EFFECTIVENESS OF THREE GRAS COMPOUNDS IN THE IN VITRO CONTROL OF TWO PENICILLIUM ITALICUM STRAINS.

One of the main etiological agents of postharvest citrus decay is blue mould caused by Penicillium italicum. This pathogen is currently controlled by the application of synthetic fungicides. The use of postharvest chemicals is essential if fruit is destined for storage, since postharvest life would be significantly reduced. However, concerns regarding human health and environmental risks, associated with chemicals residues in food have driven the search for alternative safe control methods. In the present study three substances as referred as GRAS (Generally Recognized as Save) compounds, previously found active against some phytopathogenic fungi, have been tested in vitro against two Penicillium italicum strains. The tested compounds were acetic acid, acetaldehyde and cinnamic aldehyde used at different concentrations (5, 10, 20 and 50 ppm) and applied as fumigation after 0, 24 and 48 h from inoculation, in order to evaluate the control of the pathogen radial growth. The effectiveness of the treatments was affected by the investigated parameters: characteristics of the GRAS, concentration and time elapsed between inoculation and treatment. The treatment performed after 0 h showed a significant difference, by the three compounds, in the control of the radial growth of the pathogen. Acetic acid revealed the main ability by completely inhibiting the pathogen development at 20 ppm, while acetaldehyde and cinnamic aldehyde were less effective in the control. The treatment applied after 24 h, in general, showed a greater capability in the control, probably related to particular pathogen sensitivity during the different vegetative stages. The treatments carried out after 48 h showed a very low effectiveness for acetaldehyde and cinnamic aldehyde in controlling the pathogen development and significantly reduced for acetic acid. Furthermore the experiment demonstrated that the pathogen strains also affected the effectiveness of the treatments.

EFFECTS OF ELECTROMAGNETICALLY SIGNALIZED MEDIA ON HOST-PATHOGEN INTERACTION.

Up to date, limited data are available about electromagnetic phase signaling effects on host-pathogen interactions during the postharvest of horticultural commodities. Inspired by the last striking works on water physics, quantum signaling through phase transfer and its impact on biological and histological structures, we studied the effect of different electromagnetic signals on pome blue mold (Penicillium expansum) pathogenesis. Tags with different electromagnetic-signals (EmS) were used to generate 3 Coherent Electro Dynamic (CED) environments. Artificially wounded 'Coscia' pears, placed onto 3 EmS tags (QF, QA and QR), were employed for the in vivo experiment. Whereas, a set of wounded-fruit placed onto an un-electromagnetic-signalized tag (QN) or kept without tag were used as blank or control, respectively. Inoculation was performed 2 or 24 h post-wounding with P. expansum conidia. The same tags placed under Petri dishes containing dot-inoculated PDA served for the in vitro experiment. Both experiments performed at 25 degrees C endured 7 days. The percentage of infected wounds was calculated and the radial growth measured in vitro. Concerning the in vivo experiment, 100% of control and blank fruit inoculated 2 h post-wounding was infected after 5 days, while, 97% after 7 days, when inoculation occurred 24 h post-wounding. Compared to control and blank, the pathogenesis in fruit placed on the EmS tags resulted inhibited, and when fruit was inoculated 2 h post-wounding, the infection degree on QF, QA and QR tags resulted 19, 52 and 64%, respectively. The degree for the same EmS tags was significantly lower when fruit was inoculated 24 h post-wounding (9, 32 and 42%, respectively). The in vitro experiment evidenced a notable inhibition of the radial growth by all EmS tags in comparison to control and blank (51 mm), while the QF tag provided the greatest inhibition (12 mm).

A novel device for the study of antimicrobial activity by vapor-contact of volatile substances on food products.

