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Aspergillus flavus may colonize hazelnuts and produce aflatoxins in the field and during storage. The main purpose of this study was to investigate the influence of drying temperature and exposure times on the viability of A. flavus and its ability to produce aflatoxins during the drying process and storage. Hazelnuts were inoculated with A. flavus and dried at different temperatures to reach 6% moisture content and a water activity (aw) of 0.71, a commercial requirement to avoid fungal development and aflatoxin contamination. Hazelnuts were dried at 30, 35, 40, 45, and 50°C and subsequently stored at 25°C for 14 days. After drying at 30, 35, and 40°C, increased amounts of A. flavus were evident, with the highest concentration occurring after drying at 35°C ([6.1 ± 2.4] × 106A. flavus CFU/g). At these temperatures, aflatoxins were detected only at 30 and 35°C. Aflatoxins, however, were present at higher levels after drying at 30°C, with concentrations of 1.93 ± 0.77 μg/g for aflatoxin B1 (AFB1) and 0.11 ± 0.04 μg/g for aflatoxin B2 (AFB2). After 14 days of storage, the highest A. flavus concentration and the highest levels of mycotoxins were detected in samples treated at 35°C ([8.2 ± 2.1] × 107A. flavus CFU/g and 9.30 ± 1.58 μg/g and 0.89 ± 0.08 μg/g for AFB1 and AFB2, respectively). In hazelnuts dried at 45 or 50°C, no aflatoxins were found either after drying or storage, and a reduction of A. flavus viable conidia was observed, suggesting that a shorter and warmer drying is essential to guarantee nut safety. The lowest temperature that guarantees the lack of aflatoxins should be selected to maintain the organoleptic quality of hazelnuts. Therefore, 45°C should be the recommended drying temperature to limit A. flavus growth and aflatoxin contamination on hazelnuts. HIGHLIGHTS

A collection of 124 isolates of Penicillium spp. was created by monitoring fresh chestnuts, dried chestnuts, chestnut granulates, chestnut flour and indoor chestnut mills. Sequencing of the ITS region, β-tubulin and calmodulin, macro-morphology and secondary metabolite production made it possible to determine 20 species of Penicillium. P. bialowiezense was dominant in the fresh chestnuts, while P. crustosum was more frequent in the other sources. A pathogenicity test on chestnut showed that around 70% of the isolates were virulent. P. corylophilum and P. yezoense were not pathogenic, while the other 18 species had at least one virulent isolate. P. expansum and P. crustosum were the most virulent. The isolates were characterized to establish their ability to produce 14 toxic metabolites in vivo: 59% were able to produce at least one mycotoxin. P. expansum was able to produce patulin, chaetoglobosin A and roquefortine, while P. bialowiezense produced C. Mycophenolic acid. Cyclopenins and viridicatins were produced by most of the P. crustosum, P. polonicum, P. solitum and P. discolour isolates. Some of the P. crustosum isolates were also able to produce roquefortine C or penitrem A. Information about the occurrence of Penicillium spp. and their mycotoxins will help producers to set up management procedures that can help to control the fungal growth and the mycotoxin production of chestnuts.

Bakanae, one of the most important diseases of rice, is caused by the fungal pathogen Fusarium fujikuroi. The elongation of internodes is the most common symptom induced by the pathogen, and it is related to the production of gibberellins. Despite this, the pathogenicity mechanism of F. fujikuroi is still not completely clear, and there are some strains inducing stunting instead of elongation. Even if there are relatively many genomes of F. fujikuroi strains available in online databases, none of them belongs to an isolate of proven non-virulence, and therefore there has been no comparative genomics study conducted between virulent and non-virulent strains. In the present work, the genomes of non-virulent strain SG4 and scarcely virulent strain C2S were compared to the ones of 12 available virulent isolates. Genes present in the majority of available virulent strains, but not in the non-virulent one, underwent functional annotation with multiple tools, and their expression level during rice infection was checked using pre-existing data. Nine genes putatively related to pathogenicity in F. fujikuroi were identified throughout comparative and functional analyses. Among these, many are involved in the degradation of plant cell wall, which is poorly studied in F. fujikuroi-rice interactions. Three of them were validated through qPCR, showing higher expression in the virulent strain and low to no expression in the low virulent and non virulent strains during rice infection. This work helps to clarify the mechanisms of pathogenicity of F. fujikuroi on rice.

