Workshop Parasitic Plant Management in Sustainable Agriculture Final …cost849.ba.cnr.it/Abstracts...

Workshop Parasitic Plant Management in Sustainable Agriculture Final meeting of COST849

23-24 November 2006, ITQB Oeiras-Lisbon, Portugal

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Local organizer Dr M.C. Vaz Patto Scientific Comittee: J. Verkleij, M. Vurro, D. Joel, A. Murdoch & D. Rubiales



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Germination physiology as a target for Orobanche control

Wegmann, K.

Tübingen, Germany Dormancy break and germination of Orobanche (and other root parasitic angiosperms) are rather complex processes and specifically controlled by the host plant. A survey shall be presented and approaches for Orobanche control shall be reported. Deficiencies in research shall be pointed out. (1) Conditioning of the seeds. During the conditioning period under warm and humid conditions gibberellin is synthesized in the seed tissue, which is a precondition for germinability. Only when this presumption is reached, germination is stimulated by chemical compounds exuded by the host plant root. Hence, inhibitors of gibberellin biosynthesis suppress the germination of Orobanche seeds. (2) Germination stimulation. The dormancy of conditioned seeds is broken by germination stimulants exuded by the host plant roots. Most effective natural germination stimulants are the strigolactones: strigol, sorgolactone, alectrol, orobanchol. Most root exudates contain several active compounds, many of them are not yet known. The germination inducing activity is concentration-dependent and parasite species-specific (Hauck). Therefore germination induction depends on the amount of exuded germination stimulant by the host root, and by the distance of the seed to the host root, and possibly by the composition of the active components. According to Wegmann’s hypothesis the composition can explain host specificity, this is similar to the bouquet of pheromones with insects. Nothing is known about microbial modifications of germination stimulants in the soil. Can the host specificity change? Yoneyama et al. using HPLC and tandem-mass spectrometry demonstrated and quantified the presence of several, partly unidentified strigolactones in root exudates. (3) Suicide germination by natural germination stimulants. Due to their low concentrations strigolactones cannot be isolated and used for field application in order to induce suicide germination in the absence of a host. Strigolactones, however, are exuded also by non-host plants (trap crops). Cultivation trap crops is a means for reducing the Orobanche seed bank in the soil, e.g. Linum usitatissimum for seed bank reduction of Orobanche ramosa. Due to specificity of root exudates more research is required to identify specific trap crops for each Orobanche species. Besides strigolactones other natural germination stimulants (for Orobanche minor and Striga hermonthica) have been identified: the fungal metabolites cotylenins and fusicoccins, and the phytohormone methyljasmonate (Yoneyama). Can the presence of the fungus in the absence of a host plant cause suicide germination? (4) Suicide germination by synthetic germination stimulants. Structure analogues of the sorgolactones have been synthesized by several working groups (Johnson, Welzel, Zwanenburg). GR 24 still is used as a reference substance in germination tests. GR 24 originally had been synthesized for Orobanche control by suicide germination (Patent application No. 37759/76 in Great Britain by A.W.Johnson & A.Hassanali-Walji), however, due to the instability of GR 24 in the soil experiments were not as successful as expected. Nijmegen-1 (Zwanenburg) in a formulation for field application showed a reduction of the seed bank (Benvenuti) and in several (but not all) cases a considerable reduction of Orobanche ramosa attack on tobacco. Many active germination stimulants with very different structures have been synthesized in Zwanenburg’s group. A general impediment for the



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application of a synthetic chemical in agriculture are the high costs of several million € for the registration and release by the authorities. In this context a report by Economou et al. (2006) on suicide germination of O. ramosa by the application of an Ascophyllum nodosum preparation (Algit Super®) may provide a new aspect. It cannot be expected that one application of a germination stimulant or a trap crop will eliminate all the parasite seeds. However, suicide germination appears a useful component in integrated Orobanche control. (5) Germination inhibitors. Little attention was given to the role of germination inhibitors. Germination inhibitors might be exuded by associated flora, e.g. weeds (allelopathy). Inhibitors might also be found among the numerous synthetic compounds (e.g. Zwanenburg’s group) with low or lacking stimulant activities; they have not been tested so far. (6) Exoenzyme inhibitors. Once the seed germinates, the radicle produces exoenzymes which enables it to penetrate the root tissue of the host plant. Pectinases appear the most active exoenzymes. Hence, inhibitors for these pectinases would hinder the radicle to penetrate. Pectinase inhibitors could be searched in the exudates of the associated flora, and (as resistance factor) in the root exudates of potential host plants.



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Strigolactones, signals for friends and enemies

Bouwmeester, H.

Laboratory for Plant Physiology, Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands, e-mail: [email protected]

Parasitic plants use molecules that are secreted by the roots of plants as indicators for the presence of a suitable host. These molecules, called germination stimulants, induce the germination of the seeds of Orobanche and Striga species. So far, the germination stimulant molecules that have been identified mostly belong to one chemical class of compounds, the strigolactones. Interestingly, recently the strigolactones were shown to also be important signalling molecules for arbuscular mycorrhizal (AM) fungi (Akiyama et al. 2005; Besserer et al. 2006). The strigolactones induce hyphal branching in AM fungi, a process crucial for the root colonisation by these fungi. The requirements for such signalling molecules – for parasitic plants as well as AM fungi - are that they must be specifically indicative of the presence of a plant host. If other soil-organisms would produce similar compounds and would induce branching (of AM fungi) or germination (of parastic plants) this would be detrimental for the latter two. Although the role of strigolactones for mycorrhizal colonisation should be kept in mind, strigolactone production could be an interesting target for improved control of parasitic weeds. Therefore we study the biosynthesis of the strigolactones in several host plants of mycorrhiza as well as parasitic plants and its regulation. To begin with, we have recently discovered that the strigolactones are derived from the carotenoids – and should therefore be called apocarotenoids and not sesquiterpene lactones - and as such are quite “plant-specific” (Matusova et al. 2005). Our finding is also interesting in view of the fact that mycorrhizal roots of many plant species accumulate apocarotenoids (Strack et al. 2003). Moreover, there seems to be an interaction between mycorrhiza and parasitic plants through the host plant, resulting in reduced infection by Striga of sorghum and maize that are colonised by AM fungi (Lendzemo & Kuyper 2001). The hypothesis that this interaction is mediated through the production of strigolactones will be discussed. We are further elucidating the strigolactone biosynthetic pathway, initially focusing on the first dedicated step that is probably catalysed by a carotenoid cleavage dioxygenase (Matusova et al. 2005). Our aim is to clone genes from this pathway and make transgenic plants with altered strigolactone biosynthesis. These plants will be a great tool to study the importance of these signalling molecules for the interaction of plants with friends as well as enemies. Finally, the knowledge obtained about the regulation of strigolactone formation can also be used to design control measures that should help European agriculture to deal with this important problem. References Akiyama et al. (2005) Plant sesquiterpenes induce hyphal branching in arbuscular mycorrhizal fungi.

Nature 435: 824-827 Besserer et al. (2006) Strigolactones stimulate arbuscular mycorrhizal fungi by activating mitochondria.

PLoS Biology, 4 e226. Lendzemo & Kuyper (2001) Effects of arbuscular mycorrhizal fungi on damage by Striga hermonthica

on two contrasting cultivars of sorghum, Sorghum bicolor. Agriculture, Ecosystems and Environment 87: 29-35

Matusova et al. (2005). The strigolactone germination stimulants of the plant-parasitic Striga and Orobanche spp are derived from the carotenoid pathway. Plant Physiology 139: 920-934

Strack et al. (2003). Arbuscular mycorrhiza: biological, chemical, and molecular aspects, Journal of Chemical Ecology, 29 1955-1979



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Germination stimulant(s) perception by parasitic plants

Matusova1 R. & H. Bouwmeester2

1Institute of Plant Genetics and Biotechnology, Slovak Academy of Sciences, Akademicka 2, P.O.Box 39A, 950 07 Nitra, Slovakia

2Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands The life (survival) of the parasitic plants of Orobanche and Striga spp. fully depend on presence of the host plants. The first recognition step in co-existence of these parasitic plants and their hosts is identification of the specific chemical molecules exuded from the roots of the suitable host plants. Therefore the parasitic plants developed mechanism to recognize host exuded chemical signals to ensure that the roots of host plant are present in close vicinity of the parasite. This recognition mechanism is of great importance, because the germinated seeds of parasite survive a few days only without attachment to the host plant from which they obtain all nutrients to grow.

Recently, Yoneyama and co-workers isolated and characterized several strigolactons from different host plants (Awad et al., 2006). Although biological activity of the strigolactones resides mainly in the D ring (Mangnus & Zwanenburg 1992) an interesting question is whether the small changes in the remainder of the molecule have an effect on receptor binding in the parasitic plant seeds, and hence on host-parasite specificity. There are several indications that the strigolactone composition of root exudates indeed plays a role in determining host specificity during the germination stage of the parasitic plant.

We will discuss the evidence for this and how we are studying germination stimulant(s) (strigolactones) perception by root parasitic plants of Orobanche and Striga spp. References Mangnus & Zwanenburg (1992) Tentative molecular mechanism for germination stimulation of Striga

and Orobanche seeds by strigol and its synthetic analogs. Journal of Agricultural and Food Chemistry 40: 1066-1070

Awad et al. (2006) Characterization of strigolactones, germination stimulants for the root parasitic plants Striga and Orobanche, produced by maize, millet and sorghum. Plant Growth Regulation 48:221-227



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Natural germination stimulants as a lead for parasitic weed control

Zwanenburg, B.

Department of Organic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands

In the life cycle of the parasitic weeds Striga and Orobanche spp the germination of the weed seeds is triggered by a chemical signal present in the root exudates of the host plant. The natural germination stimulants belong to the family of strigolactones of which strigol 1, sorgolactone 2, orobanchol 3 and alectrol 4 are the best known ones. The proposed structure 4 for alectrol is not correct as was demonstrated by Mori et al. The newly proposed structure 5 for this stimulant will be presented and is based on spectral evidence. 1 2 3

4 5 6 The bioactiphore of these strigolactones has been identified. Their mode of action involves a nucleophilic reaction with the α,β - enone part of the molecule. On the basis of our key feature model a series of simple strigolactone analogs have been designed, synthesized and biologically tested. A highly potent stimulant is Nijmegen-1 (6). Germination stimulants can be used in the control of parasitic weeds using the concept of suicidal germination, i.e. soil treatment with stimulant prior to sowing of the seeds of the food crop. In absence of a host plant the germinated seeds will die. Successful field experiments with Nijmegen-1 in the control of Orobanche in tobacco will be reported.

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Radboud University Nijmegen Department of Organic Chemistry

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Striga hermonthica germination in response to various Sorghum

varieties

Nielsen1, A., T.H. Nielsen2 & J.C. Streibig2 1Cheminova P:O. Box 9, 7620 Lemvig, Denmark

2Department of Agricultural Sciences, KVL, Højbakkegaard Alle 13 2630 Taastrup, Denmark - [email protected]

S. hermonthica seeds germinate when exposed to root exudates from Sorghum. Development of resistant or tolerant varieties against S. hermonthica is difficult because multi-component effects of a complex nature govern the resistant mechanisms and when found has a low heritability (Rubiales et al. 2006). The resistance depends on the ability of Striga to germinate in close proximity of the root tip and subsequent penetration of the root and induction of haustoria and forming parasite-host xylem-xylem connections. Breeding for resistance has high priority but so far with limited results, probably owing to lack of sorghum germplasms that demonstrate post-attachment resistance to Striga. Gurney et al have identified a cultivar of rice exhibiting some post-attachment resistance to Striga (Gurney et al. 2006).

We exposed Striga seeds to exudates at various distances from sorghum root-cuts as described previously (Nielsen & Streibig 2004). The logit transformed germination of Striga in response to distance could be described by a straight line. The intercept defines germination at zero distance from root-cuts; and the slopes describe the concentration gradient of the exudates. The experiment was done on 2, 3 and 4 week old Sorghum. Old plant roots initiated much lower germination of Striga than did younger plants. The regression slopes were negative indicating an effect of exudate concentrations; and the regression intercepts decreased with sorghum age.

Together with Agnes Rimondo at Natural Products Utilization Research Unit, USDA, ARS, Oxford, MS USA (Czarnota et al. 2003; Rimando et al. 2003), we are currently making bioassay directed isolation of fractions of crude exudates from Sorghum roots with thin layer chromatography (TLC) (Wegmann, 2003 Personal Communication).There were distinct differences in Striga germination in response to the compounds isolated by TLC. References Czarnota et al. (2003) Evaluation of root exudates of seven sorghum accessions. Journal of Chemical

Ecology 29, 2073-2083. Gurney et al. (2006) A novel form of resistance in rice to the angiosperm parasite Striga hermonthica.

