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Control of storage diseases of citrus by pre- and postharvest application of salts
Postharvest Biology and Technology 72 (2012) 57–63
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Control of storage diseases of citrus by pre- and postharvest application of salts Khamis Youssef a,b , Angela Ligorio a , Simona Marianna Sanzani a , Franco Nigro a , Antonio Ippolito a,∗ a b
Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, Università degli Studi di Bari “Aldo Moro”, via Amendola 165/A, 70126 Bari, Italy Agricultural Research Center, Plant Pathology Research Institute, 9 Gamaa St., 12619 Giza, Egypt
a r t i c l e
i n f o
Article history: Received 18 February 2012 Accepted 13 May 2012 Keywords: Citrus Postharvest diseases Salts Penicillium decay Citrus sinensis Citrus reticulata
a b s t r a c t The effectiveness of sodium bicarbonate (SB), sodium carbonate (SC), sodium silicate (SS), potassium bicarbonate (PB), potassium carbonate (PC), potassium sorbate (PS), calcium chloride (CC), and calcium chelate (CCh) against naturally occurring postharvest decay on ‘Comune’ clementine and ‘Valencia late’ orange fruit was investigated. Aqueous salt solutions (2%, w/v, 20 hl ha−1 ) were applied according to three strategies: (i) by spraying before harvest, (ii) by dipping after harvest, and (iii) by the combination of pre- and postharvest applications. Decay was assessed after two months at 4 ± 1 ◦ C (oranges) or 6 ± 1 ◦ C (clementines) and 95–98% RH, followed by 7 days of shelf life at 20 ± 2 ◦ C. For both species, preharvest sprays and the combination of pre- and postharvest applications were more effective in suppressing decay than postharvest dipping. With regard to preharvest application, several salts completely inhibited the incidence of decay as compared to the water control, namely, SC and PC on both species, and SS on ‘Valencia late’ oranges. In combined applications, all salts were effective in reducing the decay as compared to the water control with an efficacy varying between 66–100 and 78–100% for oranges and clementines, respectively. When salts were applied after harvest, the activity was in general less pronounced, SC and PC being the most effective on both species. In in vitro tests, the minimum inhibitory concentration (MIC) for both Penicillium digitatum and P. italicum, was achieved at 0.25% SB, SC, PB, PC, PS, and SS. The filamentous fungal population on fruit treated once in the field and with the double treatment was reduced as compared to the water control, whereas no statistical differences were observed for postharvest application. Based on these results, field application of salts can be considered a useful strategy to be included in an integrated approach for controlling postharvest diseases of citrus fruit. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Postharvest green mould, caused by Penicillium digitatum (Pers.:Fr.) Sacc., and blue mould, caused by P. italicum Wehmer, are the most important postharvest diseases affecting citrus production in arid climatic areas (Eckert and Eaks, 1989). Other pathogens can cause decay to stored citrus fruit but their incidence is generally low (Snowdon, 1990; Youssef et al., 2010a). Citrus postharvest diseases are commonly controlled worldwide by applying synthetic fungicides in packinghouses, before fruit storage. However, the development of pathogen resistance to fungicides and the growing public concern over health and environmental hazards associated with high levels of pesticide use have resulted in a considerable interest in developing alternative non-polluting control methods. Among these alternatives, the activity of several
∗ Corresponding author. Tel.: +39 0805443053; fax: +39 0805442911. E-mail addresses: [email protected] (K. Youssef), [email protected] (A. Ippolito). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.05.004
organic and inorganic salts has been comprehensively tested at 2–6% concentrations on a wide range of commodities, including citrus (Smilanick et al., 1997; Palou et al., 2008; Cunningham, 2010; Janisiewicz and Conway, 2010; Romanazzi et al., 2012). Organic and inorganic salts are “chemical means” belonging to the category of food additives or substances classified as GRAS (Generally Regarded as Safe) by the US Food and Drug Administration (FDA), and for this reason exempt from the usual Federal Food, Drug, and Cosmetic Act tolerance requirements (Senti, 1981). In spite of the interesting results obtained in laboratory and small scale experiments, salt usage limitations, i.e. inconsistent activity and limited persistence, lack of preventive effect, risk of fruit injury, and issues of disposal of exhausted salt solutions, make their postharvest commercial application still unreliable (Larrigaudiere et al., 2002; Palou et al., 2002; Smilanick et al., 2008; Smilanick, 2011). Performance of salts can be improved by combining them with other means, such as antagonistic microorganisms, hot water, sanitizers, lowdose chemical fungicides, and wax (El-Ghaouth et al., 2001; Lima et al., 2005; Palou et al., 2008; Cerioni et al., 2012; Youssef et al., 2012). However, combination may not encounter easy acceptance
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by operators because they are laborious and may need extra costs for labor, equipment, and energy (Lima et al., 2011). To overcome these problems and to attain commercially acceptable control levels, different application strategies as compared to fungicides are required. Salts have been widely investigated as postharvest treatments by dipping or spraying; very few investigations on preharvest application have been conducted to control postharvest decay (Nigro et al., 2006; Teixidó et al., 2010). This strategy could be advantageous because of lesser fruit manipulation with a consequent decrease in damage and injuries commonly occurring during any postharvest treatment (Teixidó et al., 2010). Moreover, the presence of the active ingredient as an alternative control means as the injury is inflicted, may interfere with the first phases of the infection (Ippolito and Nigro, 2000). The objective of the present research was to compare the efficacy of a selection of salts applied according to three strategies: (i) by spraying before harvest, (ii) by dipping after harvest, and (iii) by the combination of pre- and postharvest applications on citrus fruit against storage decay. In order to simulate actual commercial conditions, experiments were conducted on naturally infected fruit instead of artificially inoculated ones. Preliminary results on ‘Hernandina’ clementines and ‘Valencia late’ oranges with the use of some salts and natural substances have been reported (Youssef et al., 2008a,b).
2. Materials and methods 2.1. Salts, fungal isolates, and plant material Eight organic and inorganic salts (Table 1) were evaluated for their activity in vitro against P. digitatum and P. italicum and in vivo on different citrus species. Potassium bicarbonate (99.5%, PB) was purchased from Sigma Aldrich S.r.l (Milan, Italy), sodium bicarbonate (99.9%, SB), sodium carbonate (99%, SC), potassium carbonate (99%, PC), potassium sorbate (99%, PS), sodium silicate (72%, SS) and calcium chloride (99%, CC) from Carlo Erba Reagenti S.p.A (Rodano, Mi, Italy), and calcium chelate (CCh) from Chimica D’Agostino S.p.A. (Agrikel-Ca, div. Agridast, Ba, Italy). A standard chemical compound, DECCOZIL® 50 [50% imazalil (IMZ)], was purchased from Decco Italia s.r.l. (Belpasso, Ct, Italy). Fungal strains were isolated from decayed citrus fruit and maintained on potato dextrose agar (PDA) at 4 ± 1 ◦ C in the culture collection of the Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari “Aldo Moro”, Italy. The in vivo experiments were conducted on 7-year-old clementine (Citrus reticulata Blanco) cv. Comune, and 30-year-old sweet orange [Citrus sinensis (L.) Osbeck] cv. Valencia late trees, located in Basilicata region, Southern Italy.
