 North Bunn Road Fax No.: +54 2266-439101 Prosser, WA 99350 Lotus glaber. Breeding, Germplasm, Forage U.S.A. production, Utilization, seed production. [email protected] entry last revised Dec 2 2003

154 Lotus researchers. http://www.forage.prosser.wsu.edu Phone: +1-509-7869265 Ana María de Haro Fax No.: +1-509-7869370 Professor All Lotus spp. Germplasm curator for Lotus Facultad de Agronomía collection in USDA National Plant Germplasm Universidad Nacional del Centro de la System. Provincia de Buenos Aires entry last revised Nov 21 2003 Departamento de Producción Vegetal Av. República Italia 780

C.C. 47 Peter Gresshoff (7300) Azul Professor/Director Provincia de Buenos Aires The University of Queensland and ARC Centre Argentina of Integrative Legume Research [email protected] ARC Centre of Excellence for Integrative http://www.faa.unicen.edu.ar/ Legume Research Phone: +54-2281-433292 John Hines Plant Science Building Fax No.: +54-2281-433292 The University of Queensland St Lucia, Qld 4067 Lotus glaber. Entomology. entry last revised Apr 23 2004 Australia [email protected] http://www.legumecentre.cilr.uq.edu.au Nate Hartwig Phone: +61-7-33653550 Pennsylvania State University Fax No.: +61-7-33653559 Department of Agronomy Lotus japonicus. We conduct research into 116 ASI Bldg. Rhizobium-induced nodulation and utilize University Park, PA 16802 mutagenesis and transgenics to create the U.S.A. necessary diversity needed to determine [email protected] physiological and biochemical processes. Lotus corniculatus. Forage, utilization, Specifically we have used promoter trapping, management. Cover crops, living mulches in transfer of the Arabidopsis ethylene corn and soybean. insensitivity (via AtEtrl-l) and non-nodulation entry last revised July 20 2004 mutants. entry last revised Oct 13 2003 Makoto Hayashi Assistant Professor Mette Grønlund Osaka University Post.doc Department of Biotechnology, Graduate University of Aarhus School of Engineering Lab. of Gene Expression 2-1, Yamada-Oka Science Park Suita, Osaka 565-0871 Gustav Wieds Vej 10 C Japan DK-8000 Aarhus C [email protected] Denmark Phone: +81-6-6879-7417 [email protected] Fax No.: +81-6-6879-7418 Phone: +45-89425008 Lotus japonicus. Symbiosis, Genetics, Fax No.: +45-86123178 Physiology Lotus japonicus. Genetics, Biology, entry last revised July 1 2004 Physiology, Pathology, Tissue culture,

Molecular biology. entry last revised Oct 15 2003

Lotus researchers. 155

José A. Herrera-Cervera Assistant Research Professor Alberto A. Iglesias Facultad de Ciencias. University of Granada Profesor Asociado UNL Departamento de Fisiología Vegetal Investigador Principal CONICET Campus de FuenteNueva s/n. 18071 Grupo de Enzimología Molecular Granada Bioquímica Básica de Macromoléculas Spain Facultad de Bioquímica y Ciencias Biológicas [email protected] Universidad Nacional del Litoral Phone: +34-958243382 Paraje "El Pozo", CC 242 Fax No.: +34-958248995 S3000ZAA Santa Fe Lotus japonicus. Genetics, Physiology, Argentina Pathology, Tissue Culture, Molecular Biology, [email protected] Microbiology. entry last revised Nov 30 2003 entry last revised Oct 14 2003

Pedro Insausti Ann M. Hirsch Profesor Adjunto Professor Institute for Agricultural Plant Physiology and UCLA Ecology Molecular, Cell and Developmental Biology CONICET – Faculty of Agronomy (University 405 Hilgard Avenue, Los Angeles, CA 90095- of Buenos Aires – Argentina) 1606 Av. San Martín 4453 USA 1417 Buenos Aires [email protected] Argentina http://www.mcdb.ucla.edu/Research/Hirsc [email protected] h/ http://www.ifeva.edu.ar/ Phone: +1-310-206-8673 Phone: +54-11-4524-8070 to 71 Ext 8126 Fax No.: +1-310-206-5413 Fax No.: +54-11-4514-8730 Mostly Lotus corniculatus. Biology, Molecular Lotus glaber and Lotus corniculatus. Biology, Genetics Ecophysiology. entry last revised June 22 2004 entry last revised Mar. 17 2004

Steve Hughes Sachiko Isobe Curator Australian Temperate Pasture Genetic National Agricultural Research Center for Resource Centre Hokkaido Region South Australian Research & Development Forage Legume Breeding Lab. Institute Hitsujigaoka 1 University of Adelaide Waite Campus Toyohira GPO Box 397 Sapporo, 062-8555 Adelaide Japan South Australia 5001 [email protected] Australia http://cryo.naro.affrc.go.jp/sakumotu/mame [email protected] ka/etop.htm Phone: +61 8 8303 9408 Phone: +81-11-8579272 Fax No.: +61 8 8303 9607 Lotus japonicus. Breeding, Genetics. Annual and perennial Lotus species. Genetic entry last revised Oct 17 2003 Resources, Plant introduction, Germplasm,

Characterisation, Taxonomy entry last revised Dec 20 2004

156 Lotus researchers.

Euan James Research Scientist Sangho Jeong University of Dundee Postdoc School of Life Sciences University of California, San Diego (UCSD) Centre for High Resolution Imaging & Yanofsky Lab Processing(CHIPs) Section of Cell and Developmental Biology MSI/WTB Complex La Jolla, CA 92093-0116 Dundee DD1 5EH USA UK Phone: +1-858-534-7298 [email protected] Fax No.: +1-858-822-1772 Phone: +44-1382-344741 [email protected] Fax No.: +44-1382-345893 Lotus japonicus. Development, Genetics, Lotus corniculatus, Lotus uliginosus, Lotus Molecular Biology. japonicus. Physiology, structure (especially of entry last revised Jan 7 2004 nodules). entry last revised Oct 18 2003 Qunyi Jiang Laboratory manager Martín Jaurena The University of Queensland Research Assistance ARC Centre of Excellence for Integrative Ministry of Livestock Agriculture and Legume Research Fisheries John Hines Building Department of Soil Microbiology Brisbane Uruguay Qld 4072 Phone: ++598 2 2038152 Australia Fax No.: ++598 2 2038152 [email protected] [email protected] www.legumecentre.cilr.uq.edu.au http://fp.chasque.net:8081/microlab/LMSCI Phone: +61-7-33657227 /LMSCI.htm Fax No.: +61-7-33653556 Lotus corniculatus, Lotus glaber, Lotus Lotus japonicus. Genetics, genomics and uliginosus. Genetics, Germplasm, Taxonomy, molecular biology. entry last revised Nov 21 2003 Ecology, Microbiology. entry last revised Aug 2 2004

