The effect of nitrogenous compounds on in vitro germination of Orobanche crenata Forsk.

The effect of nitrogen (ammonium, urea and nitrate), applied during a conditioning or germination phase, on germination of Orobanche crenata Forsk, seeds was investigated in vitro. Ammonium sulphate (8 mM) applied during conditioning in combination with the nitrification inhibitor nitrapyrin, reduced germination of seeds from 46% to 26%. A lower concentration of 4 mM ammonium sulphate or supplying nitrogen as 8 mM urea or 16 mM nitrate did not inhibit germination. Applied after conditioning during the germination phase, 4 mM ammonium sulphate strongly inhibited germination, especially in combination with a nitrification inhibitor: germination was reduced to less than 5%. Urea (8 mM) reduced germination to only a limited extent (from 58% to 40%). When a urease inhibitor (carbon disulfide) was additionally applied, germination percentage was not affected. Nitrate had no effect. It is hypothesized that the inhibition by ammonium of O. crenata germination is connected with a reduced ability to detoxify ammonium, owing to the reduced activities of the enzymes GS and GOGAT. The inhibitory effect of urea may be indirect, occurring through conversion to ammonium. The effect of a short-term exposure to ammonium was largely reversible and no increase in mortality could be detected. The presence of 6 mM phosphate, Hepes or Mops buffer was found to intervene with the inhibition by ammonium.     

Seasonal changes in germination response of buried seeds of Orobanche crenata Forsk.

Orobanche crenata seeds, collected in Syria, Egypt and Spain, were buried in the field in Syria (all three seed lots) and Spain (only Spanish seeds) and at regular intervals exhumed and tested for germination, to investigate whether the seeds exhibit an annual dormancy/non-dor- mancy cycle. When exposed directly to the synthetic germination stimulant GR24 for 7 days at 20°C, seeds only germinated in autumn after the first rains and to a limited extent in winter. When the seeds were conditioned for 11 days at 20°C prior to exposure to GR24, germination occurred during summer and autumn, but seeds were dormant in winter and early spring. The observed seasonal pattern in germinability, in relation to rainfall and soil temperature, was largely consistent with the results of an in vitro experiment by Van Hezewijk et al. (1993), investigating the effect of conditioning temperature and conditioning period on germination capacity and the development of secondary dormancy. Moisture and temperature can therefore be considered the major factors regulating induction and alleviation of dormancy in buried O. crenata seeds. There were no basic differences in response owing to site of collection of O. crenata seeds, nor to the location where they were buried. Variations saisonnières des exigences de germination de graines enfouies d'Orobanche crenata Forsk. Des graines d'Orobanche crenata récoltées en Syrie, en Égypte et en Espagne ont été enfouies au champ en Syrie (les 3 lots) et en Espagne (seules les graines d'Espagne) puis ont été exhumées a intervalles régulier pour que leur aptitude à la germination soil évaluée. Le but était de déterminer si les graines possédaient un cycle annuel dormance/non dormance. Quand elles étaient directement exposées au stimulant de germination synthétique GR24 pendant 7 jours à 20°C, les graines ne germaient qu'à l'automne après les premières pluies et peu en hiver. Quand les graines restaient pendant 11 jours à 20°C avant leur exposition au GR24, la germination seproduisait en été et à l'automne mais les graines restaient dormantes en hiver et au début du prin-temps. Les variations saisonnières d'aptitude à la germination, liées aux précipitations et à la temperature du sol, étaient en accord avec les résultats d'une expérience in vitro de Van Hezewijk et al. (1993) concernant l'effet de la température et de la durée pendant laquelle elle est appliquée, sur l'aptitude à la germination et le développement de la dormance secondaire. L'humidité du sol et sa température peuvent ainsi être considérées comme les principaux facteurs qui induisent et lèvent la dormance de graines de O. crenata enfouies. On n'observait pas de différences importantes dues au lieu de récolte ou à l'endroit oü elles étaient enfouies. Jahreszeitliche Änderungen der Keimung von vergrabenen Samen von Orobanche crenata Forsk. Proben von in Syrien, Ägypten und Spanien gesammelten Orobanche-crenata-Samen wurden in Syrien und Proben nur spanischer Herkunft in Spanien im Freiland im Boden ausgelegt und in regelmäßigen Zeitabständen ausgegraben und auf ihre Keimfähigkeit getestet, um zu untersuchen, ob die Samen einen jährlichen Dormanz-Zyklus haben. Beim direktem Auslegen in dem synthetischen Keimungsmittel GR24 öber 7 d bei 20°C keimten die Samen nur im Herbst nach den ersten Regenfällen und in beschränktem Umfang im Winter. Wenn die Samen för 11 d bei 20°C vor dem Auslegen in GR24 vorbehandelt worden waren, keimten sie im Sommer und Herbst, aber im Winter und fröhen Fröhjahr waren sie dormant. Das jahreszeitliche Verhalten der Keimfähigkeit in Abhängigkeit von Niederschlag und Bodentemperatur stimmte weitgehend mit den Ergebnissen eines In-vitro-Versuches von Van Hezewijk et al. (1993) öber die Wirkung einer Wärmevorbehandlung und Vorbehandlungszeit auf die Keimfähigkeit und die Ausprägung sekundärer Dormanz öberein. Bodenfeuchte und -temperatur können deshalb als die wichtigsten Faktoren för die Induktion und Aufhebung der Dormanz von Orobanche-crenata-Samen im Boden angesehen werden. Herkunft und Versuchsort hatten keinen erheblichen Einfluß auf die Ergebnisse.     