Table 1 Minimum inhibitory concentration (MIC) of salts and imazalil on Penicillium digitatum and P. italicum in a colony growth assay. Salt
Na bicarbonate (NaHCO3 ) Na carbonate (Na2 CO3 ) K bicarbonate (KHCO3 ) K carbonate (K2 CO3 ) Ca chloride (CaCl2 ) Ca chelate (C10 H12 N2 O8 CaNa2 ·2H2 O) K sorbate (C6 H7 KO2 ) Na silicate (Na2 Ox 2SiO2 ·2H2 O) Imazalil
MIC (%, w/v) P. digitatum
P. italicum
0.25 0.25 0.25 0.25 > 2.0 1.0 0.25 0.25 0.025
0.25 0.25 0.25 0.25 > 2.0 > 2.0 0.25 0.25 0.025
The diameter of P. digitatum and P. italicum was determined after 6 days incubation at 24 ± 1 ◦ C on PDA amended with different salt concentrations (0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2%, w/v).
2.2. Effect of salts and IMZ on in vitro growth of Penicillium spp. The effect of salts and IMZ on the mycelial growth of P. digitatum and P. italicum was evaluated. An aqueous solution of the salts and IMZ was sterilized by filtration (0.45 m) and added to molten (45 ◦ C) PDA before pouring into 90 mm Petri dishes, to achieve 0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0% (w/v) final concentration; PDA without salts or IMZ served as a control. Dishes were seeded in the center with a 5 mm mycelial plug taken from the edge of actively growing colony of the pathogens and incubated for 6 days at 24 ± 1 ◦ C. For each salt/concentration, five Petri dishes were utilized as replicates and the entire experiment was repeated twice. Colony diameter was calculated as the average of the longest and the shortest diameter; the results were expressed as minimum inhibitory concentration (MIC). 2.3. In vivo trials Salt solutions (2%, w/v) were prepared at room temperature dissolving 300 g for each salt in 15 L of tap water. The pH of all the solutions was measured using a pH meter (Jenway 3510 bench, Staffordshire, UK). The application strategies used were: (i) preharvest treatment; (ii) pre- and postharvest treatments; and (iii) postharvest dipping. In all the trials, fruit treated with water served as a negative control and fruit treated after harvest with IMZ were included as a standard chemical control (Table 2). For preharvest treatments, trials were arranged in a completely randomized block design with 3 replicates, containing 3 plants each. Plants were selected for uniformity of fruit development and absence of evident symptoms of diseases and disorders, and sprayed with salt solutions (approximately 5 L plant−1 , 20 hl ha−1 ). Treatments were made 7 days before harvest using a commercial motor-driven back sprayer (Fox Motori mod. 320, Poviglio, Re, Italy). At commercial maturity fruit were harvested from treated plants and placed into covered plastic boxes (5 boxes per plant), each containing 40–50 fruit for clementines and 20–25 fruit for oranges. Fruits were stored for two months at 4 ± 1 ◦ C (oranges) and 6 ± 1 ◦ C (clementines) and 95–98% RH, followed by 7 days of shelf life at 20 ± 2 ◦ C. These conditions of storage were used to simulate actual commercial conditions. For the combination of pre- and postharvest treatments, fruit from plants treated 7 days before harvest in the field were placed in plastic boxes (5 boxes per plant). Then, in the laboratory fruit from each group of five boxes were dipped (45 boxes per treatment) for 10 min in a solution of the same salt used in the field. After dipping, fruit were left to dry at room temperature for 2 h, then placed again in the same boxes and stored as described for preharvest treatment. Regarding postharvest dipping, clementines and oranges harvested from untreated plants of the same orchard used for preharvest treatment were thoroughly mixed and arranged in 45 boxes per treatment in a completely randomized design. The remaining procedures were the same described above for combined treatments. At the end of shelf life, the incidence of decay was assessed and expressed as percentage of fruit infected by fungal pathogens. Pathogens were visually identified and in case of doubt, isolation and morphological identification were carried out. 2.4. Epiphytic populations occurring on citrus surface For the three application strategies, at the end of storage, epiphytic populations of filamentous fungi, yeasts and yeast-like fungi occurring on the fruit surface were evaluated according to Ippolito et al. (2005) with minor modifications. In particular, for each treatment, three replicates of 5 fruit each were weighed and shaken in 200 mL of sterile distilled water on a rotary shaker at 150 rpm for
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Table 2 Salts, their pH and concentration, and treatment scheme applied for controlling postharvest decay on oranges and clementines. Treatment
pH
Na bicarbonate (NaHCO3 ) Na carbonate (Na2 CO3 ) K bicarbonate (KHCO3 ) K carbonate (K2 CO3 ) Ca chloride (CaCl2 ) Ca chelate (C10 H12 N2 O8 CaNa2 ·2H2 O) K sorbate (C6 H7 KO2 ) Na silicate (Na2 SiO3 ) Water control (H2 O) Imazalil
8.4 11.4 8.4 11.4 6.5 7.7 8.7 11.4 7.7 10.6
Concentration (%) 2 2 2 2 2 2 2 2 – 0.1
Preharvest
Postharvest
Pre/postharvest
+ + + + + + + + + (*)
+ + + + + + + + + +
+ + + + + + + + + (*)
(*) Chemical control consisted of fruit harvested from un-treated plants and treated by dipping with imazalil after harvest.
30 min. The rinse water was diluted and plated (0.1 mL plate−1 ) on malt extract agar. Three plates for each replication were incubated at 24 ± 1 ◦ C and, after 4–6 days, the number of colony forming units (CFU) was recorded separately and converted into log10 CFU g−1 of fruit. Yeast and yeast-like fungi were grouped together as well as all the filamentous fungi.
preharvest applications of SC and PC were the most effective in reducing decay (Fig. 2A). SB, PB, CCh, PS, and SS were also significantly effective in reducing the incidence of decay by 94, 90, 60, 44, and 76%, respectively. Conversely, preharvest application of CC was not effective in reducing the incidence of decay on both citrus species.
2.5. Statistical analysis 8
Data on decay and on epiphytic population were subjected to analysis of variance using Statistica 6.0 software. Percentage data were arcsine transformed to normalize variance. Mean values of treatments were compared by using Fisher’s protected least significant difference (LSD) test and judged at P ≤ 0.05 level. For each citrus species experiments were repeated over a 3 year period on plants belonging to the same orchard; data from each experiment were combined since statistical analysis determined homogeneity of variances. Data in the graphs are untransformed percentage of decayed fruit.
7
5
ab
4 3 2 b
1
7
Fruit decay (%)
3.1. Effect of salts and IMZ on in vitro growth of Penicillium spp.
b
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0 8
3. Results
The results of in vitro tests showed a similar effect on the growth of P. digitatum and P. italicum for all salts, except for those containing calcium (Table 1). After 6 days of incubation, SB, SC, PB, PC, PS, and SS at 0.25% completely inhibited growth of both pathogens. For CC, in both fungal species MIC was not determined since growth was observed till 2% (the maximum concentration used); for CCh, a complete growth inhibition was evidenced at 1% on P. digitatum, whereas no MIC was observed on P. italicum. The chemical control IMZ at 0.025% completely inhibited the growth of both pathogens.
