1,721,030 research outputs found
Aspergillus flavus: controllo della biosintesi di aflatossina e strategie di biocompetizione
No full textL'aflatossina B1 è considerata una delle micotossine più pericolose per la salute dell'uomo e degli animali. La contaminazione di alimenti e mangimi che può verificarsi, a seguito dell’infezione fungina, in ogni fase del loro processo di trasformazione (dal campo alla tavola) è causa di gravi perdite economiche. Una delle strategie che attualmente è considerata tra le più promettenti nel contenere il "rischio aflatossine" consiste nell’uso della bio-competizione quale mezzo di prevenzione dell'infezione fungina delle colture in campo. Il biocontrollo realizzato tramite l’inibizione competitiva ad opera di ceppi atossigenici (afla-) di Aspergillus flavus ha dimostrato di essere un metodo efficace per il contenimento aflatossine in arachidi, mais e semi di cotone. Le popolazioni naturali di afla- sono considerate i serbatoi da cui selezionare i biocompetitori più forti; tuttavia, selezionare i ceppi migliori non è facile, sia per la scarsità di ceppi afla- efficaci che per le diverse condizioni ambientali che possono influenzare la loro efficacia in campo. Occorrono pertanto procedure “high throughput” in grado di vagliare un numero molto elevato di isolati per identificare i buoni competitori. Le prove in campo necessarie per valutare la loro efficienza una volta rilasciati nell’ambiente sono costose e laboriose, ma esperimenti di ricostruzione condotti in laboratorio possono essere utili sia per analizzare i meccanismi biologici alla base dell'efficacia dei ceppi afla- nel prevenire la biosintesi di aflatossina, sia per ottenere indicazioni preliminari sulle prestazioni degli stessi sulle colture.
In questo lavoro è stata messa a punto una procedura semplice ed economica, che sfrutta fluorescenza UV-indotta della tossina, per valutare il potenziale di ceppi afla-, che colonizzano i campi di mais della pianura padana, nel ridurre l'accumulo di aflatossina; attraverso il processo di screening è stato isolato un ceppo afla- che si è dimostrato efficace nel contenere l’accumulo di aflatossina nei confronti di tutti i ceppi aflatossigenici (afla+) isolati. Il profilo genetico del buon competitore è stato caratterizzato, e la sua efficacia testata in campo nel corso di un trial di tre anni. Sono stati analizzati alcuni dei parametri che si sospetta siano coinvolti nell'efficacia della biocompetizione: i dati riportati dimostrano che sia il momento di inoculo del ceppo afla- che il rapporto tra afla- e afla+ sono cruciali. L'effetto della concentrazione dei conidi è stato invece valutato con esperimenti in micromanipolazione in cui spore singole afla- e afla+ sono state inoculate fianco a fianco sulla superficie di terreno agarizzato.
La procedur, che si avvale di un lettore di fluorescenza di piastre multipozzetto, è stata utilizzata anche per valutare l'efficacia e la cinetica di azione di composti antiossidanti (o di miscele) sull'accumulo aflatossine: è stato testato l’effetto sia di composti puri (come acido α-lipoico, acido ascorbico e N-acetil-cisteina), sia di estratti vegetali (estratti organici di radici, stelo e foglia di Citrullus colocynthis), sia di estratti naturali commerciali (estratto di semi d’uva e di Ratania, Indena®).
Infine, è stata dimostrata l'efficacia biocompetitiva di un ceppo naturale di Aspergillus oryzae, isolato da mais campionato in Madagascar, contro ceppi aflatossigenici di A. flavus
Biocompetition as a tool to cope with the problem of aflatoxin contamination of food and feed commodities: may phenotype microarray technology contribute to the individuation of "good" competitors?
No full textAspergillus flavus is a well known plant pathogen responsible of economic losses and food safety concern due to the ability of mycotoxin production. Aflatoxin contamination of crops is a significant problem worldwide, and many Countries have set more or less strict limits for mycotoxin presence in feed and food commodities. An interesting strategy to cope with aflatoxin contamination in susceptible crops involves the use of intraspecific competition to interfere with mycotoxin production by the relevant Aspergillus flavus strains. Atoxigenic A. flavus strains (afla-), unable to produce the relevant mycotoxin, have been already used as bio-competitors to decrease aflatoxin accumulation on cotton, maize and peanuts fields. Selecting a strain performing as a strong bio-competitor is not a straightforward task since it depends on previous assessment of various interacting factors conditioning the relative fitness of the strains in a given ecological niche. Reconstruction experiment have been generally performed in laboratory conditions to uncover the biological mechanisms underlying the efficacy of atoxigenic strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the field.
