Davide Spadaro

State of the art and future prospects of the biological control of postharvest fruit diseases.

Synthetic fungicides are the primary means to control postharvest diseases of fruits. Biological control has emerged as one of the most promising alternatives to chemicals. During the last 20 years, several biological control agents have been widely investigated for use on different pathogens and fruit crops. Many biological control mechanisms have been suggested for use on fruit including antibiosis, parasitism, induced resistance in the host tissue and competition. With the aim of extending the use of the biofungicides, there have been many studies on the application of various combinations of control agents, and on the application integrated with chemical and physical means of protection. The formulation and application methods are key issues for the efficacy and successful outcome of the commercial product. Genetic engineering may provide a useful tool in the enhancement of the biological control efficacy. Since biofungicides are usually not as effective as pesticides, this approach should be viewed as an important component of an integrated disease management scheme given that a significant and permanent reduction of pesticide use is our goal.

The redox switch: dynamic regulation of protein function by cysteine modifications.

Reactive oxygen intermediates (ROIs) and reactive nitrogen intermediates (RNIs) have now become well established as important signalling molecules in physiological settings within microorganisms, mammals and plants. These intermediates are routinely synthesised in a highly controlled and transient fashion by NADPH-dependent enzymes, which constitute key regulators of redox signalling. Mild oxidants such as hydrogen peroxide (H(2)O(2)) and especially nitric oxide (NO) signal through chemical reactions with specific atoms of target proteins that result in covalent protein modifications. Specifically, highly reactive cysteine (Cys) residues of low pK(a) are a major site of action for these intermediates. The oxidation of target Cys residues can result in a number of distinct redox-based, post-translational modifications including S-nitrosylation, S-glutathionylation; and sulphenic acid, sulphinic acid and disulphide formation. Importantly, such modifications precisely regulate protein structure and function. Cys-based redox switches are now increasingly being found to underpin many different signalling systems and regulate physiological outputs across kingdoms.

Development of a microcantilever-based immunosensing method for mycotoxin detection

Mycotoxins, such as aflatoxins and ochratoxin A, are presently considered as the most important chronic dietary risk factor, more than food additives or pesticide residues. Therefore, the serious health and economic consequences of mycotoxin contamination have created the need for rapid, sensitive, and reliable techniques to detect such dangerous molecules within foodstuffs. We here report on the development of an innovative immunosensing method for mycotoxin detection, based on antibody-immobilized microcantilever resonators, a promising label free biosensing technique. A considerable part of the work is devoted to show the effect on microcantilever resonance frequency of the composition of the incubation buffer, as well as of the washing and drying procedure. We show the feasibility of using microcantilever resonator arrays to effectively identify total aflatoxins and ochratoxin A, at low concentrations (3 ng/mL and less than 6 ng/mL, respectively), with relatively low uncertainty (about 10%) and good reproducibility for the same target concentration. Furthermore, the developed immunosensing method shows a limited cross-reactivity to different mycotoxins, paving the way to a highly specific technique, able to identify different mycotoxins in the sample. To our knowledge, this work represents the first example in literature of successfully immunodetection of low concentrations of multiple mycotoxins by microcantilever resonator arrays.

First report of brown rot of stone fruit caused by Monilinia fructicola in Italy.