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Postharvest rots on clementines and oranges were mainly due to P. digitatum, the incidence of P. italicum being very low. The occurrence of decay caused by other pathogens (Alternaria spp., Botrytis spp., Phytophthora spp., etc.) was almost negligible (data not shown). In all cases the standard chemical control (IMZ) completely suppressed decay, whereas salts showed variable effects, depending on the applied strategies and citrus species. After two months storage at 4 ◦ C followed by 7 days of shelf life at 20 ± 2 ◦ C, preharvest application of several salts on ‘Valencia late’ orange significantly reduced the incidence of decay as compared to the water control (Fig. 1A). In particular, SC, PC, and SS were the most effective, completely inhibiting the development of decay (100%) as the standard chemical control. Also SB, PB, CCh, and PS significantly reduced the percentage of rotted fruit by approximately 89% (Fig. 1A). Similarly, in the trials conducted with clementines,
C
ab
6
ab
5 4
abc abc
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3 2
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1 0
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Fig. 1. Decay incidence on ‘Valencia late’ oranges preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 4 ± 1 ◦ C, followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
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8 7
A
irregular brown spots on clementines. In addition, after two months storage and one week shelf life, the general external appearance of fruit was not affected by different treatments.
6 5
3.3. Epiphytic population occurring on the fruit surface
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bc cd
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1 e
0
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Fruit decay (%)
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At the end of storage, the naturally occurring microbial population on the fruit surface was recorded. The microorganisms most commonly isolated were yeasts (mainly white and red yeasts), yeast-like fungi (mainly Aureobasidium pullulans), and several filamentous fungi (Penicillium spp., Cladosporium spp., Colletotrichum spp., Aspergillus spp., and Rhizopus spp.). On the whole, the highest population values were generally observed on water treated fruit and the lowest on IMZ treated fruit. In preharvest and combined applications, all salts, except CC, significantly reduced the number of filamentous fungi, whereas no effect was observed on postharvest application. In particular, the population density of filamentous fungi on ‘Valencia late’ orange ranged between 1.3 and 5.7 (preharvesttreated fruit, Fig. 3A), 1.9 and 6.5 (combined application, Fig. 3B) and 1.0 and 5.6 (postharvest-treated fruit, Fig. 3C) log10 units. The density of yeasts and yeast-like fungi ranged between 3.8 and 6.7 log10 units on fruit treated in the field, 2.0 and 6.8 on fruit treated twice, and 3.8 and 6.6 log10 units on fruit treated postharvest (Fig. 3A–C); salt application did not significantly influence the population of yeasts and yeast-like fungi, as compared to the water control. In the trials conducted on ‘Comune’ clementines, the density of filamentous fungi ranged between 2.2 and 4.3 (preharvest-treated fruit, Fig. 4A), 2.3 and 5.3 (combined application, Fig. 4B) and 3.0 and 3.7 (postharvest-treated fruit, Fig. 4C) log10 units. Preharvest treatments did not significantly affect the density of yeasts and yeast-like fungi, whereas it was reduced by five salts (SB, SC, PB, PC, and PS) on fruit treated twice and by SS on those treated after harvest (Fig. 4 A–C).
d
4. Discussion
Fig. 2. Decay incidence on ‘Comune’ clementines preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 6 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
In combined applications (pre and postharvest), all the tested salts significantly reduced the incidence of postharvest decay on both citrus species, as compared to the water control. The percentage of reduction varied between 66–100 and 78–100% for oranges and clementines, respectively. In particular, SC and PC completely suppressed the disease on both species (Figs. 1B and 2B). When salts were applied by dipping after harvest the activity was in general less pronounced. With respect to the trials carried out on oranges, in most cases no significant differences were observed between treated fruit and water control (Fig. 1C). Only SC and PC were effective in reducing decay by approximately 71 and 73%, respectively, as compared to the water control. In the trials performed with clementines, SC, PB, and PC significantly reduced the percentage of rotted fruit by 80, 50, and 70%, respectively (Fig. 2C). The other salts (SB, CC, CCh, PS, and SS) did not influence decay development. In the postharvest treatment, none of the salt solutions had a phytotoxic effect on both citrus species. However, under particular environmental conditions characterized by high and persistent humidity, preharvest application of SC and PC induced little
Due to the high economic value of the harvested commodity, efficient disease control under commercial conditions is the most important requirement for a postharvest treatment, including control methods alternative to synthetic fungicides. Organic and inorganic salts are among the most promising substitutes for fungicides but, as reported above, some unsolved constraints still impede their practical use (Smilanick et al., 2008; Palou et al., 2008; Teixidó et al., 2010). Taking into account these considerations, we tested a strategy different from the one used for synthetic fungicides, based on pre- and postharvest application of salts. Since the trials were conducted on naturally occurring infections, disease incidence in the control treatments was not very high, even as an average of experiments conducted over a 3 year period. This could generate uncertainties in the interpretation of the data. In this regard, it is worth mentioning that in the area where trials were conducted the climatic conditions are generally not very conducive to Penicillium rots and a low level of disease incidence is normal. Moreover, it is well known that ‘Valencia late’ fruit are tough, ripening in a dry period, and therefore not prone to postharvest decay; clementines are more susceptible to decay but not as winter ripening oranges. In addition, with naturally occurring infection it is possible to test the efficacy of the control methods on every kind of infection, namely latent, quiescent, and incipient infections, not only on wound infections. Assessing the efficacy of candidate control means on natural infections is necessary and important when researchers are looking for a commercial application (Smilanick et al., 1997). Finally, to dispel all uncertainties it has to be considered that preharvest and double applications of some
K. Youssef et al. / Postharvest Biology and Technology 72 (2012) 57–63
8 Yeasts and yeast-like
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Filamentous fungi
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Log10 (CFU/g fruit)
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Log10 (CFU/g fruit)