Factors affecting the activity of GR7 in stimulating germination of Striga hermonthica (Del.) Benth.

Experiments with the alkaline heavy clays of the Sudan Gezira showed that when the strigol analogue GR7 was applied to moist soil prior to subjecting Striga hermonthica seeds to moist conditions (‘conditioning’) for 7–14 days, there was no significant increase in germination. Conditioning of seeds in soils treated with GR7 had an adverse effect on the responsiveness of seeds when GR7 was added again after conditioning. Under ideal conditions, when GR7 was applied to conditioned seeds in moist soil, up to 75% germination could be induced by 2.0 kg/ha incorporated to 2–3 cm depth. Residual activity of GR7 persisted in air-dry soil, but at 50% moisture there was complete loss within 1 day. However, when conditioned seeds were put into the soil immediately after GR7 application its activity increased with increasing soil moisture.     

Fenugreek root exudates show species-specific stimulation of Orobanche seed germination

Various Orobanche species are weedy and cause severe reduction in the yields of many important crops. The seeds of these parasitic weeds may remain dormant in the soil for many years until germination is stimulated by the release of a chemical signal from a host plant. In order to determine the effects of fenugreek root exudate on the induction of Orobanche crenata, Orobanche ramosa and Orobanche foetida seed germination, root exudate was collected from hydroponically grown fenugreek seedlings. Fractionation patterns obtained from column and thin layer chromatography of the fenugreek root exudate showed a set of metabolites differing in their polarity with stimulatory activity on Orobanche seed germination. The crude root exudate stimulated both O. ramosa and O. crenata seed germination to the same level caused by the synthetic germination stimulant GR24 at 10 mg L^?1. It also stimulated O. foetida seed germination which did not respond to GR24. Active fractions of root exudate stimulated the germination of Orobanche species differentially.     

Influence of soil moisture on activity and persistence of the strigol analogue GR 24

Germination of Striga hermonthica (Del.) Bentth seeds in response to the strigol analogue GR 24 was strongly influenced by soil moisture prior to, at, or after, stimulation. Exposure of seeds to excessive soil moisture contents (70% w/w) during conditioning resulted in a low response to GR 24. Transfer of Seeds from soil at 70% moisture to a lower moisture level (47%) for 2 days or more improved the response to GR 24 (37–58% germination), white air-drying restored germination (88%). In Gezira soil the optimum soil moisture contents for maximum response to GR 24 were 44, 38 and 40% during conditioning, stimulation and germination, respectively. The corresponding figures for the sandy soil were 20, 22 and 22%, respectively. Persistence of GR 24 was similar in both soil types, but was strongly influenced by soil moisture content. The stimulant was stable in air-dry soil, but a rapid loss was encountered in moist soil.     