Our present goal is 1) characterization of A. flavus population colonizing the corn field of the Po valley and 2) setting a strategy for the identification of “good” competitors among afla- strains isolated from the above mentioned A. flavus population. For this purpose we are performing competition experiments in laboratory conditions and designing “high throughput” procedures to test as many as possible environmental conditions that may affect competition efficacy. Moreover, afla- strains are generally considered to be derived from aflatoxin producer strains (afla+) that have lost the capacity to produce the relevant mycotoxin as a result of mutation(s) affecting gene(s) belonging to the Aflatoxin biosynthetic cluster. afla- strains are obviously expected to be altered in one or more steps of secondary metabolism. However the story might be more complex than expected, since regulatory interactions between pathways of primary and secondary metabolism have already been described. In this respect, Phenotype MicroArray technology may represent an additional value for our goal if the specific metabolic features of different afla- strains could be correlated to their competitive ability. In the present study we have performed, as a preliminary approach, experiments to verify if afla+ and afla- strains may be differentiated by the use of the Biolog FF physiological identification kit
Development of a simple and high-throughput method for detecting aflatoxins production in culture media.
No full textAims: To develop a simple, high-throughput and inexpensive procedure to detect and quantify aflatoxins into the culture media of growing mycelia. Methods and Results: Fungal conidia (Aspergillus flavus) were inoculated into the wells of a microplate containing 200mul of different formulations of coconut-derived liquid medium. Time-dependent production of aflatoxins in the culture media was evaluated by a procedure relying on the UV-induced fluorescence emission by the toxin, using a microplate reader. These data were validated by comparison with the outputs of a conventional HPLC-based procedure. Determinations of aflatoxin concentration, according to the fluorimetric procedure, were performed either by withdrawing samples from the plates or by direct 'in situ' readings, the latter method reinforcing the high-throughput feature of the procedure. Fluorescence enhancers (cyclodextrins) did not ameliorate the sensitivity of the procedure to low concentrations of the toxin into the medium. The efficacy of the procedure was also validated by testing the effect on toxin yield of adding an antioxidant agent (alpha-lipoic acid) to the medium. Conclusions: We give evidence that our improved procedure is reliable and suitable to analyse aflatoxin accumulation time course in coconut-derived culture medium. Significance and Impact of the Study: This study shows that our procedure may profitably be used to give insights into the mechanisms of regulation of mycotoxin production and, consequently, to implement different strategies for the containment of aflatoxin contamination of food and feed commodities
Editorial: Through thick and thin - the army of secondary metabolites in plant-fungi interactions
No full textThe "Recovery" phenomenon in Grapevine (Vitis vinifera) cv. Chardonnay infected by "Bois Noir" phytoplasma: investigation of proteome modifications by 2DE and Western Blot analysis
No full textThe grapevine (Vitis vinifera) disease caused by Bois Noir (BN) phytoplasma, a plant phloem-limited pathogens belonging to the class Mollicutes, is an important concern to European vinery economy. Phytoplasmas have a broad range of plant hosts among monocots and dicots and diseases of many important crops have already been associated with these pathogens. Leaf curling and discoloration, uneven or total lack of lignification of canes, flower abortion and berry withering are the typical symptoms of phytoplasma infection in grapevine. An interesting aspect of the pathogen–plant interaction is “recovery” phenomenon, a spontaneous remission of symptoms in previously symptomatic plants. In this study, we are using 2-DE and Western Blot analysis to explore the proteome changes in leaves and midribs of V. vinifera cv. 'Chardonnay' naturally infected by BN, with particular attention to recovered plants
Potential of biocontrol agents, antioxidant compounds and natural extracts on aflatoxin containment: a simple and highthroughput procedure
No full textAflatoxin B1 is considered one of the most dangerous mycotoxin for humans and animals health. Moreover severe economic losses may be encountered due to its possible contamination of food and feed during each step of their transformation process (from field to table). Preventing fungal infection/ toxin contamination of crops in the field is considered the preferred strategy to cope with the “aflatoxin risk”.
Biocontrol by competitive inhibition using atoxigenic (afla–) Aspergillus flavus strains has been shown to be an effective method in aflatoxin containment in peanuts, maize and cottonseed(1). Naturally occurring populations of afla– strains are considered reservoirs from which to select the strongest biocompetitors. However, the selection of biocontrol strains is not an easy task, due both to the small amount of afla– strains and to the various environmental conditions that may affect their efficacy in the field(2). High throughput procedures are therefore desirable to screen large amount of isolates in order to identify “good competitors”. Moreover, as field trials required to assess their efficiency are expensive and laborious, reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of afla– strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the crops.
We developed a simple and inexpensive fluorescence-based procedure that may be used: 1) to scale-up the screening process(3) and also 2) to increase knowledge on the mechanisms interfering with mycotoxin production during intraspecific competition and 3) to analyze the effect of natural and/or synthetic compounds for their possible effects on aflatoxin biosynthesis.
Here we give a report of our results concerning the evaluation of the potential of afla– strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation, and show some preliminary evidence that suggest the possible use of antioxidants as part of an additional strategy to contain aflatoxin contamination of food and feed commodities. Conversely the procedure may be used to uncover the presence of antioxidant agents in complex mixtures deriving from natural sources
Impiego di Microstation (Biolog): creazione ed applicazione di un database per differenziare Penicillium verrucosum da Penicillium nordicum.