A novel device for the study of antimicrobial activity by vapour contact of volatile substances have been designed. This "big size" system, made up in inert acrylic material, is furnished with a fan and a hot plate with the aim to have a quick evaporation of volatile substances. It is able to contain fruits or other food products under controlled atmosphere and it can simulate real condition of storage or as well real condition of food pre-treatment by antimicrobial volatile substances. Such system is suitable to perform both in vitro (disk diffusion test) and in vivo (exposure and testing of food products) experiments. To shed light on the behaviour of this chamber the concentration in the head space of several substances have been monitored by GC-MS analysis during the time. Both single (mono-terpene compounds) and mixture of terpenoids have been studied. Different behaviours have been founds depending on the starting molecules studied. Limonene, myrcene and eucalyptol, in single standard experiment, show a similar shape of head space concentration curve versus the time: the concentration increases at the beginning, then reaches a maximum and decreases until it reaches a plateau. In contrast linalool shows a head space concentration curve constant during the time, whereas mixtures of terpenes like myrcene and linalool show a concentration curve of vapour phase in agreement with Raloult's Law. The experiments carried out with Essential Oils (EOs) shows that in our system only more volatile fraction of EOs compose the vapour phase.

A synergistic effect in controlling plum postharvest diseases occurs by applying UV-C light to sodium bicarbonate treated fruit.

The effectiveness of ultraviolet-C light (UV-C; 254 nm) alone at 0, 3, 6 and 12 kJm(-2), or combined with 0.0 or 0.5% (w7v) sodium bicarbonate (SBC), to control plum (Prunus domestica cv Stanley) postharvest decay caused by Penicillium expansum L. and Botrytis cinerea was investigated. First, fruit was sanitized and in one experiment plums were artificially wound-inoculated 24 h before treatments and afterwards kept at 25 degrees C with 90% RH for 7 days. In the second experiment, treatments were applied before fruit was spray-contaminated with conidia and then stored for 4 weeks at 5 degrees C and 90% RH (storage conditions). In both experiments, the highest degree of decay caused by the two pathogens was monitored when fruit stayed untreated (control), and a negligible reduction was achieved by treating with the sole salt or with a 3 kJm(-2) UV-C light. Compared to control (89 +/- 3% decay), the treatment of wound-inoculated fruit with 6 kJm(-2) provided a 35 and 38% reduction of P. expansum and B. cinerea decay, respectively. Meanwhile, 12 kJm(-2) provided an additional decrease of 25 and 27%, respectively. In both experiments, the best control of decay was attained when treatments with SBC and UV-C light were combined and the efficacy depended upon the sequence of application. Synergistic effects were found by applying the salt before UV-C light. When 6 or 12 kJm(-2) were employed following the 2% SBC treatment, no disease symptoms developed for either pathogens in both experiments. The same combination with 3 kJm(-2) resulted in a nearly 5 fold increase of efficacy compared to the sole light treatment. The combined treatments controlled the two pathogens to valuable levels and, since no quality losses were observed during storage, they could be considered as a suitable approach to contain postharvest losses of this fruit.

Native pears of Sardinia affect Penicillium expansum pathogenesis.

Penicillium expansum causes blue mould rot, a serious post-harvest disease of pome fruits and is the main producer of the mycotoxin patulin. The occurrence of natural resistance against different hostpathogens, has been evidenced in some pear accessions of the Sardinian germoplasm. The aim of this research was to correlate P. expansum growth and patulin production on these indigenous pear accessions. In vitro and in vivo experiments were carried out with seven accessions ('Sarmentina', 'Vacchesa', 'De Puleu', 'De su Duca', 'Natalina', 'Oliena', 'Laconi 5') belonging to the CNR-ISPA ex situ collection and one national control cultivar ('Abate'). A wild type P. expansum from our collection was isolated from blue mould-decayed Sardinian pear fruit and selected for its aggressiveness and patulin production. The in vivo assay was carried out using 5 x 2 cm (Ø x thickness) sterilized fruit discs wounded and inoculated by a 10(5)UFC/mL concentration of P. expansum. Fruit discs were incubated at 23 degrees C for 7 days before analysis. The in vitro experiments, aimed at monitoring over time P. expansum mycelial growth and patulin accumulation, were performed with a standard medium (PDA) and a pear puree Agar Medium (PAM). Petri dishes with PDA and PAM were inoculated centrally with P. expansum conidia (10(5)UFC/ml) and then incubated at 23 degrees C for 7 days. Mycelial growth on Sardinian PAMs was inhibited in comparison to 'Abate' PAM and PDA. In particular, the accessions 'Sarmentina' and 'Vacchesa' showed the maximum inhibitory activity both in vitro and in vivo. Patulin production was detected by high-pressure liquid chromatography-mass spectrometry. The mycotoxin concentration in Sardinian PAMs was lower than that detected in PDA medium, pointing out a positive correlation between fungal growth inhibition and patulin production. The lowest concentration of patulin was found in 'Sarmentina' PAM. Based on these findings, some of Sardinian pear accessions seems to affect P. expansum pathogenesis and inhibit patulin production. Further researches are necessary to assess the mechanism of this biocontrol activity.

