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The toxic activity of 2-deoxy-D-glucose (2-DG) alone or combined with the biocontrol yeast Candida saitoana strain 8C was evaluated in vitro and in vivo against the postharvest fungal pathogen Penicillium digitatum. In order to assess the effect of the 2-DG on both the biocontrol yeast and fungal pathogen, in vitro tests were performed in Petri dishes containing potato dextrose agar amended with different concentrations (1.5, 3.0, 6.0, 15.0, 30.0, 60.0 mM) of the sugar. The plates were then seeded with 25 microl of a P. digitatum conidial suspension at 10(5) conidia/mL. Result of the assays showed an enhanced inhibitory activity as concentration increased from 15.0 to 60.0 mM. Corroborated by SEM observations showing a reduced growth and the appearance of damaged hyphae were found. At 60 mM of 2-DG, a total inhibition occurred while concentrations from 1.5 to 6.0 mM resulted ineffective. The same tests evidenced no adverse effects on the yeast 8C at all tested concentrations. In vivo assays were carried out on orange fruit cv 'Biondo comune', wounded in 5 sites around the calyx. Each wound (2.5 wide and 3.4 mm depth) was first filled with 25 microl of a 0, 3.0, 6.0, 15.0, 30.0 or 60.0 mM 2-DG-water solution alone or combined with the yeast 8C at 10(8) cells/mL and then a 25 microl of the P. digitatum conidial suspension was added. Each treatment consisted of 3 replicates of 8 fruit (5 wounds/fruit) for a total 120 wounds per treatment. Oranges were maintained at 20 degrees C and high RH (95-98%) for up to 5 days, during which infection was monitored and the inhibitory activity calculated. The tests in vitro evedenced a significant slowing of the pathogen growth with the highest concentrations of 2-DG (15.0, 30.0 and 60.0 mM) with respect to the control; while at lower concentrations (1.5, 3.0, 6.0 mM) the development of the fungi was not significantly reduced. C. saitoana was resistant to all the doses employed to the abovementioned compound. In vivo the yeast alone was more effective compared to the sugar alone up to 6.0 mM while, at higher concentrations an additive effect was founded.

The biocontrol yeast Rhodotorula glutinis, isolate 21A, obtained from tomato fruit was used to control Penicillium digitatum, P. italicum and Botrytis cinerea on artificially wounded citrus fruit. Orange and satsuma mandarin fruit were treated with the biocontrol yeast, inoculated with the pathogens and stored for 7 days at 23 degrees C. On orange fruit the antagonist compared to the control reduced decay by 92.2, 88.4 and 96.2% for P. digitatum, P. italicum and B. cinerea, respectively. On satsuma mandarin fruit the same pathogens were inhibited by 96.2, 91.2 and 90.0%, respectively. Scanning electron microscope observations, focusing on the antagonist-pathogen interactions, revealed a fast colonization of the growing fungal mycelia by the yeast cells, but no sign of lytic activity on hyphae was observed. Moreover, the fruit accumulated the phytoalexins scoparone and scopoletin into artificial wounds previously treated by the yeast and either inoculated or uninoculated with the pathogen. The concentration of scoparone, which showed higher accumulation in fruit tissues, varied significantly in relation to the time lag between the application of the antagonist and the inoculation with the pathogen. In particular, the concentration of scoparone 4 days after application of the sole yeast was 69.0 microg x g(-1) fresh weight (FW), 6.3 times higher than in the uninoculated wounded tissues (11.0 microg x g(-1) FW). The phytoalexin accumulation was low (13.0 microg x g(-1)FW) applying the yeast jointly with P. digitatum into wounds, while it increased consistently (74.0 microg x g(-1)FW) when the antagonist was applied 24 h before the pathogen.

The combination of the yeast Candida oleophila strain '13L' with ultraviolet-C irradiation evidenced a synergistic effect in reducing P. digitatum mould and only 11% of the artificially inoculated wounds were infected. Adversely, when the bacteria Bacillus subtilis strain 'B160' was combined with ultraviolet-C irradiation no synergistic effect was achieved. By using only yeast, bacteria or ultraviolet-C treatments the decay percentage was reduced by 79.6, 55 and 75%, respectively. The phytoalexin scoparone accumulation was high in all treatments where UV-C was applied but the highest values were found when combined with the yeast. Population growth of bacteria in vivo was halved when fruit was irradiated, whereas direct irradiation of bacteria did not affect their growth in vitro. An inhibitory effect of the phytoalexin toward the bacteria is suggested as the reason for the growth inhibition in vivo when the bacterial treatment was combined with UV-C irradiation.

