Genetic relationships among Orobanche species as revealed by RAPD analysis

RAPD markers were used to study variation among 20 taxa in the genus OROBANCHE: O. alba, O. amethystea, O. arenaria, O. ballotae, O. cernua, O. clausonis, O. cumana, O. crenata, O. densiflora, O. foetida, O. foetida var. broteri, O. gracilis, O. haenseleri, O. hederae, O. latisquama, O. mutelii, O. nana, O. ramosa, O. rapum-genistae and O. santolinae. A total of 202 amplification products generated with five arbitrary RAPD primers was obtained and species-specific markers were identified. The estimated Jaccard's differences between the species varied between 0 and 0.864. The pattern of interspecific variation obtained is in general agreement with previous taxonomic studies based on morphology, and the partition into two different sections (Trionychon and Orobanche) is generally clear. However, the position in the dendrogram of O. clausonis did not fit this classification since it clustered with members of section TRIONYCHON: Within this section, O. arenaria was relatively isolated from the other members of the section: O. mutelii, O. nana and O. ramosa. Within section Orobanche, all O. ramosa populations showed a similar amplification pattern, whereas differences among O. crenata populations growing on different hosts were found. Orobanche foetida and O. densiflora clustered together, supporting the morphological and cytological similarities and the host preferences of these species.     

Sunflower sesquiterpene lactone models induce Orobanche cumana seed germination

Six sunflower sesquiterpene lactone models which share structural features of the lactone rings of strigol and its synthetic analogues, the GR family, with different conformational flexibilities were tested as Orobanche cumana germination stimulants. Among them, parthenolide and 3,5-dihydroxydehydrocostus-lactone significantly increased O. cumana germination, presenting higher activity than GR-24, used as a standard in the germination bioassay. The effect of these two compounds is species-specific, showing no germination stimulant activity on other Orobanche spp. tested (O. crenata, O. ramosa and O. aegyptiaca). Data presented are discussed in terms of a structure-activity relationship.     

Molecular Studies on Orobanche

The optimization of polymerase chain reaction (PCR) for random amplified polymorphic DNA (RAPD) analysis in Orobanche was investigated and the results were applied to analysis of O. aegyptiaca, O. oxyloba, O. ramosa and three O. crenata collections gathered from different locations in Egypt, Genotypes were compared using 20 random primers. The polymorphisms detected were only between the tested species, while identical banding patterns were generated in collections of the same species.
Genetic similarity among species was estimated on the basis of the percentage of common bands between species and a dendogram was constructed using the unweighted pair grouping method.
Two primers were chosen as specific PCR primers to differentiate between the studied Orobanche species. The results of this study show that RAPDs can be used as a method for clarifying relationships between species of Orobanche .     

An improved axenic system for studying pre-infection development of the parasitic plant Orobanche ramosa

BACKGROUND AND AIMS: Broomrapes (Orobanche spp.) are holoparasitic weeds that cause devastating losses in many economically important crops. The molecular mechanisms that control early stages of host infection in Orobanche are poorly understood, partly due to the lack of experimentally tractable in vitro systems that allow the efficient application of molecular tools. Here an improved axenic system for the analysis of pre-infection stages in O. ramosa in the absence of the host plant is described. METHODS: An optimized protocol for seed disinfection, based on formaldehyde, was developed. Orobanche ramosa seeds were conditioned in Petri dishes with filter paper, stimulated by addition of the synthetic strigol analogue GR24, and the percentage of germination as well as attachment-organ formation was determined. KEY RESULTS: Treatment of O. ramosa seeds with tobacco-root exudate or with GR24 resulted in highly reproducible germination rates around 70 %. A conditioning period of 8 d was both necessary and sufficient to allow optimal germination in response to GR24. Conditioned seeds that were dehydrated for several months remained fully responsive to GR24 without the need of a new conditioning period. Treatments as short as 5 min with GR24 were sufficient to fully and irreversibly induce the seed germination response. Approximately half of the germinated seeds initiated attachment-organ development. Similar rates of attachment organ induction were also detected in the rare cases of seeds that had germinated spontaneously on water. CONCLUSIONS: The results suggest that the conditioning period produces persistent changes in the seeds required for responsiveness to external stimulants. The rapid action of GR24 suggests that it may act via a receptor-mediated signalling mechanism. While germination in O. ramosa is induced by exogenous stimuli, attachment organ differentiation appears to be triggered by unknown endogenous signals. The new in vitro culture system will have useful applications for the molecular analysis of early stages of parasitic development in Orobanche.     

