Taxonomy of the Chinese Orobanche and Its Relationships with Related Genera

The morphological characters in the genus Orobanche were evaluated from the taxonomic point of view. The author finds that the plants of this genus are relatively similar to each other in respect to characters of vegetative organs, fruits and seeds. But the differences in the floral structures can be served as a basis for delimitating infrageneric taxa. The seed coat of 18 species and pollen grains of 6 species were also examined under scanning electron microscope (SEM). They seem to have little significance for distinguishing species. The result supports G. Beck’s (1930) division of the genus Orobanche into 4 sections, of which 2 occur in China, based on the characters of the inflorescence, bracteoles and calyx. The author considers that some characters, such as anther hairy or not, upper lip of corolla entire or not, lower lip longer or shorter than the upper one, the state of corolla-tube inflec tion and the hair type of filaments and plants, are important in distinguishing Chinese species. A key to the species of Orobanche in China is given. This genus consists of about 100 species, and is mostly confined to Eurasia, with over 60 species found in Caucasus and Middle Asia of USSR, where may be the mordern distribu tional centre. Orobanche L. in China is represented by 23 species, 3 varieties and l forma. As shown in Table 1, most species (12 species) are found in Xinjiang, which clearly shows a close floristic relationship between this region and Middle Asia of USSR. 6 species are endemic to China, of which 4 are confined to the Hengduan Mountains (Yangtze-Mekong-Salwin divide). The relationships between this genus and related ones of Orobanchaceae are also discussed. The author holds the following opinions: the genus Phelypaea Desf. should be considered as a member of Orobanche L. Sect. Gymnocaulis G. Beck, the monotypic genus, Necranthus A. Gilli endemic to Turkey, is allied with Orobanche L. Sect. Orobanche, the monotypic genus, Platypholis Maxim, endemic to Bonin Is. of Japan, is far from Orobanche L. in relation and should be regarded as a separate genus. The 11 OTU’s, including all the sections of Orobanche L. and 7 genera of Orobanchaceae, and 15 morphological characters were used in the numerical taxonomic treatment to test the above-mentioned suggestions. After standardization of characters, the correlation matrices were computerized. The correlation matrices were made to test the various clustering methods. At last the UPGMA clustering method was chosen and its result is shown in a phenogram. The result of numerical analysis is basically in accordance with the suggestions.     

Phylogeny and intraspecific variability of holoparasitic Orobanche (Orobanchaceae) inferred from plastid rbcL sequences

The rbcL sequences of 106 specimens representing 28 species of the four recognized sections of Orobanche were analyzed and compared. Most sequences represent pseudogenes with premature stop codons. This study confirms that the American lineage (sects. Gymnocaulis and Myzorrhiza) contains potentially functional rbcL-copies with intact open reading frames and low rates of non-synonymous substitutions. For the first time, this is also shown for a member of the Eurasian lineage, O. coerulescens of sect. Orobanche, while all other investigated species of sects. Orobanche and Trionychon contain pseudogenes with distorted reading frames and significantly higher rates of non-synonymous substitutions. Phylogenetic analyses of the rbcL sequences give equivocal results concerning the monophyly of Orobanche, and the American lineage might be more closely related to Boschniakia and Cistanche than to the other sections of Orobanche. Additionally, species of sect. Trionychon phylogenetically nest in sect. Orobanche. This is in concordance with results from other plastid markers (rps2 and matK), but in disagreement with other molecular (nuclear ITS), morphological, and karyological data. This might indicate that the ancestor of sect. Trionychon has captured the plastid genome, or parts of it, of a member of sect. Orobanche. Apart from the phylogenetically problematic position of sect. Trionychon, the phylogenetic relationships within sect. Orobanche are similar to those inferred from nuclear ITS data and are close to the traditional groupings traditionally recognized based on morphology. The intraspecific variation of rbcL is low and is neither correlated with intraspecific morphological variability nor with host range. Ancestral character reconstruction using parsimony suggests that the ancestor of O. sect. Orobanche had a narrow host range.     