No full textLaboratory tests for assessing efficacy of atoxigenic Aspergillus flavus strains as biocontrol agents
No full textA simple and highthroughput procedure to screen for biocontrol agents and to assess the efficacy of antioxidant compounds and natural extracts on aflatoxin containment
No full textAflatoxin B1 is considered one of the most dangerous mycotoxin for humans and animals health. Moreover severe economic losses may be encountered due to its possible contamination of food and feed during each step of their transformation process (from field to table). Preventing fungal infection/ toxin contamination of crops in the field is considered the preferred strategy to cope with the “aflatoxin risk”.
Biocontrol by competitive inhibition using atoxigenic (afla–) Aspergillus flavus strains has been shown to be an effective method in aflatoxin containment in peanuts, maize and cottonseed(1). Naturally occurring populations of afla– strains are considered reservoirs from which to select the strongest biocompetitors. However, the selection of biocontrol strains is not an easy task, due both to the small amount of afla– strains and to the various environmental conditions that may affect their efficacy in the field(2). High throughput procedures are therefore desirable to screen large amount of isolates in order to identify “good competitors”. Moreover, as field trials required to assess their efficiency are expensive and laborious, reconstruction experiments have been generally performed under laboratory conditions to investigate the biological mechanisms underlying the efficacy of afla– strains in preventing aflatoxin production and/or to give a preliminary indication of strain performance when released in the crops.
We developed a simple and inexpensive fluorescence-based procedure that may be used: 1) to scale-up the screening process(3) and also 2) to increase knowledge on the mechanisms interfering with mycotoxin production during intraspecific competition and 3) to analyze the effect of natural and/or synthetic compounds for their possible effects on aflatoxin biosynthesis.
Here we give a report of our results concerning the evaluation of the potential of afla– strains, colonizing the corn fields of the Po Valley, in reducing aflatoxin accumulation, and show some preliminary evidence that suggest the possible use of antioxidants as part of an additional strategy to contain aflatoxin contamination of food and feed commodities. Conversely the procedure may be used to uncover the presence of antioxidant agents in complex mixtures deriving from natural sources.
We also anticipate preliminary data concerning the possible use of our procedure to assess the aflatoxin binding efficacy of bacterial and yeast cells
Analisi proteomica nella diagnosi precoce di fitoplasmosi in Vitis vinifera
No full textI fitoplasmi, eubatteri privi di parete appartenenti alla classe dei Mollicutes e patogeni intrafloematici obbligati, sono considerati agenti eziologici di numerose patologie di piante ornamentali e alberi da frutto. In Vitis vinifera le fitoplasmosi prendono genericamente il nome di “giallumi” (GY), ma sono provocate da fitoplasmi appartenenti a diversi gruppi: ad esempio, in Italia la diffusione delle fitoplasmosi viene attribuita principalmente alla flavescenza dorata (FD) e al legno nero (BN). In quanto endemici e non curabili, i giallumi risultano particolarmente dannosi per la produttività dei vigneti colpiti; per questo motivo esiste una Direttiva nazionale che mira a ridurre l'impatto dei GY attraverso l’incenerimento delle colture infette, l'impiego di materiale di propagazione sano e il controllo degli insetti vettori come Scaphoideus titanus per FD e Hyalestes obsoletus per BN.
Approcci di genomica e trascrittomica sono stati recentemente utilizzati con successo per analizzare le interazioni pianta-fitoplasma negli individui infetti, permettendo anche di indagare il fenomeno del recovery, una remissione spontanea dei sintomi che non implica necessariamente la scomparsa del patogeno dalla pianta ospite (1).
Obiettivi di questo progetto sono la messa a punto di metodi diagnostici sensibili e l’approfondimento della comprensione dell’interazione fitoplasma/ospite (2,3): nello specifico, ci si propone di individuare variazioni nel proteoma di piante infette e recovered, con particolare attenzione alla biosintesi di PR-proteins. In questo senso, è stato testato un metodo di estrazione rapida che consente di processare limitatissime quantità di tessuto e permette di ottenere estratti cellulari molto concentrati senza la necessità di ricorrere a precipitazione. Su tali estratti si è proceduto a una valutazione preliminare dell’immunoreattività, tramite ibridazione con un anticorpo anti-GDH (glutammato deidrogenasi) specifico per V. vinifera. In estratti di lamina fogliare e di nervatura centrale provenienti da individui sani, sintomatici (BN) e recovered, è stata quindi stimata l’abbondanza relativa di PR-2 (beta-1,3-glucanasi) e PR-3(chitinasi) tramite western blot: i risultati ottenuti dimostrano che negli individui recovered la quantità delle due PR-protein è intermedia rispetto a quella evidenziata negli individui sani e infetti.
Lo stesso tipo di analisi è stata poi estesa a tessuti degli stessi individui campionati sia durante la stagione vegetativa precoce (giugno) sia durante una stagione vegetativa più tardiva (settembre).
La possibilità di associare l’espressione di PR-protein alla gravità dei sintomi ascrivibili alla infezione sembrerebbe una strategia promettente che, sfruttando la proteina come “indice quantitativo” del livello di infezione, potrebbe consentire una diagnosi precoce della presenza del fitoplasma in uno stadio di sviluppo della pianta normalmente privo di sintomi
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