Conclusions of session 1 Taxonomy in Parasitic Plants ...

COST ACTION 849: Parasitic plant management in sustainable agriculture Thematic meeting "GENETIC DIVERSITY OF PARASITIC PLANTS"

19-21 February 2004, Córdoba, Spain

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Conclusions of session 1: Taxonomy in Parasitic Plants Chairperson: Drs. A. Pujadas & G. M. Schneeweiss Recent use of molecular phylogenetic techniques in the study of parasitic plants has greatly advanced our knowledge on the often difficult taxonomy of these groups. The presentation of G. M. Schneeweiss “Taxonomy and phylogeny in Orobanche” highlighted our current understanding of the phylogenetic relationships of Orobanche in the framework of the newly defined family Orobanchaceae as well as the complex phylogenetic relationships within this genus. Most notably, there is strong molecular and karyological evidence that this genus is not monophyletic but comprises two phylogenetically independent lineages, the Orobanche- and the Phelipanche-group. In both lineages, economically important aggressive weed races occur, O. crenata and O. cumana in the Orobanche-group and O. ramosa and O. aegyptiaca in the Phelipanche-group. The presentation by M. A. García “Taxonomy and systematics of Cuscuta L. (Convolvulaceae)” provided the according overview for the genus Cuscuta. Recent molecular phylogenetic studies have clearly shown that Cuscuta nests within Convolvulaceae and does not constitute its own family. The author presents results of his thorough taxonomic revision of the Old World Cuscuta subgenus Cuscuta, which includes agronomically important species such as O. epithymum. Based also on the results of molecular phylogenetic investigations, a new classification scheme was outlined. This is grossly congruent with previous systems, most changes concerning taxa at the species level. This study set the standards for according investigations in the other subgenera of Cuscuta.

A second part of this session covered the important topic how to incorporate the new phylogenetic-taxonomic knowledge into existing data bases. Stephen Jury outlined in his talk “A new system for Orobanche taxonomy in Europe” the problems connected with the genus Orobanche in the EC-sponsored project Euro+Med PlantBase. This genus has been treated rather pragmatically in standard floras (Flora Europaea) and checklists (MedChecklist). Data for Orobanche are currently scrutinized taxonomically and are thus prepared to be checked afterwards by regional specialists. Recent taxonomic insights should then be incorporated via an Internet interface. Once Orobanche has experienced a full taxonomic treatment, other databases concerning, e.g., conservation or chromosome numbers, can be linked.

The remaining presentations covered some of the regional research activities currently undertaken. Results of a floristic survey of Orobanche species in the northwestern part of the Iberian Peninsula was presented by L. Carlón (“Taxonomic, chorological and iconographical contributions to the knowledge of genus Orobanche (Orobanchaceae) in the north of the Iberian Peninsula”). The importance of such studies was nicely shown by the first records for some Orobanche species for North Spain. The thoroughly conducted studies in the field also allow to clearly address future taxonomic questions, e.g. in the O. artemisiae-campestris aggregate. An excellent photo documentation of the species greatly aids identification purposes and sets standards for other taxonomic-floristic surveys. The importance of thorough floristic research on a regional scale was also shown by the presentation of O. Lyshede, “Orobanche in Denmark”. In contrast to the situation in the Mediterranean regions, Orobanche in



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Denmark is of concern for species protection activities, and a sound monitoring of the distribution of this species is therefore important. This is illustrated by the recent detection of O. flava in Denmark, being the by far northernmost locality of this species so far known only from the central and South European mountain ranges. Other species, e.g., O. lucorum, have been intentionally introduced by humans, but are now established in the Danish flora.

The presentation by L. Cagan dealt with “Distribution of Orobanche in Slovakia”. In the years 2002 and 2003, more than 50 localities have been checked for the occurrence of Orobanche species. This not only adds to our knowledge of the distribution of different broomrape species in Slovakia, some of them of nature conservation concerns, but also allows to name species putatively suitable as hosts for phytophagous animals and pathogens, which could be used against Orobanche weeds in the southern part of Slovakia. Current studies on Orobanche in Sicily have been presented by G. Domina, focussing on O. canescens, a species described from Sicily (“Orobanche canescens C. Presl in Sicily. Distribution and taxonomic notes”). This species belongs to the taxonomically very difficult O. minor-aggregate. The author presents the results of exhaustive herbarium studies as well as studies in the field, which allow a better and more precise circumscription of this species. Discriminating characters towards other species, such as O. minor and O. pubescens, have been worked out. The ecology and distribution of this species in Sicily and neighboring smaller islands has been clarified.



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A new system for Orobanche taxonomy in Europe

S.L. Jury1 and F.J. Rumsey2

1Centre for Plant Diversity and Systematics, The School of Plant Sciences, The University of Reading, Whiteknights, Reading, RG6 6AS, U.K.

2Department of Botany, The Natural History Museum, Cromwell Road, London, SW7 5BD, U.K.

The genus Orobanche was revised as an ‘in-house job’ by A.O. Chater and the late D.A. Webb (no specialist author could be found at the time) for volume three of Flora Europaea published in 1973. The account stood the test of time, although supplemented by an enumeration in Med-Checklist for the countries bordering the Mediterranean Sea in 1989 and a number of regional works.

The EU-sponsored Euro+Med PlantBase has electronically merged databases containing Flora Europaea, Med-Checklist and the Flora of Macaronesia Checklist of vascular plants by Hansen & Sunding. This new combined dataset has been augmented by the addition of the accepted names used in over 100 European North African and Middle Eastern Floras published since Flora Europaea and Med-Checklist. Data for the genus Orobanche is presently being scrutinised taxonomically before checking by regional specialists using the successful Flora Europaea methodology for gaining consensus. Unlike Flora Europaea, this is an internet-based project. The Secretariat has been based in Reading with software developed in Berlin and Edinburgh which will enable the taxonomy to be updated over the internet, as well as regional advisers able to edit occurrence data remotely. The Euro+Med project plans to add Orobanche to the list of genera which have been given a full taxonomic treatment, and to which other datasets concerning distribution, conservation and karyology in Helsinki (Finland), Gaggenau (Germany) and Patras (Greece) have so far been linked.

The project is searching for further funding for finding other means of displaying further data concerning host plants and other possible aids to identification (including illustrations) in the light of recent developments in systematics.