salts gave the same results as postharvest application of chemical fungicide, i.e. complete suppression of Penicillium rot. On the whole, for both citrus species, our results demonstrated that preharvest and combined applications of salts provided a significant reduction of the disease. In particular, some of them (sodium bicarbonate, sodium carbonate, and potassium carbonate) achieved the same protection level as with the standard chemical control (IMZ) applied after harvest. Also calcium chelate, potassium sorbate, and sodium silicate were effective when applied once (before harvest) or twice (before and after harvest). The efficacy of preharvest salts applications was very high, and from a statistical point of view it was not improved by further applications after harvest, except for calcium chloride. Salts applied in the field prior to harvest had a longer time to interact with the pathogen/fruit, as compared to the salts applied after harvest, thus affecting the pathogen inoculum density on fruit surface, the environment in the wound niche, and probably tissue resistance. Regarding the inoculum density, the filamentous fungal population on fruit treated
61
ab
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Fig. 3. Populations of yeast and yeast-like fungi and filamentous fungi on the surface of ‘Valencia late’ oranges preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 4 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Population was evaluated at the end of storage. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
Fig. 4. Populations of yeast and yeast-like fungi and filamentous fungi on the surfaces of ‘Comune’ clementines preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 6 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Population was evaluated at the end of storage. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
once in the field and with the double treatment had significantly lower values as compared to the water control, whereas no differences were observed for both species when salts were applied after harvest. This behavior can be ascribed to a direct activity of salts, as confirmed by our in vitro trials, except for those containing calcium. In these trials a MIC of 0.25% (w/v) was recorded for almost all salts, 10 times higher than the one recorded for IMZ, but equally interesting considering that salts are much cheaper and eco-friendly. The antifungal activity of carbonate, bicarbonate, sorbate, and silicate is well known (Palou et al., 2002; Smilanick et al., 2005; Ippolito et al., 2005; Nigro et al., 2006; Guo et al., 2007; Gregori et al., 2008; Youssef et al., 2010b), as well as the direct activity of carbonates and bicarbonates in inhibiting spore germination, germ tube elongation, and production of pectinolytic enzymes in several pathogens (Hervieux et al., 2002; Smilanick et al., 2005). Moreover, results reported herein seem to confirm that pH of salt solutions did not have a dominant role in the observed activity, as
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previously reported by Nigro et al. (2006). In fact, high disease reduction was obtained with pre- and postharvest applications of PC and SC (pH 11.4), but also with PB and SB (pH 8.4), and CCh (pH 7.7). The osmotic stress caused by the presence of salts in field applications may have contributed to the decrease in fungal populations (Elad and Kirshner, 1993; Ippolito et al., 2005). Osmotic stress should play a minor role in the case of postharvest application, where the RH is generally near the saturation point. The low influence of most of the tested salts on yeast and yeast-like populations is reported in the literature (Schena et al., 2003; Ippolito et al., 2005) and can be considered as an advantage since they are not pathogenic and may act as biocontrol agents (Janisiewicz and Korsten, 2002). In addition to the direct activity on the epiphytic fungal population, salts may have entered the wounds during infliction, leading to an interaction between salt residues and constituents of citrus rind, thus creating unfavorable environmental conditions for the pathogen, and possibly increasing tissue resistance. Indeed, specific experiments conducted by Youssef et al. (2010c) pointed out an indirect action of some salts mediated by tissue-induced resistance; in these trials, SB, SC, PC, and PB applied in wounds 5 mm apart from the pathogen-inoculated ones induced a significant reduction of disease severity (diameter of the lesion) as compared to the water control. Further trials have shown also an increase in the phytoalexins scoparone, scopoletin, and umbelliferone in SC and SB treated orange tissues (Youssef et al., 2011). Similarly, Venditti et al. (2005) reported that SC induced structural changes in cell wall, production of scoparone and an increase in pH of citrus rind tissues. Regarding the sodium silicate mode of action, a specific study showed that the activity of peroxidase and PAL was enhanced in treated melon tissues (Guo et al., 2007). In contrast, with regard to potassium sorbate, to the best of our knowledge, it seems that no specific studies have been conducted to verify its activity in enhancing citrus natural resistance. Our data showed that treatment with calcium chloride was effective against decay only when applied twice (before and after harvest), whereas, when applied once, either before or after harvest, it did not influence decay incidence as compared to the water control. The reduced efficacy of CC in preharvest applications was unexpected since its field application is effective against a number of pathogens on different hosts, including kiwifruit, cactus, pears, table grapes, and sweet cherries (Gerasopoulos et al., 1996; Schirra et al., 1999; Ippolito et al., 2005; Nigro et al., 2006). It should be considered that CC has a limited direct action on the pathogen (Nigro et al., 2006; Latifa et al., 2011) and its activity is mainly related to the stabilization of the host cell wall and middle lamella (Gerasopoulos et al., 1996; Miceli et al., 1999). It can be hypothesized that CC applied once on ripening fruit did not accumulate enough in the citrus peel to exert its activity. In the case of calcium chelate, which was effective for both species when applied before harvest and in double application, its particular chemical structure (chelate) may have allowed an accumulation in rind tissue sufficient to enhance its natural resistance against the pathogen (Lester and Grusak, 2004). In conclusion, our results have demonstrated that the incidence of postharvest decays on citrus fruit can be reduced by applying, a few days before harvest, a number of salts which are safe for consumers and the environment. This strategy may be a way of overcoming one of the main limitations of most of the alternative control means, i.e. the lack of curative effects (Spadaro and Gullino, 2004; Palou et al., 2008). In addition, their preventive effect can be of interest for packinghouses, avoiding heavy decay when harvested fruit are kept at room temperature before sorting and packaging. Salts can be applied using the same equipment as for synthetic fungicides, and being common food additives marketed at a convenient price, their use should be easily accepted by