Pistachio (Pistacia vera) is an important nut for its economic, nutritional and health aspects but it can be contaminated by aflatoxigenic fungi in the field and during storage. Biological control could be considered as an alternative to chemical treatment. In this study, we evaluated the antifungal and anti-mycotoxigenic capability of two Bacillus spp. both in vitro and on pistachio kernels. In in vitro conditions, both strains were able to reduce the mycelial growth and they were able to degrade the four aflatoxins during the first three days after inoculation. AFG1 and AFG2 were rapidly degraded within two days of incubation with the bacterial strains. No aflatoxin was found in the bacterial cell walls, permitting exclusion of mycotoxin adsorption and hypothesis of an in vitro biodegradation as a mode of action. The cultivar of pistachio most susceptible to fungal colonization was ‘Ahmad-Aghaei’, selected among four main Iranian cultivars. A. parasiticus was able to grow and produce aflatoxins on pistachios, but at longer inoculation periods, a natural decrease of aflatoxins was registered. Both strains were able to reduce the fungal incidence and number of spores on pistachio with a stronger effect during the first 5 dpi. The effect on aflatoxin content in vivo was less pronounced than in vitro, with a maximum effect at 8 dpi. At longer times, there was a contrasting effect due to the lower activity of Bacillus spp. in stationary phase and higher growth of Aspergillus species. This consideration could explain the lack of aflatoxin reduction at 12 dpi. Both bacterial strains showed good antifungal activity and aflatoxin reduction in in vitro conditions and on pistachio kernels. Altogether, these results indicate that Bacillus species could be considered as potential biocontrol agents to combat toxigenic fungal growth and subsequent aflatoxin contamination of nuts and agricultural crops in practice.

Experimental trials have been carried out in order to evaluate the efficacy of preventative treatments based on plant defense activator products, biocontrol agents, a microbial complex with arbuscular mycorrhizal fungi, and Brassica carinata pellets against Fusarium oxysporum f. sp. lactucae race 1 on lettuce and Fusarium oxysporum f. sp. raphani on cultivated rocket under greenhouse conditions. These products were compared with fungicides known for their ability to induce host resistance (phosethyl-Al and acibenzolar-S-methyl), and with azoxystrobin. Three and four applications of the tested products were carried out on lettuce and rocket seedlings grown in nursery conditions. Treated and untreated plants were transplanted into soil infested with Fusarium wilt agents to obtain an average disease severity (DS) of 65.6–69.2 and of 56.9–62.1 on the untreated lettuce and rocket plants, respectively. The best Fusarium wilt biocontrol was obtained after four applications of Bacillus subtilis Qst713 and with the Glomas microbial complex (42 and 46.7%, efficacy, respectively). B. carinata pellets provided a consistent control when applied 14 days before the rocket and lettuce were transplanted into the infested soil. Acibenzolar-S-methyl, applied at 0.025 g/Liter, showed a DS reduction in F. oxysporum f. sp. lactucae from 36 to 61% and of F. oxysporum f. sp. raphani from 54 to 73%, thus showing statistically similar results to those of azoxystrobin, which was used as a reference (DS reduction from 59 to 65%). Although the Fusarium wilt control provided by such products was not complete in the present experimental conditions, these products can be considered interesting components for an integrated pest management of the Fusarium wilt of leafy vegetables, starting from nursey applications. Moreover, the tested BCAs could become potentially useful, especially for plant monocultures. This study has been produced new information on the effects of potassium phosphite, applied at the nursery level, on reducing lettuce and rocket fusarium wilt. An average efficacy of 69.5% was observed for lettuce, while an average efficacy of 65.2% was observed for cultivated rocket. The good fungicidal activity of the phosphite-based product, coupled with the positive effect on plant biomass, is of special interest.

Kiwifruits produced from plants infected by Pseudomonas syringae pv actinidiae ( Psa ), the agent of bacterial canker, do not show visible disease symptoms and can be commercialized. Firmness, total soluble solids (TSS), and titratable acidity (TA) of Actinidia deliciosa ‘Hayward’ fruit, infected or not by Psa, were quantified, for two years, at harvest, at 90 days storage, and during shelf life. Analysis of dry matter (DM) and calcium at harvest, and postharvest rots, caused by Botrytis cinerea , during shelf life were also assessed. DM was higher and calcium content was lower in the kiwifruit from orchards infected by bacterial canker. During storage, firmness decreased and TSS increased rapidly in fruit from infected orchards. These parameters, together with the lower TA, favoured the higher susceptibility to grey mould rots. Besides the incidence of bacterial canker, climate conditions throughout the cropping seasons greatly affected the postharvest quality of fruit. Also the storage conditions modified the fruit quality: controlled atmosphere (CA) and 1-MCP treatment delayed fruit softening compared to normal atmosphere. TSS on fruit from healthy orchards was not influenced by the storage conditions. Fruit from Psa diseased orchards showed significantly lower TSS in CA storage and after 1-MCP treatment, compared with normal atmosphere storage. In this work, we showed that the presence of bacterial canker affects postharvest quality, shelf life, and susceptibility to postharvest rots of fruit.

