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The meadow spittlebug Philaenus spumarius L. is the vector for the bacterium Xylella fastidiosa subspecies pauca, involved in olive quick decline syndrome (OQDS) in Salento (Italy). Control of P. spumarius is key to limiting transmission of the bacterium, and an innovative approach can be based on effective natural compounds and biocontrol agents. Entomopathogenic fungi are an important source of bioactive natural molecules that play a role in the relationship between microorganisms and insects.Pathogenicity bioassays, performed by dipping adults of P. spumarius in either fungal culture suspension (120 mg mLT. chlorosporum GJS 91-150 affected the survival of P. spumarius adults. The lethal effect was not associated with the development of mycelium on the cuticle, but seems due, at least partly, to fungal metabolites released in the culture medium. The fungus tested here has good potential for the development of effective low-environmental impact control strategies for P. spumarius and suppression of X. fastidiosa. © 2022 Society of Chemical Industry.

The antibiotic and nematocidal activities of extracts from two coastal lichen species collected on Lampedusa Island (Sicily), Ramalina implexa Nyl. and Roccella phycopsis Ach., were tested. Methyl orsellinate, orcinol, (+)-montagnetol, and for the first time 4-chlororcinol were isolated from Roccella phycopsis. (+)-Usnic acid was obtained from Ramalina implexa. The crude organic extract of both lichen species showed strong antibiotic activity against some bacterial species and nematocidal activity. Among all the pure metabolites tested against the infective juveniles (J2) of the root-knot nematode (RKN) Meloydogine incognita, (+)-usnic acid, orcinol, and (+)-montagnetol had significant nematocidal activity, comparable with that of the commercial nematocide Velum® Prime, and thus they showed potential application in agriculture as a biopesticide. On the contrary, methyl orsellinate and 4-chlororcinol had no nematocidal effect. These results suggest that the substituent pattern at ortho-para-position in respect to both hydroxyl groups of resorcine moiety, which is present in all metabolites, seems very important for nematocidal activity. The organic extracts of both lichens were also tested against some Gram-positive and Gram-negative bacteria. Both extracts were active against Gram-positive species. The extract of Ramalina implexa showed, among Gram-negative species, activity against Escherichia coli and Acinetobacter baumannii, while that from Roccella phycopsis was effective towards all test strains, with the exception of Pseudomonas aeruginosa. The antimicrobial activity of (+)-usnic acid, methyl orsellinate, and (+)-montagnetol is already known, so tests were focused on orcinol and 4-chlororcinol. The former showed antibacterial activity against all Gram positive and Gram-negative test strains, with the exception of A. baumannii and K. pneumoniae, while the latter exhibited a potent antibacterial activity against Gram-positive test strains and among Gram-negative strains, was effective against A. baumannii and K. pneumonia. These results suggest, for orcinol and 4-chlororcinol, an interesting antibiotic potential against both Gram-positive and Gram-negative bacterial strains.

The inhibitory action of 20 antagonistic

Ligninolytic enzymes from white-rot fungi, such as laccase (Lac) and Mn-peroxidase (MnP), are able to degrade aflatoxin B1 (AFB1), the most harmful among the known mycotoxins. The high cost of purification of these enzymes has limited their implementation into practical technologies. Every year, tons of spent mushroom substrate (SMS) are produced as a by-product of edible mushroom cultivation, such as Pleurotus spp., and disposed at a cost for farmers. SMS may still bea source of ligninolytic enzymes useful for AFB1 degradation. The in vitro AFB1-degradative activity of an SMS crude extract (SMSE) was investigated. Results show that: (1) in SMSE, high Lac activity (4 U g&minus, 1dry matter) and low MnP activity (0.4 U g&minus, 1dry matter) were present, (2) after 1 d of incubation at 25 &deg, C, the SMSE was able to degrade more than 50% of AFB1, whereas after 3 and 7 d of incubation, the percentage of degradation reached the values of 75% and 90%, respectively, (3) with increasing pH values, the degradation percentage increased, reaching 90% after 3 d at pH 8. Based on these results, SMS proved to be a suitable source of AFB1 degrading enzymes and the use of SMSE to detoxify AFB1 contaminated commodities appears conceivable.