Effect of different GRAS compounds in the control of apples blue mould.

The most important pathogen for apples is Penicillium expansum that is the causal organism of blue mould. Postharvest losses are controlled with chemical fungicides such as TBZ but a growing concern for human health and a greater awareness for environmental conservation have multiplied the studies on new ecological technologies. In the search of new environment and consumer friendly technologies that can reduce toxic residues, the use of GRAS compounds represent a valid alternative to the use of synthetic postharvest fungicides. The aims of the present work were: (1) To evaluate the effectiveness of different GRAS compounds in the control of P. expansum in artificially inoculated fruit; (2) To assess the capability of injured and treated fruit with GRAS compounds, used alone or combined, to heal the wounds in order to resist to infection. Fruit was injured with a steel rod and after 1 hour was (1) Inoculated with the pathogen and after 24 hours treated or (2) Treated and after 24 hours inoculated. Treatments were performed with the following compounds: sodium bicarbonate (SBC), boric acid (BA) and calcium chloride (CC) by using a 1% solution for all of them. After 9 or 14 days fruit lesion diameters were assessed. In the trial (1) the combined treatment with BA and SBC was the most effective reducing the lesion diameter by 86.5% with respect to untreated fruit, after 9 days from infection. A good pathogen control was also obtained with BA used alone or combined with CC. When the treatment was performed before infection the best results were achieved with the combination of SBC and CC, with 87% of reduction of the lesion diameter. The addition of CC also reduced the lesion if combined with BA (66.8%). These preliminary results showed that GRAS compounds can be effective in reducing blue mold by a direct effect on the pathogen, and by modulating fruit responses enhancing host resistance.

Natural resistance against pre- and post-harvest pathogens in Sardinian pears germoplasm.

Natural resistance against Venturia pirina and Botrytis cinerea was investigated within the Sardinian pears germplasm. The natural occurrence of V. pirina was monitored at the orchard level for 5 years, while resistance against B. cinerea was evaluated by a bioassay using methanolic extracts of the fruit rind or by artificially inoculation. Methanolic extracts of the leaves were employed for the Cladosporium bioassay on TLC plates. Among the accessions, 8 resulted sensitive to V. pirina, vegetation and fruit are severely affected every year. Seven showed an intermediate behaviour and the sole vegetation was affected slightly in two out of the 5 years. Two inhibition spots, in the methanolic extract of the leaves, were clearly evidenced in 55 accessions and a positive correlation was found between the presence of these antifungal compounds in the leaves and the resistance to V. pirina in the field. The artificial inoculation of fruit with B. cinerea evidenced a great variability in resistance, with about 12 accessions that could be considered having a good resistance. The fruit rind methanolic extracts evidenced several inhibition spots in most accessions, but no correlation could be found with fruit resistance to B. cinerea. In addition, the activity of the polygalacturonase inhibiting protein (PGIP), determined with an agarose diffusion bioassay, indicate a positive correlation between the PGIP activity evidenced in the core tissue and the infection degree by B. cinerea.

Import risk analysis of fruit from Spain to Italy.