The downy mildew of basil, which is caused by Peronospora belbahrii, is one of the most devastating basil diseases throughout the world, and management programmes with a limited use of fungicides are necessary to control it. In order to develop efficient integrated disease management programmes, the efficacy of alternative products (calcium oxide, zeolite, orange oil with ascorbic acid, potassium phosphites and bicarbonate), applied in different spray programmes in trials carried out under artificial and natural infestations with the pathogen, has been tested. Various combinations of the treatments were chosen, on the basis of the efficacy of the single products, for further testing in experiments carried out in the presence of natural pathogen infestation and under a reduced crop density. The effect of the tested programmes on downy mildew severity (% of affected leaf area), disease development (AUDPC) and yield (fresh weight of plants) was evaluated. Calcium oxide, K-phosphite and orange oil with ascorbic acid generally provided the most consistent disease control (58%, 40% and 26% efficacy, respectively), when used alone under severe disease pressure, compared to the inoculated control. Spray programmes based on calcium oxide, applied 2 to 4 times, in succession to one application of azoxystrobin, and under a reduced crop density, were the most efficient treatment in the presence of a natural infestation by the pathogen; this IPM programme reduced downy severity as much as 80%, providing the same effect as a conventional chemical programme based on the alternation of azoxystrobin, fluopicolide + propamocarb and mandipropamid.

A new leaf spot of wild rocket (Diplotaxis tenuifolia), caused by the seed transmitted pathogen Plectosphaerella cucumerina, is causing severe losses on this crop. Disease management is complicated, as few chemicals are registered for use on rocket. Four experimental trials using fungicides, resistance inducers, and a biocontrol agent against P. cucumerina were carried out on two cultivars of wild rocket, under greenhouse conditions, in order to evaluate the efficacy of the different treatments. In the presence of a disease severity ranging from 5.8 to 26.6 in the control plots of wild rocket (cvs Grazia and Rucola selvatica), one foliar application of azoxystrobin, boscalid alone or combined with pyraclostrobin provided a significant reduction in disease severity, compared with the untreated control, at values ranging between 48.5% and 84.8%. The phosphites-based products and acibenzolar-S-methyl provided the most constant results when applied in three treatments, with a disease severity reduction ranging between 34%–82% and 45%–70.3%, respectively. The highest disease reduction in disease severity was observed by using two sprays of mandipropamid at 18.7 mL/100 L with an efficacy ranging between 64% and 92%. Among the copper-based products, the most effective treatments compared with the untreated control plants were the copper hydroxide and terpenic alcohols. Thiram provided a significant reduction in disease severity compared with the untreated control plants at values ranging from 53% to 78%; however, the plants generally did not improve in biomass compared with the untreated control. The most effective treatment in terms of disease severity reduction did not always provide a better response in plant biomass. Streptomyces griseoviridis provided only a partial, but consistent, reduction of leaf spot on wild rocket when applied in three treatments at seven day-intervals, with results statistically different from those observed in the untreated control plants. Considering the difficulty in reaching a complete disease management level against P. cucumerina, an integrated approach should be considered.

Metschnikowia fructicola strain AP47 is a yeast antagonist against postharvest pathogens of fruits. The yeast was able to produce chitinase enzymes in the presence of pathogen cell wall. A novel chitinase gene MfChi (GenBank accession number HQ113461 ) was amplified from the genomic DNA of Metschnikowia fructicola AP47. Sequence analysis showed lack of introns, an open reading frame (ORF) of 1098 bp encoding a 365 amino acid protein with a calculated molecular weight of 40.9 kDa and a predicted pI of 5.27. MfChi was highly induced in Metschnikowia fructicola after interaction with Monilinia fructicola cell wall, suggesting a primary role of MfChi chitinase in the antagonistic activity of the yeast. The MfChi gene overexpressed in the heterologous expression system of Pichia pastoris KM71 and the recombinant chitinase showed high endochitinase activity towards 4-Nitrophenyl β- d -N,N′,N″-triacetylchitotriose substrate. The antifungal activity of the recombinant chitinase was investigated against Monilinia fructicola and Monilinia laxa in vitro and on peaches. The chitinase significantly controlled the spore germination and the germ tube length of the tested pathogens in PDB medium and the mycelium diameter in PDA. The enzyme, when applied on peaches cv. Redhaven, successfully reduced brown rot severity. This work shows that the chitinase MfChi could be developed as a postharvest treatment with antimicrobial activity for fruit undergoing a short shelf life, and confirms that P. pastoris KM71 is a suitable microorganism for cost-effective large-scale production of recombinant chitinases.