Aflatoxin B-1 (AfB(1)) is a carcinogenic mycotoxin that contaminates food and feed worldwide. We determined the AfB(1)-adsorption capability of non-viable Pleurotus eryngii mycelium, an edible fungus, as a potential means for removal of AfB(1) from contaminated solutions. Lyophilized mycelium was produced and made enzymatically inert by sterilization at high temperatures. The material thus obtained was characterized by scanning electron microscopy with regard to the morpho-structural properties of the mycotoxin-adsorbing surfaces. The active surfaces appeared rough and sponge-like. The AfB(1)-mycelium system reached equilibrium at 37 degrees C, 30 min, and pH 5-7, conditions that are compatible with the gastro-intestinal system of animals. The system remained stable for 48 h at room temperature, at pH 3, pH 7, and pH 7.4. A thermodynamic study of the process showed that this is a spontaneous and physical adsorption process, with a maximum of 85 +/- 13% of removal efficiency of AfB1 by P. eryngii mycelium. These results suggest that biosorbent materials obtained from the mycelium of the mushroom P. eryngii could be used as a low-cost and effective feed additive for AfB(1) detoxification.

Biochar (BC) and hydrochar (HC) are carbonaceous products obtained through, respectively, pyrolysis and hydrothermal carbonization processes of biomass. Both materials are multi-functional soil amendments. Ligninolytic fungi are primary decomposers of recalcitrant lignocellulosic material in nature through their extensive hyphal network and enzymes. In this work, two BC samples from red spruce pellets (BCSP) and grapevine pruning residues (BCGV) and two HC samples from urban pruning residues (HCUP) and the organic fraction of solid urban wastes (HCSU) were tested at concentrations of 0.4% and 2% (w/v) on the growth and enzyme activity of Trametes versicolor, Pleurotus ostreatus and Pleurotus eryngii. In all treatments with the lower concentration, BC and HC significantly stimulated fungal growth (up to about 90% increase for HCSU on T. versicolor), whereas at the higher dose some inhibition was observed on T. versicolor by BCSP and P. ostreatus by BCSP, BCGV and HCUP. The two materials, especially HC, at both doses noticeably increased the activity of laccase from T. versicolor and P. eryngii, up to 21 and 13 times, respectively, for HCUP compared to controls. The activity of manganese peroxidase from P. ostreatus was also greatly stimulated by BC and HC, especially when added at the higher concentration. The overall results obtained in this study suggest potential benefits for ligninolytic fungi from the presence of these materials in soil at adequate dose of application.

Background So far, biocontrol agent selection has been performed mainly by time consuming in vitro confrontation tests followed by extensive trials in greenhouse and field. An alternative approach is offered by application of high-throughput techniques, which allow extensive screening and comparison among strains for desired genetic traits. In the genus Trichoderma, the past assignments of particular features or strains to one species need to be reconsidered according to the recent taxonomic revisions. Here we present the genome of a biocontrol strain formerly known as Trichoderma harzianum ITEM 908, which exhibits both growth promoting capabilities and antagonism against different fungal pathogens, including Fusarium graminearum, Rhizoctonia solani, and the root-knot nematode Meloidogyne incognita. By genomic analysis of ITEM 908 we investigated the occurrence and the relevance of genes associated to biocontrol and stress tolerance, providing a basis for future investigation aiming to 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 species recently 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 active enzymes (CAZYmes), proteins involved in secondary metabolites production, peptaboils, epidithiodioxopiperazines and siderophores potentially involved in parasitism, saprophytic degradation as well as in biocontrol and antagonistic activities. This abundance is comparable to other Trichoderma spp. in the T. harzianum species complex, but broader than in other biocontrol species and in the species T. reesei, known for its industrial application in cellulase production. Comparative analysis also demonstrated similar genomic organization of major secondary metabolites clusters, as in other Trichoderma species. Conclusions Reported data provide a contribution to a deeper understanding of the mode of action and identification of activity-specific genetic markers useful for selection and improvement of biocontrol strains. This work will also enlarge the availability of genomic data to perform comparative studies with the aim to correlate phenotypic differences with genetic diversity of Trichoderma species. Electronic supplementary material The online version of this article (10.1186/s12864-018-5049-3) contains supplementary material, which is available to authorized users.

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.

Competition is one of the potential mechanisms of the antagonistic action of Trichoderma harzianum against 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 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, totally inhibited the growth of Th908 on PDA containing 120 μg of FA per gram in 3-day trials. Through UV-C irradiation and subsequent selection of mutants able to grow on PDA supplemented with culture extracts of Fo2797 or FA, one stable tolerant mutant (Th908-5) with unaltered physiological features and rhizosphere competence was isolated. The biocontrol capability of the UV-mutant Th908-5 was compared to that of the wild-type strain Th908 on tomato plants grown in a substrate heavily infested with Fo2797 in two separate trials. The reduction of the disease by Th908-5 was highly (P

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/l). 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 T. 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.