operators and consumers. Less contaminated/infected fruit on packing lines should also reduce the demand for sanitizers and water during washing procedures. Practical preharvest application of salts needs to be further optimized as the obtainable level of protection is affected by various factors, including the chemical composition of the salt, citrus variety, environmental conditions prevailing during their application, etc. Regarding the last aspect, the slight phytotoxicity on clementine fruit treated in the field with sodium and potassium carbonate, observed only when heavy dew followed salt application, is difficult to explain. Probably the phytotoxicity should be ascribed to the prolonged contact and imbibitions of rind tissues with solutions having a high pH. Long interaction times and/or osmotic stress associated with preharvest treatments should have affected weight loss and fruit rind quality but the high RH during storage and the short shelf life have probably masked these effects. Finally, although our experiments clearly demonstrate the advantage of preharvest application of alternative control means, generally, farmers are not interested in the storage life of products once fruit are sold on the plant. In this regard, more than in the past, an integrated control approach along the entire production chain is necessary to obtain a high quality product. Acknowledgments The research has been realized with the contribution of the Emilia-Romagna Region (leader partner) within the interregional project “Frutticoltura post-raccolta” (L. 499/99) coordinated by CRPV. Thanks are expressed to the “Azienda Agricola Sperimentale Pantanello, ALSIA” Metaponto – (MT), Italy, directed by Dr. C. Mennone, in which the trials were conducted. References Cerioni, L., Rodriguez-Montelongo, L., Ramallo, J., Prado, F.E., Rapisarda, V.A., Volentini, S.I., 2012. Control of lemon green mold by a sequential oxidative treatment and sodium bicarbonate. Postharvest Biology and Technology 63, 33–39. Cunningham, N.M., 2010. Combinations of treatments to replace the use of conventional fungicides for the commercial control of postharvest diseases of citrus fruit. Stewart Postharvest Review 1, 1–8. Eckert, J.W., Eaks, I.L., 1989. Postharvest disorders and diseases of citrus fruits. In: Reuter, W., Calavan, E.C., Carman, G.E. (Eds.), The Citrus Industry, vol. 5. Univ. California Press, Berkeley, pp. 179–260. Elad, Y., Kirshner, B., 1993. Survival in the phylloplane of an introduced biocontrol agent (Trichoderma harzianum) and populations of the plant pathogen Botrytis cinerea as modified by biotic conditions. Phytoparasitica 21, 303–313. El-Ghaouth, A., Smilanick, J.L., Brown, G.E., Ippolito, A., Wilson, C.L., 2001. Control of decay of apple and citrus fruits in semicommercial tests with Candida saitoana and 2-deoxy-d-glucose. Biological Control 20, 96–101. Gerasopoulos, D., Chouliaras, V.E., Lionikas, S., 1996. Effects of preharvest calcium chloride sprays on maturity and storability of Hayward kiwifruit. Postharvest Biology and Technology 7, 65–72. Gregori, R., Borsetti, F., Neri, F., Mari, M., Bertolini, P., 2008. Effects of potassium sorbate on postharvest brown rot of stone fruit. Journal of Food Protection 71, 1626–1631. Guo, Y., Liu, L., Zhao, J., Bi, Y., 2007. Use of silicon oxide and sodium silicate for controlling Trichothecium roseum postharvest rot in Chinese cantaloupe (Cucumis melo L.). International Journal of Food Science and Technology 42, 1012–1018. Hervieux, V., Yaganza, E.S., Arul, J., Tweddell, R.J., 2002. Effect of organic and inorganic salts on the development of Helminthosporium solani, the causal agent of potato silver scurf. Plant Diseases 86, 1014–1018. Ippolito, A., Nigro, F., 2000. Impact of preharvest application of biocontrol agents on postharvest diseases of fresh fruit and vegetables. Crop Protection 19, 723–725. Ippolito, A., Schena, L., Pentimone, I., Nigro, F., 2005. Control of postharvest rots of sweet cherries by pre- and postharvest applications of Aureobasidium pullulans in combination with calcium chloride or sodium bicarbonate. Postharvest Biology and Technology 363, 245–252. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruits. Annual Review of Phytopathology 40, 411–441. Janisiewicz, W.J., Conway, W.S., 2010. Combining biological control with physical and chemical treatments to control fruit decay after harvest. Stewart Postharvest Review 6, 1–16. Larrigaudiere, C., Pons, J., Torres, R., Usall, J., 2002. Storage performance of clementines treated with hot water, sodium carbonate, and sodium bicarbonate dips. Journal of Horticultural Science & Biotechnology 77, 314–319.
K. Youssef et al. / Postharvest Biology and Technology 72 (2012) 57–63 Latifa, A., Idriss, T., Hassan, B., Amine, S.M., El Hassane, B., Abdellah, A.B.A., 2011. Effects of organic acids and salts on the development of Penicillium italicum: the causal agent of citrus blue mold. Plant Pathology Journal 10, 99–107. Lester, G.E., Grusak, M.A., 2004. Field application of chelated calcium: postharvest effects on cantaloupe and honeydew fruit quality. HortTechnology 14, 29–38. Lima, G., Spina, A.M., Castoria, R., De Curtis, F., De Cicco, V., 2005. Integration of biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum. Journal of Food Protection 68, 2100–2106. Lima, G., Castoria, R., De Curtis, F., Raiola, A., De Cicco, V., 2011. Integrated control of blue mould using new fungicides and biocontrol yeasts lowers levels of fungicide residues and patulin contamination in apples. Postharvest Biology and Technology 60, 164–172. Miceli, A., Ippolito, A., Linsalata, V., Nigro, F., 1999. Effect of preharvest calcium treatment on decay and biochemical changes of table grape during storage. Phytopathologia Mediterranea 38, 47–53. Nigro, F., Schena, L., Ligorio, A., Pentimone, I., Ippolito, A., Salerno, M.G., 2006. Control of table grape storage rots by preharvest applications of salts. Postharvest Biology and Technology 42, 142–149. ˜ I., 2002. Evaluation of food Palou, L., Usall, J., Smilanick, J.L., Aguilar, M.J., Vinas, additives and low-toxicity compounds as alternative chemicals for the control of Penicillium digitatum and P. italicum on citrus fruit. Pest Management Science 58, 459–466. Palou, L., Smilanick, J.L., Droby, S., 2008. Alternatives to conventional fungicides for the control of citrus postharvest green and blue moulds. Stewart Postharvest Review 2, 1–16. Romanazzi, G., Lichter, A., Gabler, F.M., Smilanick, J.L., 2012. Recent advances on the use of natural and safe alternatives to conventional methods to control postharvest gray mold of table grapes. Postharvest Biology and Technology 63, 141–147. Schena, L., Nigro, F., Pentimone, I., Ligorio, A., Ippolito, A., 2003. Control of postharvest rots of sweet cherries and table grapes with endophytic isolates of Aureobasidium pullulans. Postharvest Biology and Technology 30, 209–220. Schirra, M., Inglese, P., La Mantia, T., 1999. Quality of cactus pear fruit in relation to ripening time, CaCl2 pre-harvest sprays and storage conditions. Scientia Horticulturae 81, 425–436. Senti, F.R., 1981. Food additives and food safety. Industrial & Engineering Chemistry Research 20, 237–246. Smilanick, J.L., Mackey, B.E., Reese, R., Usall, J., Margosan, D.A., 1997. Influence of concentration of soda ash, temperature, and immersion period on the control of postharvest green mold of oranges. Plant Diseases 81, 379–382. Smilanick, J.L., Mansour, M.F., Margosan, D.A., Mlikota Gabler, F., Goodwine, W.R., 2005. Influence of pH and NaHCO3 on effectiveness of imazalil to inhibit germination of Penicillium digitatum and to control postharvest green mold on citrus fruit. Plant Diseases 89, 640–648.
