Angela Boari

Natural metabolites for parasitic weed management

Compounds of natural origin, such as phytotoxins produced by fungi or natural amino acids, could be used in parasitic weed management strategies by interfering with the early growth stages of the parasites. These metabolites could inhibit seed germination or germ tube elongation, so preventing attachment to the host plant, or, conversely, stimulate seed germination in the absence of the host, contributing to a reduction in the parasite seed bank. Some of the fungal metabolites assayed were very active even at very low concentrations, such as some macrocyclic trichothecenes, which at 0.1 microM strongly suppressed the germination of Orobanche ramosa L. seeds. Interesting results were also obtained with some novel toxins, such as phyllostictine A, highly active in reducing germ tube elongation and seed germination both of O. ramosa and of Cuscuta campestris Yuncker. Among the amino acids tested, methionine and arginine were particularly interesting, as they were able to suppress seed germination at concentrations lower than 1 mM. Some of the fungal metabolites tested were also able to stimulate the germination of O. ramosa seeds. The major findings in this research field are described and discussed.

Inuloxins A-D, phytotoxic bi-and tri-cyclic sesquiterpene lactones produced by Inula viscosa: potential for broomrapes and field dodder management.

Four phytotoxic bi- and tri-cyclic sesquiterpene lactones, named inuloxins A-D, were isolated together with the known α-costic acid, from the aerial parts of Inula viscosa (family Asteraceae), a widespread Mediterranean plant well known for its content of pharmacologically active metabolites. The structures of inuloxins A-D were established by spectroscopic and chemical methods and determined to be: (4E,7R*,8R*,10S*)-3-oxo-germacra-4,11(13)-dien-8β-12-olide (A), its 11,13-dihydro analogue (B), (5R*,7R*,8R*,10R*)-1,15-methylene-5β-hydroxy-eudesm-1(15),11(13)-dien-8β-12-olide (C), and (7R*,8R*)-1,4-dimethyl-4-hydroxy-secoeudesm-5(10),11(13)-dien-8β-12-olide (D). The S absolute stereochemistry at C-5 of 5-hydroxyhexan-2-yl side chain of inuloxin D was assigned by applying an advanced Moshers method. The phytotoxic activity of inuloxins A-D, that of the diazo and monoacetyl derivatives (of inuloxin A and C, resply), as well as that of α-costic acid was evaluated against two parasitic plant species, i.e. crenate broomrape (Orobanche crenata) and field dodder (Cuscuta campestris). Inuloxins A, C and D were the most active on both parasites and caused up to 100% inhibition of the seed germination. Inuloxin B was less active on Cuscuta and completely inactive against Orobanche. The main metabolite α-costic acid had a suppressive effect on the dodder seed germination but had a stimulating action on the broomrape seed germination. These preliminary results allowed to suppose some structure-activity relationships.

Gulypyrones A and B and Phomentrioloxins B and C Produced by Diaporthe gulyae, a Potential Mycoherbicide for Saffron Thistle (Carthamus lanatus).

A virulent strain of Diaporthe gulyae, isolated from stem cankers of sunflower and known to be pathogenic to saffron thistle, has been shown to produce both known and previously undescribed metabolites when grown in either static liquid culture or a bioreactor. Together with phomentrioloxin, a phytotoxic geranylcyclohexenetriol recently isolated from a strain of Phomopsis sp., two new phytotoxic trisubstituted α-pyrones, named gulypyrones A and B (1 and 2), and two new 1,O- and 2,O-dehydro derivatives of phomentrioloxin, named phomentrioloxins B and C (3 and 4), were isolated from the liquid culture filtrates of D. gulyae. These four metabolites were characterized as 6-[(2S)2-hydroxy-1-methylpropyl]-4-methoxy-5-methylpyran-2-one (1), 6-[(1E)-3-hydroxy-1-methylpropenyl]-4-methoxy-3-methylpyran-2-one (2), 4,6-dihydroxy-5-methoxy-2-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-2-enone (3), and 2,5-dihydroxy-6-methoxy-3-(7-methyl-3-methyleneoct-6-en-1-ynyl)cyclohex-3-enone (4) using spectroscopic and chemical methods. The absolute configuration of the hydroxylated secondary carbon of the 2-hydroxy-1-methylpropyl side chain at C-6 of gulypyrone A was determined as S by applying a modified Moshers method. Other well-known metabolites were also isolated including 3-nitropropionic, succinic, and p-hydroxy- and p-methylbenzoic acids, p-hydroxybenzaldehyde, and nectriapyrone. When assayed using a 5 mM concentration on punctured leaf disks of weedy and crop plants, apart from 3-nitropropionic acid (the main metabolite responsible for the strong phytotoxicity of the culture filtrate), phomentrioloxin B caused small, but clear, necrotic spots on a number of plant species, whereas gulypyrone A caused leaf necrosis on Helianthus annuus plantlets. All other compounds were weakly active or inactive.

