toxins Article Phytotoxic Metabolites Isolated from Neufusicoccum batangarum, the Causal Agent of the Scabby Canker of Cactus Pear (Opuntia ficus-indica L.) Marco Masi 1 , Francesco Aloi 2,3, Paola Nocera 1, Santa Olga Cacciola 3,* , Giuseppe Surico 4 and Antonio Evidente 1,* 1 Dipartimento Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy; [email protected] (M.M.); [email protected] (P.N.) 2 Dipartimento di Scienze Agrarie, Alimentari, Forestali e Ambientali, Università di Palermo, V. le delle Scienze 4, 90128 Palermo, Italy; [email protected] 3 Dipartimento di Agricoltura, Alimentazione e Ambiente, Università di Catania, Via Santa Sofia 100, 95123 Catania, Italy 4 Dipartimento di Scienze e Tecnologie Agrarie, Alimentari, Ambientali e Forestali, Sez. Patologia vegetale ed entomologia, Università di Firenze, Piazzale delle Cascine 28, 50144 Firenze, Italy; giuseppe.surico@unifi.it * Correspondence: [email protected] (S.O.C.); [email protected] (A.E.); Tel.: +39-095-7147371 (S.O.C.); +39-081-2539178 (A.E.) Received: 24 January 2020; Accepted: 14 February 2020; Published: 18 February 2020 Abstract: Six phytotoxins were obtained from the culture filtrates of the ascomycete Neofusicoccum batangarum, the causal agent of the scabby canker of cactus pear (Opuntia ficus-indica L.) in minor Sicily islands. The phytotoxins were identified as ( )-(R)-mellein (1); ( )-botryoisocoumarin A (2); − ± ( )-(3R,4R)- and ( )-(3R,4S)-4-hydroxymellein (3 and 4); ( )-terpestacin (5); and (+)-3,4-dihydro-4,5,8- − − − trihydroxy-3-methylisocoumarin, which we named (+)-neoisocoumarin (6). This identification was done by comparing their spectral and optical data with those already reported in literature. The absolute configuration (3R,4S) to (+)-neoisocoumarin (6) was determined using the advanced Mosher method. All six metabolites were shown to have phytotoxicity on the host (cactus pear) and non-host (tomato) plants, and the most active compounds were ( )-botryoisocoumarin A (2), ± ( )-terpestacin (5), and (+)-neoisocoumarin (6). − Keywords: cactus pear; scabby cankers; Neofusicoccum batangarum; phytotoxins Key Contribution: Neofusicoccum batangarum, the causal agent of the scabby canker of cactus pear (Opuntia ficus-indica L.) in minor Sicily islands, was grown in vitro to investigate its capacity to produce phytotoxic secondary metabolites. The bioguided purification of its organic extract allowed for the isolation of five low molecular weight compounds that showed phytotoxicity on host (cactus pear) and non-host (tomato) plants. 1. Introduction Cactus pear (Opuntia ficus-indica (L.) Mill.), Cactaceae family, is believed to be native of Mexico [1], and after the discovery of America, it was introduced into the Mediterranean Basin where it is now naturalized [2]. In Sicily, it has become an economically important fruit crop and a characteristic feature of the landscape. Cactus pear is also cultivated, mostly as productive living fences, in minor islands around Sicily, and as a fruit crop in Sardinia, Apulia, Calabria, and Basilicata Italian regions. Toxins 2020, 12, 126; doi:10.3390/toxins12020126 www.mdpi.com/journal/toxins Toxins 2020, 12, 126 2 of 13 Countries where cactus pear is also cultivated include Mexico, United States of America, Chile, Brazil, NorthToxins Africa, 2020, 12, South x FOR PEER Africa, REVIEW Middle East, Turkey, Tunisia, Malta, etc. 2 of 13 Recently, a severe infectious disease of the plant was reported in some minor islands of Sicily: LampedusaRecently, and a Linosasevere infectious of the Pelagie disease archipelago; of the plant Favignanawas reported of in the some Aegadian minor islands archipelago; of Sicily: and Ustica,Lampedusa a small and island Linosa in the of Tyrrhenianthe Pelagie Seaarchipelago; [3]. Symptoms Favignana of thisof the disease, Aegadian which archipelago has been; namedand scabbyUstica, cankers, a small were island visible in the on Ty cladodesrrhenian andSea included[3]. Symptoms radially of this expanding, disease, crusty,which has concentric, been named silvery, perennialscabby cankers, were with visible a leathery, on cladodes brown halo and (Figureincluded1). radially Characteristically, expanding, ancrusty, abundant, concentric, milky, silvery, perennial cankers, with a leathery, brown halo (Figure 1). Characteristically, an abundant, viscous exudate, caking on contact with air, leaked from cankers and formed strips or cerebriform milky, viscous exudate, caking on contact with air, leaked from cankers and formed strips or masses (Figure1). With time, the exudate in the central part of the canker became black, giving the cerebriform masses (Figure 1). With time, the exudate in the central part of the canker became black, cankers an appearance