Preliminary host ranges of some strains of economically important broomrapes (Orobanche)

Strains ofOrobanche crenata, O. aegyptiaca, O. minor, O. ramose andO. cernua from different geographical areas and from different host species were potted with tobacco, tomato and other hosts.Orobanche cernua, O. aegyptiaca andO. Ramose from tobacco and from tomato did equally well on either host.Orobanche crenata grew vigorously on broadbean even when the original host was safflower or coleus.Orobanche minor has apparently developed several distinct strains.     

Medicago truncatula as a model for nonhost resistance in legume-parasitic plant interactions

Crenate broomrape (Orobanche crenata) is a root parasitic weed that represents a major constraint for grain legume production in Mediterranean and West Asian countries. Medicago truncatula has emerged as an important model plant species for structural and functional genomics. The close phylogenic relationship of M. truncatula with crop legumes increases its value as a resource for understanding resistance against Orobanche spp. Different cytological methods were used to study the mechanisms of resistance against crenate broomrape of two accessions of M. truncatula, showing early and late acting resistance. In the early resistance accession (SA27774) we found that the parasite died before a tubercle had formed. In the late resistance accession (SA4327) the parasite became attached without apparent problems to the host roots but most of the established tubercles turned dark and died before emergence. The results suggest that there are defensive mechanisms acting in both accessions but with a time gap that is crucial for a higher success avoiding parasite infection.     

Synthesis and seed germination stimulating activity of some imino analogs of strigolactones

Strigolactones are germination stimulants for seeds of the root parasitic weeds, Striga and Orobanche spp. The imino analog of GR24 showed moderate germination stimulating activity against the seeds of S. hermonthica. The seed germination stimulating activity of some phenyliminoacetates and phenyliminoacetonitriles was also examined. The degree of activity of the phenyliminoacetate was less than that of the phenylacrylates. On the other hand, the degree of activity of the phenyliminoacetonitrile was comparable to that of the phenylacrylonitriles. Among the tested compounds, the 3-pyridyliminoacetonitrile showed higher activity against the seeds of O. crenata than GR24. These findings demonstrate that it is not always essential to have the Michael acceptor of the C-D ring junction moiety which has been proposed to react with nucleophilic species presented at the target site to enhance the activity.     

IAA production during germination of Orobanche spp. seeds

Broomrapes (Orobanche spp.) are parasitic plants, whose growth and development fully depend on the nutritional connection established between the parasite and the roots of the respective host plant. Phytohormones are known to play a role in establishing the specific Orobanche-host plant interaction. The first step in the interaction is seed germination triggered by a germination stimulant secreted by the host-plant roots. We quantified indole-3-acetic acid (IAA) and abscisic acid (ABA) during the seed germination of tobacco broomrape (Orobanche ramosa) and sunflower broomrape (O. cumana). IAA was mainly released from Orobanche seeds in host-parasite interactions as compared to non-host-parasite interactions. Moreover, germinating seeds of O. ramosa released IAA as early as 24 h after the seeds were exposed to the germination stimulant, even before development of the germ tube. ABA levels remained unchanged during the germination of the parasites' seeds. The results presented here show that IAA production is probably part of a mechanism triggering germination upon the induction by the host factor, thus resulting in seed germination.     