The combination of the yeast Candida oleophila strain '13L' with ultraviolet-C irradiation evidenced a synergistic effect in reducing A digitatum mould and only 11% of the artificially inoculated wounds were infected. Adversely, when the bacteria Bacillus subtilis strain 'B160' was combined with ultraviolet-C irradiation no synergistic effect was achieved. By using only yeast, bacteria or ultraviolet-C treatments the decay percentage was reduced by 79.6, 55 and 75%, respectively. The phytoalexin scoparone accumulation was high in all treatments where UV-C was applied but the highest values were found when combined with the yeast. Population growth of bacteria in vivo was halved when fruit was irradiated, whereas direct irradiation of bacteria did not affect their growth in vitro. An inhibitory effect of the phytoalexin toward the bacteria is suggested as the reason for the growth inhibition in vivo when the bacterial treatment was combined with UV-C irradiation.

Health and environmental concerns have point out the need to improve or change several manufacturing steps in the food chain. In this context, particular attention should be given to the technologies involved in fruits and vegetables production. Nearly all fresh fruit and vegetables are subjected to different periods of storage and/or shelf-life before of their consumption. This implies the need to protect the commodities from microbial spoilage. Some Citrus species (e.g. lemon and grapefruit) may be stored for several months before consumption and then postharvest treatments are essential to contain green (Penicillium digitatum) and blue (P. italicum) moulds. Alternative approaches to chemicals usually have a lower efficacy in containing rots but fulfil the consumer's expectation. Among the alternative strategies, the improvement of host natural resistance is promising. In this regard, we report some results concerning the use of biotic (yeast) and abiotic agents as inducers of phytoalexin (i.e. scoparone and/or scopoletin) accumulation in Citrus rind and its importance in the control of fungal decay. In all experiments, the inducers were applied on fruits before or 24 h after inoculation with P. digitatum and the rot severity was monitored 7 days later. The accumulation of phytoalexins was monitored according to a standard methodology by HPLC. In all experiments a positive correlation was found between increase of the phytoalexin scoparone in host tissue and reduction of decay.

One of the Citrus species is “honey murcott” (Citrus reticulata cv murcott), contained 34 constituents (accounting for 97,11% of total volatiles) which were identified by GC-MS analyses. The variation of essential oil constituents stored under different temperatures has been determined to find possible optimal conditions to preserve integrity of essential oil. The cold storage trials analyses showed that a few variation in the composition of the essential oil were obtained using cyclic temperature corresponding to lot C, also it was observed that all chemical compounds are sensitive to low temperature. In lots A (constant temperature) and B (cyclic temperature) we could found an option to our purpose.

Three commercial tests were conducted in 2000-2001 in two commercial packing-houses (Muravera and Villacidro) located in Sardinia, Italy, to evaluate the efficacy of biological, chemical and integrated treatments against Penicillium digitatum and P. italicum on naturally inoculated orange fruit. Damage caused by the packing-house processing line was also assessed. Treating orange fruits with the yeast Pichia guilliermondii (strain 5A) in the processing line generally led to a significant reduction of post-harvest decay compared to the processed control, while the commercial product Aspire®, based on Candida oleophila, was ineffective in inhibiting the pathogen when applied alone. The integrated application of thiabendazole or imazalil with the biocontrol agents significantly improved the control of fruit decay. Using thiabendazole at concentrations of 0.1 and 1.2 g l-1, led to similar results in inhibiting fruit decay in two trials. Both yeasts were equally able to actively colonise the fruit during storage. Passing fruits through the packing line caused a significant increase in fruit decay.

