Biological degradation of mycotoxins is an emerging strategy for detoxification of agricultural commodities. In particular, enzymatic degradation of aflatoxin B1 (AFB1), the most harmful among the mycotoxin known which may occur as contaminant of most of food and feed, has lately raised considerable scientific interest. Ligninolytic enzymes, such as laccase and peroxidise, from white-rot fungi have been proven to be able to break the highly stable molecule of AFB1. However, the high cost of production and purification of these enzymes have limited their implementation into practical technologies aimed at reduction of aflatoxin contamination in the food and feed chains. Among the white-rot fungi there are also cultivable edible mushrooms, such as Pleurotus spp. Every year tons of spent mushroom substrate (SMS) are produced as a by-product of mushroom cultivation and disposed at a cost for farmers. However, SMS may still be a source of bioactive compounds, including ligninolytic enzymes potentially useful for degradation of aflatoxin. We investigated the AFB1-degradative activity of a crude extract (CE) of SMS and undertook a study for characterization of enzyme content and stability of the extracts. CE of SMS was obtained by an extraction buffer (sodium phosphate buffer 0.1 M, pH 7.3) and the extract was incubated with 1 ?g/ml of AFB1 at 25 °C, under continuous shaking at 120 rpm for 1, 3 and 7 days; then the aflatoxin content was determined by ultra performance liquid chromatography (UPLC). After 1 day of incubation, the CE was able to degrade more than 50% of AFB1 and after 3 and 7 days of incubation the percentage of degradation reached the values of 75% and 90%, respectively. The CE contained a high level of laccase activity, quantified in 4 Units per gram of SMS dry weight (U/g DW) and low level of Mn-peroxidase (0.4 U/g DW), as determined by spectrophotometric assays. The enzymatic activity of the CE had its optimum at temperature ranging between 5 and 25 °C and at pH 4, and it was stable at +4 °C for about 60 days. Heat treatment at 100 °C for 10 minutes completely destroyed the degradative activity of CE, and freeze drying resulted in a decrease by 35% of the laccase activity. Based on these preliminary results, SMS proved to be a suitable source of aflatoxin-degrading enzymes and the use of SMS and/or their CE for detoxification of aflatoxin-contaminated commodities, particularly those intended for feed, appears as a coinceivable technology for aflatoxin-free feed production. Further research is needed to improve the stability of SMS extracts and to implement their use in the pipeline of feed processing.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin that occurs as natural contaminant of agricultural commodities, particularly maize. Practical solutions for detoxification of contaminated staples and reduction of agricultural wastes are scarce. We investigated the capability of the white-rot and edible fungus Plerotus eryngii (king oyster mushroom) to degrade AFB1 both in vitro and in a laboratory-scale mushroom cultivation, using a substrate similar to that routinely used in mushroom farms. In malt extract broth, degradation of AFB1 (500 ng/mL) by nine isolates of P. eryngii ranged from 81 to 99% after 10 days growth, and reached 100% for all isolates after 30 days. The growth of P. eryngii on solid medium (malt extract-agar, MEA) was significantly reduced at concentrations of AFB1 500 ng/mL or higher. However, the addition of 5% wheat straw to the culture medium increased the tolerance of P. eryngii to AFB1 and no inhibition was observed at a AFB1 content of 500 ng/mL; degradation of AFB1 in MEA supplemented with 5% wheat straw and 2.5% (w/v) maize flour was 71-94% after 30 days of growth. Further, AFB1 degradation by P. eryngii strain ITEM 13681 was tested in a laboratory-scale mushroom cultivation. The mushroom growth medium contained 25% (w/w) of maize spiked with AFB1 to the final content of 128 ?g/kg. Pleurotus eryngii degraded up to 86% of the AFB1 in 28 days, with no significant reduction of either biological efficiency or mushroom yield. Neither the biomass produced on the mushroom substrate nor the mature basidiocarps contained detectable levels of AFB1 or its metabolite aflatoxicol, thus ruling out the translocation of these toxins through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1- contaminated corn through the exploitation of the degradative capability of P. eryngii and its bioconversion into high nutritional value material intended for feed production.

Dalla ricerca micologica nuovi prodotti a basso impatto ambientale per la difesa biologica delle colture