Bjarne Jochimsen Erik Østergaard Jensen Associate professor Associate Professor IMSB-Lab. of Gene Expression University of Aarhus University of Aarhus Dept. Molecular Biology. Lab. of Gene Department of Molecular Biology Expression C.F. Møllers Alle Science Park Gustav Wieds Vej 10C Gustav Wieds Vej 10 C DK-8000 Aarhus C DK-8000 Aarhus C Denmark Denmark [email protected] [email protected] Phone: +45-89-42-50-39 http://130.225.13.27/~eoj/ Fax.no.: +45-86-12-31-78 Phone: +45-89425014 Lotus japonicus. Seed development. Entry last revised Jan 7 2004 Lotus japonicus. Molecular biology, physiology, organ development. entry last revised Nov. 7 2003

Lotus researchers. 157

Bodil Jørgensen Iera Odos 75 Senior scientist 11855 Athens Danish Institute of Agricultural Science Greece Depart. Plant Biology, Biotechnology Group [email protected] Thorvaldsensvej 40, opg. 8, 2 sal Phone: +30-210-5294314 1871 Frederiksberg C Fax No.: +30-210-5294314 Denmark Lotus japonicus. Molecular Biology, [email protected] Biochemistry, Physiology. Phone: +45-35282578 entry last revised Oct 22 2003

Fax No.: +45-35282589

Lotus japonicus. Tissue culture, Tony Kavanagh transformation, Molecular biology, Professor, Head of Department biochemistry. entry last revised Nov 25 2003 Trinity College Dublin Smurfit Institute Department of Genetics Dublin 2 Adem Kamalak Ireland Assistant Professor [email protected] Kahramanmaras Sutcu Imam University Phone: +353-1-6081035 Faculty of Agriculture Fax No.: +353-1-6714968 Department of Animal Science Lotus japonicus. Genetics. Kahramanmaras, 46040 entry last revised Oct 14 2003 Turkey [email protected] Masayoshi Kawaguchi Phone: +903442237666 Associate Professor Fax: +903442230048 University of Tokyo Lotus corniculatus. Forage Production, Graduate School of Sciences Utilization. entry last revised Jul 28 2004 Department of Biological Sciences Hongo Bunkyo-ku Norihito Kanamori Tokyo 113-0033 National Food Research Institute Japan Food Engineering Division [email protected] Kannondai 2-1-12 Fax No.: +81-3-58414458 Tsukuba-city Lotus japonicus, Lotus burttii. Molecular basis Ibaraki 305-8642 of symbiosis and systemic regulation of Japan nodule development [email protected] entry last revised Oct 21 2003 Phone: +81-29-8388047

Fax No.: +81-29-8391552 Walter Kelman Lotus japonicus. Breeding and Molecular Research Scientist Biology. entry last revised Nov 5 2003 CSIRO Division of Plant Industry Institute of Plant Production and Processing GPO Box 1600 Panagiotis Katinakis Canberra ACT 2601 Professor Australia Agricultural University of Athens [email protected] Botanikos Phone: +61-2-62465083

158 Lotus researchers.

Fax No.: +61-2-62465255 Lotus uliginosus, Lotus corniculatus. Cultivar Tatiana E. Kramina breeding and genetic analysis of agronomic Scientific Researcher traits. Moscow State University entry last revised Nov 7 2003 Biological Faculty

Higher Plants Department

Vorobyevy Gory, 1, building 12, Peter Kemp 199992 Moscow Associate Professor Russia Institute of Natural Resources [email protected] Massey University Phone: +7-095-9391603 Private Bag 11 222 Fax No.: +7-095-9391827 Massey University Section Lotus, Lotus corniculatus group. Palmerston North Taxonomy. Natural hybridization. New Zealand entry last revised Oct 13 2003 [email protected] Lotus corniculatus. Ecology, forage production, grazing management, animal Lene Krusell responses. University of Aarhus entry last revised Aug 4 2004 Department of Molecular Biology

Lab. of Gene Expression

Science Park Kyung-Nam Kim Gustav Wieds Vej 10 C Assistant Professor DK-8000 Aarhus C Sejong University Denmark Department of Molecular Biology [email protected] 98 Gunja-Dong Phone: +45-89425008 Gwangjin-Gu Lotus japonicus. Seoul 143-747 entry last revised Aug 26 2005 Korea [email protected] Phone: +82-2-34083647 Alejandro La Manna Fax No.: +82-2-34083661 Researcher Arabidopsis thaliana. Lotus japonicus. Instituto Nacional de Investigacion Molecular Biology. Agropecuaria (INIA) entry last revised Nov 20 2003 Dairy Department

Ruta 50 km 11 CC 39173

70000 Colonia Joseph H. Kirkbride Uruguay USDA-Agricultural Research Service [email protected] Systematic Botany & Mycology Laboratory http://www.inia.org.uy/ Bldg 011A, Rm 304 Phone: +598-574-8000 BARC-West Fax No.: +598-574-8012 Beltsville, MD 20705-2350 Lotus corniculatus, Lotus uliginosus. Animal U.S.A. Nutrition, nutrient management and [email protected] environmental issues. Ecology, Biology, Phone: +1-310-5049447 Forage Production, Utilization, Seed Fax No.: +1-310-5045810 Production. All Lotus spp. Taxonomy. Systematics of Lotus entry last revised March 24 2004 spp. entry last revised Oct 9 2003