Bioassay-led isolation of Myrothecium verrucaria and verrucarin A as germination inhibitors of Orobanche crenata

A total of 188 fungal isolates was obtained from the rhizosphere of Vicia faba grown in an Egyptian soil heavily infested with Orobanche species. Agar cultures of 58 isolates inhibited the germination of conditioned seed of Orobanche crenata exposed to the germination stimulant, GR24. Filtrates of inhibitory fungi grown in liquid medium for 9–15 days were also assayed and those of five isolates, which were morphologically similar, inhibited germination even when diluted 16-fold. The fungus was identified as Myrothecium verrucaria (Alb. & Schwein.) Ditmar by its morphology and the nucleotide sequence of the ITS1 and ITS2 regions of the ribosomal repeat unit. Purification of the inhibitor to homogeneity was accomplished by solvent partitioning, flash chromatography on silica gel, semi-preparative HPLC on a reversed phase C18 column, solid phase extraction and tlc on silica gel. The inhibitor was identified as verrucarin A by nuclear magnetic resonance spectroscopy and comparison of the spectra with those of an authentic sample of the compound. A preliminary experiment demonstrated that infection of V. faba by O. crenata could be prevented by addition of spores of the fungus to soil infested by the parasite.     

Germination response of Orobanche seeds subjected to conditioning temperature, water potential and growth regulator treatments

Broomrapes (Orobanche spp.) are parasitic weeds that cause significant losses of crop yield. Experiments were conducted to investigate the seed response to the artificial germination stimulant GR₂₄ in three species of Orobanche subjected to preconditioning under various temperatures, water potentials and with plant growth regulators. The highest germination percentages were observed in Orobanche ramosa, Orobanche aegyptiaca and Orobanche minor seeds conditioned at 18°C for 7 days followed by germination stimulation at 18°C. With the increase of the conditioning period (7, 14, 21 and 28 days), the germination percentage of O. ramosa and O. aegyptiaca progressively decreased. When conditioned at −2 MPa, the germination percentage was lower than at 0 and −1 MPa, especially at 13 and 28°C. Orobanche minor seeds could retain relatively high germination if conditioned at 18, 23 or 28°C, even after significantly extended conditioning periods (up to 84 days). GA₃ (30–100 mg L⁻¹), norflurazon and fluridone (10–100 mg L⁻¹), and brassinolide (0.5–1.0 mg L⁻¹) increased seed germination, while 0.01 mg L⁻¹ uniconazole significantly reduced germination rates of all three Orobanche spp. The promotional effects of GA₃ and norflurazon and the inhibitory effect of uniconazole were evident, even when they were treated for 3 days. Germination of Orobanche seeds was much lower when the unconditioned seeds were directly exposed to GR₂₄ at 10⁻⁶ m . This early GR₂₄-induced inhibition was however alleviated or even eliminated by the inclusion of GA₃ or norflurazon (10–50 mg L⁻¹) in the conditioning medium. On the contrary, the inclusion of uniconazole increased the inhibitory effect of GR₂₄, particularly in the case of O. ramosa.     

Behaviour of hard and permeable seeds of Abutilon theophrasti Medic. (velvetleaf)

Experiments were carried out on seeds of Abutilon theophrasti (velvetleaf) to examine the germination behaviour of hard (impermeable) and soft (permeable) seeds and to test various physical and chemical treatments against hardseededness. There was no evidence, in fresh or in old seeds, of leachable germination inhibitors or of leachable compounds affecting hardseededness. Marked differences were found between hard and soft seeds in respiration and leakage. Oxygen uptake was negligible in hard seeds. A rapid efflux of K⁺ ions from hard and soft seeds was detected during the first minutes of immersion. Subsequently, the rate of leakage of hard seeds decreased markedly. After 4 h of imbibition, conductivity measurements differentiated individual hard and soft seeds. Abrasion, percussion and immersion in sodium hypochlorite had no effect on hard seeds, but mechanical scarification and immersion in sulphuric acid reduced hardseededness. Gamma radiation did not affect the hard coat but damaged the embryo. A. theophrasti seeds were exposed to organic solvents such as acetone. ethanol and methanol, and to pesticides such as butylate, EPTC, metham-sodium, vernolate and methyl bromide, in liquid or gaseous phase. Some of the treatments reduced germination of soft seeds, but none, even methyl bromide, had any significant effect on the number or viability of hard seeds, proving their complete impermeability.     