Bakanae disease, caused by Fusarium fujikuroi , is the most important seedborne disease of rice. Biological control and physical treatments can be effective tools to control seedborne diseases. Sixty-two isolates of yeasts and yeast-like fungi were obtained from different rice seeds. Four yeast isolates were selected in dual culture assays for mycelial growth inhibition, and in seed tests for reduction of infection rate. The isolates R23 and R26 were identified as Metschnikowia pulcherrima , the isolate R9 as Pichia guilliermondii , and the isolate SB1 as Sporidiobolus pararoseus . Rice seeds treated with P. guilliermondii R9, M. pulcherrima R23 and R26 significantly reduced the infection rate of F. fujikuroi, compared to some commercial biofungicides. The four selected yeasts reduced the bakanae disease severity in rice plants grown in greenhouse trials. Antagonist seed dressing resulted in reduction of the disease index from 93.0% in the untreated control to 20.0% in P. guilliermondii R9 treated seeds, and to 28.5% in M. pulcherrima R23 treated seeds. Selected antagonists were also used in combination with thermotherapy, which contributed to increase their efficacy. Thus, P. guilliermondii R9 and M. pulcherrima R23 combined with thermotherapy at 60 °C for 10 min decreased the bakanae disease index below 5%, and improved the seed germination rate compared to the single treatments, showing a seed priming effect. This is the first report about the use of antagonistic yeasts for seed dressing of rice to control F. fujikuroi on rice seeds, and biological treatment may be improved through combination with thermotherapy.

A total of 130 spice samples coming from India, China, South America, USA, Northern Africa, Europe and Sub-Saharan Africa were collected in different stores of Northern Italy. They were analysed for aflatoxins (AFs: AFB1, AFB2, AFG1, AFG2) and ochratoxin A (OTA) content by liquid chromatography with mass spectroscopy and positive electrospray ionization (LC/ESI-MS/MS), and HPLC with fluorescence detector (FLD), respectively. The analysis showed that 20 (15.4%) and 31 (23.8%) out of 130 samples were contaminated with AFs and OTA, respectively. A low level of total AFs contamination was found in the positive samples, the average concentration was 0.64 ng g−1, far below the maximum threshold admitted by the European legislation (5 ng g−1 for AFB1, and 10 ng g−1 for total aflatoxins (AFB1, AFB2, AFG1 and AFG2). A higher incidence of OTA was found in chili (60.0%) more than in pepper (13.3%), ranging from 2.16 to 16.35 ng g−1, and from 1.61 to 15.85 ng g−1, respectively. Moreover, three spice samples (2.3%) contaminated by OTA trespassed the threshold admitted by the European Regulation (EC, 2010). The co-occurrence of OTA and AFs in spices was detected in 6 out of 130 samples (4.6%), ranging from 1.61 to 15.85 ng g−1 and from 0.57 to 3.19 ng g−1, respectively.

A new method for the detection of alternariol (AOH), alternariol monomethyl ether (AME), altenuene (ALT), tentoxin (TEN), and tenuazonic acid (TeA), five alternaria toxins (ATs) was developed by liquid chromatography-triple quadrupole mass spectrometry equipped with atmospheric pressure chemical ionisation (APCI). A single extraction was used to recover the five ATs by apple juices, beers, tomato sauces, olives and dried basil. Different Solid Phase Extractions (SPE) and clean-up were selected to optimise the purification step for each food matrix. Limits of detection and quantification were, respectively, in the range 0.16-12.31 and 0.54-41.04 ng g(-1). Recovery rates were generally above 70%, except for dried basil and olives. Thirty out of 70 samples analysed (7 apple juices, 14 beers and 9 tomato sauces) resulted positive to at least one alternaria toxin investigated. AOH was the most common AT (14 samples), followed by ALT (10 samples). The highest concentration of ATs was found in commercial apple juices (35.33 ng g(-1)).