The International trade has to ensure food security and preserve both human health and the diffusion of animal and plant diseases among different countries. While organic horticultural production and trade are regulated by global guidelines, no specific restrictions relay to conventional commodities and almost free import-export occurs among UE countries. Consequently, the safety characteristics of imported fresh crops have become an important issue. On the basis of these considerations, the aim of the present study is to monitor the epiphytic microflora (particularly yeasts and fungi) carried by fresh fruit, imported from Spain to Italy. Particular attention was given to pathogenic microrganisms and to natural antagonists. In addition, the resistance to a few postharvest fungicides was determined for the isolated strains and compared to the local ones. Apples, Citrus fruits, stone fruits, and persimmon fruit were sampled at arrival of the container from Spain at the wholesale market in Sassari (Italy), this to prevent fruit contamination by local strains of microrganisms. The isolation was performed by rinsing and shaking (30 min at 100 rpm) the fruit in a beaker with 500 mL of sterile water. After concentration (7.5 mL), 100 microl of the rinse water was plated on potato dextrose agar (PDA). Isolation of pure colonies was performed by multiple streaking on plates, until unicellular cultures were obtained. Fifty three microrganisms, mainly fungi and yeasts, have been isolated and assayed in vitro and in vivo. Pathogenic behavior of isolated fungi was tested on fruits artificially wounded and only 7 strains out of 18 isolates caused decay. The resistance to different concentrations of imazalil (IMZ), tiabendazolo and azoxystrobin were tested in vitro for the pathogenic isolates. All isolates were completely inhibited with 1000 ppm IMZ evidencing that no resistant strains were present on the imported fruit. Since the baseline resistance, found for all isolates, was similar to the indigenous strains, we may conclude from this risk analysis that the import of the studied fruits produced in the 4 geographical areas of Spain does not increase the local baseline resistance.

Effect of acetic acid repeated treatments on post-harvest quality of "Taloppo" table grape.

The most important pathogen for table grapes is Botrytis cinerea which causes a rapid deterioration of fruit. Postharvest losses are controlled with SO2 fumigations carried out every 7 or 10 days, but the use of this gas is becoming more difficult to justify because of undesirable effects on the fruit and the increasing concern for human health. Acetic acid, classified as a GRAS compound, can be employed with no restriction as preservative and represents a possible substitute to sulphur dioxide. The aims of the present work were: (1) to evaluate if repeated treatments with AAC during storage preserve table grapes fruit quality; (2) to verify the effectiveness of 3 different concentrations and time intervals between each treatment and compare the effects with SO2 treatment; The amounts of AAC used in each fumigation, performed for 15 minutes, were 30, 50 and 75 microL/L, and treatments were carried out 5, 3 and 2 times respectively during storage, in order to have the same final concentration (150 microL/L). Table grapes were also fumigated with SO2. Fruit was stored for 8 weeks at 5 degrees C and 95% of RH, followed by 4 days of a simulated shelf-life (SSL) at 20 degrees C and 85% RH. At the end of experiment decay, weight loss and visual assessment were evaluated. After eight weeks of storage the incidence of grey mould, with respect to untreated fruit, was reduced in all treatments. The comparison among the different treatments did not show significant differences between the fumigations performed 3 and 2 times, with 24.9% and 27.2% of rots respectively. A better decay control was achieved with 5 fumigations carried out every 2 weeks, (18.1% of rots), while decay in fruit treated with SO2 was 26.2%. During the SSL period no particular differences were observed among all treatments. None of the treatments affected weight loss, as well as no differences were found in the score attributed for the external quality (rachis browning and berries appearance). The results showed that a good control of grey mould could be achieved on table grapes by repeated fumigations during storage. AAC could be a promising compound to be used as alternative to SO2 in keeping fruit quality.

Ultrasound application for the control of decay on apple at different stage of ripening.

Ultrasonic technology is known for many years and is used for several purposes such as sonochemistry, extraction of natural compounds, degassing of solvent, inactivation of enzymes and microorganism. In postharvest ultrasound is applied in not destructive analysis of crop for the determination of the maturation stage. Until now, however, the potential of the sonication as a physical mean for not conventional postharvest treatment of fresh fruit has been little investigated. Here we report on the results obtained with apple fruit (Malus domestica Borkh. cv San Giovanni, from Sardinian germplasm) dipped in a solution of potassium sorbate with or without sonication. Treatment was carried out with an ultrasonic processor (1500 W, 20Khz) connected to a 25 mm phi probe immersed in 10 L of deionized water placed in a steel vessel. Fruit at different ripening stages, inoculated or not inoculated with Penicillium expansum, was sonicated before or after the inoculation. Following the treatment, fruit was left to dry, put into boxes and cold-stored. Results showed that ultrasound alone enhanced the natural resistance of ripe fruit when inoculated after sonication. No effect was observed when the ultrasound application was carried out after inoculation with P. expansum on un-ripe or ripe apples. Potassium sorbate showed to be ineffective in controlling the decay regardless of the ripening stage and the time of inoculation. On the contrary, a significant enhance in decay control was observed when the application of potassium sorbate was performed in the presence of ultrasound.