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Taxonomy and phylogeny in Orobanche

G.M. Schneeweiss Institute of Botany, University of Vienna

The genus Orobanche is the largest among the holoparasitic members of

Orobanchaceae and comprises about 170 species. Following the latest monographer of the genus, Beck-Mannagetta (1930), Orobanche is traditionally divided into four sections Gymnocaulis (New World), Myzorrhiza (New World), Trionychon (Old World), and Orobanche (Old Word). The monophyly of this group has been indirectly questioned by the taxonomic treatment of Teryokhin et al. (1993). These authors treat sect. Trionychon and sect. Orobanche as separate genera Phelipanche and Orobanche, respectively, and put the latter together with the two genera Cistanche and Diphelypaea into subtribe Orobanchinae, while Phelipanche constitutes its own subtribe Phelipanchinae.

Recent molecular phylogenetic analyses using nuclear ITS-sequences indicate that Orobanche in its current circumscription is not monophyletic (see Figure), thus supporting the view of Teryokhin and colleagues. The molecular data suggest a close relationship of O. sects. Gymnocaulis, Myzorrhiza, and Trionychon, informally called the Phelipanche-group, and a close relationship of O. sect. Orobanche and the small SW Asian genus Diphelypaea, informally called the Orobanche-group. This contradicts inferences from plastid sequences, where a closer relationship of the two Old World sections is proposed, but is in congruence with the distribution of basic chromosome numbers: the Phelipanche-group has x = 12, while the Orobanche-group has x = 19.

Intrasectional structurings proposed by Beck-Mannagetta for sect. Orobanche are not supported by the molecular data, indicating that most of the morphological used for delimitation of subsections, such as coloration or shape of the corolla, are homoplaseous. Additionally, O. macrolepis and O. anatolica/colorata, traditionally grouped in sect. Orobanche, constitute phylogenetically independent lineages within the Orobanche-group (see Figure).

Orobanche crenata groups with species of the taxonomically difficult O. minor-aggregate (O. minor, O. artemisiae-campestris, etc.), while O. cernua (incl. O. cumana) constitute a basal lineage of the core-group of sect. Orobanche. Economically important species of sect. Trionychon, O. aegyptiaca and O. ramosa, fall in one clade together with a series of mostly East-Mediterranean and SW. Asian species (O. pulchella, O. bungeana, O. oxyloba, etc.).

In many groups, analyses of ITS sequences do not provide enough resolution, and other markers, such as fingerprinting techniques (e.g., AFLPs), will ultimately have to be used.



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Taxonomy and systematics of Cuscuta L. (Convolvulaceae)

M.A. García Real Jardín Botánico (CSIC). Plaza de Murillo 2. 28014-Madrid. SPAIN

The genus Cuscuta L. comprises approximately 170 species of hemi or

holoparasitic plants worldwide distributed. They are twining herbs with reduced vegetative organs, attached to the host by stem haustoria. Its floral morphology is similar to Convolvulaceae and the relation with this family was early proposed. Some authors have included Cuscuta within Convolvulaceae and others have segregated the genus in its own family Cuscutaceae closely related to Convolvulaceae. Recent studies based on multiple molecular data sets shows that Cuscuta was generated within Convolvulaceae and therefore should be included in the family.

The three most important monographers of the genus considered Cuscuta as a member of Convolvulaceae. Since George Engelmann’s work in 1859 the genus was divided in three groups based on the morphology of styles and stigmas. Subgenus Monogyna is considered the most primitive and it is characterized by the totally or partially joined styles; it includes about seven species from the Old World and one from North America. Subgenus Grammica is the most diversified group and includes about 130 species most of them from the New World; it is characterized by the free styles and flattened to globose and not elongated stigmas. Subgenus Cuscuta comprises about 22 species from the Old World characterized by the free styles and elongated stigmas.

During the preparation of a taxonomic revision of subgenus Cuscuta, 6,300 herbarium specimens have been studied. A total of 28 species in three sections are recognized. Three of those species are newly described. Keys, descriptions, plates and distribution maps have been produced for the monograph. Molecular analysis based on nuclear and chloroplast gene sequences reveal that the three sections are monophyletic and well supported except for C. babylonica which appears related to section Epistigma instead of section Cuscuta. A well supported clade includes tropical African species (i.e C. somaliensis or C. rhodesiana) within section Cuscuta that have been considered varieties of C. planiflora in regional floras.



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Orobanche canescens C. Presl in Sicily. Distribution and taxonomic notes

G. Domina

Laboratorio di Sistematica, Fitogeografia ed Ecologia vegetale, Dipartimento di Scienze Botaniche, Università degli Studi di Palermo. Via Archirafi, 38 – 90123 Palermo, ITALY.

The genus Orobanche in Sicily is known mostly from literature and herbarium

sources dating back to the XIX century. In fact, the most comprehensive floras (Greuter & al., 1989; Pignatti, 1982; Chater & Webb, 1972) on the whole refer to Beck (1890, 1930), who in turn based his treatment largely on Lojacono specimens and literature data. In addition, no reliable information is given in the recent local floristic surveys. Consequently, in order to fill this gap, the revision of this genus for Sicily and the surrounding islands has been started.

Begining from the study of the original material (PRC) and other specimens housed in CAT, P, PAL, PRC and W and after field trips, it has been possible to distinguish morphologically O. canescens from the other species belonging to the O. minor group, to assess the historical and recent herbarium data and subsequently to trace its distribution.

O. canescens is morphologically distinct from O. hederae and O. amethystea by the corolla shape. The not deflexed lower lip of the corolla is useful to discriminate O. canescens from O. amethystea, O. fuliginosa, and O. picridis. Moreover in O. picridis and O. pubescens the spike is denser and the style pubescent. O. canescens and O. minor can be distinguished by the angle of insertion of the flowers in the stem, the presence of hairs at the base and, to a lesser extent, the stigma colour.

In Sicily O. canescens occurs from 0 to 900 m a.s.l. infecting several Asteraceae species. It is more frequent in coastal plains and hills in the northern part of the island and in the southern part near Palma di Montechiaro and Licata, but it has also been found in the inland south of Palermo, in the Madonie and Nebrodi mountains and in the south-eastern part near Ragusa. It occurs in the islands of Malta, the Eolie and the Pelagie, Pantelleria and also in Ustica (Guss., 1828) and in the Egadi (Francini & Messeri, 1956). Further work, especially on molecular and caryological basis, is programmed in order to assess the taxonomic position of O. canescens in comparison with the other species belonging to the group of O. minor, in particular with O. minor itself. References Beck Mannagetta G. (1890) Biblioth. Bot., 19. Beck Mannagetta G. in Engler A. (1930) Pflanzenr., 96, Leipzig. Chater A. O. & Webb D. A. in Tutin & al. (1972) Flora Europaea 3, 286-294, Cambridge. Francini E. & Messeri A. (1956) Webbia 11, 607-846. Greuter G., Burdet H. M. & Long G. (1989) Med-Checklist 4, Genéve. Gussone G. (1828) Florae Siculae Prodromus 2, Napoli. Pignatti S. (1982) Flora d'Italia, Bologna.