New Phytologist 169, 199-208. Nielsen & Streibig (2004) Root exudates from Sorghum responsible for Striga hermonthica (Del.) Benth

germination. Cost Action 849, Parasitic Plant Management in Sustainable Agriculture, Naples October 29-31 , 10.

Rimando et al. (2003) PSII inhibitory activity of resorcinolic lipids from Sorghum bicolor. Journal of Natural Products 66, 42-45.

Rubiales et al. (2006) Screening techniques and sources of resistance against parasitic weeds in grain legumes. Euphytica 147, 187-199.



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Root exudates compounds of Sorghum bicolor varieties inducing

germination of Striga hermonthica

Nielsen1, T.H., A.M. Rimando2, J.C. Streibig1 & V. Leth1 1Department of Agricultural Sciences, KVL, Thorvaldsensvej 40, DK-1871 Frederiksberg,

Denmark 2Natural Products Utilization Research Unit, Agricultural Research Service, U.S.D.A., P.O. Box

8048, MS38677 Root stimulants of a potential host plant is needed for S. hermonthica to germinate. When screening for S. bicolor varieties resistant to S. hermonthica a low stimulant production is considered as a resistant trait (Kroschel 2001; Berner et al. 1997; Rao 1985).

Thin Layer Chromatography (TLC) may have a potential as a method to screen for resistant S. bicolor varieties. It is a low cost technology that does not demands expensive and well equipped laboratories. Therefore it also has the potential as a screening method in developing countries.

Screening for resistant varieties has previously been carried out using the crude root exudates of the hosts as the germination agents (Berner et al., 1997). In order to obtain information about the composition of the root exudates of sorghum, we used TLC, a method which makes separation and isolation of the individual stimulants possible.

The main objective of this study is to develop a TLC screening method for Striga germination stimulating compounds found in the root exudates of “resistant” and susceptible varieties of S. bicolour. By this method we were able to distinguish resistant from susceptible sorghum varieties.

References Berner et al., (1997) Striga Research Methods – A manual. 2 edition. International Institute of Tropical

Agriculture. Internet<URL:http://www.iitaorg/info/striga.pdf>. Kroschel (2001) A Technical Manual for Parasitic Weed Research and Extension. Kluwer Academic

Publishers, Dordrecht, pp. 1-6, 36-39, 91-92. Rao (1985) Techniques for Screening Sorghums for Resistance to Striga: Information Bulletin No 20.

International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), pp. 1-16.



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Orobanche especies specific responses to Trigonella foenum-graecum

root exudates

Fernández-Aparicio1, M., A. Andolfi2, A. Evidente2 & D. Rubiales1. 1Instituto Agricultura Sostenible, CSIC, Apdo. 4084, 14080 Córdoba, Spain

2Universitá Napoli Federico II, Via Universitá 100, 80055 Portici, Napoli, Italy

Fenugreek (Trigonella foenum-graecum L.) is an erect annual legume with a long history of cultivation both for culinary and medicinal uses. It was one of the spices the Egyptians used for embalming, and the Greeks and Romans used it for cattle fodder. It is still particularly popular in North African countries but also in India and USA. The steroidal saponins account for many of the beneficial effects of fenugreek, particularly the inhibition of cholesterol absorption and synthesis. The seeds are rich in dietary fiber, which may be the main reason they can lower blood sugar levels in diabetes.

Some studies have suggested that intercropping fenugreek with faba bean can reduce Orobanche crenata infection. The objectives of the present work were to study allelopathic effects of fenugreek root exudates on Orobanche spp. germination.

Seedlings of fenugreek were grown during two days on hydroponic sterile deionizated water and the root exudate dissolved in the water substrate was collected and dry cooled. The resulting dry powder was extracted with ethyl acetate and the resulting extract was fractioned using a silica gel chromatography column using as a solvent chloroform and isopropanol in the ratio 95:5. As a result, 9 groups of homogeneous combined fractions were obtained. The stimulatory effect of the root exudate, the extract and the residue of the fraction groups was tested in vitro on seeds of O. crenata, O. foetida and O. ramosa. Both the root exudate and the extract inhibited O. crenata germination but stimulated O. ramosa and O. foetida. Two fractions showed inhibitory activity on O. crenata germination, whereas one fraction stimulated germination. None of the fractions inhibited O. ramosa or O. foetida germination. However, three fractions stimulated O. ramosa germination and two stimulated O. foetida germination. This is most relevant as the germination of O. foetida is atypical, not responding to GR24, generally believed to be an universal germination stimulant.

The main metabolite isolated in pure form from these fractions, showing the inhibition of O. crenata was submitted to spectroscopic investigations (essentially 1H and 13C 1D and 2D-NMR, MS) to determine its stereostructure. Preliminary data suggested the presence of a polycyclic strongly oxygenated carbon skeleton in the structure of this metabolite.



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Does the germination stimulant for O. cumana differ from

strigolactones?

Chaudhuri1, S.K., D.M. Joel2 & J.C. Steffens1 1Department of Plant Breeding and Biometry, Cornell University, Ithaca, U.S.A.

2Department of Plant Pathology and Weed Research, ARO, Newe-Ya'ar Research Center, 30095 Ramat-Yishay, Israel

Germination of obligate root parasites of the genus Orobanche depends on chemical stimuli from roots of potential hosts. A series of natural germination stimulants, collectively termed strigolactones, stimulate the germination of weedy Orobanche species, but do not the elicit germination of O. cumana, a specific parasite of sunflower. In the present study we identified the natural germination stimulant from root exudates of cultivated sunflower by bioassay-driven purification.

Petroleum ether extracts of cultivated sunflower root exudates were purified on Si gel column chromatography followed by preparative TLC and finally HPLC, isolating the germination stimulant for O. cumana. The most active fraction, induced half-maximal germination of O. cumana seeds at the very low concentration of 5.99 x 10-6 mg/ml.

The chemical structure of the stimulant, elucidated by 1H and 13C NMR, including two-dimensional (1H-1H COSY, HMQC and HMBC)-NMR, GC/FT-IR, and GC/MS, was found to differ from strigolactones, and was identified as dehydrocostus lactone (DCL), which is known from plant tissues of various other members of the Asteraceae.

Whereas O. cumana seeds germinate after exposure to low concentrations of DCL, the germination of O. aegyptiaca seeds with these stimulant concentrations do not exceed the spontaneous germination that is induced by water alone, which clearly indicates the specificity of DCL as a stimulant of O. cumana.

Similar compounds have previously been shown to induce O. cumana germination, but this is the first indication of a guaiane skeletal sesquiterpene lactone that is exuded by the root and induces the germination of an Orobanche species.



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Initial identification of tomato root stimulants inducing Orobanche

seed germination

Goldwasser1, Y., T. Yasaku2, Y. Takeuchi2, T. Yokota3 & K. Yoneyama2 1R.H. Smith Institute of Plant Sciences & Genetics in Agriculture Faculty of Agriculture, Food

& Environmental Sciences, The Hebrew University of Jerusalem Rehovot 76100, Israel. [email protected]

2Weed Science Center, Utsunomiya University, Japan. 3Department of Biosciences, Teikyo University, Japan.

Tomato (Solanum lycopersicum) is highly susceptible to the root parasites Orobanche ramosa and Orobanche aegyptiaca. Tomato fields infested by Orobanche have been reported in The Middle East, North Africa, Southern and Eastern Europe, Australia, North Central and South America. In the Mediterranean region infestations have spread rapidly and cause extreme tomato yield and quality losses, and in many cases endanger the continuation of tomato crop production.

Germination of Orobanche seeds is induced only after specific environmental conditions and their exposure to stimulants in exudates produced by plant roots. In recent years a group of strigol-related natural stimulants from plant root exudates that induce parasitic weed seed germination have been identified and termed as strigolactones. Strigolactones are abundant in plants from a wide range of botanical families, hosts and non-hosts of parasitic plants. Furthermore, these stimulants have recently been identified as the arbuscular mycorrhizal fungi branching signaling molecules produced by host plant roots. Elucidation of the specific factors determining parasitic plants seed germination is an important factor for understanding the initial steps of parasitism and for developing control measures based on manipulation of this process. The aim of this research was to determine the stimulants produced by tomato roots that cause induction of Orobanche seed germination.

Tomato seedlings (cv. 'Sekai Ichi' and cv. 'M-82') were grown hydroponically in containers containing periodically replenished tap water or liquid growth medium, and their root exudates were collected daily or absorbed on activated charcoal cartridges. Collected growth medium was extracted with ethyl acetate, purified and submitted to GC/MS analysis. Exudates collected on activated charcoal were extracted with ethyl acetate and concentrated in vacuo. The tomato root exudate crude extracts were analyzed using gas chromatography-mass spectrometry, or high performance liquid chromatography connected to tandem mass spectrometry. All chemical analyses were coupled with parallel Orobanche seed germination bioassays.

In preliminary studies with cv. 'Sekai Ichi', reversed phase and phenyl column HPLC and GC/MS analysis of the root exudates revealed the presence of at least 7 biologically active strigolactones. LC/MS/MS analyses of cv. 'M-82', extracts revealed the presence of orobanchol, dehydro-strigol and tetradehydro-strigol isomers.

These results reveal strigolactones as O. minor and O. aegyptiaca germination stimulants. In these studies, orobanchol has been identified while other strigolactones require further precise identification.



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Potential use of Nijmegen-1 and smoke water solutions to deplete

Orobanche ramosa seed banks in Greece

Chachalis1, D. & A. Murdoch2 1 National Agricultural Research Foundation (NAGREF), Plant Protection Institute of Volos,

P.O. Box 1303, Fitoko, Volos 38001, Greece, [email protected] 2 Department of Agriculture, The University of Reading, Earley Gate, P.O. Box 237, Reading

RG6 6AR, UK. Laboratory and field experiments were conducted to study the effect of using stimulants to deplete Orobanche ramosa seed banks. This abstract describes the results of laboratory tests. At time of writing, field trials are still in progress and preliminary results are described. In laboratory conditions, seeds were preconditioned for one week on moist filter paper at 23ºC and then placed for germination at the same temperature. The synthetic germination stimulant Nijmegen-1 (NE-1) at four concentrations (10-4, 10-6, 10-8 and 10-10 mol/L of NE-1) was studied. Additionally, two types of smoke water (SW) solution (“Seed Starter” supplied by Kings Park & Botanic Garden, Perth, Australia and a comparable solution produced locally in Greece) were studied. The local smoke water was produced with methodology provided by the Kings Park utilising hay as a raw burning material. The smoke water concentrations tested were 100, 10, 1, 0.1 and 0.01% v/v aqueous solutions.

In the field experiment, five treatments (10-5, 10-6, 10-8 mol/L of NE-1 respectively, 1% v/v local SW, and 10-5 mol/L of NE-1+ 1% v/v local SW) were tested in a tobacco field. The field was initially uninfected with Orobanche and 500-1000 seeds of Orobanche ramosa seeds were placed at marked locations into which tobacco plants would be subsequently planting. The field site was kept moist for a week to allow the seeds to precondition after which the stimulant solutions were sprayed using a backpack sprayer at 93.5L/ha spray volume and 207 kPa of pressure. Plots were then kept moist for two weeks to allow seeds to germinate and die before transplanting tobacco to each location. Additionally, in each plot at least 100 seeds were placed in nylon mesh packets at 2.5 cm depth. These packets were exhumed and germination was recorded. The non-germinated seeds on exhumation were placed in Petri-dish (1wk, 23ºC) with distilled water for further estimation of germinability of seeds. In the laboratory germination tests, the NE-1 at concentration of 10-6 mol/Litre induced maximum 38% germination similar to the standard 10-6 mol/Litre GR24 stimulant. In contrast, lower and higher NE-1 concentrations exhibited much lower germination, at levels similar to water control (4% germination). The local SW showed higher stimulation (78% germination) at 1% v/v concentration than the “Seed Starter” SW (52%). At high concentrations, both SW strongly inhibited (less than 13%) germination. The “Seed Starter” SW at more diluted concentrations (0.1 and 0.01% v/v) exhibited a gradual decline of germination (45, 32% germination, respectively). In contrast, the local SW showed a 48% germination even at the lowest concentration (0.01%).

Depletion of O. ramosa seeds, as recorded in the exhumed seed packets, was well over 50% with the optimum local SW treatment compared to 38% with 10-6 mol/L NE-1. There was little difference between the other treatments (max. depletion 32%). Some caution should be exercised as germination was incomplete at the time of exhumation and was allowed to continue in petri dishes moistened with water. Germination of O. ramosa seeds in situ in the soil was ½ to 1/3 of the total seed germination.

