Dehydrocostus lactone is exuded from sunflower roots and stimulates germination of the root parasite Orobanche cumana

The germination of the obligate root parasites of the Orobanchaceae depends on the perception of chemical stimuli from host roots. Several compounds, collectively termed strigolactones, stimulate the germination of the various Orobanche species, but do not significantly elicit germination of Orobanche cumana, a specific parasite of sunflower.Phosphate starvation markedly decreased the stimulatory activity of sunflower root exudates toward O. cumana, and fluridone - an inhibitor of the carotenoid biosynthesis pathway - did not inhibit the production of the germination stimulant in both shoots and roots of young sunflower plants, indicating that the stimulant is not a strigolactone.We identified the natural germination stimulant from sunflower root exudates by bioassay-driven purification. Its chemical structure was elucidated as the guaianolide sesquiterpene lactone dehydrocostus lactone (DCL). Low DCL concentrations effectively stimulate the germination of O. cumana seeds but not of Phelipanche aegyptiaca (syn. Orobanche aegyptiaca). DCL and other sesquiterpene lactones were found in various plant organs, but were previously not known to be exuded to the rhizosphere where they can interact with other organisms.     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.     

Effects of brassinosteroids on conditioning and germination of clover broomrape (Orobanche minor) seeds

Brassinolide [2, 3, 22R,23R-tetrahydroxy-24S-methyl-B-homo-7-oxa-5--cholestan-6-one] and its related compounds, brassinosteroids, applied at the early stages of conditioning shortened the conditioning period required before clover broomrape seeds would germinate after exposure to germination stimulants, such as dl-strigol and natural stimulants from red clover (Trifolium pratense L.) root exudate. Brassinosteroids applied after conditioning increased the rate of the seed germination induced by these stimulants. The inhibitory effect of light on seed germination when it was induced by dl-strigol could be overcome by brassinosteroids. Brassinosteroids also eliminated the inhibitory effects of light and dl-strigol applied at the early stages of conditioning. GA₃ was also effective in causing seed conditioning and increased the rate of the germination induced by these stimulants. There was a relationship between brassinosteroids and GA₃ in many of the experiments conducted. These findings may have practical implications in increasing the effectiveness of applying germination stimulants in the field to soils for suicidal germination of broomrape seeds.     

Allelopathy of rice (Oryza sativa L.) root exudates and its relations with Orobanche cumana Wallr. and Orobanche minor Sm. germination

Allelopathy has been considered not only as an environmentally friendly approach for weed control but also a potential reason causing autotoxicity in crop production. In this study, the responses of seeds of lettuce, wheat, rice, clover broomrape (CB), and sunflower broomrape (SB) to the root exudates of rice cultivars were studied. Lettuce germination was promoted by root exudates of Yliangyou 3218 and I-Kung-Pao. Wheat seedling growth was inhibited by all nine rice species. I-Kung-Pao and Ganxin 203 exerted greater autotoxicity than other cultivars. Yongyou 15 and I-Kung-Pao induced the highest germination rate of CB, while Yongyou 13, Zhongzao 22, and I-Kung-Pao induced the highest germination rate of SB. A significantly correlation was noted between germination-inducing ability on broomrape seeds and allelopathic effects on target plants. It is suggested that using broomrape seeds germination is a better receptor for the identification of rice allelopathic potential.     

Germination stimulation in wild oats (Avena fatua L.) by synthetic strigol analogs and gibberellic acid

At concentrations of 0.01–1 mM, five synthetic multiring analogs of strigol were effective germination stimulants of intact and dehulled wild oat (Avena fatua L.) seeds. The effect was concentration-dependent and equaled or exceeded that produced by equimolar gibberellic acid (GA₃). The most effective strigol analog treatments induced 55–80% germination within 7 days in intact wild oat seeds and resulted in 63–86% germination and normal seedling growth over 14 days. Intact wild oat controls germinated 14% after 14 days. The stimulation of wild oat germination by these synthetic strigol analogs demonstrates that these compounds, initially developed as germination stimulants for the seeds of the parasitic weed, witchweed (Striga asiatica L. Kuntz.), have bioregulatory activity in dormant seeds of monocots, as well as dicots. None of the compounds tested significantly affected the germination of nondormant cultivated oat seeds (Avena sativa L.). The commonly used dispersal agent, Tween 20 (0.1%), was found to inhibit germination of cultivated oats, alone and in the presence of 2% acetone.Names of companies or commercial products are given solely for the purpose of providing specific information; their mention does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.     