Monilinia fructicola, causal agent of brown rot, is one of the most important fungal pathogens of stone fruit. M. fructicola is a quarantined pathogen in Europe. During the summer of 2008 in 15 orchards located in Piedmont (northern Italy), 12,500 stone fruits (cherries, apricots, peaches, nectarines, and plums) were stored in cold chambers at 4 and 6°C and monitored for 8 weeks for the presence of Monilinia spp. M. fructicola was detected on 0.5% of nectarines (cvs. Sweet Red and Orion) that originated from two orchards in Lagnasco. Symptoms appeared on the fruit during storage, starting 3 weeks after harvest. Fruit rot lesions were brown, sunken, and covered with grayish tufts. The majority of infected fruit became dry and mummified. Brown rot symptoms were similar to those caused by endemic M. fructigena and M. laxa. Symptoms began with a small, circular, brown spot, and the rot spread rapidly. At the same time, numerous, small, grayish stromata developed. Finally, the whole surface of the fruit was covered by conidial tufts. Tissues were excised from diseased stone fruits and cultured on potato dextrose agar (PDA) amended with 25 μg of streptomycin per liter. The isolates produced abundant mycelium on PDA at 20 ± 2°C. Colonies were initially gray, but after sporulation, they became hazel, showing concentric rings (sporulation is sparse in M. laxa or M. fructigena). Conidia were one-celled, ellipsoid, hyaline, 15.2 × 10.1 μm, and produced in branched monilioid chains (2). Preliminary morphological identification of fungi resembling M. fructicola was confirmed by PCR using genomic DNA extracted from the mycelia of pure cultures. The DNA was amplified with a common reverse primer and three species-specific forward primers (3) obtained from a sequence characterized amplified region and a product of 535 bp, diagnostic for the species M. fructicola, was obtained. BLAST analysis of the amplified sequence (GenBank Accession No. FI569728) showed 96% similarity to the sequence of a M. fructicola isolated from Canada (GenBank Accession No. AF506700), 15% similarity to M. laxa ATCC11790 (GenBank Accession No. AF506702), and 35% similarity to a M. fructigena sequence isolated in Italy (GenBank Accession No. AF506701). Moreover, two sequences obtained through the amplification of ribosomal region ITS1-5.8S-ITS2, showing 100% similarity to the same ribosomal sequence of M. fructicola, were deposited in GenBank (Accession Nos. FJ411109 and FJ411110). The pathogen was detected on some mummified fruit from the same orchards in November of 2008. Pathogenicity was tested by spraying 103 conidia/ml on 10 surface-sterilized artificially wounded nectarines per strain of M. fructicola. After 5 days of incubation at 20 ± 2°C, typical, brown, rot symptoms developed on inoculated fruit. M. fructicola was reisolated from the inoculated fruit on PDA. Symptoms did not appear on control fruit. To our knowledge, this is the first report of M. fructicola in Italy. Its occurrence in Europe has been reported sporadically in Austria and France, and in 2006, it was detected in Hungary and Switzerland on peaches and nectarines imported from Italy and Spain (1,4). References: (1) E. Bosshard et al. Plant Dis. 90:1554, 2006. (2) R. J. W. Byrde and H. J. Willetts. The Brown Rot Fungi of Fruit: Their Biology and Control. Pergamon Press, Oxford, 1977. (3) M. J. Coté et al. Plant Dis. 88:1219, 2004. (4) M. Petròczy and L. Palkovics. Plant Dis. 90:375, 2006.

A new method for detection of five alternaria toxins in food matrices based on LC–APCI-MS

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)).

Detection of enzymatic activity and partial sequence of a chitinase gene in Metschnikowia pulcherrima strain MACH1 used as post-harvest biocontrol agent

Two antagonistic yeast strains Metschnikowia pulcherrima MACH1 and Rhodotorula sp. PW34 were tested for their efficacy against Botrytis cinerea in vitro and in vivo on apples. Metschnikowia pulcherrima strain MACH1 showed higher inhibition of B. cinerea compared to the strain PW34 in vitro on potato dextrose broth. Further, yeast strain MACH1 showed higher efficacy in reducing grey mould on apples compared to PW34 and the untreated control. In addition, partially purified extracellular proteins from strain MACH1 showed an inhibition to B. cinerea in vitro. The antagonistic yeast strains were tested for their efficacy to produce chitinases in different liquid media, including apple juice, amended with or without cell wall preparations (CWP) of B. cinerea. The study showed a higher production of chitinases from M. pulcherrima strain MACH1 when compared to PW34. Interestingly, the strain MACH1 secreted higher chitinases in the presence of cell wall fractions of B. cinerea. For this reason, the chitinase gene of strain MACH1 was amplified using PCR reactions and the nucleotide sequence data showed high homology to chitinases of other yeast strains. The results of the current study show that M. pulcherrima strain MACH1 has the ability to secrete chitinases in different liquid media including apple juice, and the enzyme could be involved in the post-harvest biological control of B. cinerea.

Efficacy of plant essential oils on postharvest control of rots caused by fungi on different stone fruits in vivo