Characterization of the Orobanche–Medicago truncatula association for studying early stages of the parasite–host interaction

There is an increasing interest in the legume species Medicago truncatula as a model plant for structural and functional genomic studies that can be used to identify agronomically important genes in legumes. Field screening has shown very high levels of resistance to Orobanche crenata in M. truncatula. However, in vitro studies with O. crenata, Orobanche foetida, Orobanche ramosa and Orobanche minor showed useful variation among accessions at early stages of the parasite–host interaction. Significant differences were observed in the levels of germination of O. crenata and O. foetida seeds induced by different accessions of M. truncatula. Only limited germination was observed on accession SA‐4327. All accessions induced little O. ramosa and O. minor germination. Accessions also varied in the number of O. crenata and O. foetida attachments supported, with few developing on accession SA‐27774. The variation observed for induction of germination and of subsequent attachment will be useful to isolating and characterizing genes involved in the early stages of Orobanche–host plant interaction and for the study of the biosynthetic pathways of production for germination stimulants.     

Germination response of Phalaris paradoxa L. seed to different light qualities

The influence of different light regimes on the germination of Australian and English populations of Phalaris paradoxa L. (awned canary‐grass) seed was investigated to determine the impact of changing tillage practices on weed infestation. Seeds of all biotypes were highly viable, but differed in levels of innate dormancy (26–99%). In one experiment seed from a single Australian biotype, either enclosed in the spikelet glumes or having the spikelet glumes removed, were exposed to nine light treatments. Germination was stimulated by red and white light, but was inhibited by far‐red light. Time to 50% germination was less for seed enclosed in the spikelet glumes than for naked caryopses, although the final percentage of seed germinating when still enclosed in the spikelet glumes was significantly lower than for naked caryopses. In another experiment, six Australian and English biotypes with varying dormancy characteristics were exposed to eight light treatments. Red light did not stimulate germination in the deeply dormant biotype, however stimulated all other biotypes. Germination in darkness was below 20% in all biotypes except for one where germination was 51%. To overcome dormancy seeds were imbibed and placed in darkness at 16°C for either 7 or 14 days prior to exposure to red or white light for a single 15‐min period. Dormancy in all biotypes was overcome indicating that a period of burial may decrease the dormancy level and increase seed sensitivity to light. This increased light sensitivity suggests that exposure to light during tillage may stimulate germination in P. paradoxa seed.     

Moisture content and dormancy in Striga asiatica seeds

Freshly harvested Striga asiatica L. seeds will germinate in response to a stimulant only after the passage of time, an after-ripening period, and exposure to moisture at a suitable temperature, a conditioning period. To investigate the role of seed moisture content in the regulation of the after-ripening period, seeds were placed in chambers having specific relative humidity of 6%, 14%, 33%, 75% and 91% for 30, 60, 90 and 150 days. The seeds were then conditioned and germination percentage, response to tetrazolium and seed moisture contents were measured. Seeds at moisture contents less than 10% at the start of conditioning had germination of greater than 93%. Seeds at moisture contents over 10% at the start of conditioning could germinate between 60% and 3%, with germination decreasing as seed moisture content at the start of conditioning increased. The highest moisture content (17%) and lowest germination percentage (3%) occurred in seeds stored at 91% relative humidity for 150 days. There was a linear relationship of a high degree of correlation (0.997) between a positive tetrazolium test and germination capacity. Germination capacity of seeds could be changed from 90% to 3% by prolonged storage in water (dilute benomyl solution), causing `wet dormancy', then returned to 90% germination by returning to dry storage. Seed moisture content at the beginning of conditioning appears to control the responsiveness of the seeds to germination stimulants. The implications of these findings to the control of the parasite are discussed.     

Variation in resistance to Orobanche crenata in species of Cicer

Crenate broomrape (Orobanche crenata) is a major constraint for legume cultivation in Mediterranean agriculture. Field trials, pot and in vitro experiments demonstrated that resistance to O. crenata is present in chickpea and wild species of Cicer. The resistance is the result of the combination of several mechanisms, including low induction of parasite seed germination and in some accessions, either a darkening at the infection site on the host root that prevents establishment, or a reduced development of established parasite tubercles.     

