(4Z)-Lachnophyllum Lactone, a Metabolite with Phytotoxic and Antifungal Activity against Pests Affecting Mediterranean Agriculture: A New Versatile and Easy Scalable Parallel Synthesis.

A methodology for the total and modulable synthesis of (4Z)-lachnophyllum lactone (1), on a gram scale, is reported for the first time. The present work started with the design of a retrosynthetic pathway for the target compound, with the key step identified in Pd-Cu bimetallic cascade cross-coupling cyclization. (4Z)-Lachnophyllum lactone (1) is an acetylenic furanone previously isolated, in a low amount, from the organic extract of the autotrophic weedConyza bonariensis. Tested against the stem parasitic weed Cuscuta campestris in a seedling growth bioassay, (4Z)-lachnophyllum lactone (1) showed almost 85% of inhibitory activity up to 0.3 mM in comparison with the control. At the same concentration, the compound displayed radicle growth inhibitory activity of the root parasitic weeds Orobanche minor and Phelipanche ramosa higher than 70 and 40%, respectively. Surprisingly, the compound showed a high percentage of inhibition, up to 0.1 mM, on C. bonariensis seed germination too. This versatile synthetic strategy was also used to obtain two further natural analogues, namely, (4E)-lachnophyllum lactone (8) and (4Z,8Z)-matricaria lactone (9), that showed, in most cases, the same inhibitory trend with slight differences, highlighting the importance of the stereochemistry and unsaturation of the side chain. Furthermore, all of the compounds showed antifungal activity at 1 mM reducing the mycelial growth of the olive pathogen Verticillium dahliae. The design and implementation of scalable and modulable total synthesis on a gram scale of acetylenic furanones allow the production of a large amount of these natural products, overcoming the limit imposed by isolation from natural sources. The results of the present study pave the way for the development of ecofriendly bioinspired pesticides with potential application in agrochemical practices as alternative to synthetic pesticides.

Sesquiterpene Lactones Isolated from Centaurea cineraria L. subsp. cineraria Inhibit the Radicle Growth of Broomrape Weeds.

The plant Centaurea cineraria L. subsp. cineraria has been investigated as a potential source of inhibitors of broomrape radicle growth. The latter are weeds that pose a threat to agriculture and for which there are few methods available for the control of infestations. Four sesquiterpene lactones have been isolated from C. cineraria L. subsp. cineraria aerial parts and identified as isocnicin, cnicin, salonitenolide, and 11ß,13-dihydrosalonitenolide using spectroscopic, spectrometric, and optical methods. Salonitenolide and 11ß,13-dihydrosalonitenolide have been isolated for the first time from this plant. Tested at 1.0-0.1 mM against the broomrape species Phelipanche ramosa, Orobanche minor, Orobanche crenata, and Orobanche cumana, isocnicin, cnicin, and salonitenolide demonstrated remarkable inhibitory activity (over 80% in most of the cases) at the highest concentrations. Structure-activity relationship conclusions indicated the significance of the α,ß-unsaturated lactone ring. In addition, the synthetic acetylated derivative of salonitenolide showed the strongest activity among all compounds tested, with inhibitions close to 100% at different concentrations, which has been related to a different lipophilicity and the absence of H-bond donor atoms in its structure. Neither the extracts nor the compounds exhibited the stimulating activity of broomrape germination (induction of suicidal germination). These findings highlight the potential of C. cineraria to produce bioactive compounds for managing parasitic weeds and prompt further studies on its sesquiterpene lactones as tools in developing natural product-based herbicides.

Structure-Activity Relationship (SAR) Study of trans-Cinnamic Acid and Derivatives on the Parasitic Weed Cuscuta campestris.