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Taxonomic, chorological and iconographical contributions to the knowledge of genus Orobanche (Orobanchaceae) in the north of the

Iberian Peninsula

L. CARLÓN, G. GÓMEZ CASARES , M. LAÍNZ, G. MORENO MORAL AND O. SÁNCHEZ

PEDRAJA

Jardín Botánico Atlántico, Avenida del Jardín Botánico, 33394 Gijón, Spain

The recent activities of our team have yielded several relevant contributions to the

knowledge of the genus Orobanche in the north of the Iberian Peninsula. Some species previously unknown or doubtful in Spain have been found (Orobanche bartlingii Griseb. —with its closer known populations, themselves relatively isolated from the core of the species, growing in a few localities in eastern France and northern Italy—, O. artemisiae-campestris Vaucher ex Gaudin, s.str.). Geographic distribution in the Peninsula of several species (O. ramosa, s.l., “O. rosmarina” sensu Foley, O. arenaria Borkh., O. purpurea Jacq., O. cernua L., O. alba Stephan ex Willd., O. reticulata Wallr. subsp. reticulata, O. amethystea Thuill., O. minor Sm., O. hederae Vaucher ex Duby, O. caryophyllacea Sm., O. teucrii Holandre, O. elatior Sutton, O. rapum-genistae Thuill.) has been extended. O. aconiti-lycoctoni Moreno Moral, Gómez Casares, Sánchez pedraja, Carlón & Laínz has been described as a new species, whereas some taxonomic problems which could require new descriptions have been presented. We have also firmly maintained the taxonomic autonomy of the misinterpreted O. santolinae Loscos & Pardo. In addition, we have published large colour photographs of 23 of the referred species, outstanding among them those belonging to species with very poor or even unavailable illustrations (“O. olbiensis” sensu Pujadas & Crespo, “O. rosmarina” sensu Foley, O. santolinae Loscos & Pardo, “O. major L. β Ritro” (Gren. & Godr.) Willk., etc.).

This contribution here summarises and discusses the value of all these findings emphasising the background guiding our research, as well as the nature of our methods and some imminent projects. The interest of colour photographs in the progress of taxonomic and chorological knowledge of Orobanche will be stressed.



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Distribution of broomrapes (Orobanche sp.) in Slovakia

¼. Cagáò and P. Tóth Slovak Agricultural University in Nitra, Slovakia

During 2002 and 2003, the survey of distribution of wild broomrapes (Orobanche

sp.), as potential hosts of phytophagous animals and pathogens, was done in Slovakia. Wild broomrapes were distributed throughout Slovakia. 50 localities were checked and broomrapes occurred on 30 of them. The existence of five more abundant broomrape species was revealed: Orobanche alba Stephan ex Willd. infested plants from the genus Thymus L., O. flava Mart. ex F. W. Schultz attacked Petasites sp. and rarely Tussilago farfara L., O. elatior Sutton parasitized exclusively Colymbada scabiosa (L.) Holub, O. caryophyllacea J. E. Smith, where Galium sp. and Asperula sp. served as hosts and O. lutea Baumg., which occurred only rarely on Medicago falcata L. The most abundant species were O. alba (found at 13 localities) and O. flava (found at 21 localities). In south Slovakia, sunny and grassy slopes (162 - 538 m a. s. l.) were places of O. alba occurrence. Northern mountain regions of Slovakia (505 - 875 m a. s. l.), usually along the creeks were O. flava typical sites. O. alba was not found in cold climatic regions, while O. flava was not recorded in warmer regions. These two broomrapes, O. alba and O. flava, are potential reservoirs of natural enemies of O. ramosa, which parasitizes on tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum Mill.) in Slovakia.



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Summary on the Session 2: Biology and Ecology of Parasitic Plants

Chairperson: Prof. Klaus Wegmann

Five oral contributions have been presented in this session, chaired by Klaus Wegmann: Lyra D., Economou G., Avgoulas Ch. and Arapis G. (after a short introduction by Arapis presented by Lyra): Seed germination study in Orobanche populations infesting tobacco plants in Greece Lyra D., Arapis G. and Economou G. (presented by Economou) : Abiotic factors affecting the infestation in tobacco crops from Orobanche in Greece Batchvarova R.B. and Slavov S.B. (presented by Batchvarova): Morphological and genetic diversity of broomrapes in Bulgaria Wegmann, K.: Ecology and epidemiology of Orobanche ramosa in Europe Streibig J.C.: Response of Striga hermonthica (Del.) Benth. biotypes to Sorghum exsudates Orobanche ramosa, O. crenata and O. cumana are the prominent species that cause increasing problems in agriculture in Southern and South Eastern European countries. During the past decade Orobanche ramosa has largely established also in Central European countries. It parasitizes tobacco and hemp in Germany (Baden, Palatinates) and France, like in Greece, Italy, Spain, Bulgaria, Romania, and Croatia. An O. ramosa race attacks winter rapeseed in Western France. Various races are known: in Germany and France the well-known blue/violet flowering type, and a white/yellowish flowering type, in Bulgaria a small and a large type, both blue/violet flowering (and O. mutelii, also on tobacco). The adaptation to new hosts has been reported: after tobacco O. ramosa has been found on camomile (Matricaria chamomilla), carrot (Daucus carota) and parsley (Petroselinum crispum), in Bulgaria on cabbage and tomato. The extreme longevity of the seeds makes crop rotation as a control measure useless. Examples of reestablishment of O. ramosa after 50 and 60 years without a host crop on the field are documented.

Due to intensive resistance breeding in sunflower the development of races is better known with Orobanche cumana, which until now only attacks sunflower. Races A, B, C, D, E and F with increasing aggressiveness have been identified. Molecular analyses (RAPD, RFLP) on the polymorphism of Orobanche were presented by Batchvarova. Increasing aggressiveness is correlated with increasing pectinolytic activities of radicle exsudates (presented by Simier in an other session).

Geographical spreading and race development needs to study the ecological requirements for Orobanche establishment in an area. This would also help to predict possible spreading due to local climatic changes. Economou et al. studied environmental factors in various areas in Greece on O. ramosa frequency and delopment on tobacco. They found precipitation and nitrogen supply statistically significant factors.

Two papers dealt with germination stimulation. Lyra et al. studied germination stimulation of Orobanche ramosa seeds from various Greek tobacco areas by 1 mg L-1 IAA, 10 mg L-1 kinetin and 25 mg L-1 GA3. IAA was found most active, followed by GA3. Streibig reported on germination experiments with Striga hermonthica races, which differed in their host specificity (sorghum, millet) by sorgoleone. Sorgoleone consists of



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various analogues with different stimulation activities. Moreover, they inhibit photosystem II, similar to PS II inhibiting herbicides. Germination stimulation with various stimulants is used for the distinction of Striga hermonthica from various origin and hosts.