Terpenoid precursors of strigol as seed germination stimulants of broomrape (Orobanche ramosa) and witchweed (Striga asiatica)

Strigol and some of its synthetic precursors and analogs are known to be germination stimulants for broomrape (Orobanche ramosa) and witchweed (Striga asiatica). Fifteen synthetic terpenoids, similar in structure to one of the four rings of the strigol molecule, were evaluated in two bioassays as seed germination stimulants with broomrape, and nine were found to be active. Five of the more active compounds contained ester groups. Whereas the study was intended primarily to evaluate forced germination of broomrape by aqueous solutions, the results are almost qualitatively identical for broomrape and witchweed. Monocyclic compounds with chemical structures similar to two of the rings of strigol have now been shown to possess significant bioactivity as germination stimulants.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.     

Effects of Fluridone and Norflurazon on Conditioning and Germination of Striga asiatica Seeds

Inhibitors of carotenoid biosynthesis are known to prevent abscisic acid (ABA) biosynthesis and to affect germination and dormancy of seeds in many plants. In this study, the effects of three carotenoid biosynthesis inhibitors, fluridone, norflurazon and diflufenican, on the conditioning and germination of Striga asiatica seeds were examined. Fluridone and norflurazon shortened the conditioning period required before S. asiatica seeds would germinate after exposure to the germination stimulant strigol, and prevented the inhibitory effects of both light and supraoptimal temperature (40 °C) on seed germination. In addition, treatment with fluridone or norflurazon after conditioning in water induced seed germination in a manner similar to the effect of natural germination stimulants. Moreover, the seedlings developing after conditioned with fluridone formed haustorium-like structures without the involvement of haustorium inducing factors. In contrast, diflufenican had no effect on the conditioning and germination of S. asiatica seeds. These results indicate that fluridone and norflurazon have various effects on the germination of S. asiatica seeds and might be available for control of root parasites.     

Reduced Germination of Orobanche cumana Seeds in the Presence of Arbuscular Mycorrhizal Fungi or Their Exudates

Broomrapes (Orobanche and Phelipanche spp) are parasitic plants responsible for important crop losses, and efficient procedures to control these pests are scarce. Biological control is one of the possible strategies to tackle these pests. Arbuscular Mycorrhizal (AM) fungi are widespread soil microorganisms that live symbiotically with the roots of most plant species, and they have already been tested on sorghum for their ability to reduce infestation by witchweeds, another kind of parasitic plants. In this work AM fungi were evaluated as potential biocontrol agents against Orobanche cumana, a broomrape species that specifically attacks sunflower. When inoculated simultaneously with O. cumana seeds, AM fungi could offer a moderate level of protection against the broomrape. Interestingly, this protection did not only rely on a reduced production of parasitic seed germination stimulants, as was proposed in previous studies. Rather, mycorrhizal root exudates had a negative impact on the germination of O. cumana induced by germination stimulants. A similar effect could be obtained with AM spore exudates, establishing the fungal origin of at least part of the active compounds. Together, our results demonstrate that AM fungi themselves can lead to a reduced rate of parasitic seed germination, in addition to possible effects mediated by the mycorrhizal plant. Combined with the other benefits of AM symbiosis, these effects make AM fungi an attractive option for biological control of O. cumana.     

Characterization of Strigolactones, Germination Stimulants for the Root Parasitic Plants Striga and Orobanche, Produced by Maize, Millet and Sorghum

The germination stimulants for root parasitic plants Striga and Orobanche produced by sorghum (Sorghum bicolor (L.) Moench), maize (Zea mays L.), and pearl millet (Pennisetum typhoideum Rich.) were examined. Characterization of strigolactones in the root exudates from the plants grown hydroponically was conducted by comparing retention times of germination stimulants on reverse phase high performance liquid chromatography (HPLC) with those of synthetic standards, and by using HPLC linked with tandem mass spectrometry (LC/MS/MS). All the plants tested, except for a sorghum cultivar Swarna, were found to exude two major stimulants, 5-deoxy-strigol, which is known as a branching factor for arbuscular mycorrhizal (AM) fungi, and an isomer of strigol, tentatively named sorghumol. Swarna was found to exude 5-deoxy-strigol and strigol. These results imply that 5-deoxy-strigol is one of major germination stimulants of gramineous plants and that major stimulants may differ even among cultivars within the same species.     