Fusapyrone (FP), an antifungal natural compound, was tested against the three main ochratoxigenic species of the Aspergillus section Nigri . The MICs at 24 h were 6.0, 11.6, and 9.9 μg/ml for Aspergillus carbonarius , Aspergillus tubingensis , and Aspergillus niger , respectively. Strong inhibition of growth and morphological changes were still observed at half the MIC after 7 days. The application of a 100 μg/ml FP solution in a laboratory assay on artificially inoculated grapes resulted in a significant reduction (up to 6 orders of magnitude) of A. carbonarius CFU counts. Dramatic reductions of the ochratoxin A (OTA) content, compared to the content of the positive control (average amount of OTA, 112.5 ng/g of grape; three experiments), were obtained with the application of either 100 or 50 μg/ml of FP (0.6 or 5.1 ng/g of grape, respectively).

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.

Fungal biocontrol agents (BCAs) have been marketed for control of crop pests, weeds, and diseases. However, BCAs may produce toxic metabolites, whose presence in the formulated products, in the crops and in the environment should be considered along with the associated risk. Two invertebrate models, viz. Artemia salina and Daphnia magna were used to assess the acute toxicity of seven BCA metabolites, characterized by different chemical nature and mode of action, namely alamethicin (ALA), paracelsin (PCS), antiamoebin (AAM), gliotoxin (GTX), destruxin A (DA), oosporein (OOS), and elsinochrome A (EA). The two invertebrates were very sensitive to all the metabolites examined, except OOS. The LC50s after 24 and 36 h exposures showed the following toxicity ranks: A. salina, DA > ALA > EA > GTX > AAM > PCS (LC50s ranging from 9.78 to 40.84 microg/ml at 24 h and from 2.92 to 18.56 microg/ml at 36 h); D. magna, DA > GTX = EA > ALA > PCS > AAM (LC50s ranging from 0.20 to 24.41 microg/ml at 24h and from 0.16 to 11.98 microg/ml at 36 h). LC50 of OOS to D. magna increased dramatically in 36 h exposure, compared to 24 h exposures (5.84 and 68.40 microg/ml, respectively). A. salina and D. magna proved to be suitable models for rapid and inexpensive screening of toxicity of BCAs at an early stage of product development.

Fusarium semitectum is a widespread species often isolated from plants with complex disease and also known to be toxigenic. Twenty-seven isolates of F. semitectum obtained from wilted plants of Medicago sativa grown in northern Italy were analysed by Amplified Fragment Length Polymorphisms (AFLP). The isolates were divided in two distinct clusters, coded A and B. Investigations were carried out on morphology, pathogenicity on alfalfa seedlings and toxin production of 18 isolates representative of the two AFLP clusters. Isolates from cluster A showed fast growth and pale peach colour of the colonies on potato-dextrose-agar, whereas those from cluster B showed slow growth and orange pigmentation, changing to brown with age. Length of 5-septate macro-conidia was higher in isolates from cluster A and the frequency of classes of conidia septation was statistically different between the two clusters. All isolates from cluster A were pathogenic to alfalfa, whereas those from cluster B were mostly non-pathogenic. The mycotoxigenic profiles were distinct between the two clusters and similar within the same cluster: isolates from cluster A produced zearalenone, alone or together with diacetoxyscirpenol, whereas isolates from cluster B produced deoxyfusapyrone. The results showed that there is a good correspondence between the AFLP clustering and the distinction of F. semitectum isolates from alfalfa into two groups based on morphology, pathogenicity and toxin-production.

In a simple experimental system with wheat plants, interactions between the collembolan Onychiurus armatus and Trichoderma harzianum, a soil-borne fungus parasite of several plant pathogenic fungi, were studied in the presence of Gaeumannomyces graminis var. tritici , one of the most important foot and root fungal pathogens of cereals world-wide. Trichoderma harzianum was used according to two different modalities: fungal inoculum applied to seed or mixed with substrate. The isolate of T. harzianum proved to be ineffective against the pathogenic fungus, independent of the application modality, as shown in the experiments where this fungus was used alone, whereas Collembola used alone significantly reduced disease severity. However, the mode by which T. harzianum was applied significantly influenced the disease control ability of Collembola. In fact, only when T. harzianum was coated to seed were springtails able to reduce the disease caused by G. graminis var. tritici.

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.