Cuscuta campestris Yunck. is a parasitic weed responsible for severe yield losses in crops worldwide. The selective control of this weed is scarce due to the difficult application of methods that kill the parasite without negatively affecting the infected crop. trans-Cinnamic acid is secreted by plant roots naturally into the rhizosphere, playing allelopathic roles in plant-plant communities, although its activity in C. campestris has never been investigated. In the search for natural molecules with phytotoxic activity against parasitic weeds, this work hypothesized that trans-cinnamic acid could be active in inhibiting C. campestris growth and that a study of a series of analogs could reveal key structural features for its growth inhibition activity. In the present structure-activity relationship (SAR) study, we determined in vitro the inhibitory activity of trans-cinnamic acid and 24 analogs. The results showed that trans-cinnamic acid's growth inhibition of C. campestris seedlings is enhanced in eight of its derivatives, namely hydrocinnamic acid, 3-phenylpropionaldehyde, trans-cinnamaldehyde, trans-4-(trifluoromethyl)cinnamic acid, trans-3-chlorocinnamic acid, trans-4-chlorocinnamic acid, trans-4-bromocinnamic acid, and methyl trans-cinnamate. Among the derivatives studied, the methyl ester derivative of trans-cinnamic acid was the most active compound. The findings of this SAR study provide knowledge for the design of herbicidal treatments with enhanced activity against parasitic weeds.

Characterization of Conyza bonariensis Allelochemicals against Broomrape Weeds.

The study of allelopathic activity of plants and the isolation and characterization of the responsible allelochemicals can lead to the development of environment friendly alternative approaches to weed control. Conyza species are invasive weeds that use allelopathic activity as part of a successful strategy to outcompete neighboring plants. Broomrape weeds are parasitic plants that use host-induced germination and the formation of a haustorium as strategies to infect host plants. The control of broomrape infection in most affected crops is limited or non-existing. In the current study, we investigated the allelopathic activity of Conyza bonariensis organic extracts in suicidal germination and radicle growth of four broomrape species (Orobanche crenata, Orobanche cumana, Orobanche minor and Phelipanche ramosa). A bioactivity-driven fractionation of Conyza bonariensis extracts led to the identification of two germination-inducing molecules and two growth-inhibitory compounds. The germination-inducing metabolites had species-specific activity being hispidulin active on seeds of O. cumana and methyl 4-hydroxybenzoate active in P. ramosa. The growth-inhibitory metabolites (4Z)-lachnophyllum lactone and (4Z,8Z)-matricaria lactone strongly inhibited the radicle growth of all parasitic weed species studied. Some structure-activity relationships were found as result of the study herein presented.

Identification of Structural Features of Hydrocinnamic Acid Related to Its Allelopathic Activity against the Parasitic Weed Cuscuta campestris.

Cuscuta campestris is a parasitic weed species that inflicts worldwide noxious effects in many broadleaf crops due to its capacity to withdraw nutrients and water directly from the crop vascular system using haustorial connections. Cuscuta campestris control in the majority of crops affected is non-existent, and thus, research for the development of control methods is needed. Hydrocinnamic acid occurs naturally in the rhizosphere, playing regulatory roles in plant-plant and plant-microbe communities. The toxicity of hydrocinnamic acid against C. campestris was recently identified. In the present work, a structure-activity relationship study of 21 hydrocinnamic acid analogues was performed to identify key structural features needed for its allelopathic action against the seedling growth of this parasitic plant. The findings of this study provide the first step for the design of herbicides with enhanced activity for the control of C. campestris infection.

Iridoid Glycosides Isolated from Bellardia trixago Identified as Inhibitors of Orobanche cumana Radicle Growth.