The use of smoke and smoke water to stimulate germination in both laboratory and field is well-known for a wide range of species although very little work has been carried out on parasitic weeds. The potential of smoke water to deplete the Orobanche soil seed bank is therefore interesting and the implications of smoke water and other stimulants such as NE-1 to deplete O. ramosa seed banks before transplanting susceptible crops will be discussed.



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A multidisciplinary study for determining genetic diversity in

Orobanche cumana Wallr. populations from Spain

Pérez-Vich, B.1, M.L. Molinero-Ruiz1, J.M. Melero-Vara1, A.J. Pujadas-Salvà2, K. Stoyanov3, J.M. Fernández-Martínez1, L. Velasco1

1 Instituto de Agricultura Sostenible, CSIC, Apdo 4084, E-14080 Córdoba, Spain, [email protected]

2 Departamento de Ciencias y Recursos Agrícolas y Forestales, Universidad de Córdoba, Apdo 3048, E-14080, Córdoba, Spain

3 Department Botany and SOA Herbarium. Agricultural University of Plovdiv, 12 Mendeleev 4000 Plovdiv, Bulgaria

Sunflower (Helianthus annuus L.) is the only oilseed crop in Spain, playing a major role in agricultural systems in large regions such as Andalucía and Castilla-La Mancha. Sunflower future in these regions is severely threatened by the attack of sunflower broomrape (Orobanche cumana Wallr.). In the last fifteen years, efforts to introduce genetic sources of resistance in sunflower hybrids have been rapidly followed by the appearance of new races of the parasite that overcome all the known resistance genes. Due to this situation, most efforts of public and private research institutions have focused on the development and characterization of new sources of genetic resistance to the most virulent races. However, little is known about the genetic structure and variability of Orobanche cumana populations in Spain and their evolution, which is of paramount importance to develop long-term strategies for sunflower broomrape management.

The objective of this study is to investigate the genetic variability of the parasite Orobanche cumana in Spain, and the structure of its populations following different strategies: a) Evaluation and characterization of 39 broomrape populations collected on cultivated sunflower at different locations throughout major cultivation areas of Spain in the last 20 years. This will include both phenotypical and molecular characterization. b) Comparison of O. cumana populations collected in Spain with those from other countries in which broomrape is also attacking sunflower (e.g. Rumania, Bulgaria, Turkey, Israel, and others), and also with wild populations of this species collected along the Black Sea region of Bulgaria parasitizing wild compositae species (mainly Artemisia maritima group). c) Comparison of Spanish populations of O. cumana, exclusively found parasitizing cultivated sunflower, and its close relative O. cernua, found parasitizing Artemisia barrelieri, Artemisia campestris subsp. glutinosa and Launaea lanifera (Compositae).

These strategies will contribute to elucidate whether the populations of O. cumana

parasitizing sunflower in Spain, with wide variation for patogenicity, evolved in situ from a single introduction, or they are the result of a series of seed introductions. Comparison with wild populations and those from several countries will provide information on the possible origin of Spanish populations. Additionally, comparison between O. cumana and native O. cernua populations will cast light on possible gene flow between them. Knowledge on genetic structure and evolution of O. cumana populations will contribute to design more durable and sustainable strategies for sunflower broomrape control.



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Virulence of European populations of sunflower’s broomrape (O. cumana Wallr.)

Molinero-Ruiz, M.L. & J.M. Melero-Vara

Instituto Agricultura Sostenible, CSIC, Córdoba, Spain Broomrape (Orobanche cumana Wallr.) is among the most important disease of sunflower in countries such as Spain, Romania and Turkey. After the introduction of genes of resistance into the hybrids and the interaction with them, the parasite evolves to more virulent pathotypes (races). Races of O. cumana in Spain seem to be different to those described in Romania in the 80’s. Besides, the virulence of populations of O. cumana from Turkey is yet unknown. Nowadays race E, and mainly race F, are the most frequent in Spain. Contrary to the case of lower virulence races, the resistance to race F is a quantitative character. At the same time, populations of this race with significantly different virulence have been reported. The objective of this work was the study of the virulence of populations of O. cumana from Spain, Romania and Turkey, as well as the evaluation of the efficacy of resistant public lines commonly used in breeding programs. Sixteen populations of O. cumana were selected: eight from Spain (S1 to S8), two from Romania (R1 and R2) and six from Turkey (T1 to T6). They were inoculated to sunflower lines B117 (susceptible to all races), NR5 (resistant to race E and susceptible to F), L86 (susceptible to E and resistant to F) and P96 (resistant to both E and F). Seeds were sown in soil artificially infested with broomrape seeds and sunflower plants were grown in glasshouse for 14 weeks. Four sequential experiments with a completely randomised factorial design were established. Evaluation of emerged broomrapes was performed weekly until physiological maturity of sunflower plants. The virulence of the populations was determined through the Area Under the Disease Progress Curve (AUDPC), which was analyzed with ANOVAs, LSD tests and orthogonal contrasts. Population R1 from Romania was characterized as race E, since it did not cause disease on NR5. Population R2 was significantly controlled by the resistance in both L86 and P96, but not in NR5. Thus, it was determined as race F. In the case of populations from Spain, S2 was race E: it did not cause disease in NR5 nor in P96, but was pathogenic to L86 and B117. The remaining five populations were race F, since they were significantly more virulent on NR5 than on L86 and P96. Population S3 was the most aggressive of them. In some cases, the severity of the disease on NR5 was significantly higher than that on B117, suggesting the selection of genotypes of O. cumana after the use of the gene of resistance Or5. Four of the populations from Turkey (T4 to T6) overcame the resistance of P96 and were identified as race G. Population T1 was race E, since it caused significantly more disease to L86 than to NR5. The number of emerged broomrapes of population T2 was significantly higher in NR5 than in L86. Thus, this population was characterized as race F. The severity of the disease caused in NR5 by populations from Turkey was similar or lower than that in the susceptible line B117. This might be due to the diversity within them, since they have not been selected with the use of the gene of resistance Or5 (in NR5).



16

Resistance and the development of virulent Orobanche cumana races in

sunflower crop in Romania

Pacureanu-Joita1 M., E. Procopovici2 & S. Raranciuc1 1National Agricultural Research and Development Institute Fundulea, Romania

2Agricultural Research and Development Station Valul lui Traian, Constanta, Romania [email protected]

Sunflower production in south-east European and Mediterranean regions is hampered by the root parasitic weed Orobanche cumana Wallr., which may cause serious yield losses (Vranceanu 2000). It is well known that the control of this parasite is extremely difficult, breeding for resistant cultivars being the most effective method. For a long time, there were two broomrape (Orobanche cumana) races (A and B) present in the areas cultivated with sunflower (Burlov & Kostiuk 1976). Vranceanu et al. (1980) indentified five physiological races (A-E) of O. cumana in southeastern Romania, differentiating the five genes associated with the resistance to the five races (Or1-Or5). From the early nineties, virulent populations of broomrape (O. cumana) belonging to race F have overcome all resistance genes Or1 to Or5 in the cultivated sunflower (Helianthus annuus) and have spread rapidly in Spain, Romania and Turkey (Dominguez 1996; Pacureanu-Joita 1998). More recently, a new race designated race G, overcoming race F resistant lines, has been identified in south Spain. Different resistant sunflower cultivars (hybrids) have been obtained by the breeders working in the research institutes or private companies, these hybrids being cultivated in the last years in a large area infested with broomrape.

In 2006 year, in the infested area with O. cumana, cultivated with sunflower in Romania, most of the commercial hybrids lost their resistance to this parasite, some of them being attacked 80-100%, some others, 10-50%. The same behaviour have had some of these hybrids in the cultivated area with sunflower in Russia. The differentiated sunflower lines for different broomrape races, placed in two research stations, Braila and Valul lui Traian – Constanta, have showed different behaviour comparing with the last year, 2005. Some of them showed full resistance, some others segregated. The restorer line O-5548-2b was resistant.

Has the parasite developed a new race in the cultivated area with sunflower and infested with broomrape in Romania or in Russia? Will it be possible to control this new virulence of the parasite by the resistant sunflower genotypes? References Burlov & Kostiuk (1976). Proc. 7-th Intern Sunflower Conf., Krasnodar, U.S.S.R., Vol. I, 322-326. Domínguez (1996). Plant Breeding, 115(3): 203-204. Pacureanu-Joita et al. (1998). Proc. 2-nd Balkan Symp. on Field Crops. Novi Sad, Yugoslavia, Vol.I:

153-155. Vranceanu (2000). Floarea-soarelui hibrida. Edit.Ceres, Bucuresti.



17

New records of broomrape (Orobanche spp.) in Greek flora Kotoula-Syka1, E. and G. Economou2

1Democritus University of Thrace 2Agricultural University of Athens

In Greece the main crops severely affected by broomrapes are tomato, tobacco and to a lower extent - sunflower, beans, pea, chickpea and carrots. Most areas where these crops are grown have been infected by broomrape to a varying and increasing degrees, suffering considerable yield losses. Several control methods have been used to control this parasite. However all the methods are criticized since the parasite still endangers many cultivated regions in Greece. Recently, during surveys conducted in the Aegean Islands, new hosts parasitized by broomrape were discovered. In the islands Chios, Rhodos, Ikaria and Serifos we identified almond trees severely infested by Orobanche as well as broomrape infested wild oat (Avena sterilis) in the island Serifos. In Attiki region (South Greece), the weed Chrysanthemum segetum has been found infested by Orobanche, as well as the ornamental plant Mesybrianthemun spp. These new Orobanche hosts reported here for the first time, indicate that the problem is continue spreading not only in crops but also in other plants. This survey is the first systematic approach to register all the Orobanche species that infest a wide variety of crops and wild plants in the flora of Greece.



18

Distribution of Orobanche species in Slovakia and possibilities of their biological

control

Cagáň L. & P. Tóth Slovak Agricultural University, Department of Plant Protection, A. Hlinku 2, 949 76 Nitra,

Slovak Republic, e-mails: [email protected], [email protected] During 2002 - 2006, the survey of distribution of wild broomrapes (Orobanche spp.), was done in Slovakia. Together 73 localities were checked and broomrapes occurred on 52 of them. The existence of six the most abundant broomrape species was revealed: Orobanche alba Stephan ex Willd. infected plants from the genus Thymus L., O. flava Mart. ex F. W. Schultz attacked Petasites spp. and rarely Tussilago farfara L., O. elatior Sutton parasitized exclusively Colymbada scabiosa (L.) Holub, and O. purpurea Achilea spp. For O. caryophyllacea J. E. Smith, Galium spp. and Asperula spp. served as hosts and O. lutea Baumg. occurred only rarely on Medicago falcata L. The most abundant species were O. alba (found at 22 localities) and O. flava (found at 27 localities). In the south of 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.

Together 13 insect species were found in association with broomrapes in Slovakia, Lygus spp. (Sternorrhyncha: Miridae), Myzus spp. (Sternorrhyncha: Aphididae), Hypera postica (Coleoptera: Curculionidae), Isomira murina (Coleoptera: Alleculidae), Celypha lacunana, C. rivulana, Cnephasia asseclana, Cn. genitalana, Cn. stephensiana, Argyrotaenia ljungiana (Lepidoptera: Tortricidae), Diaphora mendica (Lepidoptera: Noctuidae), Chyliza extenuata (Diptera: Psilidae) and Phytomyza orobanchia (Diptera: Agromyzidae). Besides of P. orobanchia, moth feeding within flowers and seeds of various broomrapes and their importance for sustainable pest management will be discussed.



19

Distribution of Cuscuta species in Slovakia and possibilities of their

biological control

Tóth P. and L. Cagáň Slovak Agricultural University, Department of Plant Protection, A. Hlinku 2, 949 76 Nitra,

Slovak Republic, e-mails: [email protected], [email protected] During 2002 - 2006, field surveys of dodders (Cuscuta spp.) occurred at cropland were done in Slovakia. From among 217 localities surveyed, 127 have been found infested by dodders. The existence of four dodder species was revealed: Cuscuta campestris Yuncker, infested vegetable crops (potato, sugar beet, alfalfa, lentil and tobacco) and variety of weeds, C. epithymum (L.) Murr., parasited exclusively on alfalfa and accidentally on weeds growing in this crop, C. europaea L. and C. lupuliformis Krocker occurred only at field margins, along rivers and roads, where Urtica dioica L. and Rubus spp. served as hosts. Dodders were distributed throughout south of Slovakia, with maximum occurrence in the western part of state. C. campestris was not found in cold climatic regions with altitude higher than 240 m, while C. epithymum was recorded up to 700 m and C. europaea up to 800 m a.s.l. The most important and damaging dodder species within agroecosystems of Slovakia were C. campestris and C. epithymum. Only invaded species is field dodder, C. campestris. So, we point out our research activities to the species. Besides the crops mentioned above as hosts for C. campestris, there was recorded also 20 weed species. Species from the genus Polygonum (Polygonaceae) were the most important and acted as a significant reservoir of field dodder in cropland. The presence of field dodder markedly reduced both quantity and quality of crop yields, above all of sugar beet in Slovakia. For example, weight of heavily infested beets was reduced from 21.6 to 37.4% and sugar content from 12.0 to 15.2%.