A simple, very sensitive and rapid HPLC method was developed for the simultaneous quantitative analysis of both fusapyrone (FP) and deoxyfusapyrone (DFP), the two antifungal 3-substituted-4-hydroxy-6-alkyl-2-pyrones isolated from rice culture of Fusarium semitectum, in crude extracts. Such method was optimized on C-18 reverse phase column, using the isolated metabolites as standards, with a sequence of linear elution steps with a MeOH-H2O mixture and using an ultraviolet detector fixed at 285 nm, where both α-pyrones showed a characteristic absorption maximum. This method was used to quantify the bioactive metabolites in crude organic extracts from two F. semitectum strains. The recovery of FP and DFP was measured in a crude extract from a poor metabolite producer F. semitectum strain. The recovery values ranged from 84 % to 99 % for FP and from 99 % to 101 % for DFP, indicating that the method was close to quantitative recovery. Furthermore, an efficient medium pressure column chromatography and TLC combined method was developed for the isolation and purification of FP and DFP from fungal culture extracts. Copyright © 1999 John Wiley & Sons, Ltd.

Isozyme and random amplified polymorphic DNA (RAPD) analyses have been used in combination with numerical taxonomy to study the taxonomic relationships among the toxigenic species Fusarium acuminatum Ellis & Everh. sensu Gordon (subsp. acuminatum and subsp. armeniacum), Fusarium sporotrichioides Sherb. and Fusarium tricinctum (Corda) Sacc. Eight enzymes, selected among 23 enzyme systems tested initially for activity, resolution, and consistent formation of bands, were used for isozyme analysis of 75 strains. Both cluster analysis grouping by average linkage method and correspondence analysis of the isozyme data set resulted in an arrangement of the four taxa inconsistent with their classical taxonomic classification in the Fusarium sections Gibbosum and Sporotrichiella. Isolates of F. acuminatum subsp. acuminatum were more closely related to F. tricinctum than to F. acuminatum subsp. armeniacum. Correspondingly, the F. acuminatum subsp. armeniacum group was closer to F. sporotrichioides than to F. acuminatum subsp. acuminatum. Twenty-seven strains of the four taxa, representative of the variability found by isozyme analysis, were studied using a RAPD analysis with five different decamer primers. All the primers produced polymorphic amplification patterns with taxon-specific bands, in addition to individual-specific bands. Correspondence analysis of the RAPDs distinguished four compact groups corresponding to the four taxa studied. These data support the separation of the varieties of F. acuminatum into two different species, and suggest a revision of the Fusarium sections Gibbosum and Sporotrichiella may be necessary. Key words: Fusarium, isozymes, RAPDs, numerical taxonomy, mycotoxins.

Banana fruits exhibiting signs of decay were collected from markets in the United States and Italy. Fungi isolated from the lesions on the banana fruits wereFusarium moniliforme, F subglutinans, andF. semitectum var.majus. When the fungal strains were cultivated on maize kernels, the cultures did not produce zearalenone (ZON), zearalenols (á-, â-ZOH), and trichothecenes [deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), T-2 toxin (T-2), diacetoxyscirpenol (DAS)]. Fumonisins and fusarin C (FUS-C) were not detected naturally nor in bananas purchased in the U.S. and artificially infected withFusarium. Moniliformin (M) (up to 267 mg/kg) was detected in maize kernel cultures ofF. subglutinans from bananas. No mycotoxins were detected in naturally infected fruits. Although no mycotoxins were detected in the extracts from corn cultures ofF. semitectum var.majus, the extracts were toxic to brine shrimp and mice.

Twenty-nine Fusarium isolates, representing three new taxa originated by Nirenberg from F. sambucinum Fuckel sensu lato, namely: F. sambucinum Fuckel sensu stricto, F. venenotum Nirenb., and F. torulosum (Berk.Curt.) Nirenb., were tested for in vitro production of toxic secondary metabolites on autoclaved corn kernels. F. sambucinum sensu stricto was able to produce type A trichothecenes and enniatin B (EB). In particular, amongst the 14 isolates tested, 5 produced only diacetoxyscirpenol (DAS) (up to 700 micrograms/g); 1 produced only neosolaniol (NEOS) (250 micrograms/g); 2 produced T-2 toxin (T-2) + NEOS (up to 175 and 150 micrograms/g, respectively); 1 produced NEOS + DAS (300 and 100 micrograms/g, respectively); and 5 produced DAS + EB (up to 500 and 140 micrograms/g, respectively). All six isolates of F. venenotum were able to produce only DAS (up to 100 micrograms/g). F. torulosum produced no trichothecenes, but four out of nine tested isolates were able to produce EB (up to 140 micrograms/g). Zearalenones and type B trichothecenes were not found. The toxicity of the culture extracts towards Artemia salina L. was correlated in general with the occurrence of the above toxins, except for some F. torulosum strains. However, the lack of correlation between the amounts of toxins recovered and toxic activity observed in the Geotrichum candidum Link ex Pers. and A. salina assays suggested the presence of unknown toxic compounds.

