This Ph.D. thesis deals with emerging and re-emerging diseases of olive (Olea europeaea L.) caused by fungi (Colletotrichum species) and oomycetes (Phytophthora species) in southern Italy. It is aimed at clarifying some unexplored aspects of the etiology and epidemiology of these diseases, determine the host range and monitoring the distribution of pathogens responsible for them in several olive-growing areas, on other host plants (most of these pathogens are polyphagous) or different ecosystems that may be inoculum reservoires, including agricultural and less anthropized ecosystems such as nature reserves, and seek for sustainable management strategies based on use of varietal genetic resistance and application of biological control agents (BCAs). Major specific objectives include: i. To test the susceptibility of italian olive cultivars to the infections of diverse Colletotrichum species associated with olive anthracnose (OA). ii. To characterize the secondary metabolites produced by diverse Colletotrichum species on olive cultivars differing in susceptibility to OA. iii. To characterize and describe a new Phytophthora species emerging in nurseries and new commercial plantings of olive in southern and insular Italy. iv. To investigate the distribution and ecology of Phytophthora oleae (a recently described species) and other Phytophthora species in agricultural and natural ecosystems with particular emphasis on those infecting olive. v. To study the effectiveness of selected lactobacilli sourced from olives as candidate BCAs of fungal plant diseases in alternative to chemical pesticides. The studies carried out to fulfill these objectives are described more in detail as it follows. Most of the results obtained have been already published or have been submitted to scientific journals. Published scientific articles form an integral part of this thesis. i. Susceptibility of italian olive cultivars to various Colletotrichum species associated with fruit anthracnose. In this study, eight among the most popular olive cultivars from central Italy and one cultivar originating from Spain were tested for their susceptibility to five different species of Colletotrichum, including C. acutatum, C. gloeosporioides, C. godetiae, C. nymphaeae and C. karsti. In the first set of experiment four fungal isolates (one isolate per Colletotrichum species) and nine olive cultivars were tested by wounding. The results obtained, based on rAUDPC, shown a significant variability among the nine olive cultivars towards C. acutatum was detected, with a greater susceptibility of Ottobratica and Coratina cultivars and an intermediate susceptibility of all other cvs, except for Leccino and Frantoio that showed a lower susceptibility to C. acutatum. No variability was recorded with C. karsti for all olive varieties tested. While a medium-high variability was registered for all nine olive cultivars inoculated with C. gloeosporioides and C. godetiae. On the contrary, with these latter species, Leccino and Frantoio cultivars showed to be more resistant. In the second set of experiment the Colletotrichum species × olive cultivar interaction was evaluated using different isolates of the Colletotrichum species tested, and also including C. nymphaeae. Ten isolates (two for each of the five Colletotrichum species tested, C. acutatum, C. gloeosporioides, C. godetiae, C. karsti and C. nymphaeae) and four olive cultivars (Coratina, Frantoio, Leccino and Ottobratica) were included. The results of the second set of experiments confirmed the olive cultivars tested differed in susceptibility to Colletotrichum species. Coratina and Ottobratica were shown to be susceptible while Frantoio and Leccino were relatively resistant to all the Colletotrichum species tested. C. acutatum was confirmed to be the most virulent among the Colletotrichum species tested, followed by C. nymphaeae. Conversely, C. karsti was again the least aggressive. No significant difference in virulence was observed between isolates of the same Colletotrichum species. The third set of experiments evaluated the effect of both wounding and maturity stage on the response of drupes to inoculation with isolates of diverse Colletotrichum species. Both mature and green drupes of Coratina were inoculated singly with four Colletotrichum species (two diverse isolates of each species). In the third set of experiments, only the isolates of C. acutatum and C. nymphaeae induced symptoms on unwounded drupes, and exclusively on mature ones, although in the parallel test on wounded drupes, Coratina was confirmed to be very susceptible to infections by these two Colletotrichum species. Conversely, isolates of C. godetiae were the least aggressive and