Induction of seed germination in <Emphasis Type="Italic">Orobanche</Emphasis> spp. by extracts of traditional Chinese medicinal herbs

The co-evolution of Orobanche spp. and their hosts within the same environment has resulted in a high degree of adaptation and effective parasitism whereby the host releases parasite germination stimulants, which are likely to be unstable in the soil. Our objective was to investigate whether extracts from non-host plants, specifically, Chinese medicinal plants, could stimulate germination of Orobanche spp. Samples of 606 Chinese medicinal herb species were extracted with deionized water and methanol. The extracts were used to induce germination of three Orobanche species; Orobanche minor, Orobanche cumana, and Orobanche aegyptiaca. O. minor exhibited a wide range of germination responses to the various herbal extracts. O. cumana and O. aegyptiaca exhibited an intermediate germination response to the herbal extracts. O. minor, which has a narrow host spectrum, showed higher germination rates in response to different herbal extracts compared with those of O. cumana and O. aegyptiaca, which have a broader host spectrum. Methanolic extracts of many Chinese herbal species effectively stimulated seed germination among the Orobanche spp., even though they were not the typical hosts. The effective herbs represent interesting examples of potential trap crops. Different countries can also screen extracts from indigenous herbaceous plants for their ability to induce germination of Orobanche spp. seeds. The use of such species as trap plants could diminish the global soil seed bank of Orobanche.     

Potential role of winter rape weeds in the extension of broomrape in Poitou-Charentes

In the Poitou-Charentes district, among the 82 species of winter rape weeds identified, 22 displayed a strong affinity for this crop (Brassica napus L.). In fields, 50% of these weeds were parasitized by Orobanche ramosa, playing the role of host plants. Greenhouse co-cultures (weed/Orobanche ramosa) showed that weeds non-parasitized in fields could be attacked by broomrape, developing a more or less complete cycle. In vitro co-cultures (weed/Orobanche ramosa) revealed that root exudates of non-parasitized weeds, in fields or in greenhouse co-cultures, could induce Orobanche ramosa seed germination, but not attachment. These weeds could play the role of false hosts.     

Trigoxazonane, a monosubstituted trioxazonane from Trigonella foenum-graecum root exudate, inhibits Orobanche crenata seed germination

Orobanche crenata is a major threat to grain legume production. Fenugreek (Trigonella foenum-graecum) is an annual legume that has been shown to effectively reduce O. crenata infection when intercropped with grain legumes. In this paper, we point that this can be attributed to allelopathy, through inhibition of the germination of O. crenata by fenugreek root exudates. The main inhibitory metabolite was isolated and characterized. Allelopathy was demonstrated in different bioassays, by inhibition of O. crenata seeds germination both by growing fenugreek and pea plants together (intercropped), and by application of fenugreek root exudates. Fenugreek root exudates were extracted with organic solvent and fractionated giving several fractions, two of which showed moderate (27%) and strong (54%) inhibition of O. crenata seed germination, respectively. The most active metabolite is a new monosubstituted trioxazonane, characterized by spectroscopic methods as the 2-butyl-[1,4,7,2]trioxazonane and named trigoxazonane.     

Isolation of mannose 6-phosphate reductase cDNA,changes in enzyme activity and mannitol content in broomrape (Orobanche ramosa) parasitic on tomato roots

We are interested in developing a control strategy efficient at the early stages of subterranean development of Orobanche in the inhibition of mannose 6-phosphate reductase (M6PR, EC 1.1.1.224), the key enzyme of mannitol production in the parasite. We examined M6PR gene expression during pre-conditioning, germination, procaulome growth, underground shoot development and emergence of Orobanche ramosa L. attached to tomato roots, the enzyme activity at each of the above stages and the level of stored mannitol in the parasite. A 1120-pb length cDNA isolated by 3' and 5'RACE was identified as a M6PR sequence by cDNA expression in E. coli and M6PR activity measurement. Only one M6PR gene was detected in O. ramosa following southern blot analysis. M6PR expression, analysed by RT-PCR, was constant from the pre-conditioned seed to the emergence of broomrape, i.e. M6PR expression is constitutive in Orobanche . M6PR activity was also detected in pre-conditioned seeds and attachment to tomato roots resulted in a two-fold increase in enzyme activity during tubercle enlargement and crown root formation. Hexose and mannitol accumulation was strongly enhanced in the attached parasite, with accumulation primarily in the shoot. These results support the prospect of utilizing M6PR inhibitors as early applied herbicides to control this parasite in the early stages of its development.     