Germination stimulants produced by Vigna unguiculata Walp cv Saunders Upright

A germination stimulant, alectrol, for the seeds of the angiospermous root parasites Alectra vogelii Benth. and Striga gesnerioides (Willd.) Vatke has been isolated from root exudates of its genuine host plant Vigna unguiculata Walp cv Saunders Upright. Its spectroscopic data lead to a chemical structure closely related to (+)-strigol. The compound is more active than strigol in stimulating seeds of the respective parasites, Alectra vogelii and Striga gesnerioides.Abbreviations AV Alectra vogelii - OA Orobanche aegyptiaca - SA Striga asiatica - SG Striga gesnerioides - SH Striga hermonthica - TFA trifluoroacetic acid     

Effects of Temperature and dl-Strigol on Seed Conditioning and Germination of Witchweed (Striga asiatica)

Seed conditioning and germination in witchweed (Striga asiatica(L.) Kuntze) were temperature-dependent. With higher conditioningtemperatures, shorter conditioning time was required for germinationwith terminal dl-strigol (strigol) treatment at 30 °C. Maximumgermination (80–100%) was obtained by conditioning inwater at 20, 25, 30 and 35 °C for 14, 7, 5 and 3 d, respectively,and terminally treating with 10^–6 M strigol at 30 °C.Seeds conditioned in 10^–8 M strigol instead of water germinatedmuch less with the same terminal strigol treatment. Generally,conditioning was slower when seeds were conditioned in strigolrather than water. The reduction in germination rate by pretreatmentin strigol or pretreatment at low temperatures could be overcomeby increasing the terminal strigol concentration in the germinationtest. Conditioned seeds did not germinate at 10 and 15 °Cwith a terminal 10^–6 M strigol treatment but yielded closeto maximum germination at 25, 30 and 35 °C with the sameterminal strigol treatment. To obtain maximum germination, boththe minimum conditioning temperature and the minimum germinationtemperature for conditioned seeds were 20 °C. Factors suchas conditioning time, and strigol concentration and temperatureduring conditioning and/or germination determine whether seedsremain in the conditioning phase or shift to a germination phase. dl-Strigol, germination stimulation, parasitic plants, seed conditioning, seed germination, Striga asiatica, temperature, weed control     

Production of Strigolactones by Arabidopsis thaliana responsible for Orobanche aegyptiaca seed germination

The germination stimulants produced by Arabidopsis thaliana, a host of root parasitic plants Orobanche spp. but not of arbuscular mycorrhizal (AM) fungi were examined. Root exudates from the hydroponically grown A. thaliana plants were subjected to reverse phase high performance liquid chromatography (HPLC) and retention times of germination stimulants inducing O. aegyptiaca seed germination were compared with those of strigolactone standards. In addition, the root exudates were analyzed by using HPLC linked with tandem mass spectrometry (LC/MS/MS). A. thaliana was found to exude at least three different germination stimulants of which one was identified as orobanchol. This is the first report of strigolactone production by a non-mycotrophic plant. These results together with recent knowledge imply that strigolactones have other unrevealed functions in plant growth and development.     

A standardized bioassay for evaluation of potential germination stimulants for seeds of parasitic weeds

A standard bioassay for the evaluation of the biological activity of potential germination stimulants for seeds of parasitic weeds has been designed by carefully reconsidering all steps of a literature procedure. Addition of a surfactant to the solution for sterilization of the seeds gave a considerable reduction of contamination during the conditioning and stimulation process. Furthermore, it was shown that the water/seed ratio during conditioning affects the germination results. For the seed conditioning and stimulation process, the sandwich technique was introduced, whereby the seeds were placed between two layers of glass fiber filter paper discs. The standardized bioassay has been used for the evaluation of the stimulatory activity of numerous (new) synthetic analogues of strigol (the natural germination stimulant). It is essential to include a reference compound—GR24, an analogue of strigol, is recommended—in every test series, because it was observed that, although a standard bioassay was used, germination percentages obtained with GR24 solutions vary from test to test. For seeds of Striga hermonthica, seasonal effects were found in the germination percentages obtained with GR24 as stimulant. Test results with strigol analogues having modifications in ring D reveal that structural variations in this part of the molecule have dramatic effects on the biological activity of strigol analogues.     