Three Fusarium species associated with bakanae disease of rice (Fusarium fujikuroi, Fusarium proliferatum, and Fusarium verticillioides) were investigated for their ability to produce fumonisins (FB1 and FB2) under different light conditions, and for pathogenicity. Compared to darkness, the conditions that highly stimulated fumonisin production were yellow and green light in F. verticillioides strains; white and blue light, and light/dark alternation in F. fujikuroi and F. proliferatum strains. In general, all light conditions positively influenced fumonisin production with respect to the dark. Expression of the FUM1 gene, which is necessary for the initiation of fumonisin production, was in accordance with the fumonisin biosynthetic profile. High and low fumonisin-producing F. fujikuroi strains showed typical symptoms of bakanae disease, abundant fumonisin-producing F. verticillioides strains exhibited chlorosis and stunting of rice plants, while fumonisin-producing F. proliferatum strains were asymptomatic on rice. We report that F. fujikuroi might be an abundant fumonisin producer with levels comparable to that of F. verticillioides and F. proliferatum, highlighting the need of deeper mycotoxicological analyses on rice isolates of F. fujikuroi. Our results showed for the first time the influence of light on fumonisin production in isolates of F. fujikuroi, F. proliferatum, and F. verticillioides from rice.

The strain M8 of Pichia guillermondii isolated from the carbosphere of apples (cv. Golden Delicious) showed a high efficacy in controlling grey mold, caused by Botrytis cinerea, on apples under semi-commercial conditions. Moreover, Pichia guilliermondii M8 produced high amounts of active exo-1,3-β-glucanase in Lilly-Barnett minimal salt medium with different carbon sources, which greatly inhibited B. cinerea in vitro and in vivo tests. Therefore, an exo-1,3-β-glucanase gene, named as PgExg1 (GenBank accession number HQ113463 ) was cloned from the genomic DNA of the strain M8 by genome walking. The sequencing and the nucleotide BLAST analysis indicates that no introns are present inside the gene, which was confirmed by amplifying the full gene from complementary DNA (cDNA) of the yeast. An open reading frame of 1224 bp encoding a 408-amino acid (aa) protein with a calculated molecular weight (Mr) of 46.9 kDa and an isoelectric point (pI) of 4.5 was characterized. Protein BLAST and phylogenetic tree analysis of the deduced amino acid sequences from the PgExg1 gene suggested that the glucanase produced by PgExg1 gene belongs to the Glycoside Hydrolase Family 5. Expression of PgExg1 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis with enzyme assay, yielded homogeneous recombinant PgExg1. At its optimal pH of 5.0 and its optimal temperature of 40 °C, the recombinant enzyme protein showed the highest activity towards laminarin, while the highest stability was obtained when the enzyme was stored at pH of 7.0 and temperature of 4 °C.

The efficacy of Pichia guilliermondii strain M8 against Botrytis cinerea on apples was evaluated under storage conditions, and its possible modes of action were investigated both in vitro and in vivo experiments. After storage at 1 °C for 120 days, M8 reduced grey mold incidence from 45.3% (control) to 20.0%. In apple juice medium (AJM) and in wound-inoculated apples, M8 at 109 and 108 cells ml−1 inhibited the spore germination of B. cinerea and the grey mold development. When co-culturing B. cinerea in vitro or in vivo in the presence of the yeast, neither inactivated cells nor culture filtrate of the yeast had any effect on spore germination or germ tube elongation. In AJM, the spore germination was significantly recovered by the addition of 1% glucose, sucrose and fructose, or 0.5% and 1% of (NH4)2SO4, phenylalanine and asparagine. When the pathogen and the yeast were co-incubated in apple wounds with addition of the same nutrients, the inhibition of rots was significantly reduced by the supplemental nutrients. Light microscopy revealed that the yeast strongly adhered to the hyphae and spores of B. cinerea. M8 produced hydrolytic enzymes, including β-1,3-glucanase and chitinases in minimal salt media with different carbon sources. Pretreatment with M8 at 108 cells ml−1 followed by washing, significantly reduced grey mold lesions, suggesting an induction of defense responses. Direct attachment, competition for nitrogen and carbon sources, secretion of hydrolytic enzymes and induction of host resistance play a role in the biocontrol mechanism of P. guilliermondii M8 against B. cinerea.

Six antagonistic yeast strains were screened initially for their ability to degrade Ochratoxin A (OTA) in liquid medium amended with OTA (7.5 μg ml−1) and different concentrations of yeast cells at 30 °C. Highest OTA degradation was observed when the yeast cell concentration used at 1 × 108 cells/ml ranging from 7.8 to 84%. Out of six yeast strains, three strains [Metschnikowia pulcherrima (MACH1), Pichia guilliemonodii (M8) and Rhodococcus erythropolis (AR14)] were selected for further studies. In preliminary studies yeast strains were evaluated for their ability to degrade OTA at different temperature regimes (10, 15, 20, 25, 30 and 35 °C) and found that 30 °C was the optimum temperature for yeast growth and highest OTA degradation ranging from 10.4 to 83.5%. These three strains were further evaluated to test their potential on OTA degradation using different concentrations of OTA (5 and 10 μg ml−1) only at 30 °C. Three strains showed more or less same results with that of 7.5 μg ml−1 concentration ranging from 7.5 to 81%. The three yeast antagonists were further tested to confirm either degradation or cell wall adsorption of OTA at different time intervals. Among the strains, MACH1 effectively degraded the OTA (>80%) at 30 °C after 15 days incubation compared to other strains tested and few amounts of OTA adsorption was observed in the yeast cell wall. LC–MS studies revealed that no by-product like α-OT or Phenylalanine was found during the degradation process. Therefore, further studies are needed to understand the mechanism of action of these yeast strains during OTA degradation.