Alternative methods to control postharvest decay caused by Penicillium expansum in plums (Prunus domestica L.).

In the latest years, investigation on postharvest treatments has been increasingly addressed to preserve human health and environment safeguard. Several preservative compounds, physical treatments and biological control agents to restrain postharvest pathogens on horticultural products have been widely studied. Among them potassium sorbate (KS) has been generally recognized as safe for use in foods and personal care products. It acts as microbial growth inhibitor and fungistatic agent in foods, including vegetable and fruit products. The efficacy of KS, used alone or combined with heat treatments or biocontrol agents, has been demonstrated in Citrus and stone fruits. Here we report the results of 3 experiments aimed at controlling Penicillium expansum Link decay with the use of KS on a yellow ('Shiro') and a red ('Sanguigna di Bosa II', from the Sardinian germplasm) plum cultivar. An integrated approach, combining ultrasounds (US) as a physical mean and KS solutions at different concentrations, has been employed. In the first experiment, 360 fruits were wounded twice and divided into 6 sets (6 x 60), three of which were inoculated with an isolate of P. expansum (20 microl of a 10(5) cfu x mL(-1)). Then, 180 fruit (half inoculated) were treated by pipetting into each wound 20 microl of a KS solution containing 0, 1.5 or 3% (w/v), respectively. In exp. 2, all fruit (number) was wounded and inoculated, and after 24 h treated by immersion (1 min) into solutions containing 0, 1.5 or 3% (w/v) of SK, with or without the use of US. In exp. 3, wounded fruits were treated by immersion or sonication like in exp. 2, while inoculation took place after 24 h. Then, plums were kept at 25 degrees C and 75-80% RH and the infection degree was monitored after 3 and 6 days. In both cultivars, the 1.5% KS solution significantly reduced the natural infection, while the 3% KS solution resulted effective only on the red one. Moreover, the 1.5% solution was effective in controlling decay of artificially inoculated fruit, achieving a 56% reduction compared to control. Similar results were attained in exp. 2 and 3, where the combination of salt and sonication improved the efficacy, likely by increasing the salt diffusion into the wounds.

Postharvest physio-pathological disorders in table grapes as affected by UV-C light.

To gain knowledge on the influence of postharvest treatments with ultraviolet-C light upon the keeping quality of table grapes, a trail was performed employing commercially mature 'Corina', 'Dawn Seedless', 'Centennial Seedless' and 'Gran Perlon' grape cultivars (cvs). After grading, bunches were subjected to 0.0, 0.5, 1.5 or 3 kJm(-2) and then stored at 5 degrees C and 90% relative humidity (RH) for 6 weeks followed by a 2 day shelf-life at 25 degrees C and 70% RH. A weekly inspection was performed and a visual evaluation of the appearance, treatment damage, stems browning and berry shrivelling was performed. Weight loss, decay and shatter were quantified at the end of storage and shelf-life. Regardless the cv and UV-C dose, fruit appearance was acceptable until the end of storage and shelf-life. Among the cvs, the highest score was held by 'G. Perlon'. After the fourth week of storage, the berries of 'Centennial S.' turned light brown and darkened over time when treated with 3.0 kJm(-2). Stem browning was not induced by the light treatment, but resulted cv depended and was pronounced for 'Centennial S.' and 'Dawn S.'. Berry shrivelling was insignificant, while shatter was very high in 'Corina' and did not depend upon UV-C treatment. Regarding weight loss, differences could not be attributed to the light treatment and after storage it ranged from 3 up to 5%. Decay was significantly reduced by light treatment and the efficacy increased by raising the dose. Botrytis cinerea was the main cause of decay with 'Corina' being the most jeopardized, followed by 'Dawn S.' and 'Centennial S.', whereas 'G. Perlon' resulted the less affected. In conclusion, hormetic effects of postharvest light treatment on table grapes were observed in almost all cvs with 'G. Perlon' having the best performance.