Variability of interactions between barrel medic (Medicago truncatula) genotypes and Orobanche species

A number of broomrapes ( Orobanche spp.) are major threats for grain legumes in the Mediterranean area. Previous studies have shown very high levels of resistance to Orobanche crenata in Medicago truncatula with little variation among accessions hampering, therefore, its use for genetic analysis. In order to identify alternative systems for ulterior genetic and genomic analysis, we studied early stages of the interaction between M. truncatula accessions and a range of Orobanche species. We found significant differences in the induction of germination on Orobanche aegyptiaca , Orobanche foetida var. broteri , Orobanche minor , Orobanche nana and Orobanche ramosa seeds. Accessions also varied in the number of O. aegyptiaca , O. crenata , O. foetida var. broteri , O. nana and O. ramosa attachments supported. Applications of the synthetic germination stimulant GR24 increased the germination level of Orobanche cumana and O. minor . No attachments were observed for any of these species because of physical barriers typical of host resistance. On the contrary, increase of O. nana germination by GR24 led to an increase in number of attachments albeit the normal development of the nodules was stopped in a later stage. The genetic variation observed for induction of germination and subsequent attachment will be useful for isolating and characterising genes involved in early stages of Orobanche –host plant interaction and for the study of the biosynthetic pathways of production of germination stimulants.     

Pollen morphology of the genus Orobanche L. (Orobanchaceae)

A comparative palynological study of 25 taxa of the genus Orobanche L. belonging to section Trionychon Wallr. and section Orobanche L. was carried out. A clear difference between the pollen grains of section Trionychon and section Orobanche was observed. The grains of section Trionychon are typically tricolpate, radially symmetrical, more or less oblate spheroidal, with a microreticulate, or scabrate-perforate sculpture. Section Orobanche on the other hand, had spheroidal, more or less inaperturate pollen grains, although some were tricolpate, but without any clear symmetry. The basic surface sculpture of the pollen in section Orobanche was scabrate to rugulate-perforate. Palynoiogicaliy, six groups can be recognized amongst the species of the genus Orobanche , lor which a key is provided.     

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 .     

Chromosome numbers and karyotype evolution in holoparasitic Orobanche (Orobanchaceae) and related genera

Chromosome numbers and karyotypes of species of Orobanche, Cistanche, and Diphelypaea (Orobanchaceae) were investigated, and 108 chromosome counts of 53 taxa, 19 counted for the first time, are presented with a thorough compilation of previously published data. Additionally, karyotypes of representatives of these genera, including Orobanche sects. Orobanche and Trionychon, are reported. Cistanche (x = 20) has large meta- to submetacentric chromosomes, while those of Diphelypaea (x = 19) are medium-sized submeta- to acrocentrics. Within three analyzed sections of Orobanche, sects. Myzorrhiza (x = 24) and Trionychon (x = 12) possess medium-sized submeta- to acrocentrics, while sect. Orobanche (x = 19) has small, mostly meta- to submetacentric, chromosomes. Polyploidy is unevenly distributed in Orobanche and restricted to a few lineages, e.g., O. sect. Myzorrhiza or Orobanche gracilis and its relatives (sect. Orobanche). The distribution of basic chromosome numbers supports the groups found by molecular phylogenetic analyses: Cistanche has x = 20, the Orobanche-group (Orobanche sect. Orobanche, Diphelypaea) has x = 19, and the Phelipanche-group (Orobanche sects. Gymnocaulis, Myzorrhiza, Trionychon) has x = 12, 24. A model of chromosome number evolution in Orobanche and related genera is presented: from two ancestral base numbers, x(h) = 5 and x(h) = 6, independent polyploidizations led to x = 20 (Cistanche) and (after dysploidization) x = 19 (Orobanche-group) and to x = 12 and x = 24 (Phelipanche-group), respectively.     

Micromorphological studies on seeds of orobanche species from the iberian peninsula and the balearic islands, and their systematic significance

BACKGROUND AND AIMS: Previous research has made clear the intrinsic taxonomic difficulties in identifying species in the genus Orobanche. The aim of this study, therefore, was to investigate the systematic utility of seed characteristics. METHODS: Light and scanning electron microscopy was used to examine the seeds of 33 taxa of Orobanche from the Iberian Peninsula and the Balearic Islands. KEY RESULTS: Characters such as size, shape and ornamentation of the seeds were not found to be very useful in differentiation of taxa; however, other characters of the epidermal seed coat cells proved to be very helpful in this respect. Ornamentation of the periclinal walls could be used to discriminate four morphological types. Other features related to the anticlinal walls of the cells, such as thickness, presence/absence of a narrow trough, or relative depth, all contributed to the characterization of a large number of species. CONCLUSIONS: The usefulness of micromorphological studies on seeds of Orobanche in relation to differentiating taxa is demonstrated, and a key is provided to distinguish species or groups of species.     

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.     

