Maria Chiara Zonno

Recent advances in the biocontrol of Orobanche (broomrape) species

Parasitic broomrapes (Orobanche spp.) are majoruncontrolled weeds in the Mediterranean regions of Europe and the NearEast causing major losses to vegetable, grain legume, and sunflowercrops. Selective herbicides alone cannot provide persistent, season-longcontrol of these parasites, and much methyl bromide is used for theircontrol, where affordable. Thus they are excellent targets forbiocontrol. The recent progress by the COST 816 Orobancheworking group in this area is reviewed herein. Natural infestation bythe fly Phytomyza orobanchia of seed capsules of Orobanchecrenata parasitising faba bean halved Orobanche seedproduction while inundative releases of adults reduced it to 5%of viable seeds. The fungi Fusarium arthrosporioides E4a andF. oxysporum E1d, as well as strains of bacteria were isolatedfrom diseased, juvenile, Orobanche flower stalks. They arepathogenic to O. aegyptiaca, O. crenata and O. ramosaon most vegetable crops. A F. oxysporum f. sp.orthoceras was specifically pathogenic to O. cumana onsunflowers. All were used in various experiments with a modicum ofsuccess. Methods were developed to formulate isolated mycelia, whichcould eventually allow the use of transgenic hypervirulent pathogens inasporogenic (deletion) mutants (as a failsafe against spread).Mycotoxins were also isolated from different Fusarium and otherfungal species that kill Orobanche, and are being consideredfor direct use, or to augment other strategies. All threeFusarium spp. used have been transformed with gusand/or gfp genes allowing tracing their movement in theenvironment, and opening the way to future transformations tohypervirulence.

Fusaric and 9,10-dehydrofusaric acids and their methyl esters from Fusarium nygamai

Abstract Fusaric and 9,10-dehydrofusaric acids and their corresponding methyl esters were isolated from the culture filtrates of Fusarium nygamai . The methyl esters were characterized by chemical and spectroscopic methods and reported here for the first time as naturally occurring products. When assayed on tomato leaves and seedlings at 2.7 × 10 −3 and 2 × 10 −4 M, respectively, fusaric and 9,10-dehydrofusaric acids and their methyl esters showed wide chlorosis rapidly evolving into necrosis as well as a strong inhibition of root elongation, respectively. When assayed at 10 −4 M on brine shrimps ( Artemia salina ), fusaric and 9,10-dehydrofusaric acids did not prove to be toxic, while their methyl esters showed a toxicity level of 50%, expressed as mortality.

Cytochalasins Z1, Z2 and Z3, three 24-oxa[14]cytochalasans produced by Pyrenophora semeniperda

Three new cytochalasans, named cytochalasins Z1, Z2 and Z3, were isolated from the wheat culture of Pyrenophora semeniperda, a fungus proposed to biologically control grass weeds. Other cytochalasins isolated from the same organic extract were identified as the already known cytochalasins F, T, deoxaphomin and cytochalasins B, the latter being produced in very large amounts. All three new cytochalasins were characterized as 24-oxa[14]cytochalasans by extensive use of NMR and MS techniques. Cytochalasins Z1 and Z2 proved to be structurally related to cytochalasin T, whereas cytochalasin Z3 was related to cytochalasin B. When assayed on wheat and tomato seedlings, cytochalasin Z3, in comparison to the new cytochalasins, cytochalasin B, its 21,22-dihydroderivative, cytochalasin F and deoxaphomin showed a remarkable ability to inhibit root elongation. The possibility of using these metabolites in biological control strategies is discussed.

Brefeldin A and α,β-dehydrocurvularin, two phytotoxins from Alternaria zinniae, a biocontrol agent of Xanthium occidentale

Abstract Alternaria zinniae, a fungus causing leaf necrosis in Xanthium occidentale, a widespread noxious weed of Australian summer crops and pastures, produces toxic metabolites when grown in liquid culture. Extensive application of NMR techniques, HR, EIMS and X-ray analysis permitted the identification of brefeldin A (4H-cyclopent[f]oxacyclotridecin-4-one-1,6,7,8,9,11a,12,13,14,14a-decahydro-1,13-dihydroxy-6-methyl) and α,β-dehydrocurvularin (4,5,6,7-tetrahydro-11,13-dihydroxy-4-methyl-2H-3-benzoxacyclododecin-2,10 (1H)-dione), two bioactive metabolites produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora, and Phyllosticta. This is the first report on the isolation of these two phytotoxins from A. zinniae. The interesting semi-selective toxicity to X. occidentale is described here, and the possible use of fungus and toxins in integrated weed management programs is discussed.