Lotus researchers. 159

Departamento de Microbiología (Interacción Pedro Laterra Rhizobium-leguminosas) Universidad Nacional de Mar del Plata Av. Gral Paz entre Albarellos y Constituyentes Facultad de Ciencias Agrarias INTI, Edif. 24 (Colectora Gral Paz 5445) CC 276, 7620 Balcarce (1650) San Martín Prov. Buenos Aires Buenos Aires Argentina Argentina [email protected] [email protected] http://www.mdp.edu.ar http://www.iib.unsam.edu.ar Phone: +54-2266-439100 Lotus glaber, Lotus japonicus. Microbiology. Fax No.: +54-2266-439101 entry last revised Nov 7 2003 Lotus glaber. Ecology. entry last revised Oct 29 2003 Lina A.C. Lett Assitant Professor W.H. (Bill) Leakey Facultad de Agronomía Deer Creek Seed Universidad Nacional del Centro de la Box 105 Provincia de Buenos Aires Ashland, WI 54806 Ciencias Básicas Agronómicas y Biológicas U.S.A. Av. República Italia 780 [email protected] C.C. 47, (7300) Azul Phone: +1-715-2783200 Provincia de Buenos Aires. Fax No.: +1-715-2783209 Argentina Lotus corniculatus, Lotus glaber, Lotus [email protected] uliginosus. Seed. Commercial production and http://www.faa.unicen.edu.ar sale of Lotus. Phone : +54-2281-433291 to 93 entry last revised Oct 28 2003 Fax No.: +54-2281-433292

Lotus glaber, Lotus corniculatus.

Microbiology, Molecular Biology, Forage Richard H. Leep production, Genetics. Michigan State University entry last revised Nov 10 2003 Crop and Soil Sciences W. K. Kellogg Biological Station 3700 E. Gull Lake Drive Dasharath Prasad Lohar Hickory Corners, MI 49060 Research Associate U.S.A. University of Minnesota, St. Paul [email protected] Department of Plant Biology http://www.msue.msu.edu/fis/ 1445 Gortner Ave. Phone: +1-269-671-2323 250 Biological Sciences Building Fax No.: +1-269-671-2104 Saint Paul, MN 55108 Lotus corniculatus. Forage Production, U.S.A. physiology. [email protected] entry last revised Mar 3 2004 Phone: +1-612-6249230

Fax No.: +1-612-6251738

Lotus japonicus. Molecular biology, Viviana Lepek microbiology, tissue culture, biology, genetics. CONICET researcher and Assistant Professor entry last revised Oct 17 2003 of Universidad de Gral San Martín Instituto de Investigaciones Biotecnológicas IIB-UNSAM W. L. (Bill) Lowther Scientist

160 Lotus researchers.

AgResearch Departamento de Bioquímica Vegetal y Plant Breeding and Genomics Biología Molecular Invermay Agricultural Centre Facultad de Química Private Bag Apartado 553 Mosgiel 41080-Sevilla New Zealand Spain [email protected] [email protected] http://www.agresearch.co.nz Phone: +34-95-4557145 Phone: +64-3-4899053 Fax No.: +34-95-4626853 Fax No.: +64-3-4893739 Lotus japonicus. Biochemistry, Molecular Lotus uliginosus, Lotus corniculatus. Ecology, biology, Biotechnology, Nitrogen assimilation, utililization, microbiology, inoculation, Enzymology, Mutagenesis rhizobia. entry last revised Oct 7 2003 entry last revised Oct 23 2003

Athole H.Marshall Lene Heegaard Madsen Principal Research Scientist Academic staff Institute of Grassland and Environmental University of Aarhus Research Dept of Molecular Biology, Lab of Gene Legume Breeding and Genetics Team Expression Plas Gogerddan Gustav Wieds vej 10 Aberystwyth DK-8000-Aarhus-C U.K. Denmark Phone: +44-1970-823171 Phone: +45 89 42 50 07 Fax No.: +44-1970-828357 Fax No.: +45 86 12 31 78 [email protected] [email protected] http://www.iger.bbsrc.ac.uk Lotus japonicus. Molecular genetics, map- Lotus corniculatus, Lotus uliginosus. based cloning, gene expression, genetic Genetics, Breeding,Forage production mapping. entry last revised Ago 4 2005 entry last revised Apr 7 2005

Douglas Martin Christina L Marley Livestock Adviser Institute of Grassland and Environmental Department of Agriculture Research Bypass Rd., Stanley Plant, Animal and Microbial Sciences Falkland Islands FIQQIZZ Department [email protected] Plas gogerddan http://www.figariculture.doa.gov.fk Aberystwyth Phone: 0050027355 Ceredigion, SY23 3EB Fax No.: 0050027352 U.K. Lotus corniculatus, Lotus uliginosus. Forage [email protected] production, utilisation, trialling new species Phone: ++44 (0)1970 823084 entry last revised Jul 30 2005 Fax No.: ++44 (0)1970 823245 entry last revised Jul 26 2005 Gabriela Martinoia Research Assitant Antonio J. Marquez Facultad de Agronomía Profesor Titular Universidad Nacional del Centro de la Provincia de Buenos Aires

Lotus researchers. 161

Departamento de Producción Vegetal http://www.inta.gov.ar/iffive/ Av. República Italia 780 Phone: +54-351-4974343 C.C. 47 Fax No.: +54-351-4974330 (7300) Azul Lotus japonicus. Physiology, Molecular Provincia de Buenos Aires biology. Argentina entry last revised Jun 22 2004 [email protected] http://www.faa.unicen.edu.ar/ Rodolfo Mendoza Phone: +54-2281-433292 Senior Research Fax No.: +54-2281-433292 CONICET Lotus glaber. Entomology. CEFYBO entry last revised Apr 23 2004 Serrano 669 1414 Buenos Aires Jim H. McAdam Argentina Principle Scientific Officer [email protected] Agriculture and Food Science Centre Phone: +54-1148570847 Applied Plant Science Research Division Fax No.: +54-1148570847 Newforge Lane Lotus corniculatus, Lotus glaber. Ecology, Belfast BT9 5PX Biology, Physiology, Forage Production, Northern Ireland Utilization, Microbiology. [email protected] entry last revised Jul 1 2004