Wegmann proposed to establish a land register for Orobanche occurrence, which could be maintained by agricultural extension officers, and completed by weather record from local weather stations, more research on catch crops for Orobanche control, and the foundation of a European Orobanche Research Station, which would be justified considering the increasing problems with Orobanche in European agriculture.



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Ecology and epidemiology of Orobanche ramosa in Europe

K. Wegmann Eberhard-Karls-Universität Tübingen, Germany

Branched broomrape (Orobanche ramosa), which in the past has been mainly

known in Southern and South Eastern Europe, now is rapidly spreading also in Central European countries. While there is a long list of suitable hosts for O. ramosa, in European countries few, but important crops are attacked, among them rapeseed, hemp and tobacco. Various forms of O. ramosa in tobacco are known in Germany, France and Bulgaria. A survey on the occurrence of O. ramosa in Europe will be presented. Farmers are concerned about continuous spreading of O. ramosa to neighboured, and sometimes distant fields, but also about its recent adaptation to new hosts. Examples will be reported.

O. ramosa was not recently introduced to Central European countries; it has been long known in tobacco and in hemp. However, due to a limited number of the parasite it did formerly rarely play an economically important role. This has changed. Examples show that O. ramosa during the past years has become more frequent and more aggressive, causing sometimes the loss of whole crops.

In order to understand this development, the environmental conditions for Orobanche development, temperature, soil structure and soil pH, soil humidity, fertilization, have to be re-considered. Knowledge on local climatic changes need to be studied. Thus, the threat of further shift of O. ramosa to Northern European countries may be estimated. Moreover, intensive work is required for a better knowledge of the host specificity or host adaptation. More attention should be given to trap crops, which can be used during crop rotation, and which meet the farmers’ economic interest. Examples will be named.

It is proposed to prepare a land register in order to follow Orobanche spreading, this could be in the responsibility of agricultural extension officers, to instruct farmers to make detailed notes on crop rotation, date and frequency of Orobanche appearance, and the amount of damage in their crops. The updated land register could be completed by weather records from local weather stations. The collection and evaluation of the data could be managed by a central European institution.



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A new race of Orobanche cumana in Israel

D. Plakhine, T. Landa, G. Akhdari, Y. Hershenhorn and D.M. Joel

ARO, Newe-Ya’ar Research Center, 30095 Ramat-Yishay, Israel

Broomrape (Orobanche cumana) is a major constraint for sunflower in various parts of Israel. In recent years several resistant lines were grown in the country and allowed a significant reduction in the damage caused by the parasite. In spite of that, more virulent Orobanche populations successfully infected the resistant lines last year in several locations.

Seeds of several virulent O. cumana populations were collected in the field, and their ability to attack resistant (var. Amber) and susceptible (var. DY3) sunflower lines was examined in the greenhouse. We found out that whereas O. cumana from Alonim failed to attack the resistant sunflower, O. cumana populations from three other fields respectively showed three levels of virulence toward the resistant sunflower plants. The population from Gadot was also more virulent than the population from Alonim on the susceptible sunflower variety.

Previous studies revealed that only race C was present in Israel, with a very low inter- and intra-specific diversity, probably due to the Founder Effect. Here we report for the first time on the occurrence of new virulent race(s) of O. cumana in Israel. Two main reasons may have allowed the occurrence of these virulent populations: they were either imported from abroad or locally developed, perhaps due to the continuous and repeated use of the available resistant varieties that are all based on a single resistance mechanism.



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Races of broomrape in Romania

M. Joita-Pacureanu1, Ch. Veronesi2, D. Nastase3 and M. Ciuca1 1ARDI Fundulea, 915200, Romania

2University of Nantes, France 3Agricultural Research Station-Braila, Romania

Orobanche sp. (Orobanche cumana Wallr. syn. Orobanche cernua Loefl.) is one

of the important factors, limiting yield in sunflower crop in Europe. The use of resistant sunflower varieties is the most reliable way to fight this parasite. A more virulent races of broomrape were reported in recent years, in Europe, in large areas cultivated with sunflower, specially in Spain, Romania and Turkey.

The study of population genetics of Orobanche sp. is of great importance since the understanding of the variability within and between pathogenic populations is essential if selection programmes need to target sources of resistance. Three Orobanche populations from Romania were studied comparing with one population from Turkey, one from Spain and one from Yugoslavia. Orobanche seeds were collected on very infested sunflowers in different areas (3 in Romania, situated in different geographic areas in country, 1 in Spain, 1 in Turkey and 1 in Yugoslavia).

Two sunflower genotypes (the differentials for broomrape races E and F) were used to be infested with Orobanche seeds from each region. The experiment was made in vitro and in vivo conditions. In vitro, were calculated the mean values of the fixation of Orobanche on root (stage 1), formation of Orobanche tubercle (stage 2), tubercle with adventive roots (stage 3) and Orobanche with stem (stage 4). The results given by the mean values in the stage 1 to stage 4 show that the isolates from these regions display differences, in all cases for the race E. For the race F, the behaviour of the broomrape in Turkey and Constanta-Romania is similar, the others 4 populations being different of these two, and different among them. The results obtained by ISSR amplification show that Orobanche population from Constanta-Romania shares several bands with Turkish Orobanche population. The population of Orobanche from Calarasi-Romania seems different from the others.

The results of in vivo experiment show differences for the virulence of broomrape on the sunflower differentials (androsterile line A and the maintainer B).



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Highly virulent populations of sunflower broomrape (Orobanche cumana)

L. Molinero and J.M. Melero-Vara

Institute of Sustainable Agriculture, C.S.I.C., Apdo. 4084, Córdoba, Spain.