C. gloeosporioides were more aggressive than isolates of C. godetiae, but less aggressive than isolates of both C. acutatum and C. nymphaeae. In general, symptoms were more severe on mature than on green drupes. Results of this study were published in Riolo et al., (2022), Plant Pathology, 00, 1– 13 https://doi.org/10.1111/ppa.13652 ii. Characterization of the secondary metabolites produced by diverse Colletotrichum species on olive cultivars differing in susceptibility to OA This study was aimed to characterize the secondary metabolites produced by four Colletotrichum species, C. acutatum, C. gloeosporioides, C. godetiae and C. karsti, both in vitro, on potato desxtrose agar (PDA) and oatmeal agar (OA), and during the infection process of fruits of four olive cultivars differing in susceptibility to anthracnose, ‘Coratina’ and ‘Ottobratica’, both susceptible, ‘Frantoio’ and ‘Leccino’, both resistant. The metabolites were extracted from axenic cultures after seven days incubation and from olives at three different times, 1, 3 and 7 days post inoculation (dpi). They were identified using the HPLC-QTOF analysis method. In total, 45 diverse metabolites were identified; of these 32 were detected on infected fruits, 24 in axenic cultures and 11 on both fruits and axenic cultures. The 45 identified metabolites comprised compounds of metabolite class, including fatty acid, miscellaneous compounds, phenolics, pyrones, sterols, terpenes. Each Colletotrichum species produced a different spectrum of metabolites and the metabolite profile of each species varied depending of the type of matrices. On artificially inoculated olives the severity of symptoms, the amount of fungal secondary metabolites and their number peaked 7 dpi irrespective of the cultivar susceptibility and the virulence of the Colletotrichum species. The metabolite profiles as represented by heat maps were the result of the interaction olive cultivar x Colletotrichum species. Results of this study have been submitted as an article to the Fungal Biology Journal (Elsevier). iii. Identification and characterization Phytophthora species in olive orchards in southern Italy. This study comprises two different lines of research:  First report of root rot caused by Phytophthora bilorbang on Olea europaea in Italy. The main aim of the present study was to identify and characterize the causative agent of root and crown rot in olive tree of the cv. Nera di Gonnos in Calabria. Based on morphological characters and sequencing of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) the oomycete was identified as Phytophthora bilorbang. Koch’s postulates were fulfilled by reproducing the symptoms on potted Olea europaea var. Nera di Gonnos saplings transplanted into infested soil. At the end of the experiment P. bilorbang was re-isolated from roots of symptomatic trees. This is the first report of P. bilorbang on O. europaea. Results of this study were published in Santilli et al., (2020), Plants, 2020, 9, 826. https://doi.org/10.3390/plants9070826.  Phytophthora heterospora sp. nov., a new conidia-producing sister species of P. palmivora. In this study, morphology, breeding system and growth characteristics of an unusual Phytophthora associated with stem lesions, root and collar rot on young olive trees in Southern Italy were examined, and multi loci phylogenetic analyses were performed. Morphologically the Phytophthora isolates, identified as Phytophthora heterospora, were characterized by the abundant production of caducous, non-papillate conidia-like sporangia compared to resembling P. palmivora. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora, thus they constitute a separate species. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in artificial stem inoculations. Results of this study were published in Scanu et al., (2021), Journal of Fungi, 2021, 7(10), 870; https://doi.org/10.3390/jof7100870. iv. Diversity of Phytophthora communities across different types of Mediterranean vegetation in a nature reserve area. The Objective of this study was to investigate and correlate the diversity and distribution of Phytophthora species with the vegetation in aquatic, riparian and terrestrial habitats within a protected area in Eastern Sicily, Southern Italy. Water and soil samples were collected from two streams running through the reserve and six different types of vegetation, including Platano-Salicetum pedicellatae, the Sarcopoterium spinosum community, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis, Oleo-Quercetum virgilianae and a gallery forest dominated by Nerium oleander. Phytophthora species isolations were performed using leaf baiting technique and the species obtained were classified on the