To determine possibilities of field dodder management in sustainable agriculture, we screened associated insect fauna. The species from four orders were found regularly feeding within dodder plants. These were aphids (Sternorrhyncha), bugs (Heteroptera), weevils (Coleoptera) and flies (Diptera). Aphids, above all Aphis fabae (Aphididae), bugs, Lygus rugilipennis (Miridae), and stem fly, Melanagromyza cuscutae (Agromyzidae), were locally common, but not harmful for field dodder. Weevils from the genus Smicronyx (Coleoptera: Curculionidae) were found to be the principal natural enemies of dodders in Slovakia. Larvae of Smicronyx spp. caused stem galls on C. campestris. Such damage prevents flowering and fruiting of field dodder vines. Together 877 Smicronyx specimens were reared from infested plants during the study. S. jungermanniae was the most abundant accounting up to 96% of the total weevils reared from field dodder galls. Possibilities of weevils for biological control of field dodder as well our scientific outputs during the project will be discussed.



20

Interaction between F. oxysporum f. sp. orthoceras and Fusarium solani - two Orobanche cumana biocontrol agents

Dor E., R. Lati & J. Hershenhorn

Dept. of Weed Research, Newe Ya'ar Research Center, ARO, Israel

Sunflower broomrape (Orobanche cumana) is the most destructive sunflower pest in Israel. Fusarium oxysporum f. sp. orthoceras (Foo) was isolated in Bulgaria from diseased inflorescences of O. cumana and is considered as a biocontrol agent of the parasite. Fusarium solani (Fs) was isolated in Israel from O. aegyptiaca and was found pathogenic to O. aegyptiaca and as a weak pathogen of O. cumana. Application of the two fungi together against sunflower broomrape significantly reduced the number and the weight of broomrapes per host plant and provided more effective control of the parasite than the combined effect of each of the fungi when applied alone (synergistic effect). The objective of this study was to clarify the interactions between Fs and Foo in the soil in order to understand the nature of this synergistic effect. Foo transformed with GFP gene and Fs marked with Gus gene were used in this study.

Both, Foo and Fs, were suppressed by the soil microflora in native soil where their developmental rate was lower than in sterile soil. On the other hand, Fs and Foo influenced the soilborn fungi, significantly reducing their soil population density. Foo and Fs were found to produce toxic compounds that inhibit the growth of other soil born fungi. PDA or PDB growth media supplemented with chloroform extract of growth medium in which Foo grew for 21 days and with ethyl acetate extract of Fs growth medium in which Fs grew for the same time, significantly inhibited the growth of some soil born fungi. Foo extract inhibited the growth of Macrophomina phaseolina and Rhizoctonia solani at concentration of 500 ppm. Fs extract was less active. Both, Fs and Foo, had a negative influence on each other when applied together to sterile soil. But in natural soil this negative effect was compensated by the positive effect of the inhibition of soil born fungi. No significant difference was found in Fs and Foo survival ability when applied together or alone to natural soil. It can be concluded that the synergistic effect between Foo and Fs is not due to the interaction between them in the saprophytic phase of their life cycle, but probably as a result of their interaction in pathogenic phase within the host plant.



21

A novel approach to parasitic weed control based on a key metabolic gene silencing in Orobanche aegyptiaca

R. Aly1, Hila Cholakh1, D.M. Joel1, B. Steinitz2, and A. Zelcer2 .

1Dept. of Weed Research, ARO, Newe-Yaar Research Center, Israel 2Dept. of Plant Genetics, ARO, The Volcani Center, Israel

The silencing approach has already been demonstrated as an effective control method against nematodes and viral disease in plants. Gene silencing provides plants with defence against various pathogens, and is a tool of immense importance for research on plant development. The introduction of double-stranded RNA (dsRNA) proved to be a powerful tool for suppressing gene expression through a process known as post-transcriptional gene silencing in plants. Gene silencing by RNA is characterized by intercellular transfer ability of the silencing agent, and by long distance systemic transport through the whole organism.

In our study we used the inverted repeat technique for gene silencing of Mannose 6-Phosphate Reductase (M6PR), a key-gene in Orobanche spp. in order to provide the host plant with resistance against the parasite. A gene construct fusing the key gene for silencing to the binary vector (pBin-19) was already transformed to tobacco and tomato host plants. By PCR and RT-PCR analysis, transgenic plants were proved to have a specific PCR fragment (286 bp) which was designated on the coding region of the O. aegyptiaca M6PR for silencing. Our results indicated that in-vitro production of small interfering RNAs (siRNAs) by introducing short double-stranded RNA molecules of the M6PR gene into O. aegyptiaca tubercles grown on tomato plants, facilitate suppression and degradation of the native M6PR mRNA, thereby reduction of total soluble solids (sugars) in the treated tubercles. Based on preliminary results, we observed that O. aegyptiaca shoots developed on transgenic tobacco plants expressing M6PR construct showed week vigour, unstable and tended to lay down as compared to normal vigour of Orobanche shoots grown on control plants.

According to our results, the pending strategy is superior to other methods in that it is effective, low cost of implementation for producers and safe for the environment.



22

Genetic variability among O. foetida populations collected in Morocco

Vaz Patto1, M.C., R. Díaz2, Z. Satovic3, B. Román4 & D. Rubiales5

1Instituto de Technologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal 2Colegio de Postgraduados, Campus Puebla, Puebla, Mexico

3Faculty of Agriculture, University of Zagreb, Zagreb, Croatia 4IFAPA, CIFA 'Alameda del Obispo', Córdoba, Spain

5CSIC-Instituto de Agricultura Sostenible, Córdoba, Spain Orobanche foetida Poir. parasitizes several herbaceous wild and cultivated legumes. It has been only considered an important threat, as agricultural parasite, on faba beans and common vetch in Tunisia. Very recently, O. foetida has been reported in Morocco also infecting common vetch. Better understanding of the evolution of this species is essential to determine the risks of appearance of a new race capable of parasitizing a particular crop where it was not observed before. Thus, the objectives of this study were to analyse molecular variability between and within different pathogenic populations of O. foetida collected in Morocco. Five populations of O. foetida were analysed by AFLP markers One population was collected from a cultivated host (Vicia sativa) while the rest was collected from wild plant species (three from Scorpiurus muricatus and one from wild Vicia sp. or Lotus sp.). Although an analysis of molecular variance (AMOVA) attributed most of the AFLP phenotypic diversity (86.25%) to differences among individuals within populations, significant φ-values among populations suggested the existence of population structure. Nevertheless, when populations were grouped according to host status (cultivated vs. wild), among-group variation was not significant. Principal coordinate analysis based on Dice's distance matrix showed clear separation of individuals belonging to population parasitizing wild Vicia sp. or Lotus sp. from the rest. The Bayesian model-based clustering method implemented in the program STRUCTURE allowed the identification of three gene pools. The gene pool A was predominant in populations infecting Scorpiurus muricatus while the gene pool B occurred exclusively in the population collected from wild Vicia sp. or Lotus sp. The gene pool C was mostly found in population parasitizing common vetch, but was also present, to a lesser extent, in two of three population infecting Scorpiurus muricatus. Observed partitioning of diversity among and within sampled populations could indicate that O. foetida is mixed-mating or outcrossing species. Newly detected O. foetida population infecting common vetch could not be unambiguously separated form populations infecting Scorpiurus muricatus implying the existence of a recent common ancestor and/or a high gene flow among them. Thus, it would seem that the parasite genotypes able to infect a cultivated species arose from naturally occurring O. foetida populations growing on wild hosts. Moreover, the results of the Bayesian analysis of underlying population structure could suggest that the host-induced differentiation process started only recently and is currently in action.



23

Application of RAPDs in identification of broomrapes collected in

rural habitats: the current state of the art

Lyra1, D., A. Katsiotis1 & G. Economou2 1Laboratory of Plant Breeding and Biometry

2Laboratory of Agronomy Agricultural University of Athens, Iera Odos 75, 11855 Athens Hellas

E-mail: [email protected]

Orobanche species are parasitic only to dicotyledonous plants, such as broad beans, tomato, sunflower, tobacco, cucumber, melon, cabbage, clover, carrot, eggplant and pea. The most troublesome species are: a) O. crenata, which mainly affects faba bean, pea and carrot, b) O. ramosa, which mainly parasitizes on tomato and tobacco, c) O. aegyptiaca, which mainly infests tobacco and d) O. cumana, which mainly affects sunflower. These species cause considerable losses to crop yields in the Mediterranean region and constitute an important problem to the farm areas planted with potential hosts in infested zones.

RAPD markers are often used to discriminate between closely related species or to detect variability within an Orobanche population (Paran, I. et al. 1997). Polymorphic amplification products were also found to be useful in distinguishing between species such as O. cumana, O. aegyptiaca, O. cernua (Joel, D. et al., 1998a).

A total of 191 specimens from 37 populations belonging to the forementioned Orobanche spp. were collected from naturally parasitized crops across Greece during the period 2002-2005. In particular, O. crenata populations were collected from infested faba bean, pea and carrot fields; O. ramosa was located in tomato and tobacco fields and O. aegyptiaca was retraced in tobacco fields.

Out of 63 primers that were screened for their amplification efficiency of the Orobanche genome, 12 primers gave robust and reproducible products and were selected for further analysis. Polymorphism among the three species was detected with all 12 primers. A total of 313 different bands were observed for the three species with an average of 26 bands per primer.

The matrix of the genetic distances and the RAPD derived phenograms clearly separated the three species: O. ramosa, O. aegyptiaca and O. crenata. The UPGMA method showed a good fit to the matrix on which it was based since the Mantel Test revealed a high and significant cophenetic correlation coefficient. The results indicated that populations were clustered according to their geographical origin. Although the O. ramosa populations analysed were grouped together, a slight divergence was found between the populations parasitizing on tomato from those on tobacco. This suggests host specificity that should be further investigated using more samples from each Orobanche population parasitizing tomato and tobacco. References Joel, D., Portnoy, V., Tzuri, G., Greenberg, R. and Katzir, N. (1998a) Molecular markers of the

identification of Orobanche species. In: Advance in Parasitic plant Research. Proceedings of the sixth International Symposium on Parasitic Weeds (eds. Moreno, M. T., Cubero, J. I., Berner, D., Joel, D. M., Musselman, L. J. and Parker, C.), 152-160. Consegeria des Agricultura y Pesca, Santa des Andelucia, Cordoba, Spain.

Paran, I., Gidoni, D. and Jacobsohn, R. (1997) Variation between and within broomrape (Orobanche) species revealed by RAPD markers. Heredity 78:68-74



24

Mechanisms of resistance to broomrape (Orobanche spp.): what have

we learned during the last five years?

Pérez-de-Luque, A. IFAPA, CIFA Alameda del Obispo, 14080 Córdoba, Apdo. 3092, Spain, [email protected]

Over 4,000 species of angiosperms are able to directly invade and parasitize other plants, but only very few of them are weedy and parasitize cultivated plants. Together with witchweeds (Striga spp.), and dodders (Cuscuta spp.), broomrapes (Orobanche spp.) affect important crops causing complete yield loses under severe infestations. Several control methods have been approached, but no one of them is completely satisfactory. Genetic resistance to parasitization remains as one of the most desirable components in a integrated control strategy. However, breeding for resistance is a difficult task. Many aspects of the host/parasite interaction remain unknown. Cytological and cytochemical analysis may provide valuable information about the mechanisms of resistance involved in the process leading to an incompatible interaction. Nevertheless, there are few detailed studies about this topic in the case of parasitic plants compared with other plant-pathogen interactions like fungi. These studies may help to understand the resistance phenomenon to manipulate resistance and to develop more successful breeding programs.

In general, two main reactions have been described in the literature associated with resistance and leading to incompatible interactions between the host and the parasitic plant. At the macroscopic level, a darkening of host tissues, what resembles a hypersensitive response (HR) has been described after invasion of various host plants by Orobanche spp. (Dörr et al. 1994; Goldwasser et al. 1997; Rubiales et al. 2003). Such darkening of host and/or parasite tissues around the point of attachment has been observed in incompatible interactions. These symptoms are more frequent in resistant than in susceptible accessions (Goldwasser et al. 1997; Rubiales et al. 2003; Pérez-de-Luque et al. 2005a; Echevarría-Zomeño et al. 2006). However, there is no conclusive evidence that a HR really occurs in these interactions (Rubiales et al. 2003; Pérez-de-Luque et al. 2005b) in a manner similar to that described for fungal attack (Heath 1999; Richael & Gilchrist 1999).