The antifungal activity of plant essential oils was evaluated as postharvest treatment on stone fruit against brown rot and grey mold rot of stone fruit caused by Monilinia laxa and Botrytis cinerea, respectively. The essential oils from basil (Ocimum basilicum), fennel (Foeniculum sativum), lavender (Lavandula officinalis), marjoram (Origanum majorana), oregano (Origanum vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), savory (Satureja montana), thyme (Thymus vulgaris), and wild mint (Mentha arvensis) were tested at two different concentrations on apricots (cv. Kyoto and cv. Tonda di Costigliole), nectarines (cv. Big Top and cv. Nectaross) and plums (cv. Italia and cv. TC Sun). The volatile composition of the essential oils tested was determined by gas chromatography-mass spectrometry analysis. The treatments containing essential oils from oregano, savory, and thyme at 1% (vol/vol) controlled both B. cinerea and M. laxa growing on apricots cv. Tonda di Costigliole and plums cv. Italia and cv. TC Sun; however, the same treatments were phytotoxic for the carposphere of nectarines cv. Big Top and cv. Nectaross. Treatments with 10% (vol/vol) essential oils were highly phytotoxic, notwithstanding their efficacy against the pathogens tested. The essential oils containing as major components α-pinene, p-cymene, carvacrol, and thymol showed similar results on stone fruit, so their antimicrobial activity and the phytotoxicity produced could be based on the concentration of their principal compounds and their synergistic activity. The efficacy of the essential oil treatments on control of fungal pathogens in postharvest depended on the fruit cultivar, the composition and concentration of the essential oil applied, and the length of storage.

Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens.

An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach.

Jasmonic Acid, Abscisic Acid, and Salicylic Acid Are Involved in the Phytoalexin Responses of Rice to Fusarium fujikuroi, a High Gibberellin Producer Pathogen.

Fusarium fujikuroi, the causal agent of bakanae disease, is the main seedborne pathogen on rice. To understand the basis of rice resistance, a quantitative method to simultaneously detect phytohormones and phytoalexins was developed by using HPLC-MS/MS. With this method dynamic profiles and possible interactions of defense-related phytohormones and phytoalexins were investigated on two rice cultivars, inoculated or not with F. fujikuroi. In the resistant cultivar Selenio, the presence of pathogen induced high production of phytoalexins, mainly sakuranetin, and symptoms of bakanae were not observed. On the contrary, in the susceptible genotype Dorella, the pathogen induced the production of gibberellin and abscisic acid and inhibited jasmonic acid production, phytoalexins were very low, and bakanae symptoms were observed. The results suggested that a wide range of secondary metabolites are involved in plant defense against pathogens and phytoalexin synthesis could be an important factor for rice resistance against bakanae disease.

Occurrence of patulin and its dietary intake through pear, peach, and apricot juices in Italy

Pear, peach, apricot, and mixed juices (125 samples) were purchased in Italian supermarkets and organic food shops and analysed for patulin content. An overall incidence of 34.4? was observed in the fruit juices, with a mean contamination level of 3.6 µg kg–1. No one sample exceeded 50 µg kg–1, the maximum permitted limit according to European legislation. However, 19 positive samples contained more than 10 µg kg–1 of patulin, which is the maximum level permitted for baby food. The incidence of patulin contamination was significantly higher in pear juices (64.1?) than in apricot, peach, or mixed juices. Statistical analysis (p = 0.002; Kruskal–Wallis test) showed a significantly higher level of patulin in pear (5.1 µg kg–1) and mixed juices (4.9 µg kg–1) than in the other juices. A slightly higher incidence of positive samples was found in conventional juices (35.7?) compared with the organic ones (29.6?). The magnitude between the mean contamination levels was similar in conventional (3.6 µg kg–1) and organic (3.3 µg kg–1) juices (p = 0.474; Mann–Whitney U-test). Samples were further divided based on the fruit content in fruit juices, fruit nectars, and fruit drinks. In fruit nectars the incidence of positive samples (37.8?) was slightly higher than in fruit drinks (27.2?), but the mean patulin contamination was higher in fruit drinks (5.2 µg kg–1) than in fruit nectars (3.4 µg kg–1). However, the magnitude between the two means was not statistically different (p = 0.734; Mann–Whitney U-test). Italian juices had a higher incidence of patulin (35.3?), with a lower mean content (3.5 µg kg–1), compared with the incidence (22.2?) and level (4.1 µg kg–1) foreign juices commercially available in Italy. The magnitude of the means between Italian and foreign juices was not statistically different, according to the Mann–Whitney U-test (p = 0.616). The estimated intake of patulin ranged from 0.1 to 1.5 ng kg–1 body weight for the whole population and from 0.3 to 5.1 ng kg–1 body weight for the consumers only. The highest patulin intake was estimated for consumers of pear juices, followed by consumers of mixed juices. The daily intake of patulin derived from the consumption of pear juice suggests that also pear juices, though a minor patulin source, could be monitored for their patulin content in order to control the mycotoxin contamination, especially in countries, such as Italy, where pear nectars are preferred as fruit drinks.