Phone: +44-28-90255275 Fax No.: +44-28-90255003 Réal Michaud Lotus uliginosus. Ecology, Utilisation. Agriculture et Agroalimentaire Canada / entry last revised Nov 21 2003 Agriculture and Agri-Food Canada Centre de recherche et de développement sur Robert L. McGraw les sols et les grandes Cultures / Soils and Associate Professor of Agronomy Crops Research and Development Centre University of Missouri 2560 Hochelaga Blvd. Department of Agronomy Ste-Foy Québec G1V 2J3 208 Waters Canada Columbia, MO 65211 [email protected] U.S.A. Phone: +1-418-6577980 ext. 261 [email protected] Fax No.: +1-418-6482402 Phone: +1-314-882-6608 Lotus corniculatus. Forage, utilization. Fax No.: +1-314-882-1467 entry last revised Oct 29 2003

Lotus corniculatus. Forage, utilization, physiology. Management strategies to improve Jorge Monza performance and persistence. Professor entry last revised Nov 30 2003 Facultad de Agronomía Departmento de Biología Vegetal, Bioquímica Mariana Melchiorre Av. Garzón 780 Post-doc student Montevideo INTA Uruguay Instituto de Fitopatologia y Fisiologia Vegetal [email protected] Córdoba http://www.fagro.edu.uy/bioquim/web Argentina Phone: +598-2-3540229 [email protected] Fax No.: +598-2-3543004

162 Lotus researchers.

Lotus corniculatus, Lotus glaber, Lotus uliginosus, Lotus japonicus. Biology, physiology, tissue culture, molecular biology, Jeremy Murray microbiology. Post Doctoral Fellow entry last revised Nov 18 2003 Southern Crop Protection and Food Research

Centre (SCPFRC), Agriculture Canada

1391 Sandford St. Ken Moore London Ont N5V 4T3 Professor Canada Iowa State University [email protected] Department of Agronomy Phone: +1-519-457-1470 Ext 643 1567 Agronomy Hall Fax No.: +1-519-457-3997 Ames Iowa 50011 Lotus japonicus, Lotus filicaulis. Genetics of U.S.A. nodulation, molecular biology. [email protected] entry last revised Jan 5 2004 http://www.public.iastate.edu/~kjmoore/ Phone: +1-515-2943160 Fax No.: +1-515-2943163 P.S. Nagar Lotus corniculatus. Forage, utilization, Coordinator, IMPLANT, Scientist ecology, physiology. IMPLANT entry last revised Oct 27 2003 Department of Biosciences Saurashtra University

Rajkot – 360005 Jorge A. Mosjidis India Professor [email protected] Auburn University http://wwwsaurashtrauniversity.edu/ Department of Agronomy and Soils Phone: +91-281-2571154 202 Funchess Hall Lotus corniculatus var. minor, Lotus Auburn, AL 36849-5412 corniculatus, Lotus garcini. Taxonomy, U.S.A. ccology, forage production, tissue culture, [email protected] molecular biology. http://www.ag.auburn.edu/ay/mosjidis entry last revised Dec 31 2003 Phone: +1-334-8443976 Fax No.: +1-334-8443945 Genetics. Valeria Negri entry last revised Nov 6 2003 Associate Professor

Universita' Degli Studi Di Perugia

Biologia Vegetale Biotecnologie John Mundy Agroambientali Professor Borgo XX Giugno 74 Institute of Molecular Biology 06100 Perugia Oester Farimagsgade 2A Italy 1353 Copenhagen K [email protected] Denmark http://www.agr.unipg.it/dbvba [email protected] Phone: +39-075-5856218 http://www.biobase.dk/~mundy Fax No.: +39-075-5856224 Phone: +45-35322131 Lotus curator. Germplasm, genetics. Fax No.: +45-35322128 entry last revised Nov 25 2003 Lotus japonicus. entry last revised Oct 16 2003

Lotus researchers. 163

C. Jerry Nelson Takuji Ohyama University of Missouri Professor 210 Waters Hall Faculty of Agriculture Columbia, MO 65211 Niigata University U.S.A. Department of Applied Biological Chemistry [email protected] 2-8050 Ikarashi Phone: +1-314-882-2801 Niigata, 950-2181 Fax No.: +1-341-882-1467 Japan Lotus corniculatus. Physiology, forage. [email protected] entry last revised Jul 15 2004 Phone: +81-25-2626643 Fax No.: +81-25-2626643

Lotus japonicus. Genetics, physiology, Minoru Niizeki symbiosis, molecular biology, microbiology. Professor entry last revised Nov11 2003 Hirosaki University Faculty of Agriculture and Life Science Plant Breeding and Genetics Felicia Oliva Tejera Hirosaki Doctoral Student Aomori-ken 036-8561 Cabildo Insular de Gran Canaria Japan Jardín Botánico Canario “Viera y Clavijo” [email protected] Apartado de Correos, 14. Phone: +81-172-393776 Tarija Alta Fax No.: +81-172-393750 35017 Las Palmas de Gran Canaria Lotus corniculatus, Lotus japonicus. Genetics, Gran Canaria, Islas Canarias breeding and molecular biology. Plant Spain breeding and genetics by using somatic cell [email protected] hybridization and somaclonal variation. Phone: +34-928219580 entry last revised Oct 18 2003 Fax No.: +34-928219581