Aflatoxin B1 (AFB1) is a major threat to human and animal health, due to its potent hepatotoxic, carcinogenic and mutagenic effects. Removal or inactivation of aflatoxin in food and feedstuff is difficult. Chemical and physical methods have been found to be effective in detoxification of AFB1 from various materials but their use in the practice is limited, due to safety issues and possible loss of nutritional value of the treated commodities. In the feed industry a proposed technology for detoxification of feedstuff is the dietary supplementation of inorganic or organic materials able to adsorb mycotoxins. Once the mycotoxin has been bound, absorption in the digestive tract of animals is strongly reduced. Some mineral adsorbents such as aluminosilicates, bentonites and activated carbons have showed good results but have some drawbacks, such as the negative impact on the nutritional quality of decontaminated feed. For this reason, interest in the use of natural microbial adsorbents has increased. In this study we investigated the capability of ground not-viable mycelium of the edible and cultivated mushroom Pleurotus eryngii to bind AFB1. P. eryngii strain ITEM 13681 was cultured on malt-extract broth for 20 days at 28 °C in the dark. For preparation of the biosorbent, the mycelium was harvested, autoclaved, lyophilized and finely ground to particles of size <= 500 µm. One hundred milligrams of the biosorbent were suspended in 5 mL of phosphate-buffer saline (PBS) pH 7 containing 2000 ng AFB1, and incubated overnight at 25 °C in a rotary shaker at 250 rpm. Then, the mycelium and the supernatant were separated by centrifugation and both the phases were analyzed for AFB1 content. Sixty-five percent of AFB1 was detected in the supernatant, while the mycelium was able to retain 6 ng of AFB1 per milligram of dry mycelium (35%). The effect of mycelium dosage was studied by testing different amounts of biomass (25, 50, 75, 100, 150, 200 mg/mL) in both acetate buffer (pH = 5) and PBS (pH = 7) containing 0.5 µg/mL AFB1. The suspensions were shaken for 90 minutes at 250 rpm, at 25 and 37 °C. Then the samples were centrifuged at 13000 rpm for 10 minutes and the pellets were washed three times and analyzed by UPLC/FLD. The efficiency of adsorption (%A) was calculated using following equation: %A = [(Ci - Cf) / Ci] x 100; where Ci was the initial and the Cf final concentration (supernatant plus washing solution) of mycotoxin. The biosorbtion showed to be both dosage- and temperature-dependant. In acetate buffer the mycelium adsorbed 44±4% at the dosage of 200 mg and no significant adsorption at 25 mg. In PBS absorption at 37 °C ranged from 7±5% at 25 mg of mycelium to 64±6% at 200 mg, and at 25 °C it ranged from 3±0 % at 25 mg of mycelium to 26±3% at 200 mg. Our results envisage a possible use of P. eryngii as a cheap and effective biosorbent for AFB1. This mechanism of AFB1-binding by P. eryngii mycelium is herein demonstrated for the first time.

Trichoderma atrobrunneum F.B. Rocha, P. Chaverri& W. Jaklitsch strain ITEM 908 (formerly known as T. harzianum ITEM 908), is a biocontrol strain that is being registered under the European Union regulation as an active ingredient for the production of commercial biopesticides. The strain ITEM 908 proved to be able to inhibit completely the formation of perithecia by Fusarium graminearum in dual cultures and to release in the agar medium metabolites that reduce the number of perithecia by over 70% (Altomare et al., poster presentation in this Congress). Therefore, ITEM 908 appears to be a good candidate biocontrol strain for prevention of Fusarium Head Blight (FHB) in the field by treatment of plant residues of the preceeding crop, thus reducing the primary inoculum. For the univocal characterization of this commercially valuable isolate and as a base for further studies aimed at elucidating its mechanisms of action and its physiological and molecular interactions with plants and target pathogens and pests, we sequenced the whole genome of the strain ITEM 908. The genome was sequenced on an Ion S5 platform generating around 7M bpand assembled using the Spades v5.0 software. The resulting genome sequence has an estimated size of 39,131,654 bp. The reference gene sequences of ITS and TEF1 were extracted from the genome assembly. The identification of the strain ITEM 908 at species level was obtained by phylogenetic analysis with Maximum likelihood (ML) analysis performed with one-hundred ITS-TEF1 manually concatenated dataset retrieved from sequences of Trichoderma spp. deposited in GenBank. The analysis placed ITEM 908 within the T. atrobrunneum group, close to T. afroaharzianum and T. guizhouense. Genome was annotated using the Augustus v3.1 software and 8649 genes were predicted. Approximately 3000 different pfam domains were detected and used to group genes within functional categories, including glycoside hydrolases, proteases and gene involved in stress tolerance and in secondary metabolites biosynthesis. Among these, 20 putative PKS, 8 putative NRPS and 5 putative PKS-NRPS were identified. Moreover, the secretome of T. atrobrunneum ITEM 908, consisting of 761 proteins, was in silico predicted by the software SignalP (http://www.cbs.dtu.dk/services/SignalP/), that detects the presence of the secretion signal peptide at the N-terminus in amino acid sequences of a protein.The preliminary analysis of predicted genes highlights the potential ability of T. atrobrunneum ITEM 908 to produce a broad range of enzymes involved in the biocontrol activity

So far, biocontrol agent selection has been performed mainly by time consuming in vitroconfrontation tests followed by extensive trials in greenhouse and field. An alternative approach is offered byapplication of high-throughput techniques, which allow extensive screening and comparison among strains fordesired genetic traits. In the genus Trichoderma, the past assignments of particular features or strains to one speciesneed to be reconsidered according to the recent taxonomic revisions. Here we present the genome of a biocontrolstrain formerly known as Trichoderma harzianum ITEM 908, which exhibits both growth promoting capabilities andantagonism against different fungal pathogens, including Fusarium graminearum, Rhizoctonia solani, and the rootknotnematode Meloidogyne incognita. By genomic analysis of ITEM 908 we investigated the occurrence and therelevance of genes associated to biocontrol and stress tolerance, providing a basis for future investigation aimingto unravel the complex relationships between genomic endowment and exhibited activities of this strain.Results: The MLST analysis of ITS-TEF1 concatenated datasets reclassified ITEM 908 as T. atrobrunneum, a speciesrecently described within the T. harzianum species complex and phylogenetically close to T. afroharzianum and T.guizhouense. Genomic analysis revealed the presence of a broad range of genes encoding for carbohydrate activeenzymes (CAZYmes), proteins involved in secondary metabolites production, peptaboils, epidithiodioxopiperazinesand siderophores potentially involved in parasitism, saprophytic degradation as well as in biocontrol andantagonistic activities. This abundance is comparable to other Trichoderma spp. in the T. harzianum speciescomplex, but broader than in other biocontrol species and in the species T. reesei, known for its industrialapplication in cellulase production. Comparative analysis also demonstrated similar genomic organization of majorsecondary metabolites clusters, as in other Trichoderma species.Conclusions: Reported data provide a contribution to a deeper understanding of the mode of action andidentification of activity-specific genetic markers useful for selection and improvement of biocontrol strains. Thiswork will also enlarge the availability of genomic data to perform comparative studies with the aim to correlatephenotypic differences with genetic diversity of Trichoderma species.