Orobanche cumana is an obligate holoparasitic plant with noxious effects in sunflower crops. Bellardia trixago is a facultative hemiparasitic plant that infects ruderal plants without noxious significance in agriculture and is known to produce a wide spectrum of bioactive metabolites. The objective of this study was to evaluate the allelopathic effects of B. trixago on the growth of O. cumana seedlings. Three different extracts using solvents of increasing polarity (n-hexane, dichloromethane and ethyl acetate) were prepared from the flowers, aerial green organs and roots of two populations, a white-flowered and a yellow-flowered population of B. trixago, both collected in southern Spain. Each extract was studied using allelopathic screenings on O. cumana which resulted in the identification of allelopathic activity of the ethyl acetate extracts against Orobanche radicles. Five iridoid glycosides were isolated together with benzoic acid from the ethyl acetate extract of aerial green organs by bio-guided purification. These compounds were identified as bartsioside, melampyroside, mussaenoside, gardoside methyl ester and aucubin. Among them, melampyroside was found to be the most abundant constituent in the extract (44.3% w/w), as well as the most phytotoxic iridoid on O. cumana radicle, showing a 72.6% inhibition of radicle growth. This activity of melampyroside was significantly high when compared with the inhibitory activity of benzoic acid (25.9%), a phenolic acid with known allelopathic activity against weeds. The ecotoxicological profile of melampyroside was evaluated using organisms representing different trophic levels of the aquatic and terrestrial ecosystems, namely producers (green freshwater algae Raphidocelis subcapitata and macrophyte Lepidium sativum), consumers (water flea Daphnia magna and nematode Caenorhabditis elegans) and decomposers (bacterium Aliivibrio fischeri). The ecotoxicity of melampyroside differed significantly depending on the test organism showing the highest toxicity to daphnia, nematodes and bacteria, and a lower toxicity to algae and macrophytes. The findings of the present study may provide useful information for the generation of green alternatives to synthetic herbicides for the control of O. cumana.

Specialized Metabolites from the Allelopathic Plant Retama raetam as Potential Biopesticides.

To cope with the rising food demand, modern agriculture practices are based on the indiscriminate use of agrochemicals. Although this strategy leads to a temporary solution, it also severely damages the environment, representing a risk to human health. A sustainable alternative to agrochemicals is the use of plant metabolites and plant-based pesticides, known to have minimal environmental impact compared to synthetic pesticides. Retama raetam is a shrub growing in Algeria's desert areas, where it is commonly used in traditional medicine because of its antiseptic and antipyretic properties. Furthermore, its allelopathic features can be exploited to effectively control phytopathogens in the agricultural field. In this study, six compounds belonging to isoflavones and flavones subgroups have been isolated from the R. raetam dichloromethane extract and identified using spectroscopic and optical methods as alpinumisoflavone, hydroxyalpinumisoflavone, laburnetin, licoflavone C, retamasin B, and ephedroidin. Their antifungal activity was evaluated against the fungal phytopathogen Stemphylium vesicarium using a growth inhibition bioassay on PDA plates. Interestingly, the flavonoid laburnetin, the most active metabolite, displayed an inhibitory activity comparable to that exerted by the synthetic fungicide pentachloronitrobenzene, in a ten-fold lower concentration. The allelopathic activity of R. raetam metabolites against parasitic weeds was also investigated using two independent parasitic weed bioassays to discover potential activities on either suicidal stimulation or radicle growth inhibition of broomrapes. In this latter bioassay, ephedroidin strongly inhibited the growth of Orobanche cumana radicles and, therefore, can be proposed as a natural herbicide.

Genetic and physiological characterization of sunflower resistance provided by the wild-derived OrDeb2 gene against highly virulent races of Orobanche cumana Wallr.

KEY MESSAGE: OrDeb2 confers post-attachment resistance to Orobanche cumana and is located in a 1.38 Mbp genomic interval containing a cluster of receptor-like kinase and receptor-like protein genes with nine high-confidence candidates. Sunflower broomrape is a holoparasitic angiosperm that parasitizes on sunflower roots, severely constraining crop yield. Breeding for resistance is the most effective method of control. OrDeb2 is a dominant resistance gene introgressed into cultivated sunflower from a wild-related species that confers resistance to highly virulent broomrape races. The objectives of this study were as follows: (i) locate OrDeb2 into the sunflower genome and determine putative candidate genes and (ii) characterize its underlying resistance mechanism. A segregating population from a cross between the sunflower resistant line DEB2, carrying OrDeb2, and a susceptible line was phenotyped for broomrape resistance in four experiments, including different environments and two broomrape races (FGV and GTK). This population was also densely genotyped with microsatellite and SNP markers, which allowed locating OrDeb2 within a 0.9 cM interval in the upper half of Chromosome 4. This interval corresponded to a 1.38 Mbp genomic region of the sunflower reference genome that contained a cluster of genes encoding LRR (leucine-rich repeat) receptor-like proteins lacking a cytoplasmic kinase domain and receptor-like kinases with one or two kinase domains and lacking an extracellular LRR region, which were valuable candidates for OrDeb2. Rhizotron and histological studies showed that OrDeb2 determines a post-attachment resistance response that blocks O. cumana development mainly at the cortex before the establishment of host-parasite vascular connections. This study will contribute to understand the interaction between crops and parasitic weeds, to establish durable breeding strategies based on genetic resistance and provide useful tools for marker-assisted selection and OrDeb2 map-based cloning.