In vitro studies on the effect of some chemicals on the growth and sporification of Penicillium expansum and Botrytis cinerea.

Penicillium expansum and Botrytis cinerea are among the pathogens most frequently affecting apples and grapes after harvest, respectively. We studied the behaviour of these moulds when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO) as a alternative method to fungicides in controlling postharvest decay of horticultural products. The experiments were performed with 5 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of the two tested chemicals. Freshly prepared conidia of B. cinerea and P. expansum were sown onto the media and then kept into an incubation chamber at 21 degrees C up to 3 and 6 days, respectively. Daily, the colony forming units (cfu), the colony diameter and the degree of sporification were monitored. Compared to the control, both chemicals affected the growth rate of the two pathogens. The P. expansum and B. cinerea cfu value was not significantly inhibited but the colony diameter and the sporification degree decreased when concentration was raised. B. cinerea cultured on DMSO showed a significant drop of sporification up to the tested concentration of 10 microL/mL, and a complete inhibition of cfu when the concentration was higher than 20 microL/mL.

Influence of curing times on the effectiveness of treatments with acetic acid on the control of P. digitatum on lemons.

The restricted number of postharvest fungicides used in packing houses is leading to the selection of resistant strains of Penicillium digitatum (citrus green mould), one of the most common and serious pathogens during storage and marketing of lemons. Furthermore a growing concern for human health and a greater awareness for environmental conservation have multiplied the studies on new ecological technologies. Among the alternatives to synthetic postharvest fungicides, the use of acetic acid (classified as GRAS) together with a physical method such as curing, have led to encouraging results. In the present study is reported the combined use of curing, performed at reduced times compared to those reported to be effective, followed by acetic acid (AAC) treatments. Lemons of the variety "Limone di Massa" artificially inoculated with P. digitatum at a concentration of 10(4) spores/mL were cured for 0, 3, 6, 12 and 24 hours and then treated with three different concentrations of AAC (25, 50 and 75 microL/L) for 15 min. Fruit was then stored at 20 degrees C and 80% relative humidity (RH) for 9 days, when the number of decayed fruits was monitored. The same combined treatments were also carried out on naturally infected lemons, stored for 6 or 8 weeks at 5 degrees C and 90% RH. After 9 days of storage the lowest percentage of infected wounds, in artificially inoculated fruit, was 0% after 6 hours of curing followed by AAC fumigation performed at 50 microL/L, while lemons untreated or cured for three hours showed the worst results with 71.4 and 61.9% of rotted fruit respectively. In naturally infected lemons the best results were achieved with curing performed for 24 hours followed by AAC fumigation at 50 microL/L. In these cases the combined treatment reduced decay by the 91.0 and 66.5% after 6 or 8 weeks of storage respectively, if compared to untreated fruit. The weight loss was not affected by any of the treatments. These results show that a good control of green mould during storage could be achieved, on lemon fruit, by combining a reduced curing time of 24 hours to the effect of AAC. The best results were obtained after 6 week of storage even if a satisfactory control was observed after 8 weeks of storage.

Sequential application of NaHCO3, CaCl2 and Candida oleophila (isolate 13L) affects significantly Penicillum expansum growth and the infection degree in apples.

The employment of biocontrol agents to restrain postharvest pathogens is an encouraging approach, although, efficacy and consistency are still below those of synthetic pesticides. Up to date, the 'integrated control strategy' seems to be the most promising way to overcome this gap. Here, we report the feasibility to control postharvest decay caused by Penicillium expansum in apples by a 2 min, single or sequential, immersion in water with an antagonistic yeast (Candida oleophila, isolate '13L'), 2% NaHCO3 (SBC) or 1% CaCl2. The treatments were carried out, on appels cv 'Miali' either un-wounded, wounded or wound-pathogen inoculated and then stored at 2 degrees C for 30 d followed by a 6 d simulated marketing period at 20 degrees C or alternatively stored only for 7 d at 20 degrees C. As a general role, the best results were attained when CaCl2 was applied with the yeast or when preceded by the SBC treatment. When the wounding and inoculation took place 24 h before the treatment, the latter application sequence of the two salts was three times more effective compared to the treatment with the sole antagonist, and one time when performed 24 h after the treatment. Interestingly, apples immersed in the sole 2% SBC solution had the highest percentage of decay during storage and when inoculated before moving to the simulated marketing period at 20 degrees C.