L'Agenda Strategica della Ricerca (ASR) riporta una panoramica completa delle priorità di ricerca e sviluppo in agricoltura biologica nel nostro paese in una prospettiva strategica ed in coerenza con quelle definite in sede europea dalla Strategic Research Agenda della Piattaforma TP Organics. L'Agenda Strategica della Ricerca individua 3 aree e 13 tematiche prioritarie a cui afferiscono 71 Temi specifici di ricerca, che investono ampi campi d'indagini con promettenti ricadute sia sul lato economico che in termini di sostenibilità, tenendo in considerazione il valore dell'agricoltura biologica anche in ragione della salvaguardia di un bene comune. In base alle considerazioni riportate, l'Agenda Strategica della Ricerca di PTBio Italia intende fornire un contributo efficace per affrontare le vaste problematiche che investono l'intero complesso agroalimentare, ritenendo che la ricerca e l'innovazione nel campo dell'agricoltura biologica aiuti anche allo sviluppo di un sistema di produzione a ridotto impatto ambientale e con minor uso di risorse, alla produzione di alimenti sicuri e di alta qualità, alla crescita di un sistema agroalimentare valido, dinamico e sostenibile ed alla diffusione di comportamenti alimentari più sani. Questo avrà ricadute importanti sulle grandi sfide globali del prossimo decennio e che riguardano l'insicurezza alimentare, l'obesità, la qualità e la salubrità del cibo, i cambiamenti climatici, la perdita della biodiversità, la degradazione del suolo e la ridotta disponibilità d'acqua.

The new challenges that will be faced by agriculture in the twenty-first century impose the adoption of strategies able to increase food production without further increase the area of arable land and with low environmental impact. Soil microorganisms are a major component of the natural fertility of soils. They can promote plant growth, increase crop productivity and contribute significantly to the mineral nutrition of crop plants. This review examines the up-to-date knowledge about the potential and existing uses of beneficial microbes as biofertilizers and gives an outline of their modes of action. Plant growth promoting microorganisms (PGPM) influence plant nutrition and growth through various mechanisms including nitrogen fixation, breakdown of organic matter, solubilization of sparingly soluble minerals, release of chelating compounds and biologically active substances such as phytohormones, vitamins and enzymes, and increase of the root system efficiency in nutrient uptake. Non symbiotic soil- or endophytic bacteria belonging to the genera Azospirillum, Azotobacter, Acetobacter, Gluconacetobacter, Azoarcus, Bacillus, Paenibacillus, Burkholderia, Herbaspirillum, Clostridium, Klebsiella, Enterobacter, Citrobacter, and Pseudomonas are able to fix atmospheric N2 and have been found to be responsible for supply of biologically fixed nitrogen to crop plants. They may also improve plant growth through production of bioactive metabolites and indirect mechanisms, such as suppression of phytopathogens or induction of resistance to pathogens in plants. Numerous species of soil and rhizosphere microorganisms may solubilize insoluble mineral phosphates, mainly through acidification and production of organic acids, and thus mobilize the enormous reserves of phosphorus (P) that are stored in most soils and are otherwise unavailable to plants. Generally, fungi exhibit greater P-solubilizing ability than bacteria. Members of the genera Aspergillus, Penicillium, and Trichoderma are particularly efficient P-solubilizers. Among bacteria, good results have been obtained with Bacillus spp. and Pseudomonas spp., especially in combination with P-solubilizing fungi and arbuscular mycorrhizal fungi (AMF). Under field conditions, the combined use of P-solubilizing microorganisms with mineral fertilizers such as rock-phosphate has often given successful results. Also, the biological activities of microorganisms in the rhizosphere can mediate the solubility, and hence the availability at root surface of micro-nutrients, of which most soils are defective due to the fast depletion resulting from intensive farming. While other elements are also involved, Fe, Mn and Zn deficiencies have the greatest impact on the yields and quality of agricultural produce. PGPM and AMF have the capability to alter soil pH and modify the equilibrium of many chemical and biochemical reactions, such as precipitation/dissolution, adsorption/desorption, complexation/dissociation, and oxidation/reduction of metal cations and thus regulate the plant uptake. Beside enhancing plant nutrition under limited or deficient conditions, they may also reduce detrimental effects of excess of micronutrients, which may occur in acid or polluted soils. In conclusion, in order to reduce the environmental and economic costs of the massive use of synthetic fertilizers and to obtain safer food, the use of PGPM as biofertilizers appears to be a concrete perspective. All the evidence summarized in this review clearly shows that beneficial soil microorganisms, either alone or in combination with mineral or organic fertilizers, may be utilized to increase crop productivity and maintain the fertility of soils without threatening the environment. These multifunctional agents are a renewable resource with low environmental impact. Therefore, beneficial soil microbes should be further studied and exploited for the development of sustainable agriculture.