Toxicity profiles of potential biocontrol agents of Orobanche ramosa

Abstract Fifty-three fungal strains belonging to 15 mainly Fusarium species were isolated from branched broomrape plants. Their virulence was assessed using a plastic bag system, and they were grown both in liquid and on solid media, extracted, and the extracts were chemically analyzed and biologically assayed to find new metabolites that inhibit germination of branched broomrape to estimate the production of fusaric and dehydrofusaric acids by Fusarium strains and evaluate their possible involvement as virulence factors and their practical use as biomarkers to make the selection of potential mycoherbicides easier, and to ascertain whether toxins affected mammals. Nine strains proved to be highly virulent and 18 strains produced fusaric and dehydrofusaric acid at concentrations from 4 to 165, and from 9 to 204 mg L−1 respectively. Fifteen extracts from solid cultures caused high mortality when assayed on brine shrimps. Five extracts from liquid cultures caused total inhibition of seed germination. Some strains may be considered as sources of new biocontrol agents but virulence was not always positively correlated to the production of phytotoxic compounds. Nomenclature: Branched broomrape, Orobanche ramosa L.; Fusarium acuminatum Ell. & Ev.; F. camptoceras Wollenw. & Reinking; F. chlamydosporum Wollenw. & Reinking; F. compactum sensu Gordon; F. equiseti (Corda) Sacc.; F. nygamai Burgess & Trimboli; F. oxysporum Schlecht.; F. proliferatum Matsushima (Nirenberg); F. sambucinum Fuckel sensu stricto; F. solani (Mart.) Appel & Wollenw.; F. verticillioides Sacc. (Nirenberg); P. tabacinum (van Beyma) M. E. Palm et al.

Higginsianins A and B, Two Diterpenoid α-Pyrones Produced by Colletotrichum higginsianum, with in Vitro Cytostatic Activity.

Two new diterpenoid α-pyrones, named higginsianins A (1) and B (2), were isolated from the mycelium of the fungus Colletotrichum higginsianum grown in liquid culture. They were characterized as 3-[5a,9b-dimethyl-7-methylene-2-(2-methylpropenyl)dodecahydronaphtho[2,1-b]furan-6-ylmethyl]-4-hydroxy-5,6-dimethylpyran-2-one and 4-hydroxy-3-[6-hydroxy-5,8a-dimethyl-2-methylene-5-(4-methylpent-3-enyl)decahydronaphthalen-1-ylmethyl]-5,6-dimethylpyran-2-one, respectively, by using NMR, HRESIMS, and chemical methods. The structure and relative configuration of higginsianin A (1) were confirmed by X-ray diffractometric analysis, while its absolute configuration was assigned by electronic circular dichroism (ECD) experiments and calculations using a solid-state ECD/TDDFT method. The relative and absolute configuration of higginsianin B (2), which did not afford crystals suitable for X-ray analysis, were determined by NMR analysis and by ECD in comparison with higginsianin A. 1 and 2 were the C-8 epimers of subglutinol A and diterpenoid BR-050, respectively. The evaluation of 1 and 2 for antiproliferative activity against a panel of six cancer cell lines revealed that the IC50 values, obtained with cells reported to be sensitive to pro-apoptotic stimuli, are by more than 1 order of magnitude lower than their apoptosis-resistant counterparts (1 vs >80 μM). Finally, three hemisynthetic derivatives of 1 were prepared and evaluated for antiproliferative activity. Two of these possessed IC50 values and differential sensitivity profiles similar to those of 1.

Phomentrioloxin, a fungal phytotoxin with potential herbicidal activity, and its derivatives: a structure-activity relationship study.

Phomentrioloxin is a phytotoxic geranylcyclohexenetriol produced in liquid culture by Phomopsis sp. (teleomorph: Diaporthe gulyae), a potential mycoherbicide proposed for the control of the annual weed Carthamus lanatus. In this study, seven derivatives obtained by chemical modifications of the toxin were assayed for phytotoxic, antimicrobial, and zootoxic activities, and the structure-activity relationships were examined. Each compound was tested on nonhost weedy and agrarian plants, fungi, Gram+ and Gram- bacteria, and on brine shrimp larvae. The results provide insights into an investigation of the structural requirements for activity. The hydroxy groups at C-2 and C-4 appeared to be important features for the phytotoxicity, as well as an unchanged cyclohexentriol ring. A role seemed also to be played by the unsaturations of the geranyl side chain. These findings could be useful for understanding the mechanisms of action of new natural products, for identifying the active sites, and possibly in devising new herbicides of natural origin.