basis of morphological and molecular characteristics. Overall, 11 Phytophthora species, were identified, including P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora and P. syringae. No Phytophthora species were found in the Sarcopoterium spinosum comm. Phytophthora asparagi, P. lacustris and P. plurivora were the prevalent species in the plant communities, but only P. plurivora was present in all of them. Overall aquatic species from clade 6 were the most common; they were recovered from all five types of vegetation, streams and riparian habitats. Phytophthora populations found in the Platano-Salicetum pedicellatae and Oleo-Quercetum virgilianae show the highest diversity, while no correlation was found with the physicochemical characteristics of the soil. The vegetation type and the aquatic or terrestrial habitat were identified as major environmental factors correlated with the diversity of Phytophthora communities in this reserve. Results of this study were published in Riolo et al., (2020), Forests, 11(8), 1–21. https://doi.org/10.3390/F11080853. v. Antifungal activity of selected lactic acid bacteria from olive drupes. In this study, different Lactobacilli (LABs) were isolated from the drupes of olive (Olea europaea) oil varieties. LABs were identified as Lactiplantibacillus plantarum, Pediococcus pentosaceus, Enterococcus faecium and Streptococcus salivarius by MALDI-TOF and sequencing of the 16S rRNA. To evaluate in vitro the antifungal activity of LABs and their cell-free fermentates (CFSs) against several plant pathogenic, including Alternaria, Aspergillus Colletotrichum, Penicillium, Plenodomus and Phytophthora, the culture overlaying and the agar diffusion tests were used. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined. The results obtained revealed that LABs showed antifungal activity against the fungi sensu lato tested. Lactobacillus plantarum and P. pentosaceus against Fusarium oxysporum, Colletotrichum species and Penicillium nordicum shown the most noticeable inhibitory activity. Chemical analysis revealed CFSs contained acid lactic and variable quantities of 14 diverse phenolic acids and 26 volatile organic compounds (VOCs). No obvious correlation was found between the metabolic profile of LABs and their antifungal efficacy. However, it is the first time that the potential of fermentates of LABs, recovered from drupes of olive oil varieties, as natural fungicides, was demonstrated. Results of this study have been submitted as an article to the LWT Journal (Elsevier)

Questa tesi di dottorato ha riguardato malattie emergenti e riemergenti dell'olivo (Olea europeaea L.) causate da funghi (specie Colletotrichum) e oomiceti (specie Phytophthora) nell'Italia meridionale. L'obiettivo è quello di chiarire alcuni aspetti inesplorati dell'eziologia e dell'epidemiologia di queste malattie, determinare l'areale d'ospite e monitorare la distribuzione dei patogeni responsabili in diverse aree olivicole, su altre piante ospiti (la maggior parte di questi patogeni sono polifagi) o su diversi ecosistemi che possono essere serbatoi di inoculo, compresi gli ecosistemi agricoli e quelli meno antropizzati come le riserve naturali, e cercare strategie di gestione sostenibile basate sull'uso della resistenza genetica varietale e sull'applicazione di agenti di controllo biologico (BCA). I principali obiettivi specifici includono: i. Testare la suscettibilità delle cultivar di olivo italiane alle infezioni di diverse specie di Colletotrichum associate all'antracnosi dell'olivo (OA). ii. Caratterizzare i metaboliti secondari prodotti da diverse specie di Colletotrichum su cultivar di olivo con diversa suscettibilità all'OA. iii. Caratterizzare e descrivere una nuova specie di Phytophthora emergente nei vivai e nei nuovi impianti commerciali di olivo nell'Italia meridionale e insulare. iv. Indagare la distribuzione e l'ecologia di Phytophthora oleae (una specie recentemente descritta) e di altre specie di Phytophthora in ecosistemi agricoli e naturali, con particolare attenzione a quelle che infettano l'olivo. v. Studiare l'efficacia di alcuni BCA candidati e di composti ecologici come alternativa ai pesticidi chimici. Gli studi condotti per raggiungere questi obiettivi sono descritti più dettagliatamente di seguito. La maggior parte dei risultati ottenuti è già stata pubblicata o è stata presentata a riviste scientifiche. Gli articoli scientifici pubblicati costituiscono parte integrante di questa tesi. i. Studio della suscettibilità di varietà olivicole italiane a diverse specie di Colletotrichum associate all' antracnosi dell’olivo. In questo studio, otto tra le più popolari cultivar di olivo dell'Italia centrale e una cultivar originaria della