Four experimental trials were carried out in northern Italy under greenhouse conditions against bacterial leaf spot of tomato, incited by Pseudomonas syringae pv. syringae , a disease recently observed in the country. The efficacy of spray programmes, based on the use of several copper-based compounds, acibenzolar-S-methyl, fosetyl Al and two biological control agents, Bacillus subtilis strain QST 713 and Pseudomonas chlororaphis MA 342, was tested. The best results were obtained with the alternation of copper oxychloride, used alone or in mixture with acibenzolar-S-methyl, with acibenzolar-S-methyl, applied for a total of two sprays. Acibenzolar-S-methyl applied alone was also effective. However, due to its possible slight phytotoxic effect, observed in our case in one trial, strategies relying on a limited number of its application are preferred. The efficacy of acibenzolar-S-methyl is higher under lower inoculum conditions. Among the tested copper-based fungicides, copper oxychloride provided the best and most consistent results, with no or limited phytotoxicity. Other copper compounds were much less effective and sometimes caused a reduction in plant height. The disease suppression achieved with B. subtilis strain QST 713 was only partial, while P. chlororaphis was not effective. The advantages of programmes based on rotation of copper compounds and acibenzolar-S-methyl are discussed.

This work reports the occurrence of suppressiveness to Fusarium oxysporum f. sp. radicis lycopersici (FORL) on recycled perlite and perlite–peat mix from closed and open soilless systems. Nine soilless systems were sampled from three different sites in Northern and Southern Italy and different parameters, including sampling site, growing period before sampling, electric conductivity of the nutrient solution, tomato cultivar, and irrigation system were considered. The effects on seed germination and FORL incidence on tomato seedlings were finally assessed with or without additional artificial inoculation of the pathogen and with or without autoclaving the samples prior to inoculation. Suppression of FORL was experimentally evaluated with a technique already adopted for rockwool. Results collected on perlite and perlite–peat confirmed the possibility to reduce FORL severity on recycled substrates. Only the composition of the substrate (perlite, perlite–peat mix) and the disinfestation did affect the incidence of FORL. Suppression of FORL was observed in not disinfected recycled perlite–peat substrates while a reduction of FORL incidence was also been recorded in disinfected and recycled perlite. Disease incidence decreased from an average ranging from 44.4% to 61.9% in new perlite to 2.5–36.3% in recycled one. Similarly disease incidence decreased from an average ranging from 35.9% to 75.2% in new perlite–peat mix to 0.4–26.4% in recycled perlite–peat mix. In conclusion the recorded data suggest the possibility to consider FORL suppression as a predictable phenomenon when recycled substrates (perlite, perlite–peat mix) are adopted.

Metschnikowia pulcherrima occurs naturally on fruits, buds and floral parts of apple trees. Some strains are effective as biocontrol agents against postharvest decay of apples and other fruits. The usefulness of the amplified fragment length polymorphism (AFLP) technique was evaluated for the genetic analysis of 26 strains of M. pulcherrima, isolated from different sources in different geographical regions. With six AFLP primer pairs, 729 polymorphic bands were scored. The technique showed a high discriminatory power. Genetic relationships between strains were also estimated using AFLP. All the isolates from the carposphere of apple, previously tested as biocontrol agents, were grouped in a single cluster with a high bootstrap value (97), indicating robustness and reproducibility. AFLP patterns could clearly distinguish the different strains and research is in progress to use some putative specific bands for single tag sequence (STS) conversion to develop isolate-specific markers.