Genetic fingerprint and diversity evaluation of halophilic Bacillus species by RAPD-PCR.

Random amplified polymorphic DNA-PCR (RAPD-PCR) is a technique successfully used to generate characteristic fingerprints of different bacteria. Bacillus is a genus that includes heterogeneous species, thus a combination of different techniques is essential for their identification. Here we used RAPD-PCR methodology to distinguish among genetically similar strains and to evaluate the genetic diversity of Bacillus species from the Salar del Hombre Muerto, in the Northwest of Argentina. The RAPD-PCR used allowed obtaining different amplification profiles for each Bacillus species and strains. By comparing the fingerprint profiles, we could observe that some of the salt flat isolates showed similar profiles than identified strains. As expected, the bacilli group isolated revealed a wide heterogeneity. RAPD-PCR was found to be a quick and reliable technique to evaluate the diversity of Bacillus strain and was successfully applied to characterize the genetic diversity present in the Salar del Hombre Muerto.

Allelopathic Effect of Quercetin, a Flavonoid from Fagopyrum esculentum Roots in the Radicle Growth of Phelipanche ramosa: Quercetin Natural and Semisynthetic Analogues Were Used for a Structure-Activity Relationship Investigation.

Allelopathic potential of buckwheat roots on the radicle growth of the broomrape weed species Orobanche cumana and Phelipanche ramosa was studied. Buckwheat root exudates induced a significant growth inhibition in P. ramosa radicles but radicles of O. cumana were not affected. Among the metabolites present in the root organic extract we identified the flavonol quercetin and the stilbene p-coumaric acid methyl ester with only quercetin showing inhibitory effect on P. ramosa. The activity of quercetin was compared with other two similar flavanoids, the flavone apigenin and the dihydroflavanol 3-O-acetylpadmatin extracted respectively from Lavandula stoechas and Dittrichia viscosa plants. In this comparative assay only 3-O-acetylpadmatin besides quercetin, showed inhibition activity of radicle growth while apigenin was inactive. These results indicated that the presence of two ortho-free hydroxy groups of C ring, like catechol, could be an important feature to impart activity while the carbon skeleton of B ring and substituents of both A and B rings are not essential. Besides reduction of radicle growth, haustorium induction was observed at the tip of P. ramosa radicles treated with quercetin which swelled and a layer of papillae was formed. Activity of quercetin on haustorium induction in P. ramosa was assayed in comparison with the known haustorium-inducing factor 2,6-dimethoxy-p-benzoquinone (DMBQ) and a three partial methyl ether derivatives semisynthetized from quercetin. Results indicated that P. ramosa haustorium was induced by DMBQ at concentrations of 1-0.5 mM and quercetin and its derivatives at concentration range 0.1-0.05 mM.

Search for Resistant Genotypes to Cuscuta campestris Infection in Two Legume Species, Vicia sativa and Vicia ervilia.

The dodders (Cuscuta spp.) are parasitic plants that feed on the stems of their host plants. Cuscuta campestris is one of the most damaging parasitic plants for the worldwide agricultural production of broad-leaved crops. Its control is limited or non-existent, therefore resistance breeding is the best alternative both economically and environmentally. Common vetch (Vicia sativa) and bitter vetch (Vicia ervilia) are highly susceptible to C. campestris, but no resistant genotypes have been identified. Thus, the aim of this study was to identify in V. sativa and V.ervilia germplasm collections genotypes resistant to C. campestris infection for use in combating this parasitic plant. Three greenhouse screening were conducted to: (1) identify resistant responses in a collection of 154 accessions of bitter vetch and a collection of 135 accessions of common vetch genotypes against infection of C. campestris; (2) confirm the resistant response identified in common vetch accessions; and (3) characterize the effect of C. campestris infection on biomass of V. sativa resistant and susceptible accessions. Most common vetch and bitter vetch genotypes tested were susceptible to C. campestris. However, the V. sativa genotype Vs.1 exhibited high resistance. The resistant phenotype was characterized by a delay in the development of C. campestris posthaustorial growth and a darkening resembling a hypersensitive-like response at the penetration site. The resistant mechanism was effective in limiting the growth of C. campestris as the ratio of parasite/host shoot dry biomass was more significantly reduced than the rest of the accessions. To the best or our knowledge, this is the first identification of Cuscuta resistance in V. sativa genotypes.