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Smilanick, J.L., Mansour, M.F., Mlikota Gabler, F., Sorenson, D., 2008. Control of citrus postharvest green mold and sour rot by potassium sorbate combined with heat and fungicides. Postharvest Biology and Technology 47, 226–238. Smilanick, J.L., 2011. Integrated approaches to postharvest disease management in California citrus packinghouses. Acta Horticulturae 905, 145–148. Snowdon, A.L., 1990. A Colour Atlas of Post-Harvest Diseases and Disorders of Fruits and Vegetables, vol. 1. Wolfe Scientific, London, pp. 102–103. Spadaro, D., Gullino, M.L., 2004. State of the art and future prospects of the biological control of postharvest fruit diseases. International Journal of Food Microbiology 91, 185–194. ˜ I., 2010. Preharvest strateTeixidó, N., Usall, J., Nunes, C., Torres, R., Abadias, M., Vinas, gies to control postharvest diseases in fruits. In: Prusky, D., Gullino, M.L. (Eds.), Postharvest Pathology, Plant Pathology in the 21st Century, vol. 2. Springer, New York, pp. 89–106. Venditti, T., Molinu, M.G., Dore, A., Agabbio, M., D’hallewin, G., 2005. Sodium carbonate treatment induces scoparone accumulation, structural changes, and alkalinization in the albedo of wounded citrus fruits. Journal of Agricultural and Food Chemistry 53, 3510–3518. Youssef, K., Ligorio, A., Pentimone, I., Sanzani, S., Donghia, A.M., Nigro, F., Ippolito, A., 2008a. Studies on application strategy of salts and chitosan for control Penicillium rot of Hernandina clementine. In: Kaiser, H.K., Kirner, R. (Eds.), Proc. Junior Scientist Conference. Vienna, Austria, pp. 189–190. Youssef, K., Ligorio, A., Pentimone, I., Nigro, F., Ippolito, A., Yaseen, T., 2008b. Studies on application strategy of salts for controlling Penicillium rot of Valencia late oranges. In: Proc. 11th International Citrus Congress, vol. 2, Wuhan, China, pp. 1276–1280. Youssef, K., Ahmed, Y., Ligorio, A., D’Onghia, A.M., Nigro, F., Ippolito, A., 2010a. First report of Penicillium ulaiense as a new postharvest pathogen of citrus fruit in Egypt. Plant Pathology 59, 1174. Youssef, K., Di Primo, P., Coniglione, M., Ippolito, A., 2010b. Evaluation of antifungal activity of Bioprotege, a mixture of sodium bicarbonate and silicium dioxide, against major postharvest fungal pathogens of fruit and vegetables. Journal of Plant Pathology 92, 104. Youssef, K., Ligorio, A., Sanzani, S.M., Nigro, F., Ippolito, A., 2010c. Evaluation of direct activity and induction of resistance of some common food additives against green mould of citrus fruit. Journal of Plant Pathology 92, 67. Youssef, K., Sanzani, S.M., Ligorio, A., Terry, L.A., Ippolito, A., 2011. Biochemical changes associated with induced resistance on citrus fruit. Journal of Plant Pathology 93, 22. Youssef, K., Ligorio, A., Nigro, F., Ippolito, A., 2012. Activity of salts incorporated in wax in controlling postharvest diseases of citrus fruit. Postharvest Biology and Technology 65, 39–43.
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Control of storage diseases of citrus by pre- and postharvest application of salts Khamis Youssef a,b , Angela Ligorio a , Simona Marianna Sanzani a , Franco Nigro a , Antonio Ippolito a,∗ a b
Dipartimento di Biologia e Chimica Agro-Forestale ed Ambientale, Università degli Studi di Bari “Aldo Moro”, via Amendola 165/A, 70126 Bari, Italy Agricultural Research Center, Plant Pathology Research Institute, 9 Gamaa St., 12619 Giza, Egypt
a r t i c l e
i n f o
Article history: Received 18 February 2012 Accepted 13 May 2012 Keywords: Citrus Postharvest diseases Salts Penicillium decay Citrus sinensis Citrus reticulata
a b s t r a c t The effectiveness of sodium bicarbonate (SB), sodium carbonate (SC), sodium silicate (SS), potassium bicarbonate (PB), potassium carbonate (PC), potassium sorbate (PS), calcium chloride (CC), and calcium chelate (CCh) against naturally occurring postharvest decay on ‘Comune’ clementine and ‘Valencia late’ orange fruit was investigated. Aqueous salt solutions (2%, w/v, 20 hl ha−1 ) were applied according to three strategies: (i) by spraying before harvest, (ii) by dipping after harvest, and (iii) by the combination of pre- and postharvest applications. Decay was assessed after two months at 4 ± 1 ◦ C (oranges) or 6 ± 1 ◦ C (clementines) and 95–98% RH, followed by 7 days of shelf life at 20 ± 2 ◦ C. For both species, preharvest sprays and the combination of pre- and postharvest applications were more effective in suppressing decay than postharvest dipping. With regard to preharvest application, several salts completely inhibited the incidence of decay as compared to the water control, namely, SC and PC on both species, and SS on ‘Valencia late’ oranges. In combined applications, all salts were effective in reducing the decay as compared to the water control with an efficacy varying between 66–100 and 78–100% for oranges and clementines, respectively. When salts were applied after harvest, the activity was in general less pronounced, SC and PC being the most effective on both species. In in vitro tests, the minimum inhibitory concentration (MIC) for both Penicillium digitatum and P. italicum, was achieved at 0.25% SB, SC, PB, PC, PS, and SS. The filamentous fungal population on fruit treated once in the field and with the double treatment was reduced as compared to the water control, whereas no statistical differences were observed for postharvest application. Based on these results, field application of salts can be considered a useful strategy to be included in an integrated approach for controlling postharvest diseases of citrus fruit. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Postharvest green mould, caused by Penicillium digitatum (Pers.:Fr.) Sacc., and blue mould, caused by P. italicum Wehmer, are the most important postharvest diseases affecting citrus production in arid climatic areas (Eckert and Eaks, 1989). Other pathogens can cause decay to stored citrus fruit but their incidence is generally low (Snowdon, 1990; Youssef et al., 2010a). Citrus postharvest diseases are commonly controlled worldwide by applying synthetic fungicides in packinghouses, before fruit storage. However, the development of pathogen resistance to fungicides and the growing public concern over health and environmental hazards associated with high levels of pesticide use have resulted in a considerable interest in developing alternative non-polluting control methods. Among these alternatives, the activity of several
∗ Corresponding author. Tel.: +39 0805443053; fax: +39 0805442911. E-mail addresses: [email protected] (K. Youssef), [email protected] (A. Ippolito). 0925-5214/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.postharvbio.2012.05.004
organic and inorganic salts has been comprehensively tested at 2–6% concentrations on a wide range of commodities, including citrus (Smilanick et al., 1997; Palou et al., 2008; Cunningham, 2010; Janisiewicz and Conway, 2010; Romanazzi et al., 2012). Organic and inorganic salts are “chemical means” belonging to the category of food additives or substances classified as GRAS (Generally Regarded as Safe) by the US Food and Drug Administration (FDA), and for this reason exempt from the usual Federal Food, Drug, and Cosmetic Act tolerance requirements (Senti, 1981). In spite of the interesting results obtained in laboratory and small scale experiments, salt usage limitations, i.e. inconsistent activity and limited persistence, lack of preventive effect, risk of fruit injury, and issues of disposal of exhausted salt solutions, make their postharvest commercial application still unreliable (Larrigaudiere et al., 2002; Palou et al., 2002; Smilanick et al., 2008; Smilanick, 2011). Performance of salts can be improved by combining them with other means, such as antagonistic microorganisms, hot water, sanitizers, lowdose chemical fungicides, and wax (El-Ghaouth et al., 2001; Lima et al., 2005; Palou et al., 2008; Cerioni et al., 2012; Youssef et al., 2012). However, combination may not encounter easy acceptance
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by operators because they are laborious and may need extra costs for labor, equipment, and energy (Lima et al., 2011). To overcome these problems and to attain commercially acceptable control levels, different application strategies as compared to fungicides are required. Salts have been widely investigated as postharvest treatments by dipping or spraying; very few investigations on preharvest application have been conducted to control postharvest decay (Nigro et al., 2006; Teixidó et al., 2010). This strategy could be advantageous because of lesser fruit manipulation with a consequent decrease in damage and injuries commonly occurring during any postharvest treatment (Teixidó et al., 2010). Moreover, the presence of the active ingredient as an alternative control means as the injury is inflicted, may interfere with the first phases of the infection (Ippolito and Nigro, 2000). The objective of the present research was to compare the efficacy of a selection of salts applied according to three strategies: (i) by spraying before harvest, (ii) by dipping after harvest, and (iii) by the combination of pre- and postharvest applications on citrus fruit against storage decay. In order to simulate actual commercial conditions, experiments were conducted on naturally infected fruit instead of artificially inoculated ones. Preliminary results on ‘Hernandina’ clementines and ‘Valencia late’ oranges with the use of some salts and natural substances have been reported (Youssef et al., 2008a,b).