A survey on the occurrence of patulin was conducted during 2005 on commercial pure apple juices (53 samples) and mixed apple juices (82 samples) marketed in Italy. The current study was undertaken to investigate the possible influence of the agro-food production process employed (conventional or organic), of the fruit percentage in the commercial product (higher or lower than 50%) and of the type of apple juice (clear or cloudy) on the occurrence and level of patulin contamination. Patulin could be quantified in 34.8% of the samples ranging from 1.58 to 55.41 μg kg −1 . With the exception of one sample, the level of patulin was lower than 50 μg kg −1 , the maximum permitted threshold in fruit juices according to the European legislation. Mean levels of patulin were significantly lower in mixed apple juices (4.54 μg kg −1 ) than in pure apple juices (9.32 μg kg −1 ). Levels of patulin contamination were comparable in clear and cloudy juices. A similar incidence of positive samples was found in conventional and organic apple based juices, and the magnitude between the mean contamination levels, although higher in organic (10.92 μg kg −1 ) than in conventional juices (4.77 μg kg −1 ), was not statistically significant ( p = 0.771; Mann–Whitney test). The magnitude between the means of patulin contamination in juices containing more than 50% fruit (11.26 μg kg −1 ) and in juices with 50% or less fruit (3.35 μg kg −1 ) was statistically significant ( p = 0.016; Mann–Whitney test).

A mixture of 1,3-dicloropropene 60.5% w/w and chloropicrin 33.3% w/w (Telone C35 EC) may be registered in Italy for soil drip fumigation. Five experiments on greenhouse tomatoes in Northern, Central and Southern Italy compared the effectiveness of this mixture in comparison with methyl bromide to find the optimum application rate in soils infested by Fusarium oxysporum f.sp. lycopersici, F. oxysporum f.sp. radicis lycopersici, Sclerotium rolfsii, Meloidogyne javanica and M. incognita.Its efficacy against F. oxysporum f.sp. radicis lycopersici and M. incognita was confirmed when applied to soils at 100, 200, 300 and 400 l/ha (132.4, 268.4, 402.6 and 536.8 kg/ha) under gas-tight films with 15–45 mm of application water (900–1200 mg Telone C35 EC/l). In sandy soils, with slight F. radicis lycopersici infections and with heavy nematode (M. incognita) attacks, the mixture, drip applied at 900 mg/l during late summer (fumigation: late summer; transplant: late-summer/autumn; last harvest: early spring), performed well up to 132.4 kg/ha (100 l/ha).In sandy loam soils with slight F. radicis lycopersici infections and severe infections of F. lycopersici and galling nematodes (M. javanica), 268.4 kg/ha (200 l/ha) of the mixture applied at 900 mg/l as a drip provided yields similar to those of methyl bromide treated plots both in spring and summer cycles. In sandy loam soils, the diseases (F. lycopersici, F. radicis lycopersici) were controlled at rates 268.4 kg/ha (containing 90 kg/ha of chloropicrin), but the mixture was ineffective against Sclerotium rolfsii occasionally observed in sandy loam soils. In both sandy and sandy loam soils, no significant relationships were found between the rates of mixture applied (132.4, 268.4, 402.6 and 536.8 kg/ha) and the degree of nematode infestation.

Technical, economical and environmental factors are forcing to adopt new sustainable methods, such as the use of microbial antagonists, for the control of soilborne pathogens. Research has mainly focused on antagonistic fungi and bacteria, often not providing consistent or satisfying results. Biocontrol agents can be combined with other chemical products, with physical methods (solarization or steam sterilization) or agronomical practices, such as enhancement of suppressive soils, use of amendments or microbial optimization in the case of soilless systems. Different biocontrol strategies should be developed for different pathogens. The use of microorganisms can play an important role in a more complex vision of crop protection, as a key element of IPM programs. Mixtures of antagonists with complementary activities could be developed, but the need for multiple registration is critical. Genetic manipulation could result in new biocontrol strains with increased production of toxic compounds or lytic enzymes, improved space or nutrient competence, wider host range or enhanced tolerance to abiotic stresses. The potential risks related to the environmental release of genetically modified microorganisms must be carefully assessed. Genes and enzymes involved in the biocontrol mechanism could be applied directly or transferred to crops. Finally, the production system, formulation and methods of application (soil and seed treatment or microbial colonization of the hydroponic nutrient solution) are crucial to maintain and improve the efficacy of microbial antagonists.

The management of soil-borne pathogens is nowadays complicated by the increasing restrictions in the usage of fumigants. Several factors need to be considered for a sustainable use of this practice such as the susceptibility of rootstocks against soil-borne pathogens which is age-dependent and the incomplete resistance of some of the rootstocks to one or more pathogens. There is an evidence of a pathogenic variation among some isolates of Phytophthora spp. on solanaceous crops and on rootstocks and the development of new diseases or the re-emergence of already known pathogens such as Colletotrichum coccodes after the phase out of methyl bromide. The critical aspects of grafting for soil-borne pathogens management are discussed.