Lotus sect. Pedrosia. Taxonomy, ecology,

molecular biology. Nazhat-Ezzamane Noureddine entry last revised Jun 2 2004 Teacher/Searcher Faculté des Sciences Biologiques Laboratoire de Biologie du Sol Fernando Olmos USTHB - BP 32 El Alia - Bab Ezzouar Assistant Researcher 16111 - Alger Instituto Nacional Investigación Agropecuaria Algeria - INIA [email protected] Pasture Ecology Phone: +213-21-24-79-50 to 64 ext. 936 Ruta 5, Km. 386 Fax No.: +213-21-24-72-17 Tacuarembo Lotus conimbricensis, Lotus creticus, Lotus Uruguay roudairei, Lotus jolyi, Lotus glinoides, Lotus [email protected] palustris (all species present in Algeria). Phone: +598-63-22407 Microbiology, symbiosis, biological nitrogen Fax No.: +598-63-23969 fixation, taxonomy, seed, utilization. Lotus corniculatus; Lotus subbiflorus; Lotus entry last revised Mar 5 2004 uliginosus. Germplasm, ecology, physiology,

forage production. entry last revised Nov 5 2003

164 Lotus researchers.

Alicia Orea Peter Paľove-Balang Postdoctoral Position Researcher Instituto de Bioquímica Vegetal y Fotosíntesis Institute of Botany SAV CSIC-Universidad de Sevilla Department of Plant Physiology Avda. Américo Vespucio, s/n Dúbravská cesta 14 41092-Seville Bratislava Spain Slovak Republic, 845 23 [email protected] [email protected] http://www.ibvf.cartuja.csic.es/ Phone: +421-2-59426127 Phone: +34-95-4489526 Fax No.: +421-2-54771948 Fax No. : +34-95-4460065 Lotus japonicus. Physiology, molecular Lotus japonicus. Physiology, nitrogen biology. assimilation. entry last revised Oct 11 2003 entry last revised Oct 31 2003

Yousef A. Papadopoulos María Cristina de Pablo Agriculture and Agri-Food Canada Profesor Adjunto 14 Fundy Drive Universidad Nacional del Centro de la Truro, NS B2N 5Z3 Provincia de Buenos Aires (UNCPBA) Canada Facultad de Agronomía [email protected] Laboratorio Regional de Análisis de Semillas y Phone: +1-902-8960400 Granos Fax No.: +1-902-8960200 Av. República Italia 780, C.C. 47 Lotus corniculatus. Genetics, breeding, forage. (7300) Azul Developing germplasm for seedling vigor and Provincia de Buenos Aires competitive ability for cultivar development. Argentina entry last revised Oct 22 2003 [email protected] http://www.faa.unicen.edu.ar Martin Parniske Phone: +54-2281-427566 Professor Fax No.: +54-2281-433292 University of Munich Lotus glaber, Lotus corniculatus. Seed Department Biology I - Genetics production and quality. entry last revised Nov 4 2003 Maria Ward Strasse 1a D-80639 Muenchen Germany Cristina Pacios-Bras [email protected] Postdoctoral position http://www.jic.bbsrc.ac.uk/science/sl/smp.h Leiden University tm Laboratoire des Proteines du Cytosquelette Phone: +49-89-21806150 41, Rue Jules Horowitz Fax No.: +49-89-1785633 F-38027 Grenoble Cedex 1 Lotus japonicus, Lotus filicaulis. Genetic and France physical mapping of plant genes. Molecular [email protected] genetic analysis of pathogenic and symbiotic Phone: +33-4-38789219 plant-microbe interactions. Nitrogen fixing root Fax No.: +33-4-38785494 nodule and arbuscular mycorrhiza symbiosis. Former Lotus japonicus research. entry last revised Aug 4 2004 entry last revised Oct 31 2003

Lotus researchers. 165

Falkland Islands Government Department of Agriculture Katharina Pawlowski Stanley, FIQQ 1ZZ Stockholm University Falkland Islands Department of Botany [email protected] 10691 Stockholm http://www.fiagriculture.doa.gov.fk/ Sweden Phone: 00500 27352 [email protected] Fax No.: 00500 27355 Phone: +46-8-16-3772 L. corniculatus, L. uliginosus. Forage Fax: +46-8-16-5525 production and utilisation by sheep and cattle entry last revised Mar 29 2005 Lotus japonicus. Physiology, Utilization, Tissue Culture, Molecular Biology entry last revised Apr 21 2005 Carsten Poulsen University of Aarhus Andrea Pedrosa-Harand Dep.Mol.Biol. - Lab.Gene Expression PostDoc Science Park Department of Cell Biology and Genetics Gustav Wieds Vej 10 C Institute of Botany DK-8000 Aarhus C University of Vienna Denmark Rennweg 14 [email protected] A-1030 Vienna http://130.225.13.27/~chp/ Austria Phone: +45-89425007 [email protected] Fax.no.: +45-86123178 Phone: +43-1-4277-54026 Lotus japonicus. Genetics, molecular biology, Fax No.: + 43-1-4277-9541 microbiology. entry last revised Oct 16 2003 Lotus japonicus, Lotus filicaulis, Lotus burttii. Genetics (cytogenetics, physical mapping, comparative genomics), taxonomy. Currently Braj Nandan Prasad no research activities with Lotus. Professor & Coordinator,Programme in entry last revised Aug 17 2004 Biotechnology Tribhuvan University Jillian Perry Programme in Biotechnology C/O Central Researcher Department of Botany John Innes Centre Kirtipru Sainsbury Laboratory P.O.Box 9782 Norwich Research Park Kathmandu Colney Lane Nepal Norwich [email protected] Norfolk, NR4 7UH Phone: +977-1-330582 United Kingdom Fax No: +977-1-4331964 [email protected] Lotus corniculatus, Lotus japonicus. http://www.lotusjaponicus.org/tillingpages/Ho Physiology and molecular biology. entry last revised Oct 18 2003 mepage.htm Lotus japonicus. TILLING, genetics, mutations entry last revised Apr. 11 2005 Daniel Real Senior Plant Breeder Andy Pollard University of Western Australia Agricultural Advisor Agronomy

166 Lotus researchers.

University Field Station Pastoral and Veterinary Institute 1 Underwood Avenue PB 105 Shenton Park Hamilton, Victoria 3300 Perth, WA 6008 Australia Australia [email protected] [email protected] Phone: +61-55-730911 Phone: +61-8-92-872831 Fax No.: +61-55-711523 Fax: +61-8-93-839907 Lotus uliginosus, Lotus corniculatus. Ecology, Lotus corniculatus, Lotus japonicus, Lotus forage, utilization, germplasm. Plant glaber, Lotus uliginosus, Lotus creticus, Lotus introduction, cultivar evaluation, animal cytisoides, Lotus marrocanus, Lotus australis, production. Lotus cruentus. Breeding and genetics. entry last revised Oct 12 2003 entry last revised Mar 4 2004