Fluridone and norflurazon, carotenoid-biosynthesis inhibitors, promote seed conditioning and germination of the holoparasite Orobanche minor

Fluridone and norflurazon, two carotenoid-biosynthesis inhibitors, shortened the conditioning period required by seeds of Orobanche minor in order to respond to the germination stimulant strigol. Neither fluridone nor norflurazon alone induced seed germination of O. minor , they promoted strigol-induced germination. In addition, these compounds restored the conditioning and germination of seeds at a supraoptimal temperature (30°C) as well as in the light. Gibberellic acid (GA₃) showed similar promotive and protective effects on the conditioning and germination of O. minor seeds. Although fluridone and norflurazon are known to prevent abscisic acid (ABA)-biosynthesis, and stresses such as supraoptimal temperatures have been reported to induce ABA accumulation in plants, the amount of ABA in the seeds or that released from the seeds into the conditioning media was not affected by the fluridone treatment and by exposure to the supraoptimal temperature. These results indicate that the promotive and protective effects of fluridone and norflurazon on the conditioning and germination of O. minor seeds would be attributed to other perturbations rather than the inhibition of ABA-biosynthesis.     

中草药浸提液对小列当种子发芽的影响

选用240种常用中草药,研究其蒸馏水和甲醇浸提液原液及其10倍、100倍和1 000倍稀释液对小列当种子发芽的影响.结果表明:小列当种子在蒸馏水浸提液刺激下发芽率超过70%的中草药有灯心草、降香和莲须,在甲醇浸提液刺激下发芽率超过70%的中草药有百部、桑椹、谷芽和萆薢;蒸馏水浸提液对小列当种子发芽抑制率超过50%的中草药有菊花、棕榈、苦杏仁和黄柏,甲醇浸提液对小列当种子发芽抑制率超过70%的有续断、地黄、肉豆蔻、地肤子、黄连、木贼和胡黄连.大部分中草药浸提液在10倍稀释液条件下对小列当种子发芽的刺激或抑制作用最明显.研究发现,一些参试中草药蒸馏水和甲醇浸提液能够刺激或抑制小列当种子萌发,并且其对种子萌发的影响程度随浸提液浓度的变化而异,不同中草药最佳处理浓度不同,而且用甲醇浸提液可筛选出更多种具有化感作用的中草药.     

Targeted mutagenesis of two homologous ATP-binding cassette subfamily G (ABCG) genes in tomato confers resistance to parasitic weed Phelipanche aegyptiaca

Journal of Plant Research - Phelipanche aegyptiaca and Orobanche spp. are obligate plant root-parasitic weeds that cause extensive damage in agricultural crop plants. Their germination requires...     

Stimulation of Orobanche ramosa seed germination by fusicoccin derivatives: a structure-activity relationship study

A structure-activity relationship study was conducted assaying 25 natural analogues and derivatives of fusicoccin (FC), and cotylenol, the aglycone of cotylenins, for their ability to stimulate the seed germination of the parasitic species Orobanche ramosa. Some of the compounds tested proved to be highly active, being 8,9-isopropylidene of the corresponding FC aglycone and the dideacetyl derivative the most active FC derivatives. In both groups of glucosides and aglycones (including cotylenol), the most important structural feature to impart activity appears to be the presence of the primary hydroxy group at C-19. Furthermore, the functionalities and the conformation of the carbotricyclic ring proved to play a significant role. The dideacetyl derivative of FC, being easily and rapidly obtainable in high yield starting by FC, could be of interest for its practical application as a stimulant of Orobanche ramosa seed germination, inducing the "suicidal germination", an interesting approach for parasitic plant management.     