The structure of paracelsin E, a new peptaibol from Trichoderma saturnisporum, has been determined primarily by fabms. The well-known paracelsins A, B, C, and D were also found in a culture of this organism.

Fourteen metabolites, isolated from phytopathogenic and toxigenic fungi, were evaluated for their in vitro antigrowth activity 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 six cancer cell lines studied, while the remaining compounds displayed weak or no activity. The data show that 1 and 5 have similar growth inhibitory activities with respect to those cancer cell lines that display certain levels of resistance to pro-apoptotic stimuli or those that are sensitive to apoptosis. Quantitative videomicroscopy analysis revealed that 1 and 5 exert their antiproliferative effect through cytostatic and not cytotoxic activity. The preliminary results from the current study have stimulated further structure-activity investigations with respect to the growth inhibitory activity of compounds belonging to the bisorbicillinoid group.

Twenty-five isolates of F. acuminatum from different sources and geographic origins were used for studying the cultural and toxigenic characteristics of the species. The isolates showed a large cultural variability, and the features ranged from slow-growing isolates with carmine-red pigmentation and relatively short 5-septate conidia to fast-growing yellow-brown isolates and long 5-septate conidia. The cultural differences corresponded to differences in production of secondary metabolites. On the basis of their metabolite production, the isolates were grouped into three categories: ( a ) enniatin B and/or moniliformin producers (up to 1000 and 2340 mg kg −1 , respectively); ( b ) T-2 toxin, HT-2 toxin and/or neosolaniol producers (up to 450, 40 and 20 mg kg −1 , respectively); ( c ) no-toxin producers. The toxicity of the culture extract towards Artemia salina could usually be related to the occurrence of the above toxins.

Orobanche ramosa is an important parasitic weed of several agriculturally important crops. A fungal strain identified as Fusarium oxysporum (named FT2) proved to be specific and highly virulent to O. ramosa plants and was proposed as a mycoherbicide for the biological control of this weed to be applied at the soil level. Considering that detecting and tracking the strain is of utmost importance to know the fate of the strain after its release, an amplified fragment length polymorphism (fAFLP) analysis was chosen and utilized with the aim to develop a molecular marker to specifically identify this strain. A wide population of F. oxysporum strains isolated from different hosts was screened against the mycoherbicide strain in order to identify specific fragments belonging to this strain. Two specific fragments were found and their DNA sequences were utilized for primer design. A primer pair (named FT2230F/FT2230R) proved to be strain–specific and it amplified a 232 bp DNA fragment of FT2. These primers were used to monitoring the presence of the F. oxysporum strain in the soil. Amplicons were detected from all the soil samples artificially infected by using known amounts of FT2 inoculum, whereas none of the primer sets amplified DNA from soils not infected by FT2.

We report the effects of some bisorbicillinoids isolated from biomass of the fungus Trichoderma citrinoviride on settling and feeding preference of the aphid Schizaphis graminum. Purification of the fungal metabolites was carried out by a combination of column chromatography and thin-layer chromatography using direct and reverse phases. Chemical identification was performed by spectroscopic methods including nuclear magnetic resonance and mass spectrometry. The identified bisorbicillinoids appeared to be bislongiquinolide, its 16,17-dihydro derivative, trichodimerol, and dihydrotrichodimerol. A feeding preference test with alate morphs of S. graminum was used to identify the active fractions. Among the four bisorbicillinoids, dihydrotrichodimerol and bislongiquinolide influenced aphid feeding preference, restraining specimens from settling on leaves treated with metabolites. Taste neurons sensitive to these compounds, particularly to bislongiquinolide, were located on tarsi of the S. graminum alate morphs.

