Orobanche species and population discrimination using intersimple sequence repeat (ISSR)

Summary Orobanche species are commonly identified using morphological characteristics. In many cases, the distinction of closely related species is difficult, and a molecular tool is more suitable to differentiate them. In this study, genomic polymorphism between morphologically distinct species was investigated through amplification by polymerase chain reaction (PCR) of intersimple sequence repeat (ISSR) regions. Five primers were used to study genetic variation in the morphologically distinct species O. hederae and O. amethystea, as well as the closely related species O. cernua and O. cumana. For the first two species, all the primers detected genetic polymorphism. Anchored primers allowed the identification of more specific molecular markers than non‐anchored tri‐ and tetranucleotide primers. Genetic polymorphism was investigated among three O. hederae populations using the two types of primer. One non‐anchored and two anchored primers detected intraspecific variation, which was not correlated with the geographical location of those populations. The primer (GATA)₄ detected polymorphism between five specimens each of O. cernua and O. cumana species collected from different countries, permitting these two closely related species to be clearly differentiated. This study demonstrated that ISSR markers can be highly reliable for precise identification of Orobanche species.     

Effects of Broomrape Parasitism on Sunflower Plants: Growth,Development, and Mineral Nutrition

Sunflower broomrape (Orobanche cumana Wallr.) is a parasitic plant that infects sunflower (Helianthus annuus L.) plants. In this work, sunflower plants were grown under greenhouse conditions in pots with the substrate infested or non-infested with broomrape seeds. At different numbers of days after sowing, plant height, internode lengths, number of leaves, head diameter, mineral composition of leaves, and potassium (K) concentration in stem were measured. The negative effects of broomrape parasitism were assessed from 57 d after sowing, when broomrape started to emerge. Parasitized plants exhibited lower shoot dry weight, height, and head diameter than control plants. The reduction in internode lengths was associated with a decrease in the gradient of K concentration from basal to apical stem. The mineral composition of leaves was also affected in parasitized plants. The concentrations of calcium (Ca), magnesium (Mg), manganese (Mn), and zinc (Zn) in leaves of parasitized plants were lower than those of the control plants, while there were few differences for K, phosphorus (P), iron (Fe), and copper (Cu). The effects of parasitism are discussed in relation to their competition for resources and to perturbations of the host physiology such as hormonal and water balance.     

Molecular analysis of resistance mechanisms to Orobanche cumana in sunflower

Resistance to the dicotyledenous parasite Orobanche cumana in sunflower is characterized by a low number of parasitic attachments and a confinement of the parasite in host tissues leading to its necrosis. To help understand what determines such resistance mechanisms, molecular, biochemical and histological approaches were employed before (early response) and after (late response) attachment of the broomrape parasite to susceptible (2603) and resistant (LR1) sunflower genotypes. The expression patterns of 11 defence-related genes known to be involved in different metabolic pathways (phenylpropanoids, jasmonate, ethylene) and/or in resistance mechanisms against microorganisms were investigated. RT-PCR and cDNA blot experiments revealed that the resistant genotype exhibited a stronger overall defence response against O. cumana than the susceptible one, involving marker genes of the jasmonate (JA) and salicylic acid (SA) pathways. Among them, the SA-responsive gene, def. (defensin), appeared to be characteristic of LR1 sunflower resistance. However, no JA accumulation and similar SA contents (250–300 ng g⁻¹ FW) were measured by GC/MS in both genotypes, parasitized or not. In addition, three cDNAs, isolated by a suppression-subtractive hybridization, were shown to be strongly induced only in the resistant genotype 8 days post-inoculation, when the first O. cumana attachments occurred. These genes, putatively encoding a methionine synthase, a glutathione S-transferase and a quinone oxidoreductase, might be involved in detoxification of reactive oxygen species, suggesting the occurrence of an oxidative burst during the incompatible interaction. Finally, host cell-wall modifications leading to parasite-confinement were correlated with more intense callose depositions in the resistant genotype, concomitant with over-expression of the callose synthase cDNA HaGSL1 .     

Direct infection of potato tubers by the root parasite Orobanche aegyptiaca

Parasitic plants of the Orobanchaceae are known as obligate root parasites that develop haustoria that connect to roots of various host plants. This article describes, for the first time, a case where the root parasite successfully connected to potato tubers, i.e. to the swollen portion of an underground stem rather than to a root. The rhizosphere of Orobanche aegyptiaca and of its host Solanum tuberosum (potato) was carefully examined. In anatomical studies, the adventitious roots were directly connected to potato tubers. Numerous secondary haustoria, which developed along the adventitious roots in close vicinity to the potato tuber, penetrated the tuber epidermis and the perimedullary tuber parenchyma and developed xylem strands that are presumably connected to the minor xylem strands within the tuber cortex. These findings indicate that parasites of the Orobanchaceae that normally attack host roots may also parasitise underground stem tubers.     