2. Materials and methods 2.1. Salts, fungal isolates, and plant material Eight organic and inorganic salts (Table 1) were evaluated for their activity in vitro against P. digitatum and P. italicum and in vivo on different citrus species. Potassium bicarbonate (99.5%, PB) was purchased from Sigma Aldrich S.r.l (Milan, Italy), sodium bicarbonate (99.9%, SB), sodium carbonate (99%, SC), potassium carbonate (99%, PC), potassium sorbate (99%, PS), sodium silicate (72%, SS) and calcium chloride (99%, CC) from Carlo Erba Reagenti S.p.A (Rodano, Mi, Italy), and calcium chelate (CCh) from Chimica D’Agostino S.p.A. (Agrikel-Ca, div. Agridast, Ba, Italy). A standard chemical compound, DECCOZIL® 50 [50% imazalil (IMZ)], was purchased from Decco Italia s.r.l. (Belpasso, Ct, Italy). Fungal strains were isolated from decayed citrus fruit and maintained on potato dextrose agar (PDA) at 4 ± 1 ◦ C in the culture collection of the Department of Environmental and Agro-Forestry Biology and Chemistry, University of Bari “Aldo Moro”, Italy. The in vivo experiments were conducted on 7-year-old clementine (Citrus reticulata Blanco) cv. Comune, and 30-year-old sweet orange [Citrus sinensis (L.) Osbeck] cv. Valencia late trees, located in Basilicata region, Southern Italy.
Table 1 Minimum inhibitory concentration (MIC) of salts and imazalil on Penicillium digitatum and P. italicum in a colony growth assay. Salt
Na bicarbonate (NaHCO3 ) Na carbonate (Na2 CO3 ) K bicarbonate (KHCO3 ) K carbonate (K2 CO3 ) Ca chloride (CaCl2 ) Ca chelate (C10 H12 N2 O8 CaNa2 ·2H2 O) K sorbate (C6 H7 KO2 ) Na silicate (Na2 Ox 2SiO2 ·2H2 O) Imazalil
MIC (%, w/v) P. digitatum
P. italicum
0.25 0.25 0.25 0.25 > 2.0 1.0 0.25 0.25 0.025
0.25 0.25 0.25 0.25 > 2.0 > 2.0 0.25 0.25 0.025
The diameter of P. digitatum and P. italicum was determined after 6 days incubation at 24 ± 1 ◦ C on PDA amended with different salt concentrations (0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2%, w/v).
2.2. Effect of salts and IMZ on in vitro growth of Penicillium spp. The effect of salts and IMZ on the mycelial growth of P. digitatum and P. italicum was evaluated. An aqueous solution of the salts and IMZ was sterilized by filtration (0.45 m) and added to molten (45 ◦ C) PDA before pouring into 90 mm Petri dishes, to achieve 0.0125, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, and 2.0% (w/v) final concentration; PDA without salts or IMZ served as a control. Dishes were seeded in the center with a 5 mm mycelial plug taken from the edge of actively growing colony of the pathogens and incubated for 6 days at 24 ± 1 ◦ C. For each salt/concentration, five Petri dishes were utilized as replicates and the entire experiment was repeated twice. Colony diameter was calculated as the average of the longest and the shortest diameter; the results were expressed as minimum inhibitory concentration (MIC). 2.3. In vivo trials Salt solutions (2%, w/v) were prepared at room temperature dissolving 300 g for each salt in 15 L of tap water. The pH of all the solutions was measured using a pH meter (Jenway 3510 bench, Staffordshire, UK). The application strategies used were: (i) preharvest treatment; (ii) pre- and postharvest treatments; and (iii) postharvest dipping. In all the trials, fruit treated with water served as a negative control and fruit treated after harvest with IMZ were included as a standard chemical control (Table 2). For preharvest treatments, trials were arranged in a completely randomized block design with 3 replicates, containing 3 plants each. Plants were selected for uniformity of fruit development and absence of evident symptoms of diseases and disorders, and sprayed with salt solutions (approximately 5 L plant−1 , 20 hl ha−1 ). Treatments were made 7 days before harvest using a commercial motor-driven back sprayer (Fox Motori mod. 320, Poviglio, Re, Italy). At commercial maturity fruit were harvested from treated plants and placed into covered plastic boxes (5 boxes per plant), each containing 40–50 fruit for clementines and 20–25 fruit for oranges. Fruits were stored for two months at 4 ± 1 ◦ C (oranges) and 6 ± 1 ◦ C (clementines) and 95–98% RH, followed by 7 days of shelf life at 20 ± 2 ◦ C. These conditions of storage were used to simulate actual commercial conditions. For the combination of pre- and postharvest treatments, fruit from plants treated 7 days before harvest in the field were placed in plastic boxes (5 boxes per plant). Then, in the laboratory fruit from each group of five boxes were dipped (45 boxes per treatment) for 10 min in a solution of the same salt used in the field. After dipping, fruit were left to dry at room temperature for 2 h, then placed again in the same boxes and stored as described for preharvest treatment. Regarding postharvest dipping, clementines and oranges harvested from untreated plants of the same orchard used for preharvest treatment were thoroughly mixed and arranged in 45 boxes per treatment in a completely randomized design. The remaining procedures were the same described above for combined treatments. At the end of shelf life, the incidence of decay was assessed and expressed as percentage of fruit infected by fungal pathogens. Pathogens were visually identified and in case of doubt, isolation and morphological identification were carried out. 2.4. Epiphytic populations occurring on citrus surface For the three application strategies, at the end of storage, epiphytic populations of filamentous fungi, yeasts and yeast-like fungi occurring on the fruit surface were evaluated according to Ippolito et al. (2005) with minor modifications. In particular, for each treatment, three replicates of 5 fruit each were weighed and shaken in 200 mL of sterile distilled water on a rotary shaker at 150 rpm for
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Table 2 Salts, their pH and concentration, and treatment scheme applied for controlling postharvest decay on oranges and clementines. Treatment
pH
Na bicarbonate (NaHCO3 ) Na carbonate (Na2 CO3 ) K bicarbonate (KHCO3 ) K carbonate (K2 CO3 ) Ca chloride (CaCl2 ) Ca chelate (C10 H12 N2 O8 CaNa2 ·2H2 O) K sorbate (C6 H7 KO2 ) Na silicate (Na2 SiO3 ) Water control (H2 O) Imazalil
8.4 11.4 8.4 11.4 6.5 7.7 8.7 11.4 7.7 10.6
Concentration (%) 2 2 2 2 2 2 2 2 – 0.1
Preharvest
Postharvest
Pre/postharvest
+ + + + + + + + + (*)
+ + + + + + + + + +
+ + + + + + + + + (*)
(*) Chemical control consisted of fruit harvested from un-treated plants and treated by dipping with imazalil after harvest.