The death of Orobanche spp. tubercles after the first developmental stages has also been described as a common indicative of resistance from the host (Dörr et al. 1994; Labrousse et al. 2001; Zehhar et al. 2003; Pérez-de-Luque et al. 2005a). As in the previous case, the precise mechanism(s) of resistance has not been yet elucidated. It is clear, however, that the presence of gel or gum-like substances within host vessels is related with the death of Orobanche spp. tubercles (Labrousse et al. 2001; Zehhar et al. 2003; Pérez-de-Luque et al. 2005b). But the exact function of these gels remains unclear. The most feasible option might be that these gels occlude the host vessels, as in the case of vascular pathogens (Vander Molen et al. 1983; Beckman 2000), and do not allow nutrient flux between host and parasite (Labrousse et al. 2001; Pérez-de-Luque et al. 2005b; 2006). However, the possibility that the composition of those gels includes toxic compounds like phytoalexins cannot be discarded, and the role of this kind of compounds in resistance to Orobanche spp. has been previously described (Serghini et al. 2000).

Both defensive reactions can take place in either resistant or susceptible accessions, being the differences quantitative: a plant allows more or less parasite infection depending on the number of these defensive reactions found in the roots (Labrousse et al. 2001; Pérez-de-Luque et al. 2005a; Echevarría-Zomeño et al. 2006).

The next question is: what are the mechanisms of resistance responsible for these two commonly observed defensive reactions? Is there only one mechanism responsible for every one of both reactions, or on the contrary many different mechanisms can lead to the observation of the same reaction?. We will try to give some answers to these questions.



25

References Beckman (2000). Phenolic-storing cells: keys to programmed cell death and periderm formation in wilt

disease resistance and in general defence responses in plants? Physiological and Molecular Plant Pathology 57: 101-110.

Dörr et al. (1994). Resistance of Helianthus to Orobanche - histological and cytological studies. In: Pieterse AH, Verkleij JAC, ter Borg SJ, eds. Proc. 3rd International Workshop on Orobanche and Related Striga Research. Amsterdam, The Netherlands: Royal Tropical Institute, 276-289.

Echevarría-Zomeño et al. (2006). Pre-haustorial resistance to broomrape (Orobanche cumana) in sunflower (Helianthus annuus): cytochemical studies. Journal of Experimental Botany, in press.

Goldwasser et al. (1997). Variation in vetch (Vicia spp.) response to Orobanche aegyptiaca. Weed Science 45: 756-762.

Heath (1999). The enigmatic hypersensitive response: induction, execution, and role. Physiological and Molecular Plant Pathology 55: 1-3.

Labrousse et al. (2001). Several mechanisms are involved in resistance of Helianthus to Orobanche cumana Wallr. Annals of Botany 88: 859-868.

Pérez-de-Luque et al. (2005a). Resistance and avoidance against Orobanche crenata in pea (Pisum spp.) operate at different developmental stages of the parasite. Weed Research 45: 379-387.

Pérez-de-Luque et al. (2005b). Interaction between Orobanche crenata and its host legumes: Unsuccessful haustorial penetration and necrosis of the developing parasite. Annals of Botany 95: 935-942.

Pérez-de-Luque et al. (2006). Mucilage production during the incompatible interaction between Orobanche crenata and Vicia sativa. Journal of Experimental Botany 57: 931-942.

Richael & Gilchrist (1999). The hypersensitive response: A case of hold or fold? Physiological and Molecular Plant Pathology 55: 5-12.

Rubiales et al. (2003). Characterization of resistance in chickpea to crenate broomrape (Orobanche crenata). Weed Science 51: 702-707.

Vander Molen et al. (1983). Pathogen-induced vascular gels: Ethylene as a host intermediate. Physiologia Plantarum 59: 573-580.

Serghini et al. (2001). Sunflower (Helianthus annuus L.) response to broomrape (Orobanche cernua Loefl.) parasitism: induced synthesis and excretion of 7-hydroxylated simple coumarins. Journal of Experimental Botany 52: 2227-2234.

Zehhar et al. (2003). Study of resistance to Orobanche ramosa in host (oilseed rape and carrot) and non-host (maize) plants. European Journal of Plant Pathology 109: 75-82.



26

Selection for resistance and characterization of its mechanisms at

GPPV-Nantes during the Cost action 849.

Thalouarn, P. Université de Nantes, Nantes Atlantique Universités, Laboratoire de Biologie et Pathologie

Végétales, EA1157, 2 rue de la Houssinière, BP 92208, Nantes, F-44000 France. Search for resistant genotypes was undertaken in several crops: colza, sunflower, tomato and hemp. Interesting results were obtained thanks to different sources of genotypes: Recombinant Inbred Lines, Introgression Lines, doubled haploid lines and classical obtentions. Methodological aspects will be discussed together with future prospects linked to each type of resistance mechanism and genetic material.



27

Molecular analysis of sunflower resistance mechanisms to Orobanche

cumana

Delavault, P., A. de Zélicourt, P. Letousey & S. Thoiron Université de Nantes, Nantes Atlantique Universités, Laboratoire de Biologie et Pathologie

Végétales, EA1157, 2 rue de la Houssinière, BP 92208, Nantes, F-44000 France. Sunflower resistance to Orobanche cumana is characterized by a low number of parasite attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed during both early and late responses of susceptible (2603) and resistant (LR1) sunflowers infected by O. cumana. The expression patterns of eleven defense-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defense response against O. cumana than the susceptible one, involving preferentially marker genes of JA and SA pathways. Among them, def. (defensin), appeared to be characteristic of the LR1 sunflower resistance. However, no correlation was observed between the expression of these molecular markers and the JA and SA contents measured by GC/MS in both sunflower roots. In addition, three cDNAs, isolated by a suppression subtractive hybridization, were shown to be strongly induced only in the resistant sunflower eight days post-infection, when the first O. cumana attachments occurred. These genes putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase might be involved in detoxification of reactive oxygen species suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell wall modifications leading to parasite confinement were correlated to more intense callose depositions in the resistant genotype concomitant with the over-expression of the callose synthase cDNA HaGSL1.



28

Identification and molecular characterization of metabolic pathways

involved in O. ramosa development

Simier P., R. Draie & S. Thoiron Université de Nantes, Nantes Atlantique Universités, Laboratoire de Biologie et Pathologie

Végétales, EA1157, 2 rue de la Houssinière, BP 92208, Nantes, F-44000 France. Broomrape tubercles are connected to the vascular system of the crop plants, through direct phloem continuity (Joel and Losner-Goshen 1994; Dorr and Kollmann 1995), and successfully compete for water and organic compounds (photoassimilates). Consequently, the metabolic processes involved in the establishment of the strong sink potential of the parasite are crucial for broomrape development and probably issue some potential targets to control Orobanche.

For several years, our activities contribute significantly to a better knowledge of processes involved in the osmotic adjustment of the parasites (Simier et al 1994a), C and N fluxes from host plants to parasitic weeds (Pageau et al 1998, 2003), and C and N metabolism in the parasites (Fer et al 1993; Simier et al 1994b; Simier et al 1998a,b; Pageau et al 2000; Simier et al 2005, 2006). Using 14C as isotopic tracer of the organic compound transferred from crop plants to broomrape tubercle, Aber et al (1983) showed that sucrose is the main host-derived compound and is subjected to degradation in tubercle. This was confirmed later by Harloff and Wegmann (1987) showing that sucrose degradation is accompanied by hexose and mannitol accumulation in tubercle. Therefore, like some other parasitic weeds including Striga (Stewart et al 1984), Thesium (Fer et al 1993; Simier et al 1994b), the harmful Orobanche species including O. cumana, O. crenata and O. ramosa accumulate hexose and mannitol as major osmotic agents. While mannitol is absent from the major host crops, mannitol content reaches up to 8% and 20% of the dry matter in O. ramosa and O. cumana, respectively (Wegmann 1986; Harloff and Wegmann 1987; Delavault et al 2002).

Mannitol metabolism is partially characterized in several mannitol-producing parasitic weeds including Thesium humile (Simier et al 1994b), Striga hermonthica (Robert et al 1999), O. ramosa and O. crenata (Wegmann 1986; Harloff and Wegmann 1987,1993). Mannose 6-phosphate reductase (M6PR, EC 1.1.1.224) is shown to be specifically involved in mannitol synthesis and corresponds to the limiting enzyme of the pathway (Figure 1). Both M6PR activity and mannitol accumulation are not detected in the major host crops of Orobanche (Simier 1994b; Robert 1997). We succeeded in purifying M6PR (Robert et al 1999) and characterizing the unique and constitutive gene, OrM6PR encoding the O. ramosa M6PR (Delavault et al 2002). Therefore, we suggest that OrM6PR is a good candidate for the proposed silencing approach.

Sucrose cleavage in plants requires either sucrose synthase or invertases (Tang et al 1999). Invertases belong to multi-enzyme family and the multiple forms contained in plants can be distinguished on the basis of pH optimum, glycosylation state and subcellular location. Two acid invertases (glycoproteins) have been described, a soluble activity residing in the vacuole and a particulate form which is associated with the cell wall. The soluble neutral or alkaline invertase resides in the cytosol and is not glycosylated. Several studies of mutants or transgenic plants aimed to determine the involvement of either sucrose synthase or invertase in major processes (Tang et al 1996; Hajirezael et al 2003; Chourey et al 2006). These proteins were shown to be active in several important functions in plants (Roitsch and Gonzales 2004) including the supply of growing tissues with energy and carbon, the establishment of sucrose concentration gradient between source and sink tissues to aid sucrose transport, the regulation of the cell turgor for cell expansion, the control of sugar composition in storage organs, and the responses to environmental factors.



29

Escape and resistance to crenate broomrape (Orobanche crenata) in

Lathyrus cicera

Fernández-Aparicio1, M., J.C. Sillero2; F. Flores3 & D. Rubiales1

1 CSIC, Instituto de Agricultura Sostenible, Apdo. 4080, 14080 Córdoba 2 CIFA “Alameda del Obispo”–IFAPA-CICE, Apdo. 3092, 14080 Córdoba

3 Escuela Politécnica La Rábida, Universidad de Huelva, 21819 Palos de la Frontera Lathyrus cicera, commonly referred to as chickling pea, is grown mainly as stock feed both as fodder and grain. As a result of the little breeding effort invested in it compared to other legumes, L. cicera cultivation has shown a regressive pattern in many areas in recent decades, however, it is gaining interest as grain legume crops Mediterranean-type environments, particularly in southern Australia. Its reintroduction in Andalusia might be hampered by crenate broomrape (Orobanche crenata) infection. Reaction to O. crenata was evaluated in a collection of Spanish L. cicera germplasm under field conditions at Córdoba in two crop seasons. Infection was found to be highly influenced by host plant vigour and precocity. In order to discern escape due to precocity and true genetic resistance, a resistance index is proposed based on residuals from the regression of infection and precocity. This allowed the identification of accessions with a substantial level of escape, and others with true resistance.



30

Mutagenesis and haploidy as means for obtaining resistant tobacco forms to patasitic weed Orobanche ramosa L.

Slavov S. & R. Batchvarova

Laboratory of Phytopathology, AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria

[email protected]

The parasitic weed Orobanche ramosa causes severe damage to tobacco and tomato in Bulgaria. Introduction of resistant cultivars are the most effective control of the parasite. Unfortunately there are no resistant plant forms to broomrape in the species within family Solanaceae, including the range of tobacco species. The aim of this study was to obtain resistant tobacco forms to O. ramosa by application of chemical mutagenesis and haploidy. Tobacco seeds were treated with EMS ant then the plants were grown in soil under controlled conditions. Haploids were regenerated from anthers and tested for resistance to broomrape in greenhouse. The noninfected haploids were dihaploidized by direct organogenesis on MS medium. Only three plants were fertile and gave seeds. Their progenies were tested for resistance. Two of them showed increased tolerance when were grown in infested soil with the parasite seeds. The combination of mutagenesis and haploidy allows the expressions of recessive mutations resulted from mutagenesis and increase the chances for catching the tolerant to broomrape tobacco forms.



31

Genetic transformation as a tool for broomrape Control

Batchvarova R. & S. Slavov Laboratory of Phytopathology, AgroBioInstitute, 8 Dragan Tzankov Blvd., 1164 Sofia, Bulgaria

[email protected] Broomrape (Orobanche ramosa L.) is the most important parasitic plant that infects tobacco (Nicotiana tabacum L.) in Bulgaria. Chemical treatment of the soil is not effective and crop rotation is not acceptable to solve this problem because of the long viability period of Orobanche seeds in the soil. Application of herbicides in the field with herbicide resistant tobacco could be a successful tool for broomrape control.