Spagna sono state testate per la loro suscettibilità a cinque diverse specie di Colletotrichum, tra cui C. acutatum, C. gloeosporioides, C. godetiae, C. nymphaeae e C. karsti. Nella prima serie di esperimenti sono stati testati per ferita quattro isolati fungini (un isolato per ogni specie di Colletotrichum) e nove cultivar di olivo. I risultati ottenuti, basati sulla rAUDPC, hanno evidenziato una significativa variabilità tra le nove cultivar di olivo nei confronti di C. acutatum, con una maggiore suscettibilità delle cultivar Ottobratica e Coratina e una suscettibilità intermedia di tutte le altre, ad eccezione di Leccino e Frantoio che hanno mostrato una minore suscettibilità a C. acutatum. Non è stata registrata alcuna variabilità con C. karsti per tutte le varietà di olivo testate. Mentre una variabilità medio-alta è stata registrata per tutte le nove cultivar di olivo inoculate con C. gloeosporioides e C. godetiae. Al contrario, con queste ultime specie, le cultivar Leccino e Frantoio si sono dimostrate più resistenti. Nella seconda serie di esperimenti l'interazione Colletotrichum specie × cultivar di olivo è stata valutata utilizzando diversi isolati delle specie di Colletotrichum testate e includendo anche C. nymphaeae. Dieci isolati (due per ciascuna delle cinque specie di Colletotrichum testate, C. acutatum, C. gloeosporioides, C. godetiae, C. karsti e C. nymphaeae) e quattro cultivar di olivo (Coratina, Frantoio, Leccino e Ottobratica) sono stati inclusi. I risultati della seconda serie di esperimenti hanno confermato che le cultivar di olivo testate differiscono nella suscettibilità alle diverse specie di Colletotrichum. Coratina e Ottobratica si sono dimostrate suscettibili, mentre Frantoio e Leccino sono risultate relativamente resistenti a tutte le specie di Colletotrichum testate. C. acutatum si è confermato il più virulento tra le specie di Colletotrichum analizzate, seguito da C. nymphaeae. Al contrario, C. karsti è risultato ancora una volta il meno aggressivo. Non sono state osservate differenze significative nella virulenza tra isolati della stessa specie di Colletotrichum. La terza serie di esperimenti ha valutato l'effetto della ferita e dello stadio di maturazione sulla risposta delle drupe all'inoculazione con isolati di diverse specie di Colletotrichum. Sia le drupe mature che quelle verdi di Coratina sono state inoculate singolarmente con quattro specie di Colletotrichum (due diversi isolati di ciascuna specie). Nella terza serie di esperimenti, solo gli isolati di C. acutatum e C. nymphaeae hanno indotto sintomi su drupe non ferite, ed esclusivamente su quelle mature, anche se nel test parallelo su drupe ferite, la Coratina si è confermata molto sensibile alle infezioni di queste due specie di Colletotrichum. Al contrario, gli isolati di C. godetiae sono stati i meno aggressivi e C. gloeosporioides è stato più aggressivo degli isolati di C. godetiae, ma meno aggressivo degli isolati di C. acutatum e C. nymphaeae. In generale, i sintomi erano più gravi sulle drupe mature che su quelle verdi. I risultati di questo studio sono stati illustrati in Riolo et al., (2022), Plant Pathology 2022, 00, 1- 13 https://doi.org/10.1111/ppa.13652 ii. Caratterizzazione dei metaboliti secondari prodotti da diverse specie di Colletotrichum su cultivar di olivo che differiscono per la suscettibilità all'OA. Questo studio ha avuto come obiettivo la caratterizzazione dei metaboliti secondari prodotti da quattro specie di Colletotrichum, C. acutatum, C. gloeosporioides, C. godetiae e C. karsti, sia in vitro, su potato desxtrose agar (PDA) e oatmeal agar (OA), sia durante il processo di infezione dei frutti di quattro cultivar di olivo diverse per suscettibilità all'antracnosi, 'Coratina' e 'Ottobratica', entrambe suscettibili, 'Frantoio' e 'Leccino', entrambe resistenti. I metaboliti sono stati estratti da colture axeniche dopo sette giorni di incubazione e da olive in tre momenti diversi, 1, 3 e 7 giorni dopo l'inoculazione (dpi). Sono stati identificati con il metodo di analisi HPLC-QTOF. In totale, sono stati identificati 45 metaboliti diversi; di questi, 32 sono stati rilevati sui frutti infetti, 24 nelle colture axeniche e 11 sia sui frutti che sulle colture axeniche. I 45 metaboliti identificati comprendevano composti della classe dei metaboliti, tra cui acidi grassi, composti vari, fenoli, pironi, steroli e terpeni. Ogni specie di Colletotrichum ha prodotto un diverso spettro di metaboliti e il profilo metabolico di ciascuna specie variava a seconda del tipo di matrice. Su olive inoculate artificialmente, la gravità dei sintomi, la quantità di metaboliti secondari fungini e il loro numero hanno raggiunto il picco a 7 dpi, indipendentemente