Several races of O. cumana have been described in sunflower crops of Spain since the late 80's. Some of them (from A to E) were controlled by the incorporation of single dominant genes (Or1 to Or4) into the host. Nowadays, the use of sunflower hybrids carrying the Or5 gene is an usual practice in all the areas of the country where the disease threatens the production. Due to the frequent appearance of more virulent populations of broomrape causing disease on resistant hybrids (races F and higher), we evaluated some of the populations overcoming the Or5 gene. The evaluation of 20 populations of O. cumana collected in Central and Southern Spain from 1997 to 2003 was conducted in three experiments. Two of them were carried out in shade house during spring-summer of 2003. Plants of the third experiment were grown under greenhouse conditions from September to December of 2003. Genotype NR5 (Or5 gene) was used as differential of race F. Besides, all the populations were inoculated to genotypes L86 and P96, both of them resistant to race F of broomrape. For every population, eight to ten plants (replications) of each of the genotypes were artificially infested with broomrape seed. Disease incidence (DI) was expressed as the percentage of plants with emerged broomrapes. The accumulated number of emerged bromrapes was recorded at weekly intervals until senescence of sunflower plants. Eighteen out of the 20 populations showed to be highly virulent on NR5, the average number of emerged broomrapes per plant ranging from 10 to 38, 22 to 33, and 15 to 38 at the end of the experiments I to III respectively. Two populations collected in Central Spain, 1 and 2, did not cause disease on NR5. This reaction was confirmed for population 1, which was tested in experiments II and III. Nevertheless, both populations were able to infect L86 genotype with DI 83 and 85% respectively. This genotype showed up to 8 broomrapes/plant when inoculated with population 2, while 6.4 broomrapes/plant were observed when 1 was used. Another broomrape population, 3, also from Central Spain, infected not only NR5 but also L86 and P96. The reaction, observed in exp. I, was confirmed in exp. III. Mean DI of 69.5 and 65% were observed in L86 and P96 respectively. The accumulated number of broomrapes per plant reached 5.4 and 3.6 for genotypes L86 and P96 respectively. Diversity of virulences among populations of sunflower broomrape from different geographical origin and the occurrence of race G are reported in Spain. These results are being confirmed by means of molecular studies.



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Morphological and genetic diversity of broomrapes in Bulgaria

R.B. Batchvarova, R. Atanasova and S.B. Slavov

AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria

According to Georgiev (1936) 25 species of Orobanche are represented in Bulgaria.

O. cumana on sunflower was first reported in 1935 (Dobrev, 1945) in the North-East part of Bulgaria. Three races of O. cumana A, B and C were described (Knyazkov, 1950; Petrov, 1970; Batchvarova, 1978) on sunflower. After 1990 a massive attack by O. cumana on resistant varieties was observed. The reason is due to appearance of new physiological races D and E of the parasite.

O. ramosa was described in tobacco fields in the South Bulgaria in 1937 by Bailov. It was found (Venkov, Bozoukov, 1994) that O. cumana from sunflower can not attack the tobacco and O. ramosa is attacking only tobacco, not sunflower.

According Georgieva and Edreva (1994) there are two Orobanche species - O. ramosa and O. mutelii found in tobacco fields. In Southern Bulgaria at least 150 000 ha of tobacco are estimated to be infested with O. ramosa.

Seeds from morphologically different broomrapes on sunflower and tobacco, from different areas in Bulgaria were collected. The host plants were grown in infested soil with seeds from each broomrape. Virulence and morphological characterization of different broomrapes were estimated. Cytological analyses of the collected broomrapes were performed. Molecular ana lyses (RAPD, RFLP) were performed for searching polymorphism between species and individual of broomrapes.



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Response of Striga hermonthica biotypes to sorghum exudates

J.C. Streibig Department of Agricultural Sciences, KVL, Thorvaldsensvej 40

DK-1871 Frederiksberg, Denmark

The germination of Striga hermonthica, a noxious root parasite of many cereal crops, is stimulated by exudates from the roots of both host and non-host trap crops. One of the major crops being heavily infested is sorghum. Striga spp. affect two-thirds of the 73 million hectare of land under cereal production in the Sahelian and savannah zones of Africa, where it threatens the livelihood of over 100 million people. The infested area continues to increase because of intensification of land use. Development of tolerant cultivars of sorghum and maize against S. hermonthica is an important area of research, and several avenues are taken (Haussmann et al. 2000; Gurney et al. 2003).

Evidently, within S. hermonthica there are host preferences that can be attributed to strains or races, some are specifically responding to millet others to Sorghum. The differential response could, among other things, be to either different stimulant(s) or concentration of stimulant(s). Wigchert et al. showed differential response of various population of Striga spp. to GR24 (Wigchert et al. 1999). Sorghum does exude various compounds from the root, some of which are biologically active and also act as germination stimulants. Sorgoleone is one of those stimulatory compounds, which consists of several analogues (Dayan et al., 2003; Rimando et al., 2003; Czarnota et al., 2003). The analogues are known to inhibit photosystem II the same way as does photosystem II inhibiting herbicides (Streibig et al. 1999). The possible differential stimulatory effect of those sorgoleone analogues, however, is unknown.

We are currently setting up screening facilities to test the germination of various “biotypes” of S. hermonthica from Burkina Faso, Nigeria and Eritrea in response to sorghum root exudates. The plan is to make bioassay directed isolation of factions of crude exudates from sorghum to unravel which compounds are affecting the germination of the parasite. The design of the assays is a modified method of that of Berner (1997) and the data are analysed by generalized linear models. References Kim 1997. PMB 5320, Ibadan, Nigeria Czarnota et al. (2003). Journal of Chemical Ecology 29, 2073-2083. Dayan et al. (2003). Journal of Biological Chemistry 278, 28607-28611. Gurney et al. (2003). New Phytologist 160, 557-568. Haussmann et al. (2000). Field Crops Research 66, 195-211. Rimando et al. (2003). Journal of Natural Products 66, 42-45. Streibig et al. (1999). Pesticide Science 55, 137-146. Wigchert et al. (1999). Journal of Agricultural and Food Chemistry 47, 1705-1710.



18

Seed germination study in Orobanche populations infesting tobacco

plants in Greece

Lyra D., G. Economou, Ch. Avgoulas and G. Arapis

Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

Orobanche ramosa and Orobanche aegyptiaca seeds were tested for

germinability, applying to them plant growth regulators such as IAA, Kinetin and Gibberellic Acid (GA3). The seeds originated from fifteen Orobanche populations, which were collected from three geographically isolated regions of Central and North Greece during summer 2002 and 2003. These regions constitute traditionally tobacco areas in Greece.

Firstly, the seeds were surface sterilized by immersion in sodium hypochlorite (3% v/v) for 3 min and then rinsed five times for 5 min with sterile distilled water before being subjected to the subsequent treatments. The seeds were, then, spread in Petri dishes on filter paper moistened with 3 ml of water and preconditioned at 21 ïC for 9 days. After this pretreatment, growth regulators were applied in the following concentrations: IAA 1 mg L-1, Kinetin 10 mg L-1, GA3 25 mg L-1. The seeds were conditioned at 25 ïC. Four separate replicates of each treatment were carried out.

The purpose of this study is to identify the differences, both in the loss of dormancy and in the germinability of the seeds. All Orobanche populations showed high level of variability in their germinability suggesting that these characteristics may depend on their adaptive ability in the local environment. Results are discussed.