Several tobacco cultivars were transformed with a mutant ahas3R gene for resistance to the herbicide chlorsulfuron (Glean®, DuPont) and the bar gene conferring resistance to the herbicide Basta. Transformed plants were selfed and the segregation of resistance was followed in the next generations. The efficiency of the herbicides for broomrape control was demonstrated in greenhouse and field trails. An Orobanche/tobacco growth system was used in order to prove the lethal effect of the herbicide to the attached broomrape plants.



32

The resistant mechanism of mutagenised tomato line

resistant to Orobanche spp.

Dor1, E., B. Alperin1, Y. Kapulnik1, S. Vininger2 & J. Hershenhorn1 1Dept. of Weed Research, Newe Ya’ar Research Center, ARO, Israel; 2Dept. of Vegetables and Natural Resources, ARO Beit-Dagan, Israel

In our ongoing effort to overcome the broomrape problem in tomato, we succeeded to develop a tomato line (ORT-10), resistant to Orobanche aegyptiaca by seed mutagenesis of the commercial tomato variety M-82. ORT-10 was found to be also resistant to O. ramosa and O. cernua.

Grafting ORT-10 and M-82 on themselves and one on another showed that the resistant stock provides almost complete resistance to the grafted plants against O. aegyptiaca regardless of the scion identity, while resistant scion grafted on sensitive stock provided partial resistance. The importance of the root system as an essential component in inducing resistance is well correlated with the results obtained in polyethylene bag tests. O. aegyptiaca seed germination induced by ORT-10 roots in polyethylene bags at temperature of 24-28o C was significantly lower than the germination induced by var. M-82 roots in the same system, while at 20o C, no difference in the germination rate induced by ORT-10 and M-82 could be observed. These results can explain the fact that the resistance of ORT-10 to O. aegyptiaca under field conditions was partially broken toward the end of growing season when lower temperature prevails. Growing ORT-10 plant in a pot surrounded with three M-82 plants turned the resistant plant to be sensitive.

ORT-10 and M-82 root extracts did not cause mortality of germinating broomrape seeds or necrosis of broomrape inflorescences.

It can be concluded that the resistance of ORT-10 to Orobanche spp. can be partially explained as a result of low broomrape germination stimulant production.



33

Laser Capture Microdissection (LCM): application to

study of gene expression in resistance to Orobanche ramosa in Medicago truncatula

Lozano-Baena1, M.D., K. Lindsey2, M.T. Moreno3, D. Rubiales1, A. Pérez-de-Luque3

1CSIC, Institute for Sustainable Agriculture, Apdo. 4084, 14080 Córdoba, Spain 2School of Biological and Biomedical Sciences, University of Durham, DH1 3LE, UK 3IFAPA-CICE (Junta de Andalucía), CIFA “Alameda del Obispo”, Área de Mejora y

Biotecnología, Apdo. 3092, 14080 Córdoba, Spain LCM is a technique by which individual cells can be isolated from tissue sections for different studies, mainly the profiling of gene expression. We use this method for RNA extraction of cells from infected tissues of Medicago truncatula with Orobanche ramosa, an important parasitic plant of legumes crops. Orobanche spp. are root parasitic angiosperms lacking in chlorophyll and totally dependent on their host for their supply of nutrients. We selected M. truncatula as a model plant in legumes to analyse the infection process and the defence mechanisms against the attack of this pathogen. O. ramosa is grown on genotypes of M. ramosa showing early or late resistance to this pathogen (accessions 196 and 53 respectively). At 25 days after inoculation, seedlings of O. ramosa are sampled at random with the corresponding attached parts of host roots. The most important problem with this method is to determine the optimal fixation process. LCM needs acceptable morphology of histological sections for isolate the correct cells but this requires fixations that affect the RNA integrity. The best process to obtain a good quality RNA needs fresh frozen tissues and cryosectioning. In our case, a brief ethanol:acetic fixation and paraffin inclusion seems the best option since our samples are mature plant cells with a big vacuole and the first process produce ice crystals in vacuoles that affect the cell morphology. Basically, in LCM technique the slide with tissue of interest is placed under an inverted microscope and the image is transferred to a computer screen. A film-coated cap is placed onto the tissue. We selected cells of interest on the basis of specific morphologic without stain. An infrared laser is pulsed at the selected region and heat generated determines the melting of the thermoplastic film from the cap with the tissue in correspondence to the demarcated region. Finally, the target region is selectively pulled away from the surrounding tissues when the cap is removed and samples are collected in a tube, now ready for molecular analysis. Previous work by other authors had analysed this interaction using the whole tissues. In this way, they make a dilution of the RNA of interest in the total plant RNA and amplified not only the RNA involved in their process but the normal RNA of plant metabolism. Plant process take place in a complex cellular environment and each cell has a unique transcriptome with differences in RNA types and levels. To understand the contribution of individual cells to the resistant process, we need efficient methods for isolating groups of specific cell types. However with this technique we try to be more specific to analyse the transcriptome of target cells involved in the pathogenic process because we select, for the first time, only the RNA of cell responding in this interaction and not all the tissue around this.



34

Precision agriculture and modeling- a novel approach in controlling

the root parasite Orobanche spp.

Eizenberg1, H., H. Hershenhorn1, T. Lande1, G. Achdari1, Y. Cohen2, V. Alchanatis2. & S. Me Tal3

1Department of Weed Research, Newe Ya'ar Research Center, ARO, Israel, [email protected]; 2The Institute of Agricultural Engineering, ARO, Israel;

3Advanced Agronomy, Israel

Site Specific Weed Management (SSWM) aims at adjusting management zones within heterogeneous fields, as a part of precision agriculture approach. This approach should lead to optimizing herbicide application rates and therefore lead to reducing the total amount of herbicides applied for Orobanche control. The state of the art in SSWM in Orobanche control is given in this abstract. Site Specific Weed Management for Orobanche control in certain crops may include:

a) Time thermal model for Orobanche development. b) Optimizing herbicide rates for chemical control of the parasite. c) Detecting the underground developmental stages of the parasite. d) Remote sensing means for spatial analysis of infestation. e)

a) Time thermal model for Orobanche development: Temperature is strongly related to the dynamics of Orobanche spp. parasitism on its hosts. In previous studies, we have described the relationship between temperature and the parasitism process of O. aegyptiaca, O. minor, and O. cumana, in tomato, red clover, and sunflower, respectively. A Growing Degree Days (GDD) model was developed to predict Orobanche parasitism in these crops. b) Means for chemical control of the parasite in certain crops and optimizing herbicide rate: Chemical control of Orobanche was achieved in several crops. In tomato, red clover, and sunflower, the parasite is effectively controlled with ALS inhibitors herbicides such as sulfosulfuron, imazamox and imazapic. Optimizing chemical control is achieved when a minimum herbicide rates are applied to the host in the most susceptible stage of the parasite. Herbicide application timing is based on the GDD model. c) In-situ technology for detecting the underground developmental stages of the parasite: The Minirhizotron provides a novel technology and facilitates the in-situ study of major aspects of the host-parasite interaction and of parasite suppression, such as parasitism dynamics, parasite growth rate, and the effect of chemical treatments on the parasite. This methodology is a non-destructive tool for detecting and monitoring parasitism over time. The minirhizotron technology is essential for verifying the suggested models and making proper decisions referring to herbicide application timing. d) Using a remote sensing for developing a spatial model for parasite infestation: The remote sensing approach was used for detecting O. aegyptiaca flowering in tomato fields. Satellite and air photos in the Infra red (NIR) and RGB ranges were acquired for O. aegyptiaca infected tomato fields Validation of Orobanche infection was made by grid sampling. Preliminary results indicate that a partial correlation exists between the scouting and the RGB aerial images.

These four components are essential tools for developing a decision support system for SSWM.



35

Integrated broomrape control – resistant lines, chemical and biological control and sanitation – can we combine them

together? Hershenhorn1, J., E. Dor1, B. Alperin1, R. Lati1, H. Eizenberg1, T. Lande1, G. Acdary1,

S. Graph2, Y. Kapulnik3 & S. Vininger3 1Dept. of Weed Research, Newe Ya’ar Research Center, ARO, Israel

2Extension Service, Ministry of Agriculture and Rural Development, Beit Dagan, Israel 3Dept. of Vegetables and Natural Resources, ARO, Beit-Dagan, Israel

In the last 5 years we have developed several approaches to control Egyptian broomrape in tomato. Those approaches include a. broomrape resistant tomato lines b. chemical control c. biological control d. sanitation. line (ORT-10), resistant to Orobanche aegyptiaca by seed mutagenesis of the commercial tomato variety M-82. ORT-10 was found to be also resistant to O. ramosa and O. cernua. Resistant lines - Resistance is considered as the preferred method for pest control. Since no genetic resources for broomrape resistance are know in tomato, we produced such genetic resistant background using fast neutron mutagenesis of M-82 seeds. Under natural field conditions with heavy broomrape seed infestation, the resistant line ORT-10 reduces the number of the broomrape infested plants by 75%. Low stimulant secretion is probably part of the resistant mechanism phenomena. Chemical control – Sulfosulfuron at a rate of 40 g. a.i./ha applied 2-3 times in two-week intervals starting two weeks after planting followed by sprinkler irrigation of 300 m3/ha delays the appearance of broomrape inflorescences above ground by 3-4 weeks. Such a delay prevents the damage caused to the yield but not the continuation of the field contamination with broomrape seeds. Addition of an imidazolinone herbicide application such as imazapic or imazamox 63-70 days after planting prevents almost completely broomrape shoot emergence and seed setting of inflorescences present in the field during herbicide application. Biological control - A synergized effect was found between Fusarium oxysporum f.sp. orthoceras and Fusarium solani on sunflower broomrape. Application of two fungi together to control sunflower broomrape was rusticated to the first weeks after application. Repeated applications are needed for adequate long season control. The development of fungal inocula application through drip irrigation system developed in Bari, Italy, opens new horizons in biological control methodology. Sanitary – Orobanche seed dispersal into new fields is mainly done with the combines and containers. Spraying the combines and combines with 10% solution of alkyl dimethyl benzyl ammonium chloride disinfected them successfully. In a broomrape integrated control approach two or more of the specified means together or in a sequence should be applied. The most promising combinations and their advantages will be discussed.



36

Effect of herbicides inhibiting amino acid biosynthesis on Cuscuta spp.

and Orobanche spp.

Rubin, B., L. Zygier, O. Korber & T. Nadler-Hassar

R H Smith Institute of Plant Sciences and Genetics in Agriculture. Faculty of Agricultural, Food and Environmental Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

([email protected])

Control of Cuscuta spp. (dodder) and Orobanche spp. (broomrape), non-specific holoparasites, is difficult. We explored the possibility to use transgenic herbicide resistant crops and herbicides that inhibit amino acid biosynthesis (AABI) to manage them. AABI herbicides were reported to affect the growth these parasites. Experiments with field dodder and Egyptian broomrape (O. aegyptiaca) and indicate that two key enzymes in the biosynthesis of amino acids (ALS, acetolactate synthase and EPSPS, 5-enolpyruvyl shikimate-3-phosphate synthase) are present in the parasite. Glyphosate inhibits the biosynthesis of aromatic amino acids by inhibition of EPSPS that participate in shikimic acid pathway, and thus causes accumulation of shikimic acid in sensitive plants. Sulfonylurea and imidazolinone, among other ALS inhibitors herbicide, inhibit the biosynthesis of branched amino acids. We have shown that C. campestris, C. gronovii and C. subenclusa seedlings growing without host plant are much more tolerant to AABI then the sorghum and transgenic glyphosate- and ALS-resistant canola and cotton; but were very sensitive to glufosinate. Greenhouse studies have shown that field dodder was unaffected by glufosinate while growing on glufosinate-resistant canola but glyphosate and imazamox inhibited the growth of the parasite growing on glyphosate and imidazolinone resistant (IMIR) canola. However, the parasite recovered after 3 weeks, suggesting that it is tolerant to these herbicides. Using 14C-amino acids and HPLC analyses of canola and dodder sap we confirmed the movement of amino acids from the host to the parasite. Being above-ground nonspecific holoparasite dodder is totally dependent on its host plant for water, assimilates and solutes. We postulated that AABI influence the transport of carbon and protein products from the host to the parasite via the phloem. Using dodder parasitizing a tobacco mutant expressing a green fluorescent protein (GFP) in the vascular tissue under the control of the Arabidopsis thaliana sucrose transporter (AtSUC2) promoter, we have shown that glyphosate inhibits the translocation of GFP and sucrose between the host and the parasite. The results support the hypothesis that glyphosate-treated field dodder is affected at least partially by the lack of assimilates and solutes from the host. These results suggest that the parasites confer the capacity to synthesize amino acids independently of the host plant, raising the question why dodder and broomrape need this enzymes particularly when assimilates and amino acids are provided by the host. The implications of these results on parasite management will be discussed.