dalla suscettibilità della cultivar e dalla virulenza della specie di Colletotrichum. I profili dei metaboliti, rappresentati dalle mappe di calore, erano il risultato dell'interazione cultivar di olivo x specie di Colletotrichum. I risultati di questo studio sono stati presentati come articolo alla rivista Fungal biology (Elsevier). iii. Identificazione e caratterizzazione delle specie di Phytophthora negli oliveti dell'Italia meridionale. Questo studio è stato sviluppato da due diverse linee di ricerca: • Prima segnalazione di marciume radicale causato da Phytophthora bilorbang su Olea europaea in Italia. Lo scopo principale del presente studio è stato quello di identificare e caratterizzare l'agente causale del marciume radicale e della chioma in un olivo della cv. Nera di Gonnos in Calabria. Sulla base dei caratteri morfologici e del sequenziamento delle regioni ITS (Internal Transcribed Spacer) del DNA ribosomiale (rDNA), l'oomicete è stato identificato come Phytophthora bilorbang. I postulati di Koch sono stati soddisfatti riproducendo i sintomi su alberelli di Olea europaea var. Nera di Gonnos trapiantati in vaso nel terreno infestato. Alla fine dell'esperimento P. bilorbang è stato nuovamente isolato dalle radici di piante sintomatiche. Si tratta della prima segnalazione di P. bilorbang su O. europaea. I risultati di questo studio sono stati illustrati in Santilli et al., (2020), Plants, 2020, 9, 826. https://doi.org/10.3390/plants9070826. • Phytophthora heterospora sp. nov., una nuova specie affine a P. palmivora che produce conidi. In questo studio sono stati esaminati la morfologia, il sistema di riproduzione e le caratteristiche di crescita di un'insolita Phytophthora associata a lesioni del fusto, marciume radicale e del colletto su giovani olivi dell'Italia meridionale e sono state eseguite analisi filogenetiche multi-loci. Morfologicamente gli isolati di Phytophthora, identificati come Phytophthora heterospora, si sono caratterizzati per l'abbondante produzione di sporangi caduchi, simili a conidi non papillati, rispetto a quelli simili a P. palmivora. Dal punto di vista filogenetico, questi isolati si sono raggruppati in un clade distinto e ben supportato, gemello di P. palmivora, costituendo così una specie separata. La nuova specie, qui descritta come Phytophthora heterospora sp. nov., si è dimostrata altamente patogena per le piante di olivo e di durian in inoculi artificiali di fusto. I risultati di questo studio sono stati illustrati in Scanu et al., (2021), Journal of Fungi, 2021, 7(10), 870; https://doi.org/10.3390/jof7100870. iv. Diversità delle comunità di Phytophthora in sei comunità vegetazionali mediterranee presenti in una riserva naturale protetta del sud Italia. L'obiettivo di questo studio è stato quello di indagare e correlare la diversità e la distribuzione delle specie di Phytophthora con la vegetazione in habitat acquatici, ripariali e terrestri all'interno di un'area protetta della Sicilia orientale, nell'Italia meridionale. Sono stati raccolti campioni di acqua e suolo da due corsi d'acqua che attraversano la riserva e da sei diversi tipi di vegetazione, tra cui Platano-Salicetum pedicellatae, la comunità Sarcopoterium spinosum, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis, Oleo-Quercetum virgilianae e una foresta a galleria dominata da Nerium oleander. Gli isolamenti delle specie di Phytophthora sono stati eseguiti con la tecnica dell'esca fogliare e le specie ottenute sono state classificate sulla base delle caratteristiche morfologiche e molecolari. Complessivamente sono state identificate 11 specie di Phytophthora, tra cui P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora e P. syringae. Non sono state trovate specie di Phytophthora nella comunità a Sarcopoterium spinosum Phytophthora asparagi, P. lacustris e P. plurivora erano le specie prevalenti nelle comunità vegetali, ma solo P. plurivora era presente in tutte. Nel complesso, le specie acquatiche del clade 6 erano le più comuni; sono state recuperate da tutti e cinque i tipi di vegetazione, dai corsi d'acqua e dagli habitat ripariali. Le popolazioni di Phytophthora trovate nel Platano-Salicetum pedicellatae e nell'Oleo-Quercetum virgilianae mostrano la più alta diversità, mentre non è stata trovata alcuna correlazione con le caratteristiche fisico-chimiche del suolo. Il tipo di vegetazione e l'habitat acquatico o terrestre sono stati identificati come i principali fattori ambientali correlati alla diversità delle comunità di Phytophthora in questa riserva. I risultati di questo studio sono stati illustrati in Riolo et al., (2020), Forests, 11(8), 1-21. ttps://doi.org/10.3390/F11080853. v. Attività antifungina di batteri lattici selezionati da