Reactions of Lotus japonicus ecotypes and mutants to root parasitic plants

Witchweeds (Striga spp.) and broomrapes (Orobanche spp.) are obligate root parasitic plants on economically important field and horticultural crops. The parasites' seeds are induced to germinate by root-derived chemical signals. The radicular end is transformed into a haustorium which attaches, penetrates the host root and establishes connection with the vascular system of the host. Reactions of Lotus japonicus, a model legume for functional genomics, were studied for furthering the understanding of host-parasite interactions. Lotus japonicus was compatible with Orobanche aegyptiaca, but not with Orobanche minor, Striga hermonthica and Striga gesnerioides. Orobanche minor successfully penetrated Lotus japonicus roots, but failed to establish connections with the vascular system. Haustoria in Striga hermonthica attached to the roots, but penetration and subsequent growth of the endophyte in the cortex were restricted. Striga gesnerioides did not parasitize Lotus japonicus. Among seven mutants of Lotus japonicus (castor-5, har1-5, alb1-1, ccamk-3, nup85-3, nfr1-3 and nsp2-1) with altered characteristics in relation to rhizobial nodulation and mycorrhizal colonization, castor-5 and har1-5 were parasitized by Orobanche aegyptiaca with higher frequency than the wild type. In contrast, Orobanche aegyptiaca tubercle development was delayed on the mutants nup85-3, nfr1-3 and nsp2-1. These results suggest that nodulation, mycorrhizal colonization and infection by root parasitic plants in Lotus japonicus may be modulated by similar mechanisms and that Lotus japonicus is a potential model legume for studying plant-plant parasitism.     

Strigolactone analogs derived from ketones using a working model for germination stimulants as a blueprint

Strigolactones are important signaling compounds in the plant kingdom. Here we focus on their germination stimulatory effect on seeds of the parasitic weeds Striga and Orobanche spp. and more particularly on the design and synthesis of new active strigolactone analogs derived from simple cyclic ketones. New analogs derived from 1-indanone, 1-tetralone, cyclopentanone, cyclohexanone and a series of substituted cyclohexanones (including carvone and pulegone) are prepared by formylation of the ketones with ethyl formate followed by coupling with a halo butenolide. Both enantiomers of the analog derived from 1-tetralone have been prepared by employing a homochiral synthon for the coupling reaction. For three other strigolactone analogs the antipodes have been obtained by chromatography on a chiral column. All analogs have an appreciable germinating activity towards seeds of Striga hermomonthica and Orobanche crenata and O. cernua. Stereoisomers having the same configuration at the D-ring as in naturally occurring strigol have a higher stimulatory effect than the corresponding antipodes. The analogs obtained from 1-indanone and 1-tetralone have an activity comparable with that of the well known stimulant GR 24. Analogs derived from 2-phenyl-cylohexanone, carvone and pulegone also have a good germinating response. The results show that the working model for designing new bioactive strigolactones is applicable.     

Gene silencing of mannose 6-phosphate reductase in the parasitic weed Orobanche aegyptiaca through the production of homologous dsRNA sequences in the host plant

Orobanche spp. (broomrape) are parasitic plants which subsist on the roots of a wide range of hosts, including tomato, causing severe losses in yield quality and quantity. Large amounts of mannitol accumulate in this parasitic weed during development. Mannose 6-phosphate reductase (M6PR) is a key enzyme in mannitol biosynthesis, and it has been suggested that mannitol accumulation may be very important for Orobanche development. Therefore, the Orobanche M6PR gene is a potential target for efforts to control this parasite. Transgenic tomato plants were produced bearing a gene construct containing a specific 277-bp fragment from Orobanche aegyptiaca M6PR-mRNA, in an inverted-repeat configuration. M6PR-siRNA was detected in three independent transgenic tomato lines in the R1 generation, but was not detected in the parasite. Quantitative RT-PCR analysis showed that the amount of endogenous M6PR mRNA in the tubercles and underground shoots of O. aegyptiaca grown on transgenic host plants was reduced by 60%–80%. Concomitant with M6PR mRNA suppression, there was a significant decrease in mannitol level and a significant increase in the percentage of dead O. aegyptiaca tubercles on the transgenic host plants. The detection of mir390, which is involved with cytoplasmic dsRNA processing, is the first indication of the existence of gene-silencing mechanisms in Orobanche spp. Gene silencing mechanisms are probably involved with the production of decreased levels of M6PR mRNA in the parasites grown on the transformed tomato lines.