Aflatoxin B1 (AFB1) is the most harmful mycotoxin produced mainly by filamentous fungi Aspergillus flavus and A. parasiticus, which can occur as natural contaminants of many agricultural commodities, including maize. Several approaches have been experimented for the removal of aflatoxin from contaminated food and feed, including microbial degradation which, however, has not been so far implemented into practical technologies due to concerns on the quality and safety of the decontaminated materials. The aim of this study was to investigate the capability of the edible and cultivated mushroom Pleurotus eryngii (king oyster mushroom) to degrade AFB1. To this purpouse, nine isolates of P. eryngii were grown on one liquid and one agar medium supplemented with 500 ng/ml of AFB1. After 30 days of growth at 30 ? 1 °C both culture media were processed and analyzed for AFB1 by ultra performance liquid chromatography (UPLC/FLD). In the liquid medium (malt extract broth) all the isolates completely detoxified AFB1. In the solid medium (malt extract-agar supplemented with corn flour and wheat, MEASM) the detoxification ranged from 65 to 84%. In the perspective of developing a technology for bioconversion of aflatoxin-contaminated cereals into added-value feeds, we carried out a pilot study at a laboratory-scale on degradation of AFB1 incorporated in a mushroom cultivation substrate similar to that used in mushroom farms. One strain of P. eryngii (ITEM 13681) was able to bioconvert up to 86% of the AFB1 initially contained in the substrate in 28 days. This value did not change significantly until the maturation of fruit bodies, 42 day post inoculation. The presence of 25% aflatoxin-contaminated maize in the mushroom substrate did not result in a significant reduction of either biological efficiency or yield of the mushroom cultivation. Finally, the analysis of mature fruit bodies did not reveal the presence of detectable amounts of AFB1 or its metabolite aflatoxicol, thus ruling out translocation or "carry-over" of AFB1 through the fungal thallus. These findings make a contribution towards the development of a novel technology for remediation of AFB1-contaminated corn through the exploitation of the degradative capability of P. eryngii.

Management of plant parasitic nematodes with nematode predators, parasites or antagonists is an eco-friendly approach that may avoid the problems arisen by the use of toxic chemicals. Fungi belonging to Trichoderma spp. are well known in literature for their role in control of plant parasitic nematodes. Root-knot nematodes (RKNs), Meloidogyne spp., are obligate parasites that cause the formation of familiar galls on the roots of many cultivated plants. The interaction between the M. incognita motile second stage juveniles (J2s) and the isolate ITEM 908 of Tricoderma harzianum was examined in its effect on the nematode infestation level of susceptible tomato plants.To gain insight into the mechanisms by which ITEM 908 interacts with nematode-infected tomato plants, the expression patterns of the genes PR1 (marker of Salycilic Acid-depending resistance signalling pathway) and JERF3 (marker of the Jasmonic Acid/Ethylene-depending resistance signalling pathway) were detected over time in: i) untrreated roots; ii) roots pre-treated with the fungus;- iii) roots inoculated with the nematode; iv) pre-treated and inoculated roots.Infestation parameters were checked in untreated plants and plants treated with the fungus to test the effect of the fungus on nematode infestation level and to compare this effect with the expression of the genes PR1 and JERF3, involved in induced resistance.

Presentazione dei risultati intermedi del progetto "Il Silver bullet contro Xylella fastidiosa (SiX)".

Obiettivo del progetto SIX è sperimentare l'efficacia di un prodotto sanitario a base di argento colloidale e di una sostanza GRAS (Generally Regarded As Safe) di origine naturale nel controllo di Xylella fastidiosa in piante di olivo. I principi attivi vengono somministrati mediante trattamenti combinati di endoterapia di nuova concezione (metodo BITE) e spray fogliare. L'efficacia dei trattamenti verrà valutata mediante ispezione visiva e determinazioni molecolari per consentire la diagnosi e quantificazione dell'agente patogeno anche in piante asintomatiche. La durata del progetto pilota è di 18 mesi