Role of cAMP in Gibberellin Promotion of Seed Germination in <Emphasis Type="Italic">Orobanche minor</Emphasis> Smith

Adenosine 3′,5′-cyclic monophosphate (cAMP) is known as a key second messenger in many living organisms, regulating a wide range of cellular responses. In higher plants the function of cAMP is poorly understood. In this study, we examined the role of cAMP in seed germination of the root parasitic plant Orobanche minor whose seeds require preincubation in warm moist environments for several days, termed conditioning, prior to exposure to germination stimulants released from roots of host plants. Accumulation of endogenous cAMP was observed in the conditioned O. minor seeds. When the seeds were exposed to light or supraoptimal temperature during the conditioning period, cAMP did not accumulate and the seeds showed low germination rates after stimulation with strigol, a germination stimulant. Addition of membrane-permeable cAMP to the medium restored the germination rates of the seeds treated with light or supraoptimal temperature during the conditioning period, suggesting that cAMP functions during the conditioning period. The endogenous cAMP levels of the seeds conditioned in the light or at a supraoptimal temperature were elevated by treatment of the seeds with gibberellin (GA) during the conditioning period. Uniconazole, a potent inhibitor of GA biosynthesis, blocked elevation of the cAMP level. Furthermore, a correlation between the endogenous cAMP level and GA level was observed during the conditioning period. These results suggest that GAs elevate the cAMP level, which is required for the germination of O. minor seeds.     

Recognition of root exudates by seeds of broomrape (Orobanche and Phelipanche) species

Background and Aims

The long co-existence of broomrapes and their hosts within the same environment has culminated in a strong adaptation and effective parasitism. As a first step of specialization in the parasitic process, seed receptors of parasitic plant species vary in their ability to recognize compounds released by their hosts. This work aims to investigate potential patterns for the reception requirements needed to activate germination within Orobanche and Phelipanche species.

Methods

Induction of the germination of seeds of nine Orobanche and Pheliphanche species by root exudates of 41 plant species was studied and subjected to biplot multivariate analysis.

Key Results

A high level of specialization in root exudate recognition was found in Orobanche densiflora, O. gracilis and O. hederae, which germinated almost exclusively in contact with root exudates from the plants they infect in nature. At the opposite extreme, Phelipanche aegyptiaca, P. ramosa and O. minor were highly generalist, germinating when in contact with the root exudates of most plant species. Orobanche crenata, O. cumana and O. foetida showed intermediate behaviour.

Conclusions

A universal germination stimulant for all broomrape species has not being identified to date. The synthetic stimulant GR24 is active against most of the weedy broomrape species, but fails with the non-weedy species tested in this study and with the very recent weedy species O. foetida. In addition, germination behaviour of broomrape species depends on the crop plant tested. Weedy broomrapes with a broad host spectrum respond better to the different exudates released by a wide range of crops and wild species than do non-weedy broomrapes, which have a narrow host spectrum and are more restricted to their host range. Root exudates of many plant species were active in stimulating germination of seeds of Orobanche and Phelipanche species for which they are not described as hosts, representing interesting examples of potential trap crops.Key words: Xenognosis, broomrape, root exudate, germination, Orobanche, Phelipanche     

Sesame genotype influences growth and phenology of Phelipanche aegyptiaca

Egyptian broomrape (Phelipanche aegyptiaca) is a parasitic weed causing major yield loss in many field and vegetable crops. Recent observations in Iran suggest that sesame (Sesamum indicum) may serve either as a trap crop or as a host for broomrape, depending on genotype. The central objective of this research was to identify sesame genotypes that could act either as potential trap crops, or sources of genetic material for development of broomrape resistant cultivars. Seeds of 11 Iranian sesame genotypes (Ardestan, Esfarayen, Oltan, Ahvaz, Borazjan, Darab‐14, Ferdous, Kalat, Naz Tak Shakhe, Varamin and Yekta) and 10 foreign genotypes (S16, S21, S23, S25, S4, S5, S6, S7, S8 and S9) were planted with broomrape seeds in pots. At the flowering stage of sesame, tubercle number and dry weight, shoot number and dry weight and shoot height of broomrape were determined. Shoot dry weight, root dry weight and seed yield of sesame were determined at the physiological maturity. The results showed that broomrape growth varied with sesame genotype. Egyptian broomrape dry weight measured from 1.6% (S23) to 35.4% (Kalat) of the dry weight of the sesame on which it grew. Broomrape total dry weight had positive and highly correlation with the root dry weight of the sesame genotype on which it grew. When broomrape planted with the S7, S8, S9, S21 and S23 genotypes produced tubercles, but no shoots on these genotypes. Such genotypes could be used as both trap crops and as breeding‐sources for development of resistant sesame genotypes.     