of carbonaceous crusts, and the cankers ceased to expand in the coldest season giving the cankers an appearance of carbonaceous crusts, and the cankers ceased to expand in the of the year. coldest season of the year. FigureFigure 1. Symptoms1. Symptoms of scabbyof scabby cankers cankers on cladodeson cladodes of pear of pear cactus ca (cO.tus ficus-indica (O. ficus-indicaL.), including L.), including radially expanding,radially expanding, crusty, concentric, crusty, concentric, silvery, perennialsilvery, perennial cankers, cankers, with a with leathery, a leathe brownry, brown halo (haloleft panel(left ); andpanel an abundant); and an milkyabundant viscous milky exudate, viscous cakingexudate, on caking contact on with contact air, which with air, leaked which from leaked cankers from and cankers and formed strips or cerebriform masses (right panel). formed strips or cerebriform masses (right panel). TheThe causal causal agent agent of theof scabbythe scabby canker canker of cactus of cactus pear foundpear found in minor in minor islands islands of Sicily of was Sicily identified was as identifiedNeofusicoccum as Neofusicoccum batangarum batangarumBegoude, Jol. Begoude, Roux &Jol. Slippers, Roux & aSlippers, fungal species a fungal not species reported not reported previously previously in Europe, whose distribution includes Africa, Brazil, and USA [4–6]. In Brazil, N. in Europe, whose distribution includes Africa, Brazil, and USA [4–6]. In Brazil, N. batangarum batangarum was reported as an aggressive pathogen of cashew (Anacardium occidentale) and cochineal was reported as an aggressive pathogen of cashew (Anacardium occidentale) and cochineal cactus cactus (Nopalea cochenillifera (L.) Salm-Dyck, syn. Opuntia cochenillifera (L.) Mill.), a relative of cactus (Nopalea cochenillifera (L.) Salm-Dyck, syn. Opuntia cochenillifera (L.) Mill.), a relative of cactus pear [6,7]. pear [6,7]. On cochineal cactus in Brazil, N. batangarum, alone or in association with other fungi, On cochineal cactus in Brazil, N. batangarum, alone or in association with other fungi, including different including different species of Bothryosphaeriaceae, causes a disease that was named cladode brown speciesspot and of Bothryosphaeriaceae, constitutes a serious threat causes to athe disease cultivation that wasof cochineal named cladodecactus as brown a fodder spot for and livestock constitutes [7– a serious10]. In pr threateliminary to the cross cultivation-pathogenicity of cochineal tests, the cactus fungus as aisolated fodder from for livestock infected cactus [7–10]. pear In preliminary plants in cross-pathogenicityminor islands of Sicily tests, was the able fungus to reproduce isolated the from disease infected symptoms cactus on pear the host plants plant in and minor to induce islands a of Sicilydisease was reaction able to reproduce on other plant the disease species symptoms, such as Aleppo on the host pine plant (Pinus and halepensis to induce), almond a disease (Prunus reaction ondulcis other), plantsweet species, orange such(Citrus as Alepposinensis), pine citrange (Pinus (Citrus halepensis sinesis), almond Poncirus (Prunus trifoliata dulcis), and), sweet holm orangeoak (Citrus(Quercus sinensis ilex),) citrangetrees. These (Citrus results sinesis show Poncirus that trifoliataN. batangarum), and holmhas a oakwide (Quercus host range ilex) trees.like many These other results showBotryosphaeriaceae that N. batangarum specieshas a, which wide hostare frequently range like manyreported other as Botryosphaeriaceaecausal agents of different species, important which are frequentlycrop diseases, reported including as causal the grapevine agents of Botryosphaeria different important dieback crop [11– diseases,15]. including the grapevine BotryosphaeriaThe purpose dieback of this [11 study–15]. was to characterize the agent of the scabby canker of cactus pear for its Theabili purposety to accumulate of this study in culture was to biologically characterize active the agent substances, of the scabbynamely canker phytotoxins, of cactus which pear may for its abilityhave to a accumulaterole in the inproduction culture biologically of disease activesymptoms. substances, The chemical namely identification phytotoxins, whichof phytotoxins, may have a roleobtained in the production from the culture of disease filtrates symptoms. of N. batangarum The chemical, and identification their toxic effects of phytotoxins, on host and obtained non-host from theplants culture are filtrates reported of. N. batangarum, and their toxic effects on host and non-host plants are reported. 2. Results2. Results and and Discussion Discussion TheThe organic organic extract extract ofof thethe N.N. batangarumbatangarum culture was