Competition is one of the potential mechanisms of the antagonistic action of Trichoderma harzianumagainst Fusarium oxysporum. The competitive capability of the T. harzianum isolate ITEM 908 (Th908)vs. an isolate of F. oxysporum f. sp. lycopersici was improved via enhancement of the tolerance togrowth-inhibitory metabolites produced by F. oxysporum. HPTLC and HPLC analyses led to the identificationof fusaric acid (FA) as the major metabolite in culture filtrate of the phytopathogenic F. oxysporumstrain ITEM 2797 (Fo2797). FA, a phytotoxin which has also been reported to be released in soil, totallyinhibited the growth of Th908 on PDA containing 120 lg of FA per gram in 3-day trials. Through UV-Cirradiation and subsequent selection of mutants able to grow on PDA supplemented with culture extractsof Fo2797 or FA, one stable tolerant mutant (Th908-5) with unaltered physiological features and rhizospherecompetence was isolated. The biocontrol capability of the UV-mutant Th908-5 was compared tothat of the wild-type strain Th908 on tomato plants grown in a substrate heavily infested with Fo2797 intwo separate trials. The reduction of the disease by Th908-5 was highly (P < 0.01) to extremely (P < 0.001)significant, while only marginally significant (P < 0.05) and inconsistent biocontrol was achieved byTh908. In addition, in non-inoculated vermiculite, Th908-5 increased the emergence and growth oftomato plants compared to the control. Th908 and Th908-5 were investigated for the expression of fivegenes (MDR ProB, MDR BrefA, MDR Protein2, Hydro II, ThPTR2) encoding proteins putatively associatedwith T. harzianum biocontrol function and involved in the mechanisms of multidrug resistance (MDR)or competition for space and nutrients. When the mutant strain was exposed to FA, the expression ofthe gene MDR ProB, encoding a protein associated with MDR was suppressed, suggesting a role for thegene in response to FA. Since UV-mutants are not regarded as genetically modified organisms (GMO)and their circulation and use is not subjected to restrictions that apply to strains derived by genetic transformation,the improved strain Th908-5 could be readily available for application in the field.

Olive quick decline syndrome (OQDS) caused by X. fastidiosa is currently causing severe damages to the production and reducing the life span of the plants in the Salento peninsula of Apulia (Italy). No effective means of control of X. fastidiosa is currently available. The objective of this study was to evaluate in vitro antimicrobial activities against X. fastidiosa (strain Salento-1) of different classes of compounds having diverse origins, i.e. traditional antibiotics, plant-derived natural products, and microbial metabolites. A preliminary bioassay, performed by the agar disc diffusion method, revealed that 17 of the 31 antibiotics tested did not affect bacterial growth at a dose of 5 ?g. Olive mill wastewaters (OMWs), which are known to possess a broad range of antimicrobial activity, are able to inhibit X. fastidiosa in vitro. Most interestingly when we analysed different OMWs derived micro, ultra and nano-filtered fractions as well as some of the single phenolic compounds that they contain, we found that the OMWs micro-filtered fraction is the most effective against the bacterium but only few phenolics are active in their pure form. Also some fungal extracts and bacteria toxins showed noteworthy inhibitory effect to strain Salento-1 growth. The possible use of some of these products for curative/preventive treating OQDS-affected or at-risk olive plants will be discussed.

Olive quick decline syndrome (OQDS) is causing severe damages to the olive trees in Salento (Apulia, Italy) and poses a severe threat for the agriculture of Mediterranean countries. DNA-Based Typing Methods have pointed out that OQDS is caused by a single outbreak strain of Xylella fastidiosa subsp. pauca referred to as CoDiRO or ST53. Since no effective control measures are currently available, the objective of this study was to evaluate in vitro antimicrobial activities of different classes of compounds against Salento-1 isolated by a OQDS affected plant and classified as ST53. A bioassay based on agar disc diffusion method, revealed that 17 out of the 32 tested antibiotics did not affect bacterial growth at a dose of 5 g disk-1. When we assayed micro-, ultra- and nano-filtered fractions of olive mill wastewaters (OMWs) we found that the micro-filtered fraction resulted the most effective against the bacterium. Moreover, some phenolics (4-methylcathecol, cathecol, veratric acid, caffeic acid, oleuropein) were active in their pure form. Noteworthy, also some fungal extracts and fungal toxins showed inhibitory effects on bacterial growth. Some of these compounds can be further explored as potential candidate in future applications for curative/preventive treating OQDS-affected or at-risk olive plants.

KEY MESSAGE: Salicylic acid-signaling pathway and ethylene biosynthesis were induced in tomato treated with Trichoderma harzianum when infected by root-knot nematodes and limited the infection by activation of SAR and ethylene production. Soil pre-treatment with Trichoderma harzianum (Th) strains ITEM 908 (T908) and T908-5 decreased susceptibility of tomato to Meloidogyne incognita, as assessed by restriction in nematode reproduction and development. The effect of T. harzianum treatments on plant defense was detected by monitoring the expression of the genes PR-1/PR-5 and JERF3/ACO, markers of the SA- and JA/ET-dependent signaling pathways, respectively. The compatible nematode-plant interaction in absence of fungi caused a marked suppression of PR-1, PR-5, and ACO gene expressions, either locally or systemically, whilst expression of JERF3 gene resulted unaffected. Conversely, when plants were pre-treated with Th-strains, over-expression of PR-1, PR-5, and ACO genes was observed in roots 5days after nematode inoculation. JERF3 gene expression did not change in Th-colonized plants challenged with nematodes. In the absence of nematodes, Trichoderma-root interaction was characterized by the inhibition of both SA-dependent signaling pathway and ET biosynthesis, and, in the case of PR-1 and ACO genes, this inhibition was systemic. JERF3 gene expression was systemically restricted only at the very early stages of plant-fungi interaction. Data presented indicate that Th-colonization primed roots for Systemic Acquired Resistance (SAR) against root-knot nematodes and reacted to nematode infection more efficiently than untreated plants. Such a response probably involves also activation of ET production, through an augmented transcription of the ACO gene, which encodes for the enzyme catalyzing the last step of ET biosynthesis. JA signaling and Induced Systemic Resistance (ISR) do not seem to be involved in the biocontrol action of the tested Th-strains against RKNs.