Switchgrass (Panicum virgatum L.) has ability to induce germination of Orobanche cumana

Orobanche cumana Wallr. (sunflower broomrape) is a devastating root parasitic weed, causing enormous crop losses worldwide. The question was whether or not switchgrass has the potential to be a ‘trap crop’ for O. cumana control. To answer this question, the field experiments, pot experiments, and laboratory experiments were conducted in this study. The ability of nine switchgrass (Panicum virgatum L.) cultivars to induce O. cumana seeds germination was tested. Results indicated that root extracts shoot extracts, rhizosphere soil, and root exudates from switchgrass induced O. cumana germination. Ability to induce germination varied significantly among growing stages, with the earlier part of growing season (grown for 2 weeks) generally inducing the highest O. cumana germination rates. The methanol was a more suitable solvent than distilled water for extracting germination stimulants from switchgrass plants. Ten-fold dilutions of the extracts generally induced higher germination rates than either undiluted or 100-fold dilutions. The germination rates of O. cumana seeds in shoot extracts treatments were positively correlated with those in the root extracts (R² = 0.6397; p < 0.01) and negatively correlated with those in the rhizosphere soil treatments ( R² = 0.4433; p < 0.05). In conclusion, it is believed that switchgrass is a potential trap crop for the control of root parasitic weed O. cumana.     

Response of Amaranthus retroflexus L. seeds to gibberellic acid, ethylene and abscisic acid depending on duration of stratification and burial

Freshly harvested, dormant seeds of Amaranthus retroflexus were unable to germinate at 25 and 35 °C. To release their dormancy at the above temperatures, the seeds were stratified at a constant temperature (4 °C) under laboratory conditions or at fluctuating temperatures in soil or by outdoor burial in soil. Fully dormant, or seeds stratified or buried (2006/2007 and 2007/2008) for various periods were treated with exogenous gibberellic acid (GA₃), ethephon and abscisic acid (ABA). Likewise, the effects of these regulators, applied during stratification, on seed germination were determined. The results indicate that A. retroflexus seed dormancy can be released either by stratification or by autumn–winter burial. The effect of GA₃ and ethylene, liberated from ethephon, applied after various periods of stratification or during stratification, depends on dormancy level. GA₃ did not affect or only slightly stimulated the germination of non-stratified, fully dormant seeds at 25 and 35 °C respectively. Ethylene increased germination at both temperatures. Seed response to GA₃ and ethylene at 25 °C was increased when dormancy was partially removed by stratification at constant or fluctuating temperatures or autumn–winter burial. The response to GA₃ and ethylene increased with increasing time of stratification. The presence of GA₃ and ethephon during stratification may stimulate germination at 35 °C. Thus, both GA₃ and ethylene can partially substitute the requirement for stratification or autumn–winter burial. Both hormones may also stimulate germination of secondary dormant seeds, exhumed in September. The response to ABA decreased in parallel with an increasing time of stratification and burial up to May 2007 or March 2008. Endogenous GA_n, ethylene and ABA may be involved in the control of dormancy state and germination of A. retroflexus. It is possible that releasing dormancy by stratification or partial burial is associated with changes in ABA/GA and ethylene balance and/or sensitivity to these hormones.     

Some Soybean Cultivars Have Ability to Induce Germination of Sunflower Broomrape

Sunflower broomrape is a noxious parasitic weed which has caused severe damage to crop ecosystems. Trap crops can release a mixture of allelochemicals to induce the germination of sunflower broomrape. We studied the allelopathic effects of soybean on sunflower broomrape. Fourteen common soybean cultivars were grown in pots. Samples were collected from soybean plants and rhizosphere soil at five growth stages (V1, V3, V5, R2, and R4). The allelopathic effects of soybean reached highest at the V3 stage. Methanolic extracts of soybean roots induced higher broomrape germination than methanolic extracts of stems or leaves. The germination rates induced by root extracts (10-fold dilution) were positively correlated with germination rates induced by stem (10-fold dilution) and leaf extracts (10-fold dilution). The broomrape germination rates induced by root extracts were also positively correlated with soybean nodule diameter and dry weight. The results indicated that soybeans could induce sunflower broomrape germination. We conclude that soybean has the potential to be used as a trap crop for sunflower broomrape.