J.S. Grant Reid Daniel Real Professor Emeritus Senior Plant Breeder University of Stirling National Institute of Agricultural Research Department of Plant Biochemistry INIA Tacuarembó Stirling FK9 4LA Ruta 5 km 386 Scotland C.C. 78086 C.P. 45000 United Kingdom Tacuarembó [email protected] Uruguay Phone: +44-1786-467762 [email protected] Fax No.: +44-1786-464994 Phone: +598-63-22407 Lotus japonicus. Physiology, molecular Lotus ornithopodioides, Lotus angustissimus, biology. Lotus conimbricensis, Lotus shoelleri. entry last revised Nov 30 2003

Breeding and genetics. entry last revised Mar 4 2004 Joel Reynaud Laboratoire de Botanique Mónica Rebuffo Faculté de Pharmacie Senior Researcher Avenue Rockefeller National Institute of Agricultural Research 69373 Lyon Cedex 08 INIA “La Estanzuela” France Ruta 50, km 11 [email protected] C.P. 70000 Colonia http://ispb.univ-lyon1.fr/cours/botanique Uruguay Teaching. No current research activity with [email protected] Lotus. http://www.inia.org.uy entry last revised Nov 30 2003 http://www.inia.org.uy/sitios/lnl/ Phone: +598-574-8000 Ext 1479 Ken Richards Fax No.: +598-574-8012 Research Manager Lotus corniculatus, Lotus uliginosus, Lotus Plant Gene Resources of Canada subbiflorus. Breeding, genetics, Lotus curator, Agriculture and Agri-Food Canada Lotus Newsletter editor. Saskatoon Research Centre entry last revised Oct 2 2003 107 Science Place Saskatoon Kevin Reed Saskatchewan S7N0X2 Department of Primary Industries Canada

Lotus researchers. 167 [email protected] Plant, Animal and Microbial Sciences http://agr.gc.ca/pgrc-rpc Aberystwyth Research Centre http://agr.gc.ca/rpc-pgrc Plas Gogerddan Phone: +1-306-956-7641 Aberystwyth Fax No.: +1-306-956-7246 Ceredigion Germplasm collection with many species of United Kingdom Lotus housed at PGRC. Germplasm [email protected] conservation, taxonomy, seed production, Phone: +44-1970-823113 genetics, breeding. Fax No.: +44-1970-823242 entry last revised Sep 30 2004 Lotus corniculatus, Lotus japonicus. Genetics, biology, physiology, molecular biology. entry last revised Jul 2 2004 Heathcliffe Riday Forage Legume Breeder US Dairy Forage Research Center (USDA- Craig A. Roberts ARS) Associate Professor 1925 Linden Drive West University of Missouri Madison Agronomy Department WI, 53706 214 Waters Hall U.S.A. Columbia, MO 65211 [email protected] U.S.A. Phone: 011-1-608-890-0077 [email protected] Fax No.: 011-1-608-890-0076 http://www.psu.missouri.edu/roberts/ Lotus corniculatus. Genetics, breeding, Phone: +1-573-8822801 germplasm, ecology, biology, physiology, Fax No.: +1-573-8844317 forage production, utilization, seed production, Mainly Lotus corniculatus, but other species as molecular biology. well. Germplasm screening, forage quality, entry last revised Dec 14 2004 biochemical defense. entry last revised Oct 22 2003

Diego F. Risso Senior Researcher William John Rogers National Institute of Agricultural Research Profesor Titular INIA “Tacuarembó” Facultad de Agronomía Ruta 5 km 386 Universidad Nacional del Centro de la C.C. 78086 C.P. 45000 Provincia de Buenos Aires Tacuarembó Ciencias Básicas Agronómicas y Biológicas Uruguay Av. República Italia 780, C.C. 47 [email protected] (7300) Azul http://www.inia.org.uy Provincia de Buenos Aires. Phone: +598-63-22407 Argentina Lotus corniculatus, Lotus uliginosus, Lotus [email protected] subbiflorus. Forage management and http://www.faa.unicen.edu.ar utilization under grazing, physiology. Phone: +54-2281-433292 entry last revised Mar 8 2004 Fax No.: +54-2281-433292 Lotus corniculatus, Lotus glaber. Genetics,

breeding, molecular biology. Mark P. Robbins entry last revised Nov 25 2003 Researcher Institute of Grassland and Environmental Research

168 Lotus researchers.

[email protected] Fax No.: +52-73-172388 Clive Ronson Lotus japonicus, Lotus corniculatus. Molecular Chair in Genetics and cell biology, cell imaging, actin University of Otago cystoskeleton, cell signaling, nodule Microbiology Department development, Rhizobium-legume interactions. P.O. Box 56 entry last revised Nov 30 2003 Dunedin

New Zealand Niels Sandal [email protected] Permanent academic staff http://microbes.otago.ac.nz/dept/STAFF/pr University of Aarhus ofile-ronsonc.html Department of Molecular Biology-Lab. of Fax No.: +64-4798540 Gene Expression Lotus japonicus, Lotus corniculatus. Gustav Wieds Vej 10 C Microbiology and genetics of the DK-8000 Aarhus C microsymbiont Mesorhizobium loti. entry last revised Oct 9 2003 Denmark [email protected] Phone: +45-89-425006 Oscar Adolfo Ruiz Fax No.: +45-86-123178 Researcher (CONICET) and Professor Lotus japonicus, Lotus filicaulis. Genetics, (UNSAM) molecular biology, map based cloning, Instituto de Investigaciones Biotecnológicas- recombinant inbred lines, symbiosis. Instituto Tecnológico de Chascomús (IIB- entry last revised Nov 22 2004 INTECh)

Unidad de Biotecnología 1 IIB-INTECh Matt A. Sanderson Casilla de Correo 164 Research Agronomist Camino de circunvalación Km 5 USDA-ARS (B7130 IWA), Chascomús Pasture Lab Building Provincia de Buenos Aires Curtin Road Argentina University Park [email protected] PA 16802-3702 [email protected] U.S.A. http://www.iib.unsam.edu.ar [email protected] Phone: +54-2241-424049/430323 General interest in Lotus. Production, Fax No.: +54-2241-424048 utilization, grazing land ecologist. Lotus corniculatus, Lotus glaber, Lotus entry last revised Aug 17 2004 japonicus. Biology, physiology, molecular biology, microbiology entry last revised Oct 6 2003 Graeme Sandral Research Officer (Plant ecology and genetics)