Synergic interactions between 2-deoxy-D-glucose and Candida saitoana enhances citrus green mould control.

The activity of 2-deoxy-D-gLucose (2-DG) alone or in combination with a biocontrol yeast (Candida saitoana, strain 8C) was evaluated in vitro and in vivo against citrus green mould (Penicillium digitatum Sacc.). The in vitro assays were performed on amended potato dextrose agar (PDA) containing 0, 1.5, 3.0, 6.0, 15.0, 30.0 or 60.0 mM of 2-DG. P. digitatum conidia were sown on the amended media and growth inhibition occurred starting from 6.0 mM. A nearly total inhibition of the growth and spore germination occurred with 60.0 mM of 2-DG. The antagonist was not affected by any of the 2-DG concentrations employed and the amended plates resulted well colonized within 2 d post-treatment. In vivo assays were carried out with 'Hamlin' oranges, inoculated with P. digitatum 24 h before treating with: the antagonist; the above reported concentrations of 2-DG, or by combining the two treatments. Seven days post-treatment the inhibition activity exerted by 3.0, 6.0, 15.0, 30.0 and 60.0 mM of 2-DG combined with the yeast was 15, 37, 42, 63 and 84%, respectively. While that exerted by the antagonist was 22% and that by the different concentrations of 2-DG were 7, 11, 27, 42 and 57%, respectively. Compared to single treatments, the co-application significantly and in a synergic mode improved the control of decay. Alterations to the hyphae were observed by SEM when the pathogen was cultured on amended media and into the wounds of inoculated oranges.

In vitro studies on the effect of some chemicals on the growth and sporification of Penicillium digitatum and P. italicum.

The behaviour of Penicillium digitatum and P. italicum was investigated when subjected to different concentrations of methanol (MeOH) and dimethyl sulfoxide (DMSO). The experiments were performed in 9 cm Petri dishes containing PDA amended with 0, 5, 10, 20, 30, 40 or 50 microL/mL of each of the single or combined chemicals. Daily, the formed colonies (cfu), the colony diameter and the degree of sporification were monitored during incubation at 20 degrees C for 5 day. Additionally, the pathogen development and its performance were studied by scanning electron microscopy (SEM). According to the chemical, the mycelium growth rate was affected differently and, compared to the control, only MeOH inhibited the expansion of the colony diameter. This effect was more pronounced for P. italicum. A nearly linear drop of cfu was observed as the concentration of the two chemicals was raised, and a complete inhibition of the two pathogens was attained with 50 microL/mL MeOH. With respect to the sporification degree the two pathogens were influenced similarly, but the tested compounds had opposite effects. Indeed, with MeOH, sporification took place earlier (24-36 h postinoculation) compared to the control (60 h), while during the whole experiment, DMSO at concentrations higher than 0.5 microL/mL, drastically inhibited the sporification. SEM observations of P. digitatum growth on DMSO amended media evidenced a marked increase of mycelium branching and alterations to the conidiophore, while MeOH reduced the mycelium length and fastened the conidiophore formation. The combination of the two compounds produced a synergistic interaction reducing by 40% the concentration required to inhibit completely the germination and growth of P. digitatum.

Immersion of 'Coscia' pear fruit in water at 55 degrees C for 60 sec controls Penicillium expansum decay and delays ripening during short storage.