19

Abiotic factors affecting the infestation in tobacco crops from Orobanche in Greece

Lyra D., G. Arapis and G. Economou

Agricultural University of Athens, Iera Odos 75, 11855 Athens, Greece

The object of this study is to correlate the rate of infestation of nine Orobanche

populations with the following abiotic factors: temperature expressed as degree-days, total precipitation, soil pH, the structure of the soil and the quantity of the total nitrogen and humidity. These populations originated from tobacco fields in two geographically isolated regions of Greece. The Orobanche plants were collected during summer 2003.

In addition, different plant characteristics were studied, in order to estimate the phenological variation among the Orobanche populations, such as the height of the central shoots of the plants, the number of the ramifications, the number of flowers per inflorescence and the length of the inflorescence. The measurements were carried out in six plants of each population.

F-values (Anova test) showed high variability in the reproductive parts of the Orobanche plants, differences that may be attributed to the microclimatic conditions, which define the local environment of the tobacco crops in each region. The other phenological traits showed no statistically significant differences. At this point, it is essential to mention that Greece has a constantly varied environment that determines different ecosystems in each sub-region. It is also remarkable the fact that the preliminary survey which was conducted to estimate the Orobanche parasitism in tobacco fields in Central and North Greece, showed strong differentiation in the infestation level among the tobacco crops from region to region.

Correlations between the rate of infestation from Orobanche and the climatic and soil parameters are analyzed for the time being. Results will be discussed.



20

Aggressiveness and pectinolytic activities within populations of Orobanche cumana Wallr. a root parasite of sunflower

Ph. Simier1, Ch. Véronési1, E. Bonnin2, H. Benharrat1, A. Fer1 and P.

Thalouarn1

1GPPV, Université de Nantes, 2 rue de la Houssinière BP 92208 44322 Nantes, France 2 URPOI, INRA, Rue de la Géraudière, BP 71627 44316 Nantes - France

Using a host differential screening method in pot and in hydroponic co-culture,

four populations of the sunflower root parasite Orobanche cumana Wallr. were studied for their pathogenicity against sunflower genotypes which are thought to carry specific resistance genes. These broomrape populations which belong to three supposed races were observed to differ by their aggressiveness (D < E < F). The Spanish supposed-race F population is the most aggressive showing the greatest number of fixations and biomass reduction on sunflower cultivars resistant to the other populations tested (Romanian supposed-race D population and both Spanish and Bulgarian supposed-race E populations). However none of the sunflowers from the differential screening was absolutely immune to every populations of broomrape. Indeed at least few broomrape attachments with sometimes necrosed seedlings were observed during some weeks on roots of the most resistant sunflower. In any case typical hypersensitive response was not observed on the host roots. Root exudates from all the screened sunflower genotypes stimulate O. cumana germination in the same extent with some variations depending on the O. cumana populations tested. When germinated in response to GR24, Orobanche excreted cell wall degrading enzymes such as pectin methylesterase (PME, EC 3.1.1.11) and polygalacturonase (PG, EC 3.2.1.15). Note that pectinolytic activities were shown to be higher in the most aggressive and recent race F.



21

Session 3: Molecular Evolution and Phylogeny of Parasitic Plants

D.L. Nickrent Department of Plant Biology, Southern Illinois University

Carbondale, IL 62901-6509 USA

The use of DNA sequences to infer evolutionary relationships in plants began less than two decades ago, yet these data have provided tremendous advancements in knowledge of these unique organisms. For parasitic plants, many long-standing questions are now being answered using these methods. The parasitic habit has evolved at least ten times in angiosperms and the positions of several groups are now well-established at the ordinal level. These include Cassytha (Lauraceae, Laurale), Lennoaceae (Boraginales), Krameriaceae (Zygophyllales), Cuscuta (Convolvulaceae, Solanales), and Orobanchaceae (Lamiales). More recently, Hydnoraceae have been placed with Aristolochiaceae in Piperales using sequence data from the nucleus, plastid, and mitochondrion. The large order Santalales remains part of a large polytomy at the base of the eudicot clade, however, relationships among the 158 genera and 2300 species are being clarified using molecular data. Olacaceae and Santalaceae are paraphyletic whereas Misodendraceae, Loranthaceae, Opiliaceae and Viscaceae are monophyletic. Arial parasitism (the mistletoe habit) evolved at least five times in the order and two of these independent events occurred in Santalaceae. Using nonparametric rate smoothing, calibrated with fossil evidence of Anacolosidites, Santalales evolved ca. 125 mybp (Barremian of the Cretaceous). Subgeneric phylogenetic studies have been conducted on all species of Arceuthobium using nuclear and chloroplast sequences. The plastid trnT-L-F region of Arceuthobium demonstrates homoplastic deletions in at least two species pairs, thus showing that caution should be exercised in using such structural features as phylogenetic markers. Phylogenetic placement within angiosperms of the holoparasitic Balanophoraceae, Cynomoriaceae, and Rafflesiaceae s. lat. remain controversial. These families lack the chloroplast genes that have been most often used to reconstruct broad-scale phylogenies. Using nuclear small-subunit rDNA sequences, Cynomoriaceae appear related to Saxifragales and Balanophoraceae to Santalales, although these positions have little bootstrap support. Nuclear SSU rDNA sequences have been obtained for 11 genera of Balanophoraceae and the topology of the maximum parsimony tree indicates three southern hemisphere genera (Dactylanthus, Hachettea, and Mysropetalum) are basal, thus suggesting an ancient Gonwanan origin for the family. Sequences of clones of Cynomorium plastid 23S rDNA demonstrate the existence of extensive heteroplasmy in this taxon. Despite the extensive elevation of substitution rates, these plastid rRNA molecules may be functional given that most mutations are present in regions that vary in other organisms. Three genera of Rafflesiaceae were recently analyzed using mitochondrial matR sequences (Barkman et al. 2004) which allied Rafflesia and Rhizanthes with Malpighiales and Mitrastema with Ericales. These positions are in conflict with nuclear small-subunit rDNA data that place all Rafflesiaceae in Malvales. Current attention is focused upon discovering the sources of these conflicting signals.



22

Parasitism and Evolution of The Plastid Genome

P. Letousey, P. Thalouarn and P. Delavault GPPV, Université de Nantes, 2 rue de la Houssinière BP 92208 44322 Nantes, France

With the exception of legumes, the plastid genome of angiosperms is highly

conserved in structure as well as in gene composition and arrangement. Before the detailed studies of Epifagus virginiana, it was thought that the plastid genome of parasitic angiosperms, mainly holoparasite that are devoid of chlorophyll and have consequently lost photosynthetic capacity, was absent or nun-functional. In the 90's, several works revealed in fact that, in parasitic plants, this plastid DNA is conserved and is potentially functional although highly reduced in size and lacking some genes for photosynthesis. A dramatic size reduction of the plastid genome has taken place in the holoparasitic species, which have lost most, if not all, photosynthetic genes.