University of Western Australia and NSW Federico Sanchez Agriculture Professor University Field Station Universidad Nacional Autónoma de México 1 Underwood Drive, Shenton Park Instituto de Biotecnología Perth, WA 6009 Plant Molecular Biology Department Australia Av. Universidad 2001 [email protected] Col. Chamilpa. 62210 [email protected] Cuernavaca, Morelos http://www1.crcsalinity.com/ México

Lotus researchers. 169

Phone: +61-8-92-872-851 Facultad de Ciencias Agrarias, Instituto de Fax No.: +61-8-93-839-907 Botánica del Nordeste Mobile: + 61-040-922-6235 Sargento Cabral 2131 Perennial Lotus species known to the CC: 209 Macaronesian Islands (Azores, Madeira’s, CP: 3400, Corrientes. Canary Islands and Cape Verde) and the more Argentina cultivated species such as Lotus corniculatus [email protected] and Lotus glaber. Plant breeding, Phone: +54 3783 427589 ext. 146 phylogenetics, taxonomy, ecology. Fax No.: +54 3783 427131 entry last revised Mar 24 2004 Lotus glaber. Physiology, Tissue Culture,

Molecular Biology. entry last revised Dec 7 2004 Juan Sanjuán Research Scientist Consejo Superior de Investigaciones Sindhu Sareen Científicas (CSIC) Indian Grassland and Fodder Research Institute Estación Experimental del Zaidin CSKHPKV Campus Departamento de Microbiologia del Suelo y Palampur 176062 (H.P.) Sistemas Simbióticos India Prof. Albareda 1 [email protected] E-18008 Granada entry last revised Oct 9 2003

Spain [email protected] Shusei Sato http://www.eez.csic.es/inves/grupos/D3.htm Researcher Phone: +34-958-181600 Ext 259 Kazusa DNA Research Institute Fax No.: +34-958-129600 Plant gene research group Lotus japonicus, Lotus corniculatus, Lotus 2-6-7 Kazusa-Kamatari glaber, Lotus uliginosus. Microbiology. entry last revised Oct 13 2003 Kisarazu, 292-0818 Japan [email protected] Analia Sannazzaro http://www.kazusa.or.jp/lotus/ CONICET doctoral fellow Phone: +81-438-523935 IIB-INTECh. Sede Chascomús Fax No.: +81-438-523934 UB1 Lotus japonicus. Genomics, molecular Camino Circunvalacion Laguna Km 6 genetics. CC 164, (7130) Chascomús entry last revised Nov 24 2003 Argentina

Phone: +54-2241-430323 Leif Schauser [email protected] IMSB-Lab. of Gene Expression Lotus glaber. Genetics, Biology, Physiology, University of Aarhus Tissue Culture, Molecular Biology, Science Park Microbiology entry last revised Apr 19 2005 Gustav Wieds Vej 10 C DK-8000 Aarhus C Denmark Pedro Alfonso Sansberro [email protected] Professor/Scientist Fax No.: +45 86 12 31 78 Universidad Nacional del Nordeste and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) Simone Meredith Scheffer-Basso Professor

170 Lotus researchers.

Universidade de Passo Fundo Lotus corniculatus. Forage production, Instituto de Ciências Biológicas utilization. Departamento Biologia entry last revised Nov 21 2003

Rua Silva Jardim, 303 apto. 701

99010-240 Passo Fundo Marina Sisterna Rio Grande do Sul Teacher- Researcher Brazil Comisión de Investigaciones Científicas (Prov. [email protected] Bs. As.) – Facultad de Ciencias Agrarias y http://www.upf.tche.br Forestales (Universidad Nacional de La Plata) Phone: +55-316-8100 Ext. 8326 Departamento de Fitopatología Lotus corniculatus. Morphophysiology, calle 60 y 119 evaluation of forage production (cutting- (1900) La Plata grazing tolerance, growth habit, nutritive value, Buenos Aires competition, mixtures with grasses). entry last revised Oct 16 2003 Argentina [email protected] Phone: +54 0221 423 6758 ext 423 Ms Anne Schneider Fax No.: +54 0221 424 0997 Executive Secretary Delegate Lotus glaber. Pathology. AEP, European Association for Grain Legume entry last revised May 14 2005

Research

Executive Secretariat 12 Avenue George V Richard R. Smith 75 008 Paris Retired Research Geneticist France USDA-ARS, University of Wisconsin [email protected] US Dairy Forage Research Center http://www.grainlegumes.com 1925 Linden R. West Phone: +33-1-40694909 Madison WI 53706 Fax No.: +33-1-47235872 U.S.A. The AEP is an international multidisciplinary [email protected] network of scientists and end-users concerned Phone: +1-608-2645240 with grain legumes. The AEP office is Fax No.: +1-608-2645147 interested in being informed about the progress Lotus corniculatus. Genetics, breeding, forage, in Lotus investigations for establishing cultivar evaluation. Selection for improved connection with other legumes research seedling establishment and improved activities. persistence entry last revised Nov 20 2003 entry last revised Nov 11 2003

Philippe Seguin Dmitry Sokoloff Assistant Professor Lecturer McGill University, Macdonald Campus Higher Plants Department Department of Plant Science Biological Faculty 21,111 Lakeshore Rd. Moscow State University Ste. Anne de Bellevue 119992 Moscow Quebec H9X 3V9 Russia Canada [email protected] [email protected] http://www.herba.msu.ru/ http://www.mcgill.ca/plant/faculty/seguin/ Phone: +7-095-9391603 Phone: +1-514-3987851 Fax No.: +7-095-9392777 Fax No.: +1-514-3987897

Lotus researchers. 171

Lotus australis and related taxa, Lotus Macdonald campus arabicus and related taxa, Lotus discolor and 21,111 Lakeshore Rd related taxa, Lotus creticus and related taxa. Ste.-Anne-de-Bellevue Taxonomy. Generic limits of Lotus, taxonomy QC, H9X 3V9 of its segregate genera, sectional system of Canada Lotus, taxonomy of some white-, red- and [email protected] pink-flowered species. Cladistic analyses. http://www.mcgill.ca/plant/faculty/stromvik Flower development in Lotus corniculatus. Phone: +1-514-398-8627 entry last revised Oct 9 2003 Fax No.: +1-514-398-7897