Il pre-trattamento radicale di piante di pomodoro con i due ceppi di Trichoderma harzianum ITEM 908 (T908) e T908-5 ha indotto una minore suscettibilità delle piante al nematode galligeno Meloidogyne incognita, indicata da una limitazione della riproduzione e del numero di nematodi che si sono sviluppati nelle radici. La possibilità che tale minore suscettibilità potesse essere causata da un'induzione delle difese della pianta è stata verificata misurando localmente nelle radici e sistemicamente nelle foglie l'espressione dei geni PR-1 e JERF3, marcatori delle vie molecolari di segnalazione dipendenti rispettivamente dall'acido salicilico (SA) e dall'etilene/acido jasmonico (ET/JA),. Nella normale reazione compatibile tra piante non trattate e nematodi si registrava una forte soppressione dell'espressione del gene PR-1, sia localmente che sistemicamente, e una maggiore espressione del gene JERF3, anche se solo nelle radici. Al contrario, quando le piante erano pre-trattate con i ceppi di Trichoderma, l'espressione del gene PR-1 non veniva più repressa dall'infezione dei nematodi, mentre non si osservava nessun effetto sull'induzione dell'espressione di JERF3. Nelle piante trattate con T908-5 veniva osservato addirittura un aumento dell'espressione di PR-1 dopo l'infezione. Negli stadi più precoci della colonizzazione delle radici da parte di Trichoderma (un giorno dopo il trattamento) e in assenza di infestazione di Meloidogyne si registrava un' inibizione sistemica di entrambe le vie di segnalazione. Al 5° giorno dopo il trattamento la via di segnalazione SA-dipendente era ancora inibita, mentre quella legata a ET/JA non era differente rispetto alle piante non trattate. Dal momento che l'infezione del nematode a radici di piante pre-trattate con i funghi antagonistici induce invece che reprimere l'espressione del gene PR-1, è pensabile che in presenza dei Trichoderma la pianta risponda all'attacco del nematode attivando meccanismi di Resistenza Sistemica Acquisita (SAR). E' ipotizzabile che l'interazione iniziale tra radici di pomodoro e i ceppi di Trichoderma usati in questo studio inizializzi (effetto "priming") le piante per la SAR, con la conseguenza che la difesa contro l'attacco del nematode risulta più rapida ed efficace che nelle piante non trattate, limitando così l'attacco dei nematodi. Tale immunizzazione dura nel tempo, dato che l'effetto del trattamento si protrae sino all'attacco della seconda generazione di larve che si schiude nel terreno 30 giorni dopo l'inoculo della prima generazione. La seconda generazione è in grado di penetrare nelle radici e svilupparsi, ma non è giunta a riproduzione negli esperimenti condotti, in quanto le piante sono raccolte 40 giorni dopo l'inoculo. La totalità delle larve penetrate e sviluppate nelle radici è stata calcolata essere il 30-35% inferiore nelle piante trattate con i due ceppi di Trichoderma rispetto a quelle non trattate.

Fusarium head blight (FHB) is a world-wide occurring disease of wheat and other grain crops that causes yearly considerable losses in terms of yield and quality of grains. The severity of the disease is aggravated by intensive crop management and some cultural practices, such as monocropping and conservation tillage. Moreover a further increase of FHB is expected in temperate areas as a result of the global climate change. The infection of wheat heads is primarily caused by spores of Fusarium graminearum (teleomorph: Gibberella zeae) that infect the spikes at flowering and impair formation of the embryos and accumulation of starch in the endosperm of the developing kernels. Besides being small, shrunk and whitened, the infected kernels may also contain mycotoxins produced by F. graminearum (mainly deoxynivalenol and zearalenone), which enter the food and feed chains and pose safety concerns for human and animal health. The main source of inoculum for flowers infection are the ascospores, which are formed inside perithecia, the flask-shaped fruiting bodies of the fungus that are developed by the overwintering mycelium on the infected plant debris of previous susceptible crops. Since chemical control is difficult and raises environmental and safety concerns, prevention of perithecia formation and ascospore release appears a feasible means for FHB control. We investigated the capability of seven Trichoderma spp. strains to inhibit perithecia formation in dual culture tests. One isolate of F. graminearum was challenged with the antagonistic Trichoderma spp. strains on carrot-agar medium; after 7 days of co-culture the mycelium was peeled off the plates and production of perithecia was induced by fertilization of cultures. After 7 more days of incubation at 25 °C, the number of perithecia formed was assessed in the plate sectors that were pre-colonized by either Trichoderma or Fusarium. In the Trichoderma pre-colonized sectors, perithecia formation was inhibited by 80 to 100%. In the Fusarium pre-colonized sectors, perithecia formation was totally inhibited by 3 out of 7 tested Trichoderma isolates, while the other 4 isolates showed not significant perithecia inhibition. Further investigations on the mechanism of perithecia inhibition showed that the Trichoderma strains released unidentified metabolites that were able to reduce the number of perithecia formed. The reduction of number of perithecia formed by F. graminearum colonies exposed to Trichoderma cell-free metabolites ranged from 27% to 91%, depending on the Trichoderma strain. To explore the effect of Trichoderma metabolites on the regulatory mechanisms of perithecia formation, we carried out a preliminary study of genes involved in the perithecia developmental process. This study allowed to identify a first group of genes associated with different stages of the perithecia formation, whose expression rate in response to Trichoderma metabolites is under investigation.