Putaminoxin, a phytotoxic nonenolide from Phoma putaminum

Phoma putaminum, the causal agent of leaf necrosis of Erigeron annuus, a common weed of field and pasture, produced toxic metabolites when grown in liquid culture. The main phytotoxin, named putaminoxin, was isolated and characterized using spectroscopic and chemical methods as (5S)5-hydroxy-9-propyl-6-nonen-9-olide, a new 10-macrolide. When assayed on leaves of host and non-host plants, putaminoxin showed a wide range of toxicity, with leaves of E. annuus being most sensitive.

Trans-4-aminoproline, a phytotoxic metabolite with herbicidal activity produced by Ascochyta caulina.

A phytotoxic metabolite, characterized through NMR techniques and synthetic methods as trans-4-aminoproline, was isolated from the culture filtrates of Ascochyta caulina, a promising mycoherbicide for biological control of Chenopodium album. The metabolite, which shows interesting phytotoxic properties, together with ascaulitoxin (recently characterized as N.2-beta-D-glucoside of the unusual bis-amino acid 2,4,7-triamino-5-hydroxyoctandioc acid) and another unidentified compound, compose an active fraction of A. caulina culture filtrates with promising herbicidal properties. When assayed on leaves of host and non host dicots, including wild and cultivated plants, the trans-4-aminoproline showed a wide range of toxicity, with leaves of C. album being the most sensitive. Other interesting aspects were its inefficacy on several monocots, both cultivated and wild, and its lack of antifungal, antibiotic and zootoxic activities. This is the first report on trans-4-aminoproline as naturally occurring compound and phytotoxic metabolite produced by A. caulina.

Ascaulitoxin, a phytotoxic bis-amino acid N-glucoside from ascochyta caulina

Abstract A new unusual phytotoxic bis-amino acid N-glucoside, named ascaulitoxin, was isolated from the culture filtrate of Ascochyta caulina, the causal agent of leaf and stem necrosis of Chenopodium album, a promising mycoherbicide for the biological control of this common noxious weed. Ascaulitoxin, characterized by extensive use of NMR techniques and chemical methods as N 2 - (2,4,7-triamino-5-hydroxy)-octanedioyl-β- d -glucopyranoside , showed phytotoxic activity against host and non-host plants.

Chenopodolin: A Phytotoxic Unrearranged ent-Pimaradiene Diterpene Produced by Phoma chenopodicola, a Fungal Pathogen for Chenopodium album Biocontrol

A new phytotoxic unrearranged ent-pimaradiene diterpene, named chenopodolin, was isolated from the liquid culture of Phoma chenopodicola, a fungal pathogen proposed for the biological control of Chenopodium album, a common worldwide weed of arable crops such as sugar beet and maize. The structure of chenopodolin was established by spectroscopic, X-ray, and chemical methods as (1S,2S,3S,4S,5S,9R,10S,12S,13S)-1,12-acetoxy-2,3-hydroxy-6-oxopimara-7(8),15-dien-18-oic acid 2,18-lactone. At a concentration of 2 mg/mL, the toxin caused necrotic lesions on Mercurialis annua, Cirsium arvense, and Setaria viride. Five derivatives were prepared by chemical modification of chenopodolin functionalities, and some structure-activity relationships are discussed.

Structure-activity relationship studies of putaminoxins and pinolidoxins: phytotoxic nonenolides produced by phytopathogenic Phoma and Ascochyta species

Putaminoxin and pinolidoxin, two structurally related nonenolides, isolated respectively from organic extracts of Phoma putaminum and Aschochyta pinodes cultures, together with some of their natural analogs and synthetic derivatives, were used in a structure-activity relationship study. Their phytotoxic, antifungal and zootoxic activities were assayed with the aim to find compounds with potential herbicidal properties. The strongest phytotoxic compounds proved to be putaminoxin and pinolidoxin, whose activity appeared to be correlated to the integrity of the nonenolide ring and to the presence of both the hydroxy groups and the unmodified propyl side chain. None of the assayed nonenolides showed antifungal activity, whereas pinolidoxin analogs and derivatives showed high to weak zootoxicity.

Septoria cirsii, a potential biocontrol agent of Canada thistle and its phytotoxin—ß-nitropropionic acid

Abstract Septoria cirsii causes leaf spot of Canada thistle (Cirsium arvense L.) and no other plat tested was susceptible to this pathogen. Because this pathogen possesses host specificity, and because it is so devastating to Canada thistle plants in the field, it is proposed for consideration as a biological control agenet. In culture, S. cirsii produced copious amounts of a known phytotoxin which was identified as s-nitropropionic acid. The toxin is inhibitory to seed germination, root elongation and causes the typical symptoms of chlorosis and necrosis on the leaves of Canada thistle which is one of the most toxin-sensitive plants of all of those tested.