Effects of Benzoquinones on Radicles of Orobanche and Phelipanche Species.

The holoparasitic broomrape weeds (Orobanche and Phelipanche species) cause severe yield losses throughout North Africa, the Middle East, and Southern and Eastern Europe. These parasitic weeds form an haustorium at the tip of their radicles to infect the crop upon detection of the host-derived haustorium-inducing factors. Until now, the haustorial induction in the broomrapes remains less studied than in other parasitic plant species. Known haustorium-inducing factors active in hemiparasites, such as Striga and Triphysaria species, were reported to be inefficient for the induction of haustoria in broomrape radicles. In this work, the haustorium-inducing activity of p-benzoquinone and 2,6-dimethoxy-p-benzoquinone (BQ and DMBQ) on radicles of three different broomrapes, namely Orobanche cumana, Orobanche minor and Phelipanche ramosa, is reported. Additional allelopathic effects of benzoquinones on radicle growth and radicle necrosis were studied. The results of this work suggest that benzoquinones play a role in the induction of haustorium in broomrapes. Although dependent on the broomrape species assayed and the concentration of quinones used in the test, the activity of BQ appeared to be stronger than that of DMBQ. The redox property represented by p-benzoquinone, which operates in several physiological processes of plants, insects and animals, is invoked to explain this different activity. This work confirms the usefulness of benzoquinones as haustorium-inducing factors for holoparasitic plant research. The findings of this work could facilitate future studies in the infection process, such as host-plant recognition and haustorial formation.

Resistance against Orobanche crenata in Bitter Vetch (Vicia ervilia) Germplasm Based on Reduced Induction of Orobanche Germination.

Bitter vetch (Vicia ervilia (L.) Willd.) is a legume well adapted to cultivation in marginal areas, being an important source of protein for animal feed in low input cropping systems. Surprisingly, it is an underutilized crop as it could be a good alternative to increase the sustainability of extensive rainfed cropping systems. In Mediterranean rainfed cropping systems, the productivity of bitter vetch is severely reduced by the parasitic weed species Orobanche crenata (Forsk). To date, few resistant bitter vetch genotypes have been identified. O. crenata infection process initiates with the recognition of germination factors exuded by roots of susceptible hosts. In this work, the interaction of a collection of bitter vetch accessions and O. crenata has been analyzed in order to discover accessions with low germination induction activity. Through a combination of field and rhizotron experiments, two bitter vetch accessions were selected showing low germination-induction activity, which resulted in less infection. In addition, in vitro germination assays revealed that the low germination activity was due to low exudation of germination factors and not due to the exudation of germination inhibitors. The selected low germination-inducers genotypes could be the basis for a new breeding program generating locally adapted alternatives with resistance to O. crenata.

Identification of Vicia ervilia Germplasm Resistant to Orobanche crenata.