Ecotoxicological characterisation of a mycoherbicide mixture isolated from the fungus Ascochyta caulina

BACKGROUND Recent studies have highlighted the possibility of using a mixture of three fungal toxins produced by Ascochyta caulina as a bioherbicide to control noxious weeds. However, to assess the commercial potential of this new bioherbicide, it is necessary to increase knowledge regarding the environmental behaviour of these toxins, and particularly their effects on non-target organisms. A negative ecotoxicological profile of this potential bioherbicide could eventually hinder its registration. RESULTS In this study, acute and chronic effects of the A. caulina toxins (ACTs) on non-target organisms were evaluated in the laboratory. The (96 h) calculated algal Er C50 and NOEC were 142.7 and <12.5 mg L(-1) respectively. For daphnids, the EC50 (48 h, acute test) was 20.1 mg L(-1) , whereas the NOEC (21 day chronic reproduction test) was 6.3 µg L(-1) . No cases of mortality, sublethal effects or abnormalities were observed in the acute and chronic tests for Brachydanio rerio at a concentration of 100 mg L(-1) , indicating a low toxicity of ACTs for this organism. The calculated LC50 (14 days, acute test) and NOEC (56 day chronic reproduction) for earthworms were >1000 and ≥250 mg kg(-1) soil respectively. CONCLUSIONS The present study has provided baseline information needed to assess the potential hazard of the fungal toxins of A. caulina. In particular, the acute and chronic effects on aquatic (algae, Daphnia and fish) and terrestrial organisms (earthworms) have been highlighted. Finally, a comparison of the inherent toxicity of ACTs with other synthetic herbicides has shown comparative ecotoxicity of the tested mixture.

On the metabolites produced by Colletotrichum gloeosporioides a fungus proposed for the Ambrosia artemisiifolia biocontrol; spectroscopic data and absolute configuration assignment of colletochlorin A

Abstract Ambrosia artemisiifolia L. is responsible for serious allergies induced on humans. Different approaches for its control were proposed during the COST Action FA1203 “Sustainable management of Ambrosia artemisiifolia in Europe” (SMARTER). Fungal secondary metabolites often show potential herbicidal activity. Three phytotoxins were purified from the fungal culture filtrates of Colletotrichum gloeosporioides, isolated from infected leaves of A. artemisiifolia. They were identified by spectroscopic and chemical methods as colletochlorin A, orcinol and tyrosol (1, 2 and 3). The absolute configuration 6’R to colletochlorin A was assigned for the first time applying the advanced Mosher’s method. When assayed by leaf-puncture on A. artemisiifolia only 1 caused the appearance of large necrosis. The same symptoms were also induced by 1 on ambrosia plantlets associated with plant wilting. On Lemna minor, colletochlorin A caused a clear fronds browning, with a total reduction in chlorophyll content.

Investigation of Amino Acids As Herbicides for Control of Orobanche minor Parasitism in Red Clover

Certain amino acids induce inhibitory effects in plant growth due to feedback inhibition of metabolic pathways. The inhibition patterns depend on plant species and the plant developmental stage. Those amino acids with inhibitory action on specific weeds could be utilized as herbicides, however, their use for weed control has not been put into practice. Orobanche minor is a weed that parasitizes red clover. O. minor germination is stimulated by clover root exudates. The subsequent seedling is an obligated parasite that must attach quickly to the clover root to withdraw its nutrients. Early development of O. minor is vulnerable to amino acid inhibition and therefore, a series of in vitro, rhizotron, and field experiments were conducted to investigate the potential of amino acids to inhibit O. minor parasitism. In in vitro experiments it was found that among a collection of 20 protein amino acids, lysine, methionine and tryptophan strongly interfere with O. minor early development. Field research confirmed their inhibitory effect but revealed that methionine was more effective than lysine and tryptophan, and that two successive methionine applications at 308 and 543 growing degree days inhibited O. minor emergence in red clover up to 67%. We investigated additional effects with potential to influence the practical use of amino acids against broomrape weeds, whether the herbicidal effect may be reversible by other amino acids exuded by host plants or may be amplified by inducing host resistance barriers against O. minor penetration. This paper suggests that amino acids may have the potential to be integrated into biorational programs of broomrape management.

Development of a rapid and sensitive HPLC method for the identification and quantification of cavoxin and cavoxone in Phoma cava culture filtrates

Abstract Cavoxin is a tetrasubstituted phytotoxic chalcone and cavoxone is the corresponding chroman-4-one, both produced in vitro by Phoma cava, a fungus isolated from chestnut. Cavoxin showed biofungicide potential against fungal species responsible for food moulding. Therefore, cavoxin has potential to be incorporated into biopolymer to generate ‘intelligent food packaging’. To reach this objective, large-scale production of cavoxin by P. cava fermentation needs to be optimized. A rapid and efficient method for cavoxin analysis, as well as of cavoxone, in the fungal culture filtrates and the corresponding organic extracts is the first experimental step. Thus, a HPLC method was developed and applied to quantify cavoxin and cavoxone production in two different fungal culture conditions. The analysis proved that cavoxin production in stirred culture filtrates is significantly higher than in static ones.