Two novel metabolites with potential antifeedant activity were isolated from cultures of the fungus Trichoderma citrinoviride strain ITEM 4484 grown in solid-state fermentation on sterile rice kernels. The producing strain was identified at species level by sequence analysis of the internal transcribed spacer regions ITS-1 and ITS-2 of the nuclear rDNA and a fragment of the translation elongation factor gene TEF-1alpha. Fractionation by column chromatography and TLC of the culture organic extract, followed by feeding preference tests on the aphid pest Schizaphis graminum (Rondani), allowed the purification of 5.8 and 8.9 mg/kg of culture of two bioactive metabolites, which were named citrantifidiene and citrantifidiol ( 1 and 2). Citrantifidiene and citrantifidiol, whose structures were determined by spectroscopic methods (NMR and MS) are a symmetrical disubstituted hexa-1,3-dienyl ester of acetic acid and a tetrasubstituted cyclohexane-1,3-diol, respectively. The pure metabolites influenced the feeding preference of S. graminum restraining individuals from feeding on wheat leaves dipped in 5% aqueous methanol solution containing 0.57 mg/mL of citrantifidiene or 0.91 mg/mL of citrantifidiol.

The pathogenicity of nine Fusarium graminearum isolates belonging to three different trichothecene-producing chemotypes, i.e. NIV + FUS, DON + 3-AcDON, and DON + 15-AcDON was tested on corn, wheat, triticale and rye. Although these compounds are known to have different toxicity, no difference in pathogenicity was observed among the mean values for chemotypes. However, within each chemotype there were isolates with different pathogenicity which was not correlated with the ability to produce trichothecenes in vitro. The pathogenicity of each isolate was almost the same for all tested cereals. The involvement of trichothecenes in pathogenesis was investigated in infected corn stalk tissues. All the expected trichothecenes were detected with the exception of NIV and FUS.

The feeding preferences of alate and apterous morphs of the aphid Schizaphis graminum (Rondani) (Homoptera: Aphididae) were evaluated using leaves treated with powdered rice cultures of four fungal isolates belonging to different species of the genus Trichoderma (Deuteromycotina: Hyphomycetes). All of the fungal isolates restrained alate morphs of S. graminum from visiting treated leaves, but only Trichoderma citrinoviride Bisset also influenced the preference of apterous morphs. Trials carried out with supernatants obtained by centrifuging aqueous suspensions of the fungal cultures showed that the feeding preference of aphids was maintained in the absence of fungal spores and mycelia, supporting the hypothesis that at least part of the fungal metabolites responsible for this effect were water-soluble compounds. Electrophysiological studies showed that the structures involved in the perception of the fungal metabolites are located on the aphid tarsomeres.

Fusapyrone (1) and deoxyfusapyrone (2) are two 3-substituted-4-hydroxy-6-alkyl-2-pyrones isolated from Fusarium semitectum that have considerable antifungal activity against molds. Because of their low zootoxicity and selective action they are potentially utilizable along with biocontrol yeasts for control of postharvest crop diseases. Seven derivatives of 1 (3 and 5-10) and one derivative of 2 (4) were obtained by chemical modifications of the glycosyl residue, the 2-pyrone ring, the aliphatic chain, or a combination thereof, and a structure-activity correlation study was carried out with regard to their zootoxicity and antifungal activity. Derivatives 7-10, as well as 1, were slightly zootoxic in Artemia salina (brine shrimp) bioassays, whereas pentaacetylation of 1 into 3, 5, and 6 resulted in a strong increase in toxicity. Compound 4, the tetraacetyl derivative of 2, was as toxic as 2. Because the structural changes of 1 that resulted in an increase of biological activity in A. salina bioassay were those that affected mainly the water solubility of the molecule, it appears that toxicity is related to hydrophobicity. Compounds 1 and 2 showed strong antifungal activity toward Botrytis cinerea, Aspergillus parasiticus, and Penicilliun brevi-compactum (minimum inhibitory concentration at 24 h = 0.78-6.25 microg/mL). Among derivatives 3-10, only compounds 7, 9, and 10 retained some activity, limited to B. cinerea and at high concentration (25-50 microg/mL). None of the compounds 1-10 inhibited the growth of the biocontrol yeasts Pichia guilliermondii and Rhodotorula glutinis at the highest concentration tested (50 microg/mL).