drupe di olivo. In questo studio sono stati isolati diversi lattobacilli (LAB) dalle drupe delle varietà di olio di oliva (Olea europaea). I LAB sono stati identificati come Lactiplantibacillus plantarum, Pediococcus pentosaceus, Enterococcus faecium e Streptococcus salivarius mediante MALDI-TOF e sequenziamento del 16S rRNA. Per valutare in vitro l'attività antifungina dei LAB e dei loro fermentati senza cellule (CFS) nei confronti di diversi patogeni vegetali, tra cui Alternaria, Aspergillus, Colletotrichum, Penicillium, Plenodomus e Phytophthora, sono stati utilizzati i test di sovrapposizione delle colture e di diffusione su agar. Sono state determinate la concentrazione minima inibitoria (MIC) e la concentrazione minima fungicida (MFC). I risultati ottenuti hanno rivelato che i LAB hanno mostrato attività antifungina contro i funghi sensu lato testati. L. plantarum e P. pentosaceus hanno mostrato l'attività inibitoria più evidente contro Fusarium oxysporum, Colletotrichum species e Penicillium nordicum. L'analisi chimica ha rivelato che i CFS contenevano acido lattico e quantità variabili di 14 diversi acidi fenolici e 26 composti organici volatili (VOC). Non è stata trovata alcuna correlazione evidente tra il profilo metabolico dei LAB e la loro efficacia antifungina. Tuttavia, è la prima volta che viene dimostrato il potenziale dei fermentati di LAB, recuperati da drupe di varietà di olio d'oliva, come fungicidi naturali. I risultati di questo studio sono stati presentati come articolo alla rivista LWT (Elsevier)

Emerging fungal and oomycete pathogens of olive with a wide host range: characterization, monitoring and management / Riolo, Mario. - (2023 Mar 10).

Emerging fungal and oomycete pathogens of olive with a wide host range: characterization, monitoring and management

2023-03-10

Abstract

This Ph.D. thesis deals with emerging and re-emerging diseases of olive (Olea europeaea L.) caused by fungi (Colletotrichum species) and oomycetes (Phytophthora species) in southern Italy. It is aimed at clarifying some unexplored aspects of the etiology and epidemiology of these diseases, determine the host range and monitoring the distribution of pathogens responsible for them in several olive-growing areas, on other host plants (most of these pathogens are polyphagous) or different ecosystems that may be inoculum reservoires, including agricultural and less anthropized ecosystems such as nature reserves, and seek for sustainable management strategies based on use of varietal genetic resistance and application of biological control agents (BCAs). Major specific objectives include: i. To test the susceptibility of italian olive cultivars to the infections of diverse Colletotrichum species associated with olive anthracnose (OA). ii. To characterize the secondary metabolites produced by diverse Colletotrichum species on olive cultivars differing in susceptibility to OA. iii. To characterize and describe a new Phytophthora species emerging in nurseries and new commercial plantings of olive in southern and insular Italy. iv. To investigate the distribution and ecology of Phytophthora oleae (a recently described species) and other Phytophthora species in agricultural and natural ecosystems with particular emphasis on those infecting olive. v. To study the effectiveness of selected lactobacilli sourced from olives as candidate BCAs of fungal plant diseases in alternative to chemical pesticides. The studies carried out to fulfill these objectives are described more in detail as it follows. Most of the results obtained have been already published or have been submitted to scientific journals. Published scientific articles form an integral part of this thesis. i. Susceptibility of italian olive cultivars to various Colletotrichum species associated with fruit anthracnose. In this study, eight among the most popular olive cultivars from central Italy and one cultivar originating from Spain were tested for their susceptibility to five different species of Colletotrichum, including C. acutatum, C. gloeosporioides, C. godetiae, C. nymphaeae and C. karsti. In the first set of experiment four fungal isolates (one isolate per Colletotrichum species) and nine olive cultivars were tested by wounding. The results obtained, based on rAUDPC, shown a significant variability among the nine olive cultivars towards C. acutatum was detected, with a greater susceptibility of Ottobratica and Coratina cultivars and an intermediate susceptibility of all other cvs, except for Leccino and Frantoio that showed a lower susceptibility to C. acutatum. No variability was recorded with C. karsti for all olive varieties tested. While a medium-high variability was registered for all nine olive