Soybean (For the moment not studying Gary Stacey Lotus). Bioinformatics, Genomics, Professor Molecular Biology. entry last revised Aug 26 2004 University of Missouri Plant Microbiology and Pathology 108 Waters Hall Norio Suganuma Columbia, MO 65203 Professor U.S.A. Aichi University of Education [email protected] Department of Life Science http://psu.missouri.edu/staceylab/index Kariya .htm Aichi 448-8542 Phone: +1-573-884-4752 Japan Fax No.: +1-573-882-0588 [email protected] Lotus japonicus. Symbiotic nitrogen fixation, Phone: +81-566-262647 genetics, microbiology, pathology. Fax No.: +81-566-262310 entry last revised Mar 23 2004 Lotus japonicus. Genetics, physiology. entry last revised Nov 5 2003

Jens Stougaard Lecturer, Group leader John Sullivan University of Aarhus Research fellow IMSB-Lab. of Gene Expression University of Otago Department of Molecular Biology Department of Microbiology Science Park New Zealand Gustav Wieds Vej 10 C [email protected] DK-8000 Aarhus C Phone: +64-3-4798373 Denmark Fax No.: +64-3-4798540 [email protected] Lotus corniculatus, Lotus japonicus. Phone: +45-89-425011 Molecular biology, microbiology. Fax No.: +45-86-21222 entry last revised Nov 20 2003

Lotus japonicus. Plant molecular genetics and general molecular biology. Genetic mapping, Krzysztof Szczyglowski map-based cloning, gene isolation, gene Agriculture & Agri-Food Canada characterisation, expression studies. entry last revised Oct 15 2003 1391 Sandford Street London, ON N5V 4T3 Canada Martina Stromvik [email protected] Assistant professor Phone: +1-519-457-1470 ext. 273 McGill University Fax.no.: +1-519-457-3997 Department of Plant Science Lotus japonicus

172 Lotus researchers. entry last revised Oct 02 2003 Lotus japonicus. Physiology, molecular biology, microbiology, symbiosis. entry last revised Oct 21 2003

Shigeyuki Tajima Professor Michael Udvardi Lab. Molecular Plant Nutrition Group Leader Faculty of Agriculture Max-Planck-Institut fur Molekulare Kagawa University Pflanzenphysiologie Miki-cho, Kita-gun Molecular Plant Nutrition Kagawa 761-0795 Am Mühlenberg 1 Japan 14476 Golm [email protected] Deutchland Phone: +81-87-8913129 [email protected] Fax.no.: +81-87-8913021 Phone: +49-331-5678149 Lotus japonicus. Genetics, molecular biology. Fax No.: +49-331-5678250 entry last revised Oct 17 2003 Lotus japonicus. Genetics, biochemistry, molecular biology, functional genomics

(transcriptomics, proteomics, metabolics). Nancy Terryn Symbiotic nitrogen fixation and molecular Researcher plant nutrition. University Gent entry last revised Oct 22 2003 Instititute Plant Biotechnology for Developing Countries K.L. Ledeganckstraat 35 Don R. Viands 9000 Gent Associate Dean and Director of Academic Belgium Programs, Professor of Plant Breeding [email protected] Dept. of Plant Breeding and Biometry http://www.ipbo.ugent.be/ 151 Roberts Hall Phone: +32-9-2645201 Ithaca, NY 14853 Fax No.: +32-9-2648795 U.S.A. Lotus japonicus, Phaseolus, Lathyrus. [email protected] Transformation, seed development, stress Phone: +1-607-255-3081 response, molecular biology of Grain legumes Fax No.: +1607-254-4613 such as Phaseolus, cowpea, Lathyrus for which Lotus corniculatus. Breeding, pathology. there is little genomic data so we look at model Breeding for resistance to crown-rot and to systems Lotus japonicus. Fusarium wilt caused by Fusarium oxysporum. entry last revised Oct 20 2003 entry last revised Oct 7 2003

Toshiki Uchiumi Osvaldo Ramón Vignolio Associate Professor Professor Kagoshima University Universidad Nacional de Mar del Plata- EEA Faculty of Science Facultad de Ciencias Agrarias Department of Chemistry and BioScience INTA Balcarce 1-21-35 Korimoto Producción Vegetal Kagoshima 890-0065 Ruta 226, Km 73.5 Japan CC 276 (7620) Balcarce [email protected] Argentina Phone: +81-99-2858164 Fax No.: +81-99-2858163

Lotus researchers. 173 [email protected] Lotus japonicus. Genetics, biology, [email protected] physiology, molecular biology, metabolism. [email protected] entry last revised Nov 4 2003

Phone: +54-2266-439100

Fax No.: +54-2266-439101 Judith Webb Lotus corniculatus, Lotus glaber. Germplasm, Research Scientist ecology, biology, physiology, forage Institute of Grassland and Environmental production, utilization, seed production. entry last revised Nov 9 2003 Research Plas Gogerddan Aberystwyth Jeffrey J. Volenec Ceredigion Professor SY23 3EB Wales Purdue University United Kingdom Department of Agronomy [email protected] Lilly Hall of Life Sciences Phone: +44-1970-823124 915 West State St. Fax No.: +44-1970-823243 West Lafayette IN 47907-2054 Lotus japonicus, Lotus corniculatus. U.S.A. Genetics, biology, physiology, tissue culture, [email protected] molecular biology, microbiology. http://www.agry.purdue.edu/staffbio/jjvbio. entry last revised Nov 9 2003 htm

Phone: +1-765-494-8071

Fax No.: +1-765-496-2926

Lotus corniculatus. Physiology, utilization, molecular biology, biochemistry. entry last revised Oct 27 2003

Trevor L. Wang Group Leader Metabolic Biology Department John Innes Centre Norwich Research Park Norwich NR4 7UH United Kingdom [email protected] http://www.jic.bbsrc.ac.uk/staff/trevor- wang/index.htm Phone: +44-1603-450283 Fax No.: +44-1603-450014