The fungitoxic activity against Penicillium digitatum, P. italicum, Botrytis cinerea and Alternaria citri of 12 essential oils (EO) distilled from medicinal plants is reported. The results of the in vitro trials show strong fungitoxic activity of Thymus capitatus (L.) Hofmgg EO, which inhibited the growth of the 4 fungi at a concentration of 250 ppm (v/v). The other 11 essences reduced the development of the fungi from 95% to 9% at 250 ppm (v/v). The fungitoxic activity of T. capitatus EO (75, 150, and 250 ppm) on healthy orange fruits, inoculated with P. digitatum (108 conidia ml-1) by spraying and placed in 10-litre desiccators, was weak at atmospheric pressure (3-10% inhibition at all 3 concentrations), while in vacuum conditions (0.5 bar) conidial mortality on the exocarp was high (90-97% at all 3 concentrations). These data proved not to be statistically different from treatments with thiabendazole-TBZ (2000 ppm). SEM observations showed that T. capitatus EO vapours altered the morphology of P. digitatum hyphae and conidia. The gas-chromatographic analyses of thyme EO indicated that carvacrol was present at 81-83%, p-cymene at 4.5-5%, Beta-terpinene at 2.6-3.3%, caryophyllene at 1.5-1.6%, Alfa-myrcene at 1.6%, linalool at 1.1-1.2%. Carvacrol proved to be the most important fungitoxic compound among the thyme EO constituents, but, unlike thyme EO, it caused alterations to the fruit at the concentration of 75 ppm.

Lemon and Satsuma fruit were sprayed with a 100.000 spore suspention of Penicillium digitatum Sacc. and placed in 10-L jars at constant temperature (20°C). Vacuum (10-1 Bar) was created and the essential oil of Thymus capitatus was vaporized by aerosol at 5 different concentrations (50, 100, 200 and 300 microL/10L) in order to determine the effect on P. digitatum pathogenesis. Viability of the pathogen spores was checked 24 hr post-treatment by plating 100 mL of fruit rinse water on PDA and growth was monitored. In 80% of the plates with un-treated fruit-rinse water growth of the pathogen was observed, while when fruit was vacuum-treated with 300 mL/10L of the essential oil, only 10% of the plates evidenced pathogen growth.

Among the alternatives to synthetic postharvest fungicides encouraging results have been reported with biocontrol agents, and on Citrus fruits, their efficacy was improved when co-applied with GRAS compounds or with physical means. Still, the reason for this increased efficacy has not been explained and therefore a study was performed using orange fruit (Citrus sinensis Osbec. cv 'Washington navel') as host, P. digitatum as the pathogen, a yeast (Pichia guiliermondii, isolate 5A) as the biocontrol agent, white 2% Na2CO3 (SC) and 1% CaCl2 were employed as GRAS compounds. When treatments were combined salts were applied sequentially, and SC preceded CaCl2 followed by the yeast. As a result of large scale trait with inoculated and un-inoculated fruit a clear beneficial interaction occurred when treatments were combined. SC exerted a direct fungistatic activity and an indirect one by inducing scoparone in host tissue. Also the isolate A5 induced the phytoalexin accumulation and when combined with SC a greater accumulation occurred within the first 7 days post-treatment. The application of CaCl2 alone had no effect on pathogenesis, while when combined with SC or with the yeast, decay was towered. The yeast growth on an amended medium was negatively affected by the addition of SC; while in vivo this effect was missing. The antagonist growth in vivo was enhanced when applied together with 1% CaCl2 also when applied with SC. The results reported improve our knowledge on the complex interactions among host, pathogen and the antagonist as affected by SC and CaCl2.

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.

Health and environmental concerns have point out the need to improve or change several manufacturing steps in the food chain. In this context particular attention should be given to the technologies involved in fruits and vegetables production. Nearly all fresh fruit and vegetables are subjected to different periods of storage and/or shelf-life before of their consumption. This implies the need to protect the commodities from microbial spoilage. Some Citrus species (e.g. lemon and grapefruit) may be stored for several months before consumption and then post-harvest treatments are essential to contain green (Penicillium digitatum) and blue (P. italicum) moulds. Alternative approaches to chemicals usually have a lower efficacy in containing rots but fulfill the consumer's expectation. Among the alternative strategies, the improvement of host natural resistance is promising. In this regard, we report some results concerning the use of biotic (yeast) and abiotic agents as inducers of phytoalexin (i.e. scoparone and/or scopoletin) accumulation in Citrus rind and its importance in the control of fungal decay. In all experiments the inducers were applied on fruits before or 24 h after inoculation with P. digitatum and the rot severity was monitored 7 days later. The accumulation of phytoalexins was monitored according to a standard methodology by HPLC. In all experiments a positive correlation was found between increase of the phytoalexin scoparone in host tissue and reduction of decay.