Fourteen metabolites, isolated from phytopathogenic and toxigenic fungi, were evaluated for their in vitro antigrowthactivity for six distinct cancer cell lines, using the MTT colorimetric assay. Bislongiquinolide (1) and dihydrotrichodimerol(5), which belong to the bisorbicillinoid structural class, displayed significant growth inhibitory activity against the sixcancer cell lines studied, while the remaining compounds displayed weak or no activity. The data show that 1 and 5have similar growth inhibitory activities with respect to those cancer cell lines that display certain levels of resistanceto pro-apoptotic stimuli or those that are sensitive to apoptosis. Quantitative videomicroscopy analysis revealed that 1and 5 exert their antiproliferative effect through cytostatic and not cytotoxic activity. The preliminary results from thecurrent study have stimulated further structure-activity investigations with respect to the growth inhibitory activity ofcompounds belonging to the bisorbicillinoid group.

In this study we report the effects of fungal metabolites isolated from cultures of the fungus Trichoderma citrinoviride ITEM 4484 on the feeding preference of the aphid Rhopalosiphum padi, a major pest of cereal crops. Different phagodeterrent metabolites were purified by a combination of direct and reverse phase column chromatography and thin-layer chromatography. Chemical investigations, by spectroscopic and chemical methods, led to the identification of different long chain primary alcohols (LCOHs) of the general formula R-OH, wherein R is a long, unbranched, unsubstituted, linear aliphatic group. LCOHs have been reported as components of lepidopteran pheromone blends, but their phagodeterrent effect to aphids is herein reported for the first time. The effects of LCOHs on R. padi were studied by behavioral and electrophysiological bioassays. Feeding preference tests that were carried out with winged and wingless morphs of R. padi showed that LCOHs had high phagodeterrent activity and restrained aphids from settling on treated leaves at a concentration as low as 0.15 mM (0.036 g/I). The results of different electrophysiological analyses indicated that taste receptor neurons located on the aphid tarsomeres were involved in the LCOHs perception. Behavioral assays carried out with some commercial agrochemicals, including azadirachtin A, pyrethrum and a mineral oil based product, in combination with 1-hexadecanol, the LCOH most abundantly produced by I citrinoviride ITEM 4484, showed that these different active principles could be applied together, resulting in a useful increase of the phagodeterrent effect. The data shown indicate that these compounds can be profitably utilized for novel applications in biotechnical control of aphid pests. Furthermore, the tested LCOHs have no chiral centers and therefore can be obtained with good yield and at low cost through chemical synthesis, as well as from natural sources.

Aphids are insect pests of great economic impact on agricultural production causing yield losses and reduction of quality products. They damage host plants in different ways, indeed during the feeding phase they subtract sap and spread virus diseases. Chemoreception in aphids plays a fundamental role in host plant selection and in host acceptance, therefore the identification of new compounds that interfere with these processes are of great interest for the development of innovative biotechnical control strategies. In this context, our studies aim to identify fungal metabolites that interfere with aphid feeding behavior and to understand the aphid sensory structures involved in their perception. Our previous studies led to the characterization of three distinct groups of structurally related metabolites, produced by an isolate of the fungus Trichoderma citrinoviride, which exhibit fagodeterrent activity towards different species of aphids. The first two are novel metabolites, characterized as a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively and named citrantifidiene and citrantifidiol (Evidente et al., 2008). Other two compounds belong to the chemical family of bisorbicillinoids, namely bislongiquinolide and dihydrotrichodimerol (Evidente et al., 2009). Further investigations led to the isolation of a fourth group of compounds. They were identified by spectroscopic, chemical and chromatographic methods as primary alcohols: hexadecanol, octadecanol, cis-9-octadecanol and trans-9-octadecanol. Feeding preferences tests showed that these long chain alcohols have a highly significant fagodeterrent activity towards aphids of the species Rhopalosiphum padi (L.), one of the major pests of cereal crops. Antennal responses, obtained by using a standard electroantennographic technique, showed that these long chain alcohols were not able to stimulate significantly the olfactory sensilla of R. padi. Results of single chemosensory cell recordings carried out with long-chain alcohols indicate that the structures involved in their perception, particularly cis-9-octadecanol and trans-9-octadecanol, are taste cells located on the aphid tarsomeres. The tested alcohols, obtained by T. citrinoviride can modify the aphid feeding activity, thus functioning as signalling molecules that can be potentially utilized for novel applications in biotechnical control of aphid.

