The fungal pathogens Cochliobolus australiensis and Pyricularia grisea have recently been isolated from diseased leaves of buffelgrass (Cenchrus ciliaris) in its North American range, and their ability to produce phytotoxic metabolites that could potentially be used as natural herbicides against this invasive weed was investigated. Fourteen secondary metabolites obtained from in vitro cultures of these two pathogens were tested by leaf puncture assay on the host plant at different concentrations. Radicinin and (10S, 11S)-epi-pyriculol proved to be the most promising compounds. Thus, their phytotoxic activity was also evaluated on non-host indigenous plants. Radicinin demonstrated high target-specific toxicity on buffelgrass, low toxicity to native plants, and no teratogenic, sub-lethal, or lethal effects on zebrafish (Brachydanio rerio) embryos. It is now under consideration for the development of a target-specific bioherbicide to be used against buffelgrass in natural systems where synthetic herbicides cause excessive damage to native plants.

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.

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 11β,13-dihydrosalonitenolide using spectroscopic, spectrometric, and optical methods. Salonitenolide and 11β,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,β-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.

Buffelgrass (Cenchrus ciliaris L.) is an invasive C4 perennial grass species that substantially reduces native plant diversity of the Sonoran Desert through fire promotion and resource competition. Broad-spectrum herbicides are essentially used for its control, but they have a negative environmental and ecological impact. Recently, phytotoxicity on C. ciliaris has been discovered for two metabolites produced in vitro by the phytopathogenic fungi Cochliobolus australiensis and Pyricularia grisea. They were identified as (10S,11S)-(-)-epi-pyriculol and radicinin and resulted in being potential candidates for the development of bioherbicides for buffelgrass biocontrol. They have already shown promising results, but their ecotoxicological profiles and degradability have been poorly investigated. In this study, ecotoxicological tests against representative organisms from aquatic ecosystems (Aliivibrio fischeri bacterium, Raphidocelis subcapitata alga, and Daphnia magna crustacean) revealed relatively low toxicity for these compounds, supporting further studies for their practical application. The stability of these metabolites in International Organization for Standardization (ISO) 8692:2012 culture medium under different temperatures and light conditions was also evaluated, revealing that 98.90% of radicinin degraded after 3 days in sunlight. Significant degradation percentages (59.51-73.82%) were also obtained at room temperature, 30 °C or under ultraviolet (254 nm) light exposure. On the other hand, (10S,11S)-epi-pyriculol showed more stability under all the aforementioned conditions (49.26-65.32%). The sunlight treatment was also shown to be most effective for the degradation of this metabolite. These results suggest that radicinin could provide rapid degradability when used in agrochemical formulations, whereas (10S,11S)-epi-pyriculol stands as a notably more stable compound.

(10S,11S)-(-)-epi-pyriculol is a phytotoxic metabolite produced by Pyricularia grisea, a fungus identified as a foliar pathogen on the invasive weed species buffelgrass (Cenchrus ciliaris) in North America. The effective control of buffelgrass has not yet been achieved, and there is a need to develop effective and green solutions. Herbicides based on natural products and the use of phytopathogenic organisms could provide the most suitable tools for the control of weeds such as buffelgrass. Thus, one of the most relevant points to study about potential suitable phytotoxins such as (10S,11S)-(-)-epi-pyriculol is its production on a large scale, either by isolation from fungal fermentations or by synthesis. For these purposes, rapid and sensitive methods for the quantification of (10S,11S)-(-)-epi-pyriculol in complex mixtures are required. In this study, a high-pressure liquid chromatography (HPLC) method for its quantification was developed and applied to organic extracts from twelve P. grisea isolates obtained from diseased buffelgrass leaves and grown in potato dextrose broth (PDB) liquid cultures. The analysis proved that the production of (10S,11S)-(-)-epi-pyriculol is fungal-isolate dependent and strongly correlated with phytotoxic activity, shown by the P. grisea organic extracts in a buffelgrass radicle elongation test. The HPLC method reported herein allowed us to select the best strain for the production of (10S,11S)-(-)-epi-pyriculol and could be useful for selecting the best cultural conditions for its mass production, providing a tool for the use of this promising metabolite as a new bioherbicide for the control of buffelgrass.

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.