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.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Starch Accumulation

The investigation dealt with starch accumulation in four species of Cuscuta (Cuscuta campestris, C. indecora, C. planiflora and C. reflexa), a leafy mistletoe (Dendrophthoe falcata) and a chlorophyll-lacking root parasite (Orobanche aegyptiaca). The highest content of starch occurred In O. aegyptiaca, with a maximum of 45 per cent of dry weight Starch in Cuscuta filaments and mistletoe leaves showed a maximum of about 10 per cent of dry weight. The starch content varied along the length of the Cuscuta vine, with a maximum in the apical region. Orobanche had a higher starch content when it was still submerged than it was fully developed. Cuscuta vines did not show any marked diurnal alteration in the starch content. The content of ethanol-soluble carbohydrate was only a tenth of the starch in Orobanche, but was relatively higher in the other parasites. the neutral sugars in Cuscuta filaments were sucrose and glucose, whereas fructose was also present in mistletoe and Orobanche. Raffinose and stachyose were absent or present only ill traces in parasite tissue. Starch granules from Cuscuta and Orobanche bad ADPG/UDPG-starch synthetase activity and homogenates starch phosphorylase activity. The former enzyme appeared to be responsible for synthesis of starch and the latter for utilization. The four different species of Cuscnta, growing on alfalfa, had more or less the same activity of starch synthetase and also of phosphorylase activity. Hosts infected by Cuscuta had significantly less starch per plant than the controls. A characteristic feature of invasion by Cuscuta and Orobanche was increased phosphorylase activity in the host tissues. The protein content of the tissues of Cuscuta and Orobanche was of a lower level than that of the host shoot system or foliage, indicating that the parasite differed from the host in having a higher carbon (of starch) to nitrogen (of protein) ratio.     

Phylogeny of holoparasitic Orobanche (Orobanchaceae) inferred from nuclear ITS sequences

Orobanche is the largest genus among the holoparasitic members of Orobanchaceae. We present the first molecular phylogenetic analysis (using nuclear ITS sequences) that includes members of all sections of Orobanche, Gymnocaulis, Myzorrhiza, Trionychon, and Orobanche. Orobanche is not monophyletic, but falls into two lineages: (1) the Orobanche group comprises Orobanche sect. Orobanche and the small Near Asian genus Diphelypaea and is characterized by a chromosome base number of x=19 and (2) the Phelipanche group contains Orobanche sects. Gymnocaulis, Myzorrhiza, and Trionychon and possesses a chromosome base number of x=12. The relationships between these two groups and to other genera such as Boschniakia or Cistanche remain unresolved. Within the Orobanche group, Orobanche macrolepis and Orobanche anatolica (including Orobanche colorata) constitute two phylogenetically distinct lineages. Intrasectional structurings proposed by some authors for O. sect. Orobanche are not confirmed by the molecular data. In most cases, intraspecific sequence divergence between accessions, if present, is negligible and not correlated with morphological or ecological traits. In a few cases, however, there is evidence for the presence of cryptic taxa.     

Alternate paths of evolution for the photosynthetic gene rbcL in four nonphotosynthetic species of Orobanche

We have determined the nucleotide sequence for the Rubisco large subunit from four holoparasitic species of Orobanche. Intact open reading frames are present in two species (O. corymbosa and O. fasciculata), whereas the remaining species (O. cernua and O. ramosa) have rbcL pseudogenes. Sequences for rbcL 5'-UTRs from species of Orobanche have few changes in the promoter and ribosome binding sites compared to photosynthetic higher plants. Comparison of rbcL 3'-UTR sequences for Nicotiana, Ipomoea, Cuscuta, and Orobanche reveal that nucleotide sequences from parasitic plants have regions capable of forming stem-loop structures, but 56–69 nt are deleted upstream of the stem-loop in the parasitic plants compared to their photosynthetic relatives. Although rbcL pseudogenes of O. cernua and O ramosa have many large and small deletions, few indels are shared in common, implying that their common ancestor probably had an intact rbcL reading frame. Intact rbcL reading frames in O. corymbosa and O. fasciculata retain a bias of synonymous over nonsynonymous substitutions and deduced protein sequences are consistent with potentially functional Rubisco large subunit proteins. A conservative model of random substitution processes in pseudogene sequences estimates that the probability is low (P<0.028) that these sequences would retain an open reading frame by chance. Species of Orobanche have either had recent photosynthetic ancestors, implying multiple independent losses of photosynthesis in this genus, or the rbcL gene may serve an unknown function in some nonphotosynthetic plants.     