Using a Long-Distance PCR strategy (LD-PCR), the organization of the 88 kb- length O. cumana plastid genome was investigated (Delavault and Thalouarn, 2002). This species contains at least two distinct rbcL sequences: one similar in size to the truncated rbcL pseudogene from O. cernua, a closely related species, and another with a size comparable to that of rbcL plastid genes from autotrophic plants. Southern-Blot and PCR Chromosome Walking experiments revealed that the second copy has been transferred to the nuclear genome.

Concerning the chlorophyllous parasitic species, two genuses, Striga and Cuscuta, were thoroughly examined. Size of the S. hermonthica plastome was estimated to 190 kb using a pulsed field electrophoresis strategy. While the SSC region has undergone a dramatic size reduction, the LSC seems to contain an extra DNA of 50 kb-length. Thanks to PCR Chromosome Walking experiments, we are investigating this region in order to identify its nature. Recently, two works clearly indicated that, in the Cuscuta genus, the degree of conservation of the plastid genome (size, gene content and coding capacity) depends on their chlorophyll content (Berg et al., 2003; Krause et al., 2003).

Thus, all these works suggest that the plastid genome is still useful in studying the evolution of parasitic plants.

References: Delavault et al, 1996. Physiol. Plant. 96:674-682. Delavault and Thalouarn, 2002. Gene 297:85-92. Berg et al., 2003. Planta 218:135-142. Krause et al., 2003. Planta 216:815-82.



23

Session 4: The study of broomrape diversity in agriculture Chaired by DM. Joel and B. Román

Five species of Orobanche attack agricultural crops around the Mediterranean and in Southern Europe. Taxonomic problems and agriculturally important biodiversity problems are not necessarily the same, e.g. the distinction between O. mutelii and O. ramosa is not important for the farmers because both have similar hosts and similar impact on crops, while O. cumana and O. cernua significantly differ in host preference though regarded by some taxonomists as one single species.

Knowledge of the levels of diversity allows prediction of the potential occurrence of new races in different geographical regions, as shown for O. crenata (Román et al. 2002). Knowledge of the variation between populations may lead to the discovery of differences in host preference, and hybridization between populations can lead to an increase in virulence (Joel, 2001). These aspects are highly important for the farmers. A suitable nomenclature is hence needed in order to assists the farmers in identifying potential damage regarding particular crops.

Therefore the study of broomrape biodiversity should focus on two levels: host-specific populations, and races that attack specific resistant crops. The available morphological characteristics are usually not sufficient for broomrape identification on these levels. Molecular markers are needed for this sake. SCAR markers for the weedy broomrape are already available, and can be used for identification of single soil-borne seeds (Joel et al. 1998).

Further research should focus on the analysis of broomrape intra-specific diversity between countries, between populations and within regions and populations, with reference to host preference and virulence. Once this is achieved, specific molecular markers for broomrape diagnosis should be developed, for use with mature plants, with seeds, and with soil samples.

Joel et al.1998. Aspects of Applied Biology 51:23-27.

Joel 200. WSSA Abstracts 41:126.

Román et al. 2002. Phytopathology 92:1262-1266.



24

Genetic diversity of Orobanche species and host range potential

D.M. Joel1, V.H. Portnoy1, I. Paran2 and D. Gidoni2

1ARO, Newe-Ya’ar Research Center, 30095 Ramat-Yishay, Israel 2 ARO, the Volcani Center, 50250 Bet-Dagan, Israel

In previous reports we demonstrated significant and consistent inter-specific

variations among the five major broomrape species in Israel. These findings provided molecular means for the identification of each species and additionally, these results further substantiated the specification of O. cumana as potentially distinct species from O. cernua.

RAPD-based analysis was used to evaluate the magnitude of intra-specific genetic variability within Orobanche species with relation to inter-specific genetic distances. Different primers revealed different extent of polymorphism, however, only 5% polymorphic bands were found in O. cumana grown on sunflower, compared to 11% in O. cernua grown on tomato.

The intra-species Genetic distance rates found in O. cumana and O. cernua were however significantly lower than those found for O. crenata and O. aegyptiaca. Additionally, most of the genetic diversity within the latter two species was found among individuals rather than between geographically distant collections of each species. The possible linkage between intra-specific genetic diversity and host-range potential in Orobanche will be discussed.



25

Genetic variability of Striga (Review)

B.I.G. Haussmann University of Hohenheim, Ins. Plant Breeding and Population Genetics, 70593 Stuttgart, Germany

The genus Striga contains about 25 species. Economically most important are the parasitic weeds S. hermonthica and S. asiatica which infest cereals in Africa and parts of Asia, and S. gesnerioides which severely attacks legumes in the same regions. The different Striga species have distinct morphological features by which they can be distinguished. In addition, RAPD and SCAR markers have been developed to separate the morphologically similar species S. hermonthica and S. aspera.

Variability within striga species is considerable. In West Africa, S. hermonthica populations with specific adaptation to sorghum or millet have been reported whereas others attack both hosts. Maize-specific S. asiatica populations were found in Benin. The strength of intercrop specialization depends on the cultivation history of the field but precise mechanisms of adaptation are unknown. Molecular marker (RAPD, AFLP) profiles were able to distinguish between geographically distinct populations of one species, such as West versus East African S. hermonthica populations and local S. asiatica populations from Benin. There is also evidence for intracrop specialization in terms of different sensitivity to germination stimulants of S. hermonthica populations from Kenya versus Mali or Niger and variable degrees of virulence among maize-parasitizing S. asiatica populations from Benin. High selection pressure, reducing the genetic variability of striga, has been reported on S. hermonthica populations parasitizing resistant sorghum varieties and supports the hypothesis of potential adaptation to resistant host cultivars. The variation between plants of individual striga populations is not well elucidated. Hardy-Weinberg-Equilibrium has been found for two iso-enzyme loci in 13 S. hermonthica populations from West Africa. Due to its outcrossing behavior, intrapopulational variation of S. hermonthica is almost as big as the interpopulational variation. On the other hand, interpopulational variation seems relatively larger, and race differentiation clearer in the autogamous S. asiatica and S. gesnerioides.