Seed viability determination in parasitic broomrapes (Orobanche and Phelipanche) using fluorescein diacetate staining

Fluorescein diacetate (FDA) was investigated as a potential viability stain for seeds of holoparasitic broomrapes Orobanche and Phelipanche (Orobanchaceae), using four weedy and two non-weedy taxa. FDA viabilities were compared with the currently used 2,3,5-triphenyl tetrazolium chloride (TTC) test, and in vitro germination induced by the strigolactone analogue GR-24. Viable FDA-stained seeds were characterised by bright yellow–green fluorescence under 450–490 nm blue light. These viable seeds could be discriminated from non-viable seeds, in which low levels of fluorescence, similar to non-stained control seeds were observed. FDA viabilities were not significantly different from those obtained using the TTC stain; however, viability values for both stains were consistently higher than levels of artificially induced germination. Positive TTC-staining showed continuous variation that was difficult to interpret. Nevertheless, the TTC test does not involve grinding seeds, and therefore probably remains a practical alternative to FDA for screening soil samples contaminated with Orobanche and Phelipanche seeds, which indicates different applications for the two viability tests. Interestingly, the strigolactone analogue GR-24 only induced germination in O. crenata , P. ramosa and O. minor , suggesting a high specificity towards this germination inducer among the species investigated. We suggest that FDA-staining provides a potential alternative to the currently used TTC test for seed viability assays for Orobanche and Phelipanche , and other parasitic weeds which are an obstacle to crop cultivation.     

Identification of PCR markers linked to different Or genes in sunflower

Broomrape is a parasitic plant that significantly decreases yield of sunflower. Breeding for resistance has proved to be the most efficient method for suppressing broomrape infestation in the field; however, new races of parasite constantly emerge, and new resistance genes need to be discovered and introduced into cultivated sunflower lines. The aim of this work was to test SSR markers from linkage group 3 (LG3) to investigate whether they could be used for identification of a particular Or gene. Twenty sunflower inbred lines were used, and polymorphism between the lines with various resistance genes and genetic background was investigated. The used markers revealed DNA polymorphism between the investigated lines. Strong association of markers from LG3 with Or₆, as well as Or₄ and Or₂ genes, was found. Identified markers could be used for introduction of these resistance genes into commercial sunflower lines and for establishment and identification of differential lines.     

Effects of arbuscular mycorrhizal fungal symbiosis for control of Egyptian broomrape (Orobanche aegyptiaca Pers.) in tomato (Lycopersicon esculentum L.) cultivation

The present study assessed the effect of arbuscular mycorrhizal fungi (AMF) on broomrape (Orobanche aegyptiaca Pers.) control to determine its effects on tomato (Lycopersicon esculentum L.) growth indices. This experiment was carried out using a randomized complete block design with four replications. The treatments included three strains of AMF (Glomus mosseae, Glomus intraradices and Glomuse hoe ) at three levels (100, 200 and 300 kg ha^?1). The control treatments (without mycorrhiza) were with and without broomrape (weed infest and weed free). The results showed that the use of G. intraradices and G. mosseae decreased broomrape seed germination, the number of nodules and the dry weight of the broomrape and increased root area and dry weight of the tomato plant when compared to G. hoe and the control treatments without broomrape. The tomato yield in G. intraradices 300 kg ha^?1 treatment increased by 10 and 205% than weed free and weed infest treatments, respectively. Finally, G. intraradices and G. mosseae at, respectively, 200 and 300 kg ha^?1 are recommended for land under tomato cultivation that has been contaminated with broomrape.     

Parasitic plant infection is partially controlled through symbiotic pathways

Legumes are unique in interacting with Rhizobium , arbuscular mycorrhizal (AM) fungi, and parasitic plants. To dissect common parts of these three plant–organism interactions, infection by Orobanche crenata was studied in mutants with altered symbiotic phenotypes of Medicago truncatula and Pisum sativum . Orobanche crenata inoculation of mutant lines carrying defective mutation in the genes dmi2 / sym19 and dmi3 resulted in an increase in O. crenata establishment. Similarly, inoculation of mutants carrying mutation in the gene sunn / sym29 that controls the autoregulation mechanism of the symbiosis, also lead to a significant increase in haustoria formation. Altogether, our results suggest that parasitic plant infection is partly controlled by both the conserved symbiotic pathway that mediates symbiont recognition and establishment and the autoregulation mechanism that regulates the extent of colonisation by Rhizobium and AM fungi.