Radi Aly

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.

Broomrape (Orobanche cumana) Control in Sunflower (Helianthus annuus) with Imazapic1

Abstract: Field trials were conducted in 1997 and 1998 at two locations in Israel to evaluate the efficacy of imazapic applied postemergence (POST) to sunflower for broomrape control under irrigated and nonirrigated conditions. Two sequential treatments of imazapic at 1.5 followed by (FB) 3.0, 3.0 FB 4.5, or 4.5 FB 6.0 g ai/ha on sunflower plants 12 ± 3 and 55 ± 5 cm tall, respectively, reduced sunflower broomrape throughout the growing season under irrigated and nonirrigated conditions. Sunflower growth was not affected by imazapic treatments. It was confirmed, in accordance with an earlier report, that when sequential treatments of imazapic included an application at the sunflower inflorescence developmental stage, the herbicide decreased seed yield in proportion to the applied rate. Nomenclature: Imazapic; broomrape, Orobanche cumana Waller; sunflower, Helianthus annuus L. Additional index words: Parasitic weed, herbicide. Abbreviations: fb, followed by; POST, postemergence; PRE, preemergence.

A peptide from insects protects transgenic tobacco from a parasitic weed

Parasitic plants present some of the most intractable weed problems for agriculture in much of the world. Species of root parasites such as Orobanche can cause enormous yield losses, yet few control measures are effective and affordable. An ideal solution to this problem is the development of parasite-resistant crops, but this goal has been elusive for most susceptible crops. Here we report a mechanism for resistance to the parasitic angiosperm Orobanche based on expression of sarcotoxin IA in transgenic tobacco. Sarcotoxin IA is a 40-residue peptide with antibiotic activity, originally isolated from the fly, Sarcophaga peregrina. The sarcotoxin IA gene was fused to an Orobanche-inducible promoter, HMG2, which is induced locally in the host root at the point of contact with the parasite, and used to transform tobacco. The resulting transgenic plants accumulated more biomass than non-transformed plants in the presence of parasites. Furthermore, plants expressing sarcotoxin IA showed enhanced resistance to O. aegyptiaca as evidenced by abnormal parasite development and higher parasite mortality after attachment as compared to non-transformed plants. The transgenic plants were similar in appearance to non-transformed plants suggesting that sarcotoxin IA is not detrimental to the host.

Technologies for Smart Chemical Control of Broomrape (Orobanche spp. and Phelipanche spp.)

Abstract Broomrapes (Orobanche and Phelipanche spp.) are obligate root parasites that spend most of their life cycle in the soil subsurface, making them hard to detect. In these underground developmental stages, broomrapes are highly sensitive to herbicides, and therefore knowledge of the dynamics of their parasitism is essential to precisely apply herbicide for their control. To address these complexities, two approaches have been proposed: (1) estimating the temporal variation in parasitism dynamics and predicting broomrape parasitism on its host by thermal time; (2) characterizing the spatial variation in infestation within and between fields by using a geographical information system and a global positioning system. In addition, the use of molecular markers to identify broomrape infestation (species and amount) in the field can contribute to determining its spatial distribution, which can then be used for site-specific weed management. In this paper, we discuss how technology can be optimized for control of the root-parasitic broomrapes. Special attention is given to the development of integrative approaches. An example of a decision support system for the rational management of Egyptian broomrape in processing tomato is given. Nomenclature: Egyptian broomrape, Phelipanche aegyptiaca Pers. (syn. Orobanche aegyptiaca) ORAAE; tomato, Solanum lycopersicon L.

Formation of Norisoprenoid Flavor Compounds in Carrot (Daucus carota L.) Roots: Characterization of a Cyclic-Specific Carotenoid Cleavage Dioxygenase 1 Gene

Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and β-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and β-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

Biological activity of natural phytoecdysteroids from Ajuga iva against the sweetpotato whitefly Bemisia tabaci and the persea mite Oligonychus perseae.

BACKGROUND Ecdysteroids are steroid hormones that control moulting and govern several changes during metamorphoses in arthropods. The discovery of the same molecules (phytoecdysteroids) in several plant species displayed a wide array of rather beneficial agricultural impact. Many representatives of the genus Ajuga plants contain phytoecdysteroids with a 5β-7-ene-6-one system exhibiting physiological activities in insects. RESULTS By means of chromatographic (silica gel column, TLC) and LC-MS, two major ecdysteroids (20-hydroxyecdysone and cyasterone) have been isolated and identified from Israeli carpet bugle Ajuga iva (L.) Schreber (Lamiales: Lamiaceae) plants. Ajuga iva extract fractionated on the silica gel column yielded two fractions that showed high activity against the sweetpotato whitefly Bemisis tabaci and the persea mite Oligonychus perseae. A dose of 5 mg AI L(-1) of the purely identified A. iva ecdysterone significantly reduced fecundity, fertility and survival of these pests, while commercial 20-hydroxyecdysone at the same dose had lesser effects. CONCLUSION The results demonstrate considerable efficacy of natural phytoecdysteroids against major agricultural pests, and suggests that these materials should be considered for potential development of friendly control agents.