Bitter vetch (Vicia ervilia L.) is an ancient grain legume used as animal feed in the Mediterranean basin. This legume has a large economical potential because of its high yield under low inputs and good protein content, as well as resistance to cold and drought. Nevertheless, its growth and production area are affected in the presence of the broomrape weed species Orobanche crenata. Due to the small bitter vetch size, infection by as few as two or three O. crenata per vetch plant can be devastating. There are no efficient methods of selectively controlling O. crenata in this crop, for which reason the development of varieties resistant and tolerant to O. crenata infection is needed. Phytogenetic resources are valuable reserves for species survival. They represent important genetic variability and allow the possibility of finding characters of interest, such as new resistance sources. A large-scale field screening of a collection of 102 bitter vetch accessions indicated that most bitter vetch accessions were susceptible but allowed us to select 16 accessions with low levels of O. crenata infection. Next, we used a combination of field and rhizotron experiments to investigate the resistant response of selected bitter vetch genotypes in detail by studying the performance and resistance mechanisms. These experiments led to the identification of three different mechanisms that block O. crenata parasitism. A pre-attachment mechanism of low induction of O.crenata germination was identified in two bitter vetch accession Ve.055 and Ve.155. In addition, a post-attachment mechanism of resistance to O. crenata penetration was identified inthe accession Ve.125. In addition, the field-resistant accession Ve.123 showed susceptible response in rhizotron, indicating that a late mechanism acting after vascular connection, most probably related with bitter vetch of escape due to fructification precocity was acting against O. crenata development.

Management of Infection by Parasitic Weeds: A Review.

Parasitic plants rely on neighboring host plants to complete their life cycle, forming vascular connections through which they withdraw needed nutritive resources. In natural ecosystems, parasitic plants form one component of the plant community and parasitism contributes to overall community balance. In contrast, when parasitic plants become established in low biodiversified agroecosystems, their persistence causes tremendous yield losses rendering agricultural lands uncultivable. The control of parasitic weeds is challenging because there are few sources of crop resistance and it is difficult to apply controlling methods selective enough to kill the weeds without damaging the crop to which they are physically and biochemically attached. The management of parasitic weeds is also hindered by their high fecundity, dispersal efficiency, persistent seedbank, and rapid responses to changes in agricultural practices, which allow them to adapt to new hosts and manifest increased aggressiveness against new resistant cultivars. New understanding of the physiological and molecular mechanisms behind the processes of germination and haustorium development, and behind the crop resistant response, in addition to the discovery of new targets for herbicides and bioherbicides will guide researchers on the design of modern agricultural strategies for more effective, durable, and health compatible parasitic weed control.

Inuloxin E, a New Seco-Eudesmanolide Isolated from Dittrichia viscosa, Stimulating Orobanche cumana Seed Germination.

A new sesquiterpenoid belonging to the subgroup seco-eudesmanolides and named inuloxin E was isolated from Dittrichia viscosa, together with the already known sesquiterpenoids inuloxins A-D and α-costic acid. Inuloxin E was characterized by spectroscopic data (essentially NMR and ESI MS) as 3-methylene-6-(1-methyl-4-oxo-pentyl)-3a,4,7,7a-tetrahydro-3H-benzofuran-2-one. Its relative configuration was determined by comparison with the closely related inuloxin D and chemical conversion of inuloxin E into inuloxin D and by the observed significant correlation in the NOESY spectrum. Both inuloxins D and E induced germination of the parasitic weed Orobanche cumana, but were inactive on the seeds of Orobanche minor and Phelipanche ramosa. The germination activity of some hemisynthetic esters of inuloxin D was also investigated.

A Novel Electron Microscopic Characterization of Core/Shell Nanobiostimulator Against Parasitic Plants.

Nanoencapsulation has proven to be an efficient route to increase significantly the solubility and bioavailability of organic compounds. This aspect of nanotechnology is illustrated for the case of phthalimide-lactone (PL), a recently synthesized strigolactone mimic whose very limited solubility in water, as a free chemical, precludes its practical use as an agrochemical in the fight against parasitic plants. Pluronic F-127 (P127) nanoparticles functionalized with PL have been synthesized and embedded in a polymeric matrix of poly(vinyl alcohol) (PVA). Low-voltage and medium voltage imaging and spectroscopic scanning electron microscopy (S(T)EM) techniques were combined to confirm the synthesis of multicore nanoparticles that were rich in nitrogen, a finding that is due to the successful encapsulation of PL. This PL@P127/PVA nanobiostimulator formulation has an impressive solubility in water, that is, 27 times higher than that of pure phthalimide-lactone. Also critical from the functional point of view, comparative bioassays clearly showed that the intrinsic stimulatory activity of this agrochemical is fully maintained in the nanoencapsulated formulation.