Seed dormancy and germination in the summer annual Galeopsis speciosa

This study examined germination and dormancy in Galeopsis speciosa (Lamiaceae), a common summer annual weed in cold‐temperate areas. Seeds collected in southern Sweden were subjected to several experiments. The seeds were dormant at maturity. Seeds sown outdoors after collection produced a small number of seedlings that emerged early in the spring. After long cold stratification or stratification outdoors over two winters, the maximum germination was 40–50%; germination occurring over a wide range of temperatures. Warm stratification preceding cold stratification had no effect on germination, but repeated warm and cold periods seemed to promote germination. Gibberellic acid (GA) stimulated germination, but full germination was only achieved after more than 2 months of incubation at the most suitable temperature regime tested. Excised embryos grew and developed into normal seedlings. With these results, the species does not fit into the currently used system for seed dormancy classifications. The response to GA and the growth of excised embryos indicate non‐deep or intermediate physiological dormancy, but dormancy alleviation by stratification was not in line with the guiding principles for these classifications. Galeopsis speciosa has a strong dormancy that is sufficiently alleviated during the winter to allow germination of only part of a seed batch each year; hence a stepwise germination pattern occurs over a period of several years.     

Effects of drying and dl-strigol on seed conditioning and germination of Striga asiatica (L.) Kuntze

Seeds of Striga asiatica (L.) Kuntze required 7 and 14 days at 30°C to become conditioned when they were immersed in water and 10^?8m dl-strigol, respectively. At least 80% of seeds so conditioned germinated with 10^?8m dl-strigol as a terminal stimulation treatment. Seeds that were first immersed in water for 2 days, then subjected to one to three drying periods of 2 days each, either separately or continuously, required only 2 days of water immersion to become conditioned. Seeds first immersed in 10^?8m dl-strigol, then subjected to one or two drying periods, required only 3–4 days of water immersion as compared to a 14 days’requirement without drying treatments. The seeds first immersed in water or 10^?8m dl-strigol and then dried, plus one or two 10^?8m dl-strigol immersions, required 4 days of water immersion to become conditioned. However, the seeds immersed in various combinations of water or 10^?8m dl-strigol solutions plus one, two or three drying periods of 2 days each required 6 days of water immersion to become conditioned. The results indicate that the interaction of prolonged drying treatments and dl-strigol immersions delay conditioning processes in the seeds. Both water immersion and short drying treatments, however, seem to enhance certain required processes that occur during conditioning, thereby reducing the time required for the seeds to become conditioned when re-immersed in water.     

Thermal and hormonal regulation of the dormancy–germination transition in Amaranthus tuberculatus seeds

The transition from seed dormancy to germination is a multi‐step process. However, distinguishing between physiological processes involved in seed dormancy alleviation and those involved in germination has been difficult. We studied the seed dormancy alleviation process in Amaranthus tuberculatus, an important weed species in midwestern USA. Using three A. tuberculatus biotypes that differ in dormancy level, it was determined that stratification reduced seed dormancy from a high to a low level. Temperature alternation alleviated low seed dormancy and triggered germination. Exogenously applied abscisic acid (ABA) and gibberellic acid (GA) had no effect on seeds with high dormancy. However, ABA and paclobutrazol (a GA biosynthesis inhibitor) significantly reduced germination of seeds with low dormancy. Hormones could not replace the effects of stratification or temperature alternation on dormancy alleviation. Based on our results, we propose a seed dormancy–germination transition model in which the dormancy of A. tuberculatus seeds is progressively reduced from a high to a low level; but environmental conditions (i.e. stratification) can accelerate the dormancy alleviation process. Under low dormancy levels, the seed is more sensitive to environmental cues that are responsible for removing dormancy and triggering germination (i.e. temperature alternation). Finally, ABA and GA regulation occurs primarily during the final transition from low dormancy to germination rather than the alleviation of high dormancy.     

Seed dormancy and periodicity of seedling emergence in Veronica hederifolia L.

Emergence of Veronica hederifolia seedlings began in mid-October and continued into spring; few appeared from June to September. Ripe seeds shed in June were dormant but wben buried in soil outdoors developed a capacity for germination initially at low temperatures (constant4 C; daily alternations of 4-10° and 4-1 5 C) and later at somewhat higher temperatures, with peak germination in September-November. During winter, spring and early summer thc germination capacity declined, to increase again in late summer and early autumn. Cyclic physiological changes thus occur in seeds of V,hederifolia present in the soil, with which lhe consistent seasonal periodicity of seedling emergence is associated. In dry storage ihe capacity for germination progressively increased, but alter 12 months there was a sharp decline in germination at 4° C. Few seeds germinated at 20° C, but moistening with GA 4/7; brought about complete germination at this temperature.     