'Coscia' is an early ripening pear with a short postharvest Life (1 month) and chemical treatments to prevent decay are generally not undertaken. This, along with the fast deterioration under shelf-life conditions, makes it difficult to contain postharvest moulds. 1-methylcyclopropene (1-MCP) has been employed with success to delay ripening and, as a co-effect, the development of decay was contained, but this treatment is not allowed for organically grown crops. Here we report the results of an alternative approach employing immersion treatments in water at 45, 50, 55 or 60 degrees C for 0 (control), 15, 30, 45 or 60 sec. Fruit was harvested after the climacteric peak, immediately subjected to the heat treatment and stored for 2 weeks at 1 degrees C followed by a 3 days of a simulated shelf-life at 17 degrees C and 75% RH. Half of the fruit was wounded (3 x 3mm) and inoculated with Penicillium expansum (20 microL of 10(4) conidia mL(-1). Decay inhibition and fruit appearance, rated from 0 to 3 (0 = excellent; 1 = good; 2 = scarce; 3 = not marketable), were monitored and compared to the control after storage and shelf-life. All heat treatments affected the mould development when performed for 45 or 60 sec. The best result in terms of decay control and appearance after storage and shelf-life occurred when fruit was immersed at 55 degrees C for 60 sec with a decay reduction of about 85% compared to control (75% decay) and with a good appearance.

Postharvest behaviour of two Sardinian apple varieties following immersion in heated sodium bicarbonate solution.

'Miali' and 'Caddina' are apple varieties of Sardinian germplasm, mainly produced under sustainable conditions. Fruit is rarely subjected to cold storage and postharvest losses are generally high. In order to prolong the marketing period and contain postharvest decay of these local varieties, we investigated on their storage behaviour and on the efficacy of combined alternative postharvest treatments. Pre-climateric fruit was harvested and immersed for 0 (control), 15, 30, 45 or 60 sec. in water at 20, 50, 55 or 60 degrees C with or without 2% (W/V) NaHCO3 (SBC). Then, fruit was stored for 4 months at 5 degrees C and 90% RH followed by a 6 day simulated marketing period (SMP) at 10 degrees C and 75% RH. Decay was monitored at the end of storage and after the SMP, while appearance and physiological disorders were evaluated after SMP. During storage 56 and 62% of the untreated 'Caddina' and 'Miali' apples rotted, respectively. During the SMP, an additional 3% of 'Caddina' and 5% of 'Miali' was lost. Among the treatments the best decay control, for both varieties, was attained when fruit was immersed in the SBC solution at 55 degrees C for 60 sec. Compared to control, decay was reduced by 91 and 95% for 'Caddina' and 'Miali', respectively. This combination induced some rind damage, mainly on 'Caddina' fruit. Superficial scald was evident on 'Caddina' and scored as medium while, cold storage induced a significant deposition of epicuticular wax in 'Miali' fruit, affecting significantly fruit appearance. A significant reduction of decay was also achieved when fruit was immersed at 60 degrees C for 30 or 45 sec., attaining for 'Caddina' a reduction of 82 and 88% of decay, respectively. Other combinations were lesser effective or produced rind damages and most decay was caused by Penicillium expansum.

Postharvest behaviour of five Sardinian prune varieties as affected by immersion in heated sodium bicarbonate solution.

Storage behaviour of 'Core', 'Core Columbu', 'Fradis' and 'Meloni' white prunes, and a black one ('Sighera') of Sardinian germplasm were evaluated following immersion for 0 (control), 15, 30, 45 or 60 sec in water at 20, 50, 55 or 60 degrees C with or without 2% (w/v) NaHCO3 (SBC). As international varieties, fruit from one white plum ('Shiro') and one black prune ('Stanly') were subjected to the same treatments. Fruit was harvested at commercial maturity, treated and then stored for 1 month at 5 degrees C and 90% RH followed by a simulated marketing period at 20 degrees C and 80% RH for 6 days. Fruit appearance, external damage, firmness and decay percentage were monitored after storage and SMP. Treatments did not induce rind damage (browning or discoloration) to any variety. SBC at 20, 45, 50 or 55 degrees C for 15 or 30 sec was not effective in controlling decay and compared to controls no improvement was observed. Immersion for 45 or 60 sec with SBC at all temperatures improved decay control with respect to controls and best results were obtained at 50 or 55 degrees C. Immersions at 60 degrees C improved decay control, but differences were not significant compared to the control attained with solutions of SBC heated at 55 degrees C. The overall appearance of 'Core', 'Core Columbu', 'Fradis' and 'Shiro' decreased significantly after the SMP period, especially when treated at 55 or 60 degrees C for 60 sec. Fruit shrivel was the main cause of the low rating. SBC did not affect shrivel indicating that heat treatment may be the probable cause. In general, local varieties were less affected by decay than other varieties and they performed well during storage.