37

Marine algae as a means for Orobanche biocontrol

Economou1, G. & D. Lyra2

1Laboratory of Agronomy 2Laboratory of Plant Breeding and Biometry

Agricultural University of Athens, Iera Odos 75, 11855 Athens Hellas E-mail: [email protected]

Broomrapes (Orobanche spp.) are obligate root parasites that cause severe damage in many important crops in Greece such as Solanaceae (tobacco and tomato), Fabaceae (faba beans) and Apiaceae (carrots). Although different control methods for broomrape have been proposed (i.e. cultivating methods, solarization, fumigation, biological and chemical control, etc.), recent research activities have been directed towards the use of natural or synthetic chemical stimulants in order to induce germination in absence of the host. This control strategy is known as the “suicidal germination” approach and has been extensively stated as a promising method for the control of the holoparasite. GR24, a synthetic strigol-analogue, has been proved the most reliable stimulant to evaluate the germinability of Orobanche seeds. Furthermore, the effect of growth regulators (i.e. cytokinins, gibberellins, auxins etc.) as germination stimulants has also been well documented. Based on this approach, the efficacy of a natural product, extracted from the alga Ascoplyllun nodosum (Algit Super), has been evaluated for the induction of germination of O. ramosa seeds.

Aqueous solutions of Algit Super were firstly evaluated for Orobanche ramosa seeds at the following concentrations: 2.5 v/v, 1.25 v/v, 0.313 v/v, 0.078 v/v, 0.019 v/v, 0.0048 v/v, 0.0012 v/v. The germination of Orobanche seeds showed a concentration dependant course, in a bell-shaped form, with a maximal germinability at 0.019 v/v, while higher and lower concentrations failed to trigger Orobanche seeds. The populations examined responded to Algit Super in a similar way compared to the reference stimulant GR24. Radicle lengths were also affected after Algit Super treatments and in some cases the values for radicle elongation were higher than those obtained from GR24 treatment.

A step forward was to assess the efficacy of Algit Super on O. ramosa, O. aegyptiaca and O. crenata seeds at the active dose of 0.019 v/v and at three incubation temperatures (18, 20 and 230C). The highest germinability was observed at 200C for all Orobanche species. However, O. ramosa seeds showed the greatest response, O. aegyptiaca seeds responded in lower level, while lower germinability was observed for O. crenata seeds. References Batchvarova et al. (1999). In vitro culture of Orobanche ramosa. Weed Research, 39(3):191-197 Joel et al. (1995). Germination of Weedy Root Parasites in Seed Development and Germination eds.J.

Kigel and G. Galili Kebreab & Murdoch (1999). A quantitative model for loss of primary dormancy and induction of

secondary imbibed seeds of Orobanche spp. Journal of Experimental Botany, 50:211-219.



38

Control of broomrape on tobacco crops in Romania

Jinga, V., H. Iliescu, V. Stanescu, & M. Gradila

Research-Development Institute for Plant Protection, Bucharest; e-mail: [email protected]; [email protected]

Broomrape is one of the most dangerous parasitic plants for many crops including tobacco. This plant causes losses both in yield and in tobacco quality. Tobacco is attacked by 5 broomrape species, among them the most spread and dangerous are Orobanche ramosa and Orobanche cumana. In order to diminish the negative effect of broomrape on tobacco yield, it were tested the chemical methods (herbicides, and effect of the diethyl sulphate).

Taking into account previous information about the use of herbicides in broomrape control, we have tested Roundup (glyphosate). The product contains 360 g of glyphosate per liter and its action consists of inhibition of amino – aromatic acids. It has no permanence in soil. The first effects of Roundup on broomrape can be seen at about 5 – 10 days after spraying as leaf welting. The complete effect of root destruction occurs after 20 – 30 days. The Roundup has reduced the parasite appearance in all experimental variants.

Taking into account the recent investigations to create some tobacco mutants with broomrape resistance we have used the diethyl sulphate, which, as compared to same other chemicals used up to now, increases the mutation frequency and their diversity 5, 10 up to 18 times. This product is an alkylating mutagen which inhibits the growing of broomrape roots both by affecting the processes at the anatomic structure of the root and the metabolism of tobacco plant.

The following concentrations were used in tobacco seed treatment: 0.05, 0.1, 0.2, 0.4% and the duration of the treatment was 12, 24 and 48 hours. The tested varieties were the tobacco forms with broad leaves Virginia 196 and Baragan 132. Effect of diethyl sulphate in tobacco seed treatment was performed at 200C. After treatment the seed was seeded in hotbed. The plants had a good evolution, diethyl sulphate exhibiting a stimulating effect. The emergence of the treated tobacco plants has occurred earlier (10 – 14 days) than the untreated control, which under cold and humidity conditions have emerged after 18 – 19 days. It is to note especially the Baragan 132 variants treated with 0.05%, 0.1% and 0.2% diethyl sulphate for 48 hours, which were at the cross phase when the untreated control was only emerging. The experiment was plotted in a field infested with broomrape (the trial plot in Urziceni, Calarasi district). The broomrape appearance has affected mainly the Virginia 196 and Baragan 132 controls, causing the plant dwarfing and early plant welting. In Virginia 196 in tobacco Baragan 132 variants with 0.05%, the broomrape is 70% controlled, and at higher concentrations (0.2 and 0.4%) broomrape does not appear. Conclusions

• Tobacco plants are attacked by 5 broomrape species. Among them the most dangerous are Orobanche ramosa and Orobanche cumana.

• The Roundup herbicide (glyphosate), when applied at 40 and 60 days from planting, has reduced the parasite appearance in all experimental variants.

• A second Roundup application at 60 days increases its concentration in tobacco roots and so it controls the broomrape plants which have later appeared.

• The use of diethyl sulphate in order to create broomrape – resistant mutants causes the limitation or inhibition of the broomrape infestation and stimulates the tobacco development.

• The diethyl sulphate in concentrations of 0.05 – 0.1% controls the broomrape at a level of 70% and the higher concentrations causes the full stop of parasite.



39

Chemical control of sunflower broomrape in Rumania by using imidazolinone

herbicides

Raranciuc, S. & M. Pacureanu-Joita Agricultural Research and Development Institute, N Titulescu, 1, Fundulea, Calarasi,

915200, Romania, [email protected] Herbicides based on imidazolinone are already known being efficient in control of broad spectrum of grass and broadleaf weeds and also parasitic weeds such as Orobanche sp. The first detection in USA of sunflower mutant gene for resistance to imidazolinone herbicide opened new prospects in Orobanche control.

Field experiments were conducted by Fundulea Research Institute in two consecutive years and three locations with a view to evaluate the herbicide Pulsar, based on imazamox, in controlling of sunflower broomrape by using a resistant hybrid to imidazolinone (commercial hybrid Rimisol). Three heribicide rates and four time of foliar application, in different sunflower stages have been tested. The herbicide efficacy in broomrape control was revealed by significant reduction in percentage of infested plants until 54 - 96 % depending on application time and rates. Based on the good experimental results in field trials, this herbicide was official registered in our country for controlling of Orobanche cumana on sunflower resistant hybrids by foliar treatment in the 6-8 pair of leaves stage. This strategy of control is highly recommended in the regions with severe infestation of Romania where the genetic strategy tends to loose their efficiency.

At present in our research institute has already started research activity for transfer of the mutant gene for imidazolinone resistance, in own sunflower germoplasm with the aim to develop commercial hybrids resistant to these herbicides.



40

In vitro experiments on the control of Orobanche ramosa L. with

glyphosate in tomato

Montemurro P., M. Fracchiolla & D. Caramia Dipartimento di Scienze delle Produzioni Vegetali – University of Bari

Via Amendola 165/A – 70037 Bari, [email protected]

Orobanche ramosa L. (broomrape) is a parasitic species that causes severe yield quality and quantity reductions in host plants. Particularly in tomato, yield losses can reach the 25% (Fracchiolla & Boari 2000) or even the 75% of total yield according to Hodosy (1981). The plant is able to produce a large amount of seeds that germinate in response to specific host root exudates; they form haustoria that are able to penetrate the root up to the vascular cylinder. In this way, this chlorophyll-lacking holoparasite can interfere with water and mineral intake in the host plant (Parker & Riches 1993). The control of broomrape is very difficult and any method (chemical, physical, biological, mechanical) is limited in effectiveness (Quasem 1998; Boari & Vurro 2004). Chemical control has been investigated in several host crops such as favabean (Miccolis & Montemurro 1999 ), potato (Goldwasser et al. 2001) and tomato (Foy et al. 1989; Nandula 1998).

Quasem (1998) evaluated, in glasshouse grown tomato, the activity of different doses of fifteen herbicides in controlling this species. Among the active ingredients tried, chlorsulfuron, pronamide and pendimethalin gave the best results. However, they were phytotoxic for tomato plants. Glyphosate showed good effectiveness and acceptable selectivity In field experiments (Montemurro et al. 2005). Treatments with the above mentioned herbicide reduced both the number of parasitized plants and the amount of shoots per plant. Particularly, good effectiveness was observed with 3 or 4 applications, regardless of the application rate.

The aim of this work was to found further confirmations on glyphosate effectiveness, under in vitro conditions. A “plastic-bag system” was adopted (Amsellem et al. 2001), using transparent plastic envelopes, A4 size. A sheet of microfiber glass filter (Whatman GF/A, 15 × 23 cm), wetted with distilled water, was put inside the bags and O. ramosa seeds (about 25 mg) were dispersed uniformly on the surface of the filter sheet. Four tomato plants, at the second-leaf stage, were placed in each bag, leaving shoots and leaves outside. Water and nutrient solution were added by a syringe through plastic pipes inserted into the bags. The plants, arranged as described above, were grown at a controlled temperature of 27 °C, supplying 10 hours of light.

Solutions (5.0 ml c.a. per plant) were sprayed on the leaves; four doses of glyphosate (22.5 – 45.0 – 90.0 and 135.0 mg of a.i. per litre of water, corresponding to 9 - 18 - 36 e 54 g ha-

1 of a.i. with a normal application water volume of 400 l ha-1) were used and each of them was applied 1 – 2 – 3 and 4 times. First treatment was made as soon as first tubercles were observed on the roots; the other treatments were planned with a two-weeks timing. Each 7 days, tubercles were counted and divided in “alive” or “dead”, in case they turned black. At the same time, phytotoxicity symptoms were assessed on plants, according to the EWRS scale ranging from 1(no symptoms) to 9 (dead plant).

A split-plot experimental design with four replications was used; all data were subjected to analysis of variance and Duncan’s test was performed to determine significant differences between means.

Data regarding the percentage of control are shown in table 1. Effects of the dose

The highest percentage of dead tubercles per plant was found with 90.0 and 135.0 g l-1 of active ingredient (90.0 % and 93.0 % respectively). These data were significantly higher than those obtained with 22.5 mg l-1 (76.8 %). Effects of number of treatments

One treatment gave the lowest percentage of dead tubercles (88.0 %).



41

Interaction between doses and number of treatments

Statistical analysis gave no significantly interaction between the two factors. Phytotoxicity

No phytotoxicity symptom was detected.

Table 1 – Effects of glyphosate doses and number of treatments on the broomrape tubercles.

Dead tubercles per tomato plant (%) Number of treatments (n.) Glyphosate rate

(mg l-1) 1 2 3 4

Mean (1)

22.5 37.0 91.1 86.4 93.0 76.8 b 45.0 83.3 94.6 90.0 95.5 88.0 ab 90.0 93.2 90.9 96.2 93.4 90.0 a

135.0 84.3 92.9 100.0 97.5 93.5 a Mean (1) 88.0 b 92.4 a 93.2 a 94.6 a

(1) Means along a row or column showing no letter in common differ significantly at 0.05 P. (Duncan Test).

Discussion

Glyphosate showed good effectiveness and acceptable tomato crop selectivity. All treatments reduced the amount of alive tubercles per plant; the herbicide was particularly effective when it was applied more than 1 time, with an application rate of 90.0 or 135.0 mg l-1.

Results obtained in vitro seam to confirm the trend found by Montemurro et al. (2005) under field conditions. References Amsellem et al. (2001). Isolation, identification and activity of mycoherbicidal pathogens from juvenile

broomrape plants. Biological Control 21: 274-284. Boari & Vurro (2004). Evaluation of Fusarium spp. and other fungi as biological control agents of

broomrape (Orobanche ramosa). Biological Control 30: 212–219. Foy et al. (1989). Recent approaches for chemical control of broomrape (Orobanche spp.). Rev. Weed Sci

4: 123-125. Fracchiolla & Boari (2000). Effetti dell'infestazione di Orobanche ramosa sulla produzione di pomodoro e

cavolfiore. Informatore Fitopatologico 2: 52-54. Goldwasser et al. (2001). Control of Orobanche aegyptiaca and O. ramosa in potato. Crop Protection, 20:

403-410. Hodosy (1981). Biological control of broomrape, Orobanche ramosa, a tomato parasite. In: Occurrence

and Adaptability of Fusarium species to Control Broomrape in Hungary, Zoldsegtermesztesi, Kutato Intezet Bulletinje, 1979/80, 14: 21–29.