In vitro infection of host roots by differentiated calli of the parasitic plant Orobanche

Root parasites of the genus Orobanche are serious weeds in agriculture. An aseptic infection system of host roots using calli of three Orobanche species was developed for the study of host-parasite interaction. The response of calli to various hormonal combinations was studied, because a requirement for infection is the differentiation of root-like protrusions, which are capable of producing haustorial connections to the host. Infectious root-like protrusions develop under the influence of 0.5-1.0 mg l(-1) IAA, and under the combination of 0.2 mg l(-1) NAA with 5.0 mg l(-1) kinetin. These protocols produced root protrusions with pad-like structures that resembled attachment organs of Orobanche seedlings, and proved effective in parasitizing host roots. Direct contact with the medium inhibited haustorium development and prevented infection. To overcome this problem, certain root portions were isolated from the medium by inserting thin glass plates underneath. Calli were then placed on the raised root portions and successfully infected the roots and developed young Orobanche tubercles with vascular system that directly connected to the host.     

Die Orobanchaceen der Türkei

A key for the Turkish species of Orobanche is given, one new species Orobanche sideana is described, and all localities from which the author has seen specimens, are enumerated.     

Fatty acids and tocochromanols in seeds of Orobanche

The evaluation of tocochromanols (tocopherols and tocotrienols) in 49 accessions from 21 Orobanche species revealed three well separated groups. The first one, characterized by high gamma-tocotrienol content, included all the accessions of sect. Orobanche. The second one, exhibiting high gamma-tocopherol content, comprised the accessions of O. arenaria Borkh. and O. purpurea Jacq. (sect. Trionychon Wallr.). All the other accessions of this section presented high delta-tocopherol content. Differences for tocochromanol derivatives within sect. Trionychon were paralleled by differences in the fatty acid profile, with the high delta-tocopherol class having also a higher oleic to linoleic acid ratio.     

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.     

Correlated evolution of life history and host range in the nonphotosynthetic parasitic flowering plants Orobanche and Phelipanche (Orobanchaceae)

Most members of the nonphotosynthetic parasitic genera Orobanche and Phelipanche (Orobanchaceae) have narrow host ranges, and, as they grow on perennial hosts, are (at least potentially) perennial themselves. A few species, however, have wide host ranges and grow on annual hosts, and are thus (at least facultatively) annuals themselves. Among the latter are the weedy species, which include economically important pest taxa such as Orobanche crenata or Phelipanche aegyptiaca. Using a phylogenetically based maximum likelihood approach, which takes phylogenetic and branch length uncertainty into account, we can show that the life trait host range and life history evolve in a correlated fashion. This supports the hypothesis that parasite specialization is associated with predictable resources (i.e. long-lived hosts) and generalism with unpredictable ones (i.e. short-lived hosts), a pattern often found in animal parasites. The mechanisms and temporal sequence of the life trait changes and their interrelations remain speculative.     

A plastid gene phylogeny of the non-photosynthetic parasitic <Emphasis Type="Italic">Orobanche</Emphasis> (Orobanchaceae) and related genera

The phylogenetic relationships of the non-photosynthetic Orobanche sensu lato (Orobanchaceae), which includes some of the economically most important parasitic weeds, remain insufficiently understood and controversial. This concerns both the phylogenetic relationships within the genus, in particular its monophyly or lack thereof, and the relationships to other holoparasitic genera such as Cistanche or Conopholis. Here we present the first comprehensive phylogenetic study of this group based on a region from the plastid genome (rps2 gene). Although substitution rates appear to be elevated compared to the photosynthetic members of Orobanchaceae, relationships among the major lineages Cistanche, Conopholis plus Epifagus, Boschniakia rossica (Cham. & Schltdl.) B. Fedtsch., B. himalaica Hook. f. & Thomson, B. hookeri Walp. plus B. strobilacea A. Gray, and Orobanche s. l. remain unresolved. Resolution within Orobanche, however, is much better. In agreement with morphological, cytological and other molecular phylogenetic evidence, five lineages, corresponding to the four traditionally recognised sections (Gymnocaulis, Myzorrhiza, Orobanche, Trionychon) and O. latisquama Reut. ex Boiss. (of sect. Orobanche), can be distinguished. A combined analysis of plastid rps2 and nuclear ITS sequences of the holoparasitic genera results in more resolved and better supported trees, although the relationships among Orobanche s. l., Cistanche, and the clade including the remaining genera is unresolved. Therefore, rps2 is a marker from the plastid genome that is well-suited to be used in combination with other already established nuclear markers for resolving generic relationships of Orobanche and related genera.     

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.