Physiological and Cytological Studies on the Inhibition of Striga Seed Germination by the Plant Growth-promoting Bacterium Azospirillum brasilense

Striga hermonthica (Del.) Benth. is an obligate parasitic weed of tropical cereals whose rhizosphere can also be colonised by bacteria of the genus Azospirillum. A previous study demonstrated that the two organisms (Azospirillum and striga) interacted during cerealroot colonisation. Two strains of A. brasilense isolated from an African sorghum rhizosphere prevented the germination of striga seeds although they were stimulated to germinate by the presence of sorghum roots. Azospirillum cells suspended in a synthetic germination stimulant (GR24) did not inhibit striga seed germination, but did block radicle elongation. Those radicles had an abnormal morphology, and contained no vacuolated cells in the root elongation zone. Lipophilic compounds extracted from the medium of bacteria in the log and stationary growth phases prevented the germination of striga seeds.     

Asian spiderflower (Cleome viscosa) germination ecology in southern Iran

Cleome viscosa is one of the most important weeds of warm‐season crops in southern Iran. Laboratory experiments were conducted to assess the impact of environmental factors on seed germination of C. viscosa . Freshly harvested seeds exhibited dormancy that was relieved (>90%) after immersion for 20 min in concentrated sulfuric acid. Regardless of the temperature regime, the final percentage of germination in light/dark (69.3%) was significantly higher than in complete darkness (58.3%). The optimum temperature for germination was 35/25°C in both light and dark. No germination was observed at constant temperatures of either 15 or 45°C. The thermal thresholds for seed germination, the base (T _b) and the mean ceiling germination temperatures (T _c(50)) were estimated to be 18.8 and 39.9°C, respectively. A base water potential ( Ψ _b(50) ) of ?0.96 MPa was identified for C. viscosa seeds. The response threshold of C. viscosa to reduce 50% of maximum germination for salinity was estimated to be 255 mM. Seeds that were placed on the soil surface had the highest percentage of seedling emergence (77.3%), and no seedlings emerged from seeds placed at a depth of 6 cm. The findings of this study could help to improve the integrated weed management strategies for this species.     

Factors affecting the activity of ethephon in stimulating seed germination of Striga hermonthica (Del.) Benth.

Ethephon (2-chloroethylphosphonic acid), which generates ethylene, stimulated the germination of pre-conditioned seeds of Striga hermonthica when it was added to the alkaline Gezira clay soil at concentrations of 2.5 to 30 mg kg^?1. As little as 5 min contact with treated soil was enough to stimulate germination. Ethephon in soil did not cause germination of unconditioned seeds for periods of up to 12 days and also had an adverse effect on seed germination when such seeds were given a second ethephon exposure after a storage period which was adequate, in untreated soil, to give the necessary pre-conditioning. Ethephon activity persisted in air-dry soil but declined over a 14 day period in moist soil. In the field ethephon at 0.6 to 4.8 kg ha^?1 decreased the number of Striga shoots and increased sorghum height and flowering.     

A competition index for Orobanche crenata Forsk effects on broad bean (Vicia faba L.)

The influence of broomrape (Orobanche crenata Forsk) infection levels on commercial broad bean (Vicia faba L.) crops was investigated and an equation relating yield losses to O. crenata density was derived. O. crenata growth was weakly and negatively related to the final height or shoot number of the broad bean plant. O. crenata decreased seed yield mainly by reducing the number of pods. The number of seeds per pod was also decreased by O. crenata infection when competition occurred at late-crop growth stages when most of the pods had already developed. Bean unit seed weight was only weakly affected by infection. Correlation coefficients between O. crenata number and broad-bean seed yield per plant were significant at P=0·05 and greater than 0·70 in most locations. On average, about four O. crenata per broad-bean plant decreased seed yield by half. An estimation of the percentage of losses in any field can be calculated from the equation: %loss = 100 × 0·124 × OcN. where OcN is the average final number of emerged O. crenata per plant.