cultivars inoculated with C. gloeosporioides and C. godetiae. On the contrary, with these latter species, Leccino and Frantoio cultivars showed to be more resistant. In the second set of experiment the Colletotrichum species × olive cultivar interaction was evaluated using different isolates of the Colletotrichum species tested, and also including C. nymphaeae. Ten isolates (two for each of the five Colletotrichum species tested, C. acutatum, C. gloeosporioides, C. godetiae, C. karsti and C. nymphaeae) and four olive cultivars (Coratina, Frantoio, Leccino and Ottobratica) were included. The results of the second set of experiments confirmed the olive cultivars tested differed in susceptibility to Colletotrichum species. Coratina and Ottobratica were shown to be susceptible while Frantoio and Leccino were relatively resistant to all the Colletotrichum species tested. C. acutatum was confirmed to be the most virulent among the Colletotrichum species tested, followed by C. nymphaeae. Conversely, C. karsti was again the least aggressive. No significant difference in virulence was observed between isolates of the same Colletotrichum species. The third set of experiments evaluated the effect of both wounding and maturity stage on the response of drupes to inoculation with isolates of diverse Colletotrichum species. Both mature and green drupes of Coratina were inoculated singly with four Colletotrichum species (two diverse isolates of each species). In the third set of experiments, only the isolates of C. acutatum and C. nymphaeae induced symptoms on unwounded drupes, and exclusively on mature ones, although in the parallel test on wounded drupes, Coratina was confirmed to be very susceptible to infections by these two Colletotrichum species. Conversely, isolates of C. godetiae were the least aggressive and C. gloeosporioides were more aggressive than isolates of C. godetiae, but less aggressive than isolates of both C. acutatum and C. nymphaeae. In general, symptoms were more severe on mature than on green drupes. Results of this study were published in Riolo et al., (2022), Plant Pathology, 00, 1– 13 https://doi.org/10.1111/ppa.13652 ii. Characterization of the secondary metabolites produced by diverse Colletotrichum species on olive cultivars differing in susceptibility to OA This study was aimed to characterize the secondary metabolites produced by four Colletotrichum species, C. acutatum, C. gloeosporioides, C. godetiae and C. karsti, both in vitro, on potato desxtrose agar (PDA) and oatmeal agar (OA), and during the infection process of fruits of four olive cultivars differing in susceptibility to anthracnose, ‘Coratina’ and ‘Ottobratica’, both susceptible, ‘Frantoio’ and ‘Leccino’, both resistant. The metabolites were extracted from axenic cultures after seven days incubation and from olives at three different times, 1, 3 and 7 days post inoculation (dpi). They were identified using the HPLC-QTOF analysis method. In total, 45 diverse metabolites were identified; of these 32 were detected on infected fruits, 24 in axenic cultures and 11 on both fruits and axenic cultures. The 45 identified metabolites comprised compounds of metabolite class, including fatty acid, miscellaneous compounds, phenolics, pyrones, sterols, terpenes. Each Colletotrichum species produced a different spectrum of metabolites and the metabolite profile of each species varied depending of the type of matrices. On artificially inoculated olives the severity of symptoms, the amount of fungal secondary metabolites and their number peaked 7 dpi irrespective of the cultivar susceptibility and the virulence of the Colletotrichum species. The metabolite profiles as represented by heat maps were the result of the interaction olive cultivar x Colletotrichum species. Results of this study have been submitted as an article to the Fungal Biology Journal (Elsevier). iii. Identification and characterization Phytophthora species in olive orchards in southern Italy. This study comprises two different lines of research:  First report of root rot caused by Phytophthora bilorbang on Olea europaea in Italy. The main aim of the present study was to identify and characterize the causative agent of root and crown rot in olive tree of the cv. Nera di Gonnos in Calabria. Based on morphological characters and sequencing of Internal Transcribed Spacer (ITS) regions of ribosomal DNA (rDNA) the oomycete was identified as Phytophthora bilorbang. Koch’s postulates were fulfilled by reproducing the symptoms on potted Olea europaea var. Nera di Gonnos saplings transplanted into infested soil. At the end of the experiment P. bilorbang was re-isolated from roots of symptomatic trees. This is the first report of P. bilorbang on O. europaea. Results of this study were published in Santilli et al., (2020), Plants, 2020, 9, 826. https://doi.org/10.3390/plants9070826.  