Biolistic transformation of Cercospora caricis a specific pathogenic fungus of Cyperus rotundus

Cercospora caricis is being considered as a bioherbicide agent for use against purple nutsedge (Cyperus rotundus), one of the worlds worst weeds. However, first its efficiency must be improved, for example by genetic transformation. By optimizing physical and biological parameters, the particle gun acceleration method (biolistics) has been adapted to transform mycelial cells of C. caricis. Two genes were expressed in C. caricis by biolistic transformation. The β-glucuronidase gene (GUS) fused to the GDP1 promoter of Cochliobolus hetrostrophus and the hygromycin B resistance gene under control of the PtrpC promoter of Aspergillus nidulans. Although the transformation frequency was not high, all transformants were stable when they were propagated on a selective medium after eight subsequent transfers.

Gene silencing of CCD7 and CCD8 in Phelipanche aegyptiaca by tobacco rattle virus system retarded the parasite development on the host

Strigolactones are phytohormones that stimulate seed germination of parasitic plants including Phelipanche aegyptiaca. Strigolactones are derived from carotenoids via a pathway involving the carotenoid cleavage dioxygenases CCD7 and CCD8. We report here identification of PaCCD7 and PaCCD8 orthologous genes from P. aegyptiaca. Expression analysis of PaCCD7 and PaCCD8 genes showed significant variation in their transcript levels in seeds and tubercles of P. aegyptiaca at different developmental stages. These two parasitic PaCCD7 and PaCCD8 genes were silenced in P. aegyptiaca using a trans-silencing approach in Nicotiana benthamiana. The transient knock-down of PaCCD7 and PaCCD8 inhibited tubercle development and the infestation process in host plants. Our results suggest an important role of the strigolactone associated genes (PaCCD7 and PaCCD8) in the parasite life cycle.

Host-Parasite-Bacteria Triangle: The Microbiome of the Parasitic Weed Phelipanche aegyptiaca and Tomato-Solanum lycopersicum (Mill.) as a Host

Broomrapes (Phelipanche/Orobanche spp.) are holoparasitic plants that subsist on the roots of a variety of agricultural crops, establishing direct connections with the host vascular system. This connection allows for the exchange of various substances and a possible exchange of endophytic microorganisms that inhabit the internal tissues of both plants. To shed some light on bacterial interactions occurring between the parasitic Phelipanche aegyptiaca and its host tomato, we characterized the endophytic composition in the parasite during the parasitization process and ascertained if these changes were accompanied by changes to endophytes in the host root. Endophyte communities of the parasitic weed were significantly different from that of the non-parasitized tomato root but no significant differences were observed between the parasite and its host after parasitization, suggesting the occurrence of bacterial exchange between these two plants. Moreover, the P. aegyptiaca endophytic community composition showed a clear shift from gram negative to gram-positive bacteria at different developmental stages of the parasite life cycle. To examine possible functions of the endophytic bacteria in both the host and the parasite plants, a number of unique bacterial candidates were isolated and characterized. Results showed that a Pseudomonas strain PhelS10, originating from the tomato roots, suppressed approximately 80% of P. aegyptiaca seed germination and significantly reduced P. aegyptiaca parasitism. The information gleaned in the present study regarding the endophytic microbial communities in this unique ecological system of two plants connected by their vascular system, highlights the potential of exploiting alternative environmentally friendly approaches for parasitic weed control.

Cucumber Mosaic Virus as a carotenoid inhibitor reducing Phelipanche aegyptiaca infection in tobacco plants.

Cucumber Mosaic Virus (CMV) is a highly infectious cucumovirus, which infects more than 800 plant species and causes major diseases in greenhouse and field crops worldwide. Parasitic weeds such as Phelipanche aegyptiaca are a major constraint to the production of many crops in the world and the parasites lifestyle makes control extremely difficult. The parasite seeds can germinate after conditioning and perceiving strigolactones secreted by the host roots. Strigolactones are rhizosphere signaling molecules in plants that are biosynthesized through carotenoid cleavage. In the present study we investigated the possibility of reducing β-carotene and then strigolactone production in the host roots by blocking carotenoid biosynthesis using CMV-infected tobacco. It was found that CMV downregulated the enzyme phytoene desaturase(PDS) and reduced significantly both carotenoid production and Phelipanche infection in tobacco host roots infected with both CMV and P. aegyptiaca. Based on our results (decrease of β-carotene and repression of PDS transcripts in tobacco roots), we hypothesized that the reduction of Phelipanche tubercles and shoots occurred due to an effect of CMV on secondary metabolite stimulators such as strigolacetones. Our study indicated that mass production of the host roots was not affected by CMV; however, most inflorescences of Phelipanche grown on CMV-infected tobacco developed abnormally (deformed shoots and short nodes). Carotenoid biosynthesis inhibitors such as CMV can be used to reduce the production of strigolactones, which will lead to decreased Phelipanche attachment. Interestingly, attenuated CMV strains may provide a safe means for enhancing crop resistance against parasitic weeds in a future plan.