Funghi appartenenti a diverse specie del genere Trichoderma sono stati fino ad ora utilizzati per il controllo di funghi fitopatogeni e il loro impiego ha confermato l'assenza di effetti negativi per la salute umana e l'ambiente. Nostri precedenti studi hanno messo in evidenza per alcune specie del genere Trichoderma una potenziale attività di controllo degli afidi. Tali insetti rappresentano uno dei più importanti gruppi di fitomizi di molte coltivazioni di notevole interesse economico. Il danno provocato alla pianta ospite non è dovuto solo alla sottrazione di elementi nutritivi e ormonali attraverso la suzione della linfa, ma anche e soprattutto, alla capacità di trasmettere virus. Test di preferenza alimentare da noi condotti, hanno evidenziato una significativa attività fagodeterrente di colture dell'isolato di T. citrinoviride ITEM 4484 nei confronti di diverse specie di afidi tra cui il polifago Myzus persicae (Sulzer) e Rhopalosiphum padi un diffuso parassita dei cereali. Ulteriori indagini condotte mediante metodi spettroscopici (essenzialmente NMR e tecniche MS) e volte ad identificare i composti fungini con attività fagodeterrente prodotti dall'isolato di T. citrinoviride hanno permesso di isolare e caratterizzare quattro distinti gruppi di metaboliti tra di loro strutturalmente correlati. I primi due metaboliti, denominati citrantifidiene e citrantifidiol, sono rispettivamente un esa-1,3-dienil estere dell'acido acetico simmetrico ed un cicloesadien-1,3-diolo tetrasostituito. Altri due composti individuati appartengono alla famiglia dei bisorbicillinoidi, e sono il bislongiquinolide e il diidrotricodimerolo.Il quarto gruppo, il quale mostra una spiccata attività fagodeterrente, è composto da diversi alcoli primari a catena lunga: esadecanolo, octadecanolo, cis-9-octadecanolo e trans-9-octadecanolo. Questi composti non hanno centri chirali e oltre ad essere reperibili in natura possono essere facilmente sintetizzati con una buona resa. Per questi alcoli a catena lunga e per altri ad essi chimicamente correlati è stata depositata una domanda di brevetto nazionale (MI2011A000831), con successiva estensione a livello internazionale (PTC IB2012/052383) per la loro attività fagodeterrente che potrà essere utilizzata per il controllo biotecnico degli afidi.

Trichoderma spp. are long-time known antagonists of fungal plant pathogens and probably the most widely-employed biological fungicides worldwide. Much is known about the mode of action of antagonistic Trichoderma strains against different plant pathogens and the mechanisms of regulation of the antagonistic activity. Considerable progress has been made with regard to understanding plant-Trichoderma interaction, casting a new light on the complex relationships among plant, pathogen and antagonist. The advancements in biomass production and formulation have allowed to obtain products that are less expensive, more durable and more effective than in the past, bridging the gap between chemical control and biocontrol with Trichoderma in terms of efficacy and reliability and making some strains of Trichoderma the most successful biological fungicides so far.In spite of this, the potential of Trichodema spp. for protection of crops from biological and abiological constraints, do not appears to be completely disclosed and exploited. Recent findings have shown the potential of some Trichoderma for biological control of insect pests. Some Trichoderma are source of bioactive metabolites that have unexpected and quite interesting activities. From cultures of T. citrinoviride we have isolated and characterized secondary metabolites that have fagodeterrent effect against aphids (PCT patent filed), insect pests of great economic importance for agriculture and one of the main causes of loss of agricultural produce and reduction of its quality.Also, some Trichoderma strains have shown the capability of to control plant parasitic nematodes. We have investigated the capability of our reference biocontrol strain T. harzianum ITEM 908 to protect tomato plants from the attack of the root-knot nematode Meloidogyne incognita. The presence of T. harzianum ITEM 908 in soil reduces all the infestation parameters of M. incognita, such as egg mass production, female fecundity and reproduction potential, on the infested tomato roots. In addition, T. harzianum ITEM 908 is able to induce resistance to M. incognita in tomato. These results open up interesting opportunities for both research and development of new Trichoderma-based products.

Nematodes of the genus Meloidogyne are obligate parasites that infect a wide range of different crops in all agricultural regions worldwide and cause significant losses of both yield and quality of produce. Trichoderma spp. have long been known to be a feasible biological alternative to chemicals for control of several soil-borne plant pathogens. More recently, it has been shown that Trichoderma spp. may be effective also in control of plant parasitic nematodes. However, very little is known on the mechanism(s) by means of which these antagonistic fungi can limit nematode infestation. In order to assess the capability of the antagonistic strain T. harzianum ITEM 908 to elicit resistance to M. incognita in tomato plants, we investigated the expression of the genes PR-1 (marker of the salicylic acid-depending resistance signalling pathway, SAR) and JERF3 (marker of the jasmonic acid/ethylene-depending resistance signalling pathway, ISR) during the interaction between ITEM 908 and M. incognita on susceptible tomato plants. Our findings suggest that jasmonic acid/ethylene-mediated resistance to M. incognita, is induced in tomato roots by treatment with T. harzianum ITEM 908, whereas, salicylic acid-mediated resistance, seems not to be involved in the interaction between this strain and tomato plants, at least in the roots. We also investigated the effect of T. harzianum ITEM 908 on infestation parameters, such as egg mass production, female fecundity and reproduction potential of M. incognita on the infested tomato roots. The presence of a suitable amount of T. harzianum ITEM 908 in soil (106 CFU/g of rhizosphere soil) significantly reduced all of the infestation parameters investigated. It is still to be established whether nematode infestation is reduced by ISR induction in roots or by a putative nemato-static and/or nemato-toxic activity of ITEM 908 in the soil, or a combination of both.

In this study we report the effects of some long-chain alcohols, obtained from an isolate of the fungus Trichoderma citrinoviride, on feeding preference of the aphid Rhopalosiphum padi, a major pest of cereal crops. Purification of these fungal metabolites was carried out by a combination of direct and reverse phase column chromatography and thin-layer chromatography. Chemical identification, performed by spectroscopic and chemical methods including nuclear magnetic resonance and mass spectrometry, led to isolation and structure determination of different primary alcohols of the general formula R-OH, wherein R is a long, unbranched, unsubstituted, linear aliphatic group. Feeding preference tests carried out with winged and wingless morphs of R. padi showed that these metabolites had a distinctly high fagodeterrent activity and thus restrained aphids from settling on treated leaves. The tested metabolites, have no chiral centers and therefore can be obtained in good yields through chemical synthesis, besides natural sources and can be potentially utilized for novel applications in biotechnical control of aphid pests.