30 min. The rinse water was diluted and plated (0.1 mL plate−1 ) on malt extract agar. Three plates for each replication were incubated at 24 ± 1 ◦ C and, after 4–6 days, the number of colony forming units (CFU) was recorded separately and converted into log10 CFU g−1 of fruit. Yeast and yeast-like fungi were grouped together as well as all the filamentous fungi.
preharvest applications of SC and PC were the most effective in reducing decay (Fig. 2A). SB, PB, CCh, PS, and SS were also significantly effective in reducing the incidence of decay by 94, 90, 60, 44, and 76%, respectively. Conversely, preharvest application of CC was not effective in reducing the incidence of decay on both citrus species.
2.5. Statistical analysis 8
Data on decay and on epiphytic population were subjected to analysis of variance using Statistica 6.0 software. Percentage data were arcsine transformed to normalize variance. Mean values of treatments were compared by using Fisher’s protected least significant difference (LSD) test and judged at P ≤ 0.05 level. For each citrus species experiments were repeated over a 3 year period on plants belonging to the same orchard; data from each experiment were combined since statistical analysis determined homogeneity of variances. Data in the graphs are untransformed percentage of decayed fruit.
7
5
ab
4 3 2 b
1
7
Fruit decay (%)
3.1. Effect of salts and IMZ on in vitro growth of Penicillium spp.
b
b b
0 8
3. Results
The results of in vitro tests showed a similar effect on the growth of P. digitatum and P. italicum for all salts, except for those containing calcium (Table 1). After 6 days of incubation, SB, SC, PB, PC, PS, and SS at 0.25% completely inhibited growth of both pathogens. For CC, in both fungal species MIC was not determined since growth was observed till 2% (the maximum concentration used); for CCh, a complete growth inhibition was evidenced at 1% on P. digitatum, whereas no MIC was observed on P. italicum. The chemical control IMZ at 0.025% completely inhibited the growth of both pathogens.
A
a
6
b
b
b
a
b
B
6 5 4 3 b
b
2 1
b
b b
0
b
b b
b
8
3.2. In vivo trials
7
a
Postharvest rots on clementines and oranges were mainly due to P. digitatum, the incidence of P. italicum being very low. The occurrence of decay caused by other pathogens (Alternaria spp., Botrytis spp., Phytophthora spp., etc.) was almost negligible (data not shown). In all cases the standard chemical control (IMZ) completely suppressed decay, whereas salts showed variable effects, depending on the applied strategies and citrus species. After two months storage at 4 ◦ C followed by 7 days of shelf life at 20 ± 2 ◦ C, preharvest application of several salts on ‘Valencia late’ orange significantly reduced the incidence of decay as compared to the water control (Fig. 1A). In particular, SC, PC, and SS were the most effective, completely inhibiting the development of decay (100%) as the standard chemical control. Also SB, PB, CCh, and PS significantly reduced the percentage of rotted fruit by approximately 89% (Fig. 1A). Similarly, in the trials conducted with clementines,
C
ab
6
ab
5 4
abc abc
abc
abc
3 2
bc
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Fig. 1. Decay incidence on ‘Valencia late’ oranges preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 4 ± 1 ◦ C, followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
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8 7
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irregular brown spots on clementines. In addition, after two months storage and one week shelf life, the general external appearance of fruit was not affected by different treatments.
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At the end of storage, the naturally occurring microbial population on the fruit surface was recorded. The microorganisms most commonly isolated were yeasts (mainly white and red yeasts), yeast-like fungi (mainly Aureobasidium pullulans), and several filamentous fungi (Penicillium spp., Cladosporium spp., Colletotrichum spp., Aspergillus spp., and Rhizopus spp.). On the whole, the highest population values were generally observed on water treated fruit and the lowest on IMZ treated fruit. In preharvest and combined applications, all salts, except CC, significantly reduced the number of filamentous fungi, whereas no effect was observed on postharvest application. In particular, the population density of filamentous fungi on ‘Valencia late’ orange ranged between 1.3 and 5.7 (preharvesttreated fruit, Fig. 3A), 1.9 and 6.5 (combined application, Fig. 3B) and 1.0 and 5.6 (postharvest-treated fruit, Fig. 3C) log10 units. The density of yeasts and yeast-like fungi ranged between 3.8 and 6.7 log10 units on fruit treated in the field, 2.0 and 6.8 on fruit treated twice, and 3.8 and 6.6 log10 units on fruit treated postharvest (Fig. 3A–C); salt application did not significantly influence the population of yeasts and yeast-like fungi, as compared to the water control. In the trials conducted on ‘Comune’ clementines, the density of filamentous fungi ranged between 2.2 and 4.3 (preharvest-treated fruit, Fig. 4A), 2.3 and 5.3 (combined application, Fig. 4B) and 3.0 and 3.7 (postharvest-treated fruit, Fig. 4C) log10 units. Preharvest treatments did not significantly affect the density of yeasts and yeast-like fungi, whereas it was reduced by five salts (SB, SC, PB, PC, and PS) on fruit treated twice and by SS on those treated after harvest (Fig. 4 A–C).
d
4. Discussion
Fig. 2. Decay incidence on ‘Comune’ clementines preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 6 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
In combined applications (pre and postharvest), all the tested salts significantly reduced the incidence of postharvest decay on both citrus species, as compared to the water control. The percentage of reduction varied between 66–100 and 78–100% for oranges and clementines, respectively. In particular, SC and PC completely suppressed the disease on both species (Figs. 1B and 2B). When salts were applied by dipping after harvest the activity was in general less pronounced. With respect to the trials carried out on oranges, in most cases no significant differences were observed between treated fruit and water control (Fig. 1C). Only SC and PC were effective in reducing decay by approximately 71 and 73%, respectively, as compared to the water control. In the trials performed with clementines, SC, PB, and PC significantly reduced the percentage of rotted fruit by 80, 50, and 70%, respectively (Fig. 2C). The other salts (SB, CC, CCh, PS, and SS) did not influence decay development. In the postharvest treatment, none of the salt solutions had a phytotoxic effect on both citrus species. However, under particular environmental conditions characterized by high and persistent humidity, preharvest application of SC and PC induced little
Due to the high economic value of the harvested commodity, efficient disease control under commercial conditions is the most important requirement for a postharvest treatment, including control methods alternative to synthetic fungicides. Organic and inorganic salts are among the most promising substitutes for fungicides but, as reported above, some unsolved constraints still impede their practical use (Smilanick et al., 2008; Palou et al., 2008; Teixidó et al., 2010). Taking into account these considerations, we tested a strategy different from the one used for synthetic fungicides, based on pre- and postharvest application of salts. Since the trials were conducted on naturally occurring infections, disease incidence in the control treatments was not very high, even as an average of experiments conducted over a 3 year period. This could generate uncertainties in the interpretation of the data. In this regard, it is worth mentioning that in the area where trials were conducted the climatic conditions are generally not very