Holm et al. (1997). World Weeds, Natural Histories and Distribution. Obligate parasitic weeds, Wiley, NY, pp. 511–530 (Chapter 61).

Linke et al. (1989). Orobanche Field Guide. F & T Mullerbader, Germany. Miccolis & Montemurro (1992). Imazaquin ed imazethapyr nel controllo dell'Orobanche crenata Forsk.

nella fava da orto. Atti Gior. Fitopat., 1992, Copanello (Cz) 21-21 Aprile 1992, 3: 71-78. Montemurro et al. (2005). Preliminary results on the control of Orobanche ramosa L. with glyphosate in

tomato. Proceedings of 13th European Weed Research Symposium (EWRS), Bari 19-23 Giugno. Nandula (1998). PhD-thesis, Virginia Polytechn. Just. And State University; 136 pp. Parker & Riches (1993). Parasitic Weeds of the World: Biology and Control. CAB International,

Wallingford. UK, pp. 332. Quasem (1998). Chemical control of branched broomrape (Orobanche ramosa) in glasshouse grown

tomato. Crop protection 17: 625-630. Vercesi (1983). Diserbanti e loro impiego. Ed. Agricole, pp. 491.



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Severity and control management of parasitic weeds in Cyprus

Vouzounis, N.

Agricultural Research Institute, Nicosia, Cyprus, [email protected]

Cuscuta and Orobanche are the most important parasitic weeds, infesting a variety of crops. Most widespread species of Cuscuta are C. campestris and C. monogyna. C. campestris attacks principally alfalfa and clover and plagues several important crops including potato, tomato, eggplant, onion, melon, watermelon and some aromatic plants. The estimated yield losses due to C. campestris averaged 10% in alfalfa, while the alfalfa area infested was estimated at 100% corresponding to 24% of the total alfalfa cultivated area. C. monogyna attacks grapes and occasionally citrus and olives. Although the vine- infested area is negligible (70 ha), total destruction of grapevines could be expected from heavy infestation of C. monogyna. Selective pre-emergence treatments against C. campestris can be carried out in many crops. For alfalfa, among other herbicides, trifluraline and pendimethaline were proved to be more effective, since they have longer soil persistence. Trifluraline and pendimethaline can also be used in many other crops. Propyzamide can be used in lettuce and endive, among other crops, while imazaquin and imazethapyr are mainly for legume crops. Effective control of C. monogyna in vineyards was achieved by keeping the vineyard free of annual and perennial weeds.

The genus Orobanche contains two economically important species, causing severe agricultural problems, i.e. O. crenata and O. ramosa. The infested by O. crenata faba bean area is estimated at 142 ha, which corresponds to 30% of the total faba bean area. O. ramosa attacks cabbages, potato, tomato, melon, watermelon and other crops. The total infested area of the above mentioned crops in Cyprus was estimated at 100 ha, which corresponds to 10% of the total cultivated area with the same crops.

Work at the Agricultural Research Institute has led to the formulation of strategies, which successfully controlled Orobanche in several vegetables. In broad beans, two sprays of glyphosate, at 45 to 90 g a.i.ha-1 controlled Orobanche effectively. In cabbage, spraying twice with glyphosate at 60 to 100 g a. i.ha-1 or imazaquin at 5 to 10 g a.i.ha-1 controlled the parasite. In celery, two applications of glyphosate at 40 to 50 g a.i.ha-1 controlled O. ramosa and allowed the celery heads to attain full size, while in tomato, eggplant, melon and watermelon Orobanche was completely controlled in the field by mulching the soil with black polyethylene sheeting on the day of transplanting. In greenhouse grown tomato, soil solarization for three and six weeks controlled Orobanche by 75% and 99%, respectively and also most annual weeds.

Work summarized in this short review indicates that the use of herbicides for some vegetable production crops can be readily replaced by soil solarization. In addition, covering the soil with black polyethylene just prior to planting vegetable seedlings gave the best results in reducing Orobanche infestation and increasing yield.



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Green manures in Tanzania: A Striga management technology whose time has come?

C.R. Riches

Natural Resources Institute, University of Greenwich, Chatham, Kent, ME4 4TB, UK

Soil fertility depletion contributes to low per capita food production and poverty in sub-Saharan Africa. Crop yields are no better now than 20 years ago. Indeed many communities in Tanzania have observed falling yields of rain-fed upland crops in living memory. Recent estimates from the International Fertiliser Development Center1 have drawn attention to the scale of plant nutrient loss through erosion and nutrient mining. While average fertilizer use in Tanzania was less than 2 kg ha-1 in 20022, loss of nitrogen, potassium and phosphorus was estimated at 61 kg ha-1, one of the highest annual rates in Africa. An associated problem, often indicative soil fertility decline in East and Southern Africa is increasing infestation of cereals by the parasitic witchweed Striga asiatica that thrives in nitrogen deficient soils. Research, district council extension and NGO staff have been working with farmers in upland rice producing areas of Kyela District, rice and maize growing villages in Matambo Division of the Ulugulu Mountains and with the maize-based system in Muheza district, Tanga Region since 2000 to identify low-cost approaches to increase agricultural productivity. Low crop vigour on Striga infested soils was identified as an important constraint by communities in each of these areas.

Farmer groups in four villages in Kyela tested legume-cereal rotations and achieved average rice yield increases in 2003 and 2004 of 1114 (108%) and 827 kg ha-1 (72%) respectively when planting after growing the green manure Crotalaria ochroleuca the previous year and an additional 119 kg ha-1 (18%) when planting rice in 2004 after pigeon pea. The disappointing result with pigeon pea was related to establishment of sparse plant populations. Knowledge of the use of legume-rice rotations was disseminated to a further five communities and farmer-managed rice crops monitored in 2005 showed mean yield gains of 791 (74%) and 487 kg ha-1 (46%) when planted after Crotalaria or pigeon pea compared to continuous rice. Following visits to Kyela by lead farmers and extension officers, farmer groups in Matambo initiated trials with the rotations for rice and maize. Across 16 sites in 2005 maize yield was increased by 180 and 153% following Crotalaria and pigeon pea respectively.

Few farmers in Muheza district apply fertilizer to maize. It is not available in village stores and farmers believe the rainfall in recent years has been too unreliable to justify such an investment. Farmers need low-cost fertility management practices in this difficult environment and tested maize in rotation with three green manures. Data from masika season maize crops on 20 farms in 2005 indicated average yield gains of 1105 kg (79%), 863 kg (62%) and 667 kg ha-1 (48%) following a one season break crop of Canavalia ensiformis, Mucuna pruriens and Crotalaria respectively. For an effective contribution to the nutrition of the subsequent maize crop at least 30 kg fertiliser equivalent nitrogen ha-1 is needed from a green manure3. Estimates of biomass dry matter, assumed to include 3% N, indicated that this was achieved at 92% of farms where Canavalia was grown, but at only 83 and 47% of sites for Mucuna and Crotalaria respectively in vuli 2004.

Green manures are nothing new, indeed there was a campaign some years ago calling on Tanzanian farmers to plant Crotalaria. What is different now that is leading to farmers to try green manures for themselves? Understanding how farmers see soil fertility challenges, involving them in the selection of “best bets” and identifying the knowledge that farmers need to adopt new methods successfully has been central to laying the foundations for up-scaling adoption. As a result of village seminars, exchange visits, field days, work with primary schools and radio programmes organised by researchers and extension there is increasing awareness of the use of green manures in the farming community. District council policy makers, councillors and field staff have also been involved from an early stage and encouraged to attend field days and workshops. As a result Kyela district council voted funds to extend training of lead farmers



44

to all villages in the district in 2006 after the project ended. There is a history of limited adoption of “package defined” soil fertility technologies in sub-Saharan Africa. Solutions clearly need to be context specific and it is therefore important to identify local circumstances that will favour adoption. In Kyela for example, farmers operate within a diversified livelihood system. With income from lowland rice, fishing, and a range of tree crops they are prepared to take land out of upland rice for a season to plant a green manure. Rice now finds a ready market with buyers from urban areas and for export in the region so providing farmers with the incentive to increase production. Ideally farmers would like to produce a grain crop every season. In Muheza farmers have started experimenting with relay-planting Crotalaria into maize at final weeding to avoid loosing a cereal season, as an adaptation of the original green manure rotational concept.

Bibliography 1 Henao, J. and Baanante, C. (2006) Agricultural production and soil nutrient mining in Africa:

Implications for resource conservation and policy development. Muscle Shoals, Alabama: International Fertiliser Development Center.

2 IFAD (2006) Tanzania Statistics: Rural Poverty Portal (www.ruralpovertyportal.org) 3 Gilbert, R A 1998 Undersowing green manures for soil fertility enhancement in the maize-

based cropping systems of Malawi. pp. 73-80, In: Soil fertility research for maize-based farming systems in Malawi and Zimbabwe. CIMMYT, Harare, Zimbabwe.



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Orobanche occurance and control in potatoes in Turkey

Demirkan, H.1, A. Uludag2, Y. Nemli1, S. Turkseven1, K. Kacan3 & F. Albay3

1Ege University, Plant Protection Department, Bornova, Izmir, Turkey [email protected] 2Agricultural Quarantine Direct., Liman Cad. No: 25, Alsancak, Izmir, Turkey [email protected]

3Bornova Plant Protection Research Institute, Bornova, Izmir, Turkey Potatoes are among important crops in Turkey. Although potatoes are host of Orobanche, it had not been recorded in potato fields in Turkey. Complaints about Orobanche infestation in potato fields was started in the 2000’s. A research program was initiated in 2003 to quantify Orobanche infestation in potatoes fields in the Aegean Region, one of the main potato producing regions of Turkey, and find out control methods proper for the region. Surveys that were carried out in 2003 and 2004 showed that Orobanche did not infest all over the region. Two species, O. ramosa L. and O. aegyptiaca Pers., were identified in mixed populations in half of the fields in the villages where Orobanche has been already determined. Potato varieties responded similarly to Orobanche infestation in pot experiments. Cabbage residues, chicken manure, cow manure and olive jift did not affect potato yield significantly although they affected the number of Orobanche emerged in 2004. Olive jift caused fitotoxicity at 30 t ha-1. Glyphosate was controlled Orobanche at both 24 and 72 g a.i. ha-1 rates; however, higher dose of glyphosate caused significant reduction in yield and lower rate did not cause any significant increase in yield. It is expected that Orobanche infestation will be widen in the region. Unfortunatelly current data are not good enough to establish an effective Orobanche control system in potatoes fields. Experiments have been going on and a new project will be effective next year aiming integrated control of Orobanche in potatoes using crop rotation, herbicides, allelopathic relations etc.



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Orobanche crenata control in Egypt

Emeran1, A.A., W.M. El-Rodeny2 & M. Fernández-Aparicio3

1Dept. Agric. Botany, Fac. Of Agric., Kafr El-Sheikh University,33516-Kafr El-Sheikh, Egypt 2Food Legumes Section, Field Crop Research Institute, Agricultural Research Center, Sakha,

Kafr El-Sheikh, Egypt 3Institute Sustainable Agriculture, CSIC, Apdo. 4084, 14080 Córdoba, Spain

Orobanche crenata is one of the major constrains limiting faba bean production in Egypt. The best methods to control Orobanche are preventive measures to avoid any spread from infested to non-infested areas. Second approach is the development of resistant cultivars. Recently, breeding program resulted in releasing three new cultivars (Giza 843, Misr 1 and Misr 2) having high level of resistance to Orobanche that were available to the Egyptian farmers.

Additional cultural measures have been tried in the same integrated control package, including 1) delayed sowing date to be from the 15th Nov. to 1st Dec.; 2) using early maturing varieties (Sakha 1 and Giza 716); 3) zero tillage; 4) intercropping; 5) rotation with trap and catch crops (berseem clover and flax); 6) irrigation treatments; 7) fertilizer application specially nitrogenous fertilizers; 8) soil solarization; 9) hand-wedding, and 10) a reduced rate of glyphosphate of 34 g a.i./ha combined with NPK (1:1:2 % applied twice at 60 and 75 days after planting).

Some of theses methods like crop rotation are not completely effective in eradicating broomrape (Orobanche sp.) because its seeds can remain viable for years. However, a reduction in seed bank in soil of Orobanche can be achieved especially when we used some trap crops in the rotation.

In the last two years, intercropping experiments represented promising results under our environments.

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