Pleurotus eryngii (DC) Quél., comunemente noto con il nome di "fungo cardoncello", oltre ad essere un fungo edule molto apprezzato per le caratteristiche organolettiche e per il valore nutritivo, viene anche studiato per la capacità di produrre enzimi ligninolitici che hanno un elevato interesse biotecnologico. Il sistema enzimatico ligninolitico di P. eryngii, costituito dagli enzimi laccasi, lignino-perossidasi e Mn-perossidasi, ha un ruolo fondamentale nella demolizione della lignina in composti a basso peso molecolare. Poiché tali enzimi non hanno specificità di substrato, possono trovare impiego nella biodegradazione di molecole aromatiche complesse e di composti recalcitranti nell'ambito di applicazioni biotecnologiche come la decolorazione di coloranti sintetici e il biorisanamento ambientale da inquinanti chimici. Inoltre, alcuni recenti studi indicano che gli enzimi laccasi e Mn-perossidasi sono anche in grado di degradare l'aflatossina B1 (AfB1), una micotossina prodotta da funghi del genere Aspergillus nel corso di processi di ammuffimento di prodotti agricoli (specialmente mais) e la cui assunzione tramite alimenti contaminati può causare gravi patologie nell'uomo e in specie zootecniche. L'obiettivo di questo studio è stato quello di valutare la capacità degradativa dell'AfB1 da parte di diversi ceppi di P.eryngii. L'analisi cromatografica mediante Ultra Performance Liquid Chromatography (UPLC) dei filtrati colturali di 9 ceppi di P. eryngii coltivati in un substrato liquido a base di estratto di malto (MEB) con l'aggiunta di 500 ng/ml di AfB1 ha evidenziato la totale scomparsa dell'AfB1 dal brodo di coltura dopo 30 giorni. Gli stessi isolati, coltivati su substrato agarizzato a base di estratto di malto e contenente paglia di grano e farina di mais (MEASM), hanno mostrato una capacità detossificante compresa tra 65% e 84%, Un ceppo di P.eryngii, selezionato tra quelli più dotati di capacità degradativa, è stato infine coltivato su un substrato idoneo alla produzione dei carpofori e costituito da paglia di grano (50%), mais contaminato con Afb1 (128 ng/g, 25%), scarti della lavorazione della barbabietola da zucchero (12,5%) e favino (12,5%). Anche su questo substrato è stata osservata una detossificazione dell'86% dell'AfB1 dopo 28 giorni dall'inoculazione del fungo, in accordo con i risultati ottenuti in vitro. Nei corpi fruttiferi non è stata rilevata la presenza di AfB1, escludendo quindi un possibile trasporto della tossina nella parte edibile del fungo. L'efficienza biologica e la resa produttiva di carpofori non hanno mostrato differenze significative rispetto al controllo. I risultati di questo studio aprono possibilità di sviluppo di tecnologie per la conversione e la valorizzazione di prodotti agricoli contaminati con AfB1, che vengono attualmente distrutti e che potrebbero invece destinarsi alla produzione di mangimi ad uso zootecnico. Ulteriori studi sono in corso per identificare i prodotti di degradazione dell'AfB

Competition has been reported as one of the mechanisms of the antagonistic action of Trichoderma harzianum against F. oxysporum. Competition between microorganisms occurs when a vital factor, such as nutrients or space, is available in limited quantity. Under these limiting conditions, production of inhibitory compounds able to stop or slow down the growth of other microorganisms confers on the producing microbe an ecological advantage over the competitor(s). The competitive capability of the T. harzianum isolate ITEM 908 (Th908) vs. F. oxysporum f. sp. lycopersici was improved via enhancement of the tolerance to growth-inhibitory metabolites produced by F. oxysporum. HPTLC and HPLC analyses led to the identification of fusaric acid (FA) as the major metabolite in culture filtrate of the phytopathogenic F. oxysporum strain ITEM 2797 (Fo2797). FA, a phytotoxin which has also been reported to be released in soil, was strongly inhibitory to T. harzianum as well to isolates of other Trichoderma species. Through UV-C irradiation and subsequent selection of mutants able to grow on PDA supplemented with culture extracts of Fo2797 or FA, two stable FA-tolerant mutants were isolated. The biocontrol capability of one selected UV-mutant with unaltered physiological features and rhizosphere competence was compared to that of the wild strain Th908 on tomato plants grown in a substrate heavily infested with Fo2797 in two separate trials. The reduction of the disease by the mutant strain was significantly (P < 0.01) higher than that achieved with the wild strain Th908. Based on the above results, the combined use of certain biocontrol strains of F. oxysporum and T. harzianum might be problematic, because of the production of FA by the biocontrol strains of F. oxysporum. Therefore, we investigated the compatibility of the FA-tolerant mutant strain of T. harzianum with biocontrol strains of F. oxysporum in microcosms wherein the two biocontrol agents were co-inoculated. Since UV-mutants are not regarded as GMO, their circulation and use is not subjected to restrictions and the improved strain could get the registration for field use in some countries more easily than strains derived by genetic transformation.

The present invention relates to the use of R-OH alcohols as phagodeterrents, and to a method for treating plants infested with aphids which comprises the administration of these R-OH alcohols.