Genetic differences among striga populations most likely contribute to the frequently highly significant genotype-environment interaction variances for striga resistance in multi- location field trials. A better understanding of the virulence variation among and within striga populations is urgently required. Standardized cross- infestation experiments, study of the genetics of striga virulence, development of co-dominant marker systems and estimation of population-genetic parameters in striga populations (outcrossing rate, heterozygosity, effective population size, gene flow, selection pressure by host cultivars on striga populations), and subsequent modeling of the evolution of virulence could be one possible approach towards a better understanding and therefore a more effective deployment of resistance genes against these noxious parasites.



26

Molecular markers for diagnosis and genetic diversity studies in Orobanche

B. Román1, Z. Satovic2, C. Martínez1, D. Rubiales3, A. Pujadas4 and JI Cubero4

1CIFA, Córdoba, Spain 2Faculty of Agriculture, University of Zagreb, Zagreb, Croatia

3IAS-CSIC, Córdoba, Spain 4UCO-ETSIAM, Córdoba, Spain

Detection of the presence and identification of the Orobanche species in a soil or

crop seed lots is of great interest for growers and quarantine officers in order to prevent new infestations or spreading of the parasite. This is often difficult via traditional methods considering the minuscule seed size of the parasite. In a previous study we determined the differences among 28 species of the genera by sequencing cp DNA fragments. Specific primers can be designed from these sequences in order to obtain PCR amplification from a target parasite species, avoiding the fragment amplification from the host.

Microsatellites have proven to be an extremely valuable tool for population genetic studies due to the high polymorphism shown and the relative ease of scoring. However, they have not been used yet in studies of the genus Orobanche. The major drawback of microsatellites is that they need to be isolated de novo from most species being examined for the first time. This is due to the fact that microsatellites are usually found in non-coding regions where the nucleotide substitution rate is higher than in coding region. Consequently, the strategy of designing "universal primers" matching conserved sequences is problematic. However the presence of highly conserved flanking regions across different species have been reported for some microsatellite loci.



27

Overcoming limitations of dominant marker data: population structure of the parasitic plant Cistanche phelypaea inferred from RAPD markers

Z. Satovic1, B. Roman2, C. Alfaro2, D. Rubiales3, J.I. Cubero4, and A. Pujadas4

1Faculty of Agriculture, University of Zagreb, Zagreb, Croatia

2CIFA, Córdoba, Spain 3IAS-CSIC, Córdoba, Spain

4UCO-ETSIAM, Córdoba, Spain RAPD and AFLP markers have been widely used to assess genetic diversity and population structure because of the rapidity and ease with which a high number of polymorphic markers can be generated. Genetic variation is represented by the presence or absence of amplified DNA fragments (bands), whose signals behave as dominant markers and the data analysis is hampered by the lack of complete genotypic information. Recent advances in statistical methods (Lynch and Milligan, 1994; Stewart and Excoffier, 1996; Zhivotovsky, 1999; Pritchard et al., 2000; Holsinger et al., 2002) make analyses of population structure based on dominant marker data possible although some problems of bias cannot be completely eliminated. The analysis of molecular variance (AMOVA) is usually used to partition the total phenotypic variance into within populations, among populations within subspecies, and between subspecies (Excoffier et al., 1992). The AMOVA can be performed on Dice distance matrix among individuals treating an RAPD (or AFLP) profile as a haplotype (Huff et al., 1993). The variance components can be tested statistically by non-parametric randomisation tests. A Bayesian method with non-uniform prior distribution of allele frequencies (Zhivotovsky, 1999) could be used assuming Hardy-Weinberg genotypic proportions in order to estimate marker allele frequencies. Genetic diversity and population genetic structure can be computed following the treatment of Lynch and Milligan (1994). Expected heterozygosity or Nei's gene diversity (Nei, 1973) can be calculated for each population and the total gene diversity (Ht), the average gene diversity within populations (Hw), the average gene diversity among populations in excess of that observed within populations (Hb), and finally Wright's Fst can be obtained. Yet another Bayesian method developed by Holsinger et al. (2002) can be used in order to estimate FIS (inbreeding coefficient) from dominant marker data. The estimates of FIS often seem to be unreliable and have to be regarded with great caution. Nevertheless, estimates of θB (= FST) can also be obtained without estimating FIS (FIS free model). Finally, the model-based clustering program STRUCTURE (Pritchard et al., 2000) can be used to estimate the underlying population structure. This Bayesian method enables identification of clusters of genetically similar individuals from multilocus genotypes without prior knowledge of their population affiliation. In this approach, it is assumed that there are K populations contributing to the gene pool of the sampled populations.



28

Two-dimensional gel electrophoresis as a tool to identify and characterize the protein profile of Orobanche spp. seeds.

M. Curto1, M.M. Castillejo2, D. Rubiales1, J.V. Jorrín2

1Institute for Sustainable Agriculture, IAS-CSIC, Córdoba 2Departament of Biochemistry and Molecular Biology, University of Córdoba.

Proteomics is becoming a powerful tool in plant biology studies (Cánovas et al.,

2004). Defined as a scientific discipline or experimental approach, deals with the cellular proteome characterization, considered in the most ample sense, from individual structural and functional protein identification, to postraductional modifications and interaction studies. Differently from the genome, the cellular proteoma is quite dynamic, depending on the specie, individual, organ, tissue, developmental stage and environmetal conditions. A typical proteomic experiment includes the following steps: protein extraction, protein separation by using mainly two-dimensional gel electrophoresis (2-DE), analysis of peptide digests by mass spectrometry and identification through genomic or protein database searching by using specific algorithms.

Our groups have just initiated a proteomics research programme for studying plant-parasitic plant interactions (Castillejo et al., 2004). We pretend to evaluate if 2-DE can be used to discriminate and identify Orobanche spp. populations or races. 2-DE have been used, with success, to detect genetic diversity (Thiellement et al., 1999).

Broomrape species, populations and races characterization and identification is still a key unsolved problem that has been approached by using morphological, biochemical, and the most recent molecular biology techniques.

In a preliminary step we are optimising a protocol for protein extraction and electrophoresis by using seeds of different Orobanche spp., including O. cumana, O. ramose, O. crenata, and O. foetida. Seeds were abundantly washed with 1:3 bleach water diluted, dried, homogenized with liquid nitrogen by using a mortar, and proteins extracted by using the TCA-acetone protocol. Proteins were resolved by 2-DE, IEF as first dimension (7 cm, 3-10 pH linear gradient), and SDS-PAGE (13% polyacrilamide gel) as second dimension. Gels were Coomassie or silver stained, captured and analysed by using the PD-Quest (Bio-Rad) software.

References Cánovas et al. (2004). Proteomics 4: 285-298. Castillejo et al.. Submitted. Thiellement et al. (1999). Electrophoresis 20: 2013-2026.

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