Trichoderma species have long been known as effective biocontrol agents of major plant diseases, and some strains have also been reported to produce toxic metabolites, such as trichothecenes. In order to clarify the molecular aspect of trichothecene biosynthesis the strain ATCC 90237 of Trichoderma harzianum, a recognised producer of the trichothecene harzianum A, was investigated. An amplification fragment of 600 bp obtained by using a degenerate PCR approach showed several degrees of identity with trichodiene synthase genes previously reported from other fungal species. Flanking genomic regions were identified through genomic walking technique. The analysis of resultant sequence allowed the identification of an open reading frame of 1226 nucleotides. The expression of the gene was confirmed by the determination of the corresponding cDNA. The comparison with the genomic sequence revealed the presence of a unique 59 bp intron that presents consensus border and internal sequence typical of filamentous fungi. The deduced putative protein of 388 amino acids displayed an identity of about 60% to the enzyme trichodiene synthase which catalyses the first step in the trichothecene pathway of other mycotoxins producing fungi. The isolation of a putative trichodiene synthase (tri5) gene required for trichothecene biosynthesis was described for the first time in Trichoderma and enabled the design of specific tri5 primers for a PCR assay that demonstrated the presence of the trichodiene synthase gene in the genomes of different Trichoderma species.

Fusapyrone (1) and deoxyfusapyrone (2), two alpha-pyrones originally isolated from rice cultures of Fusarium semitectum, were tested in several biological assays. Compounds 1 and 2 showed considerable antifungal activity against several plant pathogenic and/or mycotoxigenic filamentous fungi, although they were inactive toward yeasts isolated from plants and the Gram-positive bacterium Bacillus megaterium in disk diffusion assays. Compound 1 was consistently more active than 2. Among the tested fungi, Fusarium species were the least sensitive to the two pyrones, while Alternaria alternata, Ascochyta rabiei, Aspergillusflavus, Botrytis cinerea, Cladosporium cucumerinum, Phoma tracheiphila, and Penicillium verrucosum were the most sensitive. Compounds 1 and 2 also showed good inhibitory activity toward agents of human mycoses. Aspergilli were the most sensitive, while some species-specific variability was found among the Candida spp. In an Artemia salina larvae bioassay, 1 was not toxic at the highest concentration tested (500 microM), whereas the LC(50) of 2 was 37.1 microM (21.8 microg/mL). Neither 1 nor 2 was phytotoxic in a panel of assays that monitored plant-cell toxicity, as well as wilt-, chlorosis-, and necrosis-inducing activity. Moreover, 2 stimulated the root elongation of tomato seedlings at doses of 10 and 100 microM. In consideration of the biological activities evidenced in this study, 1 and 2 appear to be potential candidates for biotechnological applications, as well as good models for studies on mechanism(s) of action and structure-activity relationships.

We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO 2 , metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO 2 the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe 2 O 3 , MnO 2 , Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture, T. harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na 2 -bathophenanthrolinedisulfonic acid and Cu(I)-Na 2 -2,9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by Trichoderma involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.

Extracts from autoclaved maize culture of Fusarium tumidum strain R-5823 were toxic towards Artemia salina. Bioassay-guided fractionation of the organic extract led to the isolation of the toxic compound that was identified as the trichothecene toxin neosolaniol (NEOS) by H-1, C-13 nuclear magnetic resonance spectroscopy and low-resolution electronic impact mass spectrometry. The amount of NEOS produced by the strain R-5823 was 300 mg/kg maize culture, NEOS was also detected by HPLC in cultures of four out of seven additional strains of F. tumidum and Gibberella tumida with different origin, in amounts ranging from 1 to 311 mg/kg. This is the first report on the production of a trichothecene toxin by F. tumidum.

A strain of Fusarium semitectum Berk. & Rav. from maize stalk rot in southern Italy produced bioactive metabolites when cultured on autoclaved rice kernels at room temperature for 4 weeks. The organic extracts of fungal culture showed a strong antibiotic activity towards Geotrichum candidum in disk diffusion assays, but they were not toxic to Artemia salina larvae. Two antifungal metabolites were isolated and characterized by chemical and spectroscopic methods as two 3-substituted-4-hydroxy-6-alkyl-2-pyrones, in particular, the 3-(4-deoxy-β-xylo-hexopyranosyl)-4-hydroxy-6-[-hydroxy-7-hydroxymethyl-1,1,5,9,11-pentamethyl-3,5,8-heptadecatrienyl]-2H-pyran-2-one and its 6-[2-hydroxy-1,1,5,7,9,11-hexamethyl] analog, which were named fusapyrone and deoxyfusapyrone, respectively. © 1994 wiley-Liss, Inc.