Phytophthora heterospora sp. nov., a new conidia-producing sister species of P. palmivora. In this study, morphology, breeding system and growth characteristics of an unusual Phytophthora associated with stem lesions, root and collar rot on young olive trees in Southern Italy were examined, and multi loci phylogenetic analyses were performed. Morphologically the Phytophthora isolates, identified as Phytophthora heterospora, were characterized by the abundant production of caducous, non-papillate conidia-like sporangia compared to resembling P. palmivora. Phylogenetically, these isolates grouped in a distinct well-supported clade sister to P. palmivora, thus they constitute a separate species. The new species, described here as Phytophthora heterospora sp. nov., proved to be highly pathogenic to both olive and durian plants in artificial stem inoculations. Results of this study were published in Scanu et al., (2021), Journal of Fungi, 2021, 7(10), 870; https://doi.org/10.3390/jof7100870. iv. Diversity of Phytophthora communities across different types of Mediterranean vegetation in a nature reserve area. The Objective of this study was to investigate and correlate the diversity and distribution of Phytophthora species with the vegetation in aquatic, riparian and terrestrial habitats within a protected area in Eastern Sicily, Southern Italy. Water and soil samples were collected from two streams running through the reserve and six different types of vegetation, including Platano-Salicetum pedicellatae, the Sarcopoterium spinosum community, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis, Oleo-Quercetum virgilianae and a gallery forest dominated by Nerium oleander. Phytophthora species isolations were performed using leaf baiting technique and the species obtained were classified on the basis of morphological and molecular characteristics. Overall, 11 Phytophthora species, were identified, including P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora and P. syringae. No Phytophthora species were found in the Sarcopoterium spinosum comm. Phytophthora asparagi, P. lacustris and P. plurivora were the prevalent species in the plant communities, but only P. plurivora was present in all of them. Overall aquatic species from clade 6 were the most common; they were recovered from all five types of vegetation, streams and riparian habitats. Phytophthora populations found in the Platano-Salicetum pedicellatae and Oleo-Quercetum virgilianae show the highest diversity, while no correlation was found with the physicochemical characteristics of the soil. The vegetation type and the aquatic or terrestrial habitat were identified as major environmental factors correlated with the diversity of Phytophthora communities in this reserve. Results of this study were published in Riolo et al., (2020), Forests, 11(8), 1–21. https://doi.org/10.3390/F11080853. v. Antifungal activity of selected lactic acid bacteria from olive drupes. In this study, different Lactobacilli (LABs) were isolated from the drupes of olive (Olea europaea) oil varieties. LABs were identified as Lactiplantibacillus plantarum, Pediococcus pentosaceus, Enterococcus faecium and Streptococcus salivarius by MALDI-TOF and sequencing of the 16S rRNA. To evaluate in vitro the antifungal activity of LABs and their cell-free fermentates (CFSs) against several plant pathogenic, including Alternaria, Aspergillus Colletotrichum, Penicillium, Plenodomus and Phytophthora, the culture overlaying and the agar diffusion tests were used. Minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) were determined. The results obtained revealed that LABs showed antifungal activity against the fungi sensu lato tested. Lactobacillus plantarum and P. pentosaceus against Fusarium oxysporum, Colletotrichum species and Penicillium nordicum shown the most noticeable inhibitory activity. Chemical analysis revealed CFSs contained acid lactic and variable quantities of 14 diverse phenolic acids and 26 volatile organic compounds (VOCs). No obvious correlation was found between the metabolic profile of LABs and their antifungal efficacy. However, it is the first time that the potential of fermentates of LABs, recovered from drupes of olive oil varieties, as natural fungicides, was demonstrated. Results of this study have been submitted as an article to the LWT Journal (Elsevier)
10-mar-2023
Settore AGR/12 - PATOLOGIA VEGETALE
MAGNANO DI SAN LIO, Gaetano
SCHENA, Leonardo
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12318/136769
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