Botrytis cinerea is one of the pathogens resulting in the heaviest commercial losses in ornamental cut flowers, and the severity of grey mould disease partly depends on the presence of ethylene in the storage environment. The efficacy of a β-cyclodextrin-based nanosponge 1:8 (CD-NS) – 1-methylcyclopropene (1-MCP) complex was evaluated as a novel control agent in protecting carnation (Dianthus caryophyllus L. ‘Idra di Muraglia’) cut flowers against B. cinerea infection. Two concentrations of this non-volatile 1-MCP formulation (CD-NS complex, 0.25 and 0.5 μL L−1, a.i.) were compared with commercial gaseous 1-MCP (0.25 μL L−1, a.i.), and an inoculated control. A non-inoculated control was also used to assess the natural infection level. Eleven days after inoculation, the development of grey mould on carnation was significantly reduced (59.9% of flower surface) in cut stems treated with the CD-NS complex at low dosage, compared to the high dosage of the CD-NS complex (91.5%), the commercial gaseous 1-MCP formulation (76.2%) and the inoculated control (100.0%). Endogenous ethylene production was associated with symptom development. Results showed a reduced ethylene production in 1-MCP treated flowers (0.25 μL L−1, a.i., both suspended and gaseous formulation). The CD-NS complex could therefore be an effective alternative to conventional chemicals to protect ornamental cut flowers.

Soil solarization, carried out in raised concrete benches in two subsequent years, proved very effective against Fusarium oxysporum f. sp. basilici and Rhizoctonia solani on basil. The temperatures reached in mulched benches at 5 and 15 cm depth were respectively 48 and 45 °C. The efficacy of soil solarization against Fusarium wilt was generally not improved by combining with strains of Fusarium spp. antagonistic to F. o. f. sp. basilici.

The efficacy of several antifungal compounds, chosen among salts, oils, plant extracts, and of the hyperparasitic fungus Ampelomyces quisqualis , were tested in seven glasshouse trials against powdery mildew on roses ( Sphaerotheca pannosa var. rosae ) in Sanremo (N. Italy). In the presence of a high disease incidence (55% of untreated leaves infected in six out of seven trials), all the antifungal compounds and the biocontrol agent A. quisqualis , applied individually, provided satisfactory control of powdery mildew. Most treatments were as efficient as the fungicide dodemorph, which, sprayed at weekly intervals, provided a 75% average efficacy throughout the trials. Among the test compounds, KH 2 PO 2 , at 0.5 and 1%, offered good and consistent disease control, NaHCO 3 , at 0.5%, showed a satisfactory level of activity, while at 1% it was phytotoxic. Wine vinegar, applied at 5 and 10%, gave good control, but acetic acid was phytotoxic. JMS Stylet oil, the canola oil, Synertrol and neem extract provided satisfactory disease control. Fatty acids formulated as potassium salts significantly and consistently reduced powdery mildew severity, but caused some phytotoxicity. Milsana, a concentrated extract from leaves of Reynoutria sachalinensis , only partially controlled powdery mildew. Several strategies based on the rotation of different antifungal compounds, with or without the application of dodemorph, provided very good, consistent disease control. The potential of some of the antifungal compounds and of the biocontrol fungus A. quisqualis against rose powdery mildew is discussed.

Nine experiments were carried out from 1989 to 1992 in Israel and in northern Italy in greenhouse-produced tomatoes infected with Botrytis cinerea Pers., Fr. In Israel B. cinerea affected leaves, stems and fruits (both rot and ghost spot symptoms were apparent). In Italy, the only symptom of the disease was fruit rot. Several fungicides, fungicide mixtures and spraying programmes were applied. Some trials also included a biological preparation based on Trichoderma harzianum isolates T39 and TF. Although a dicarboximide-resistant population of the pathogen existed in all greenhouses, dicarboximide fungicides (iprodione or procymidone) applied alone suppressed the disease by 40–88%, as effectively as its mixture with thiram, dichlofluanid or tebuconazole. Similar disease suppression was achieved by mixtures of tebuconazole + dichlofluanid and carbendazim + diethofencarb. Trichoderma harzianum T39 alone reduced disease by 31–82% but in more than half of the cases the reduction was non-significant. Disease control achieved by the biocontrol preparations did not differ significantly (p = 0.05) from that achieved by the chemical fungicides. Adequate control was achieved when the biocontrol and the chemical products were applied alternately although the quantity of chemical sprays was reduced by one half. The consistency among treatments in the various trials with respect to percent disease control was greater in the T. harzianum-fungicide alternation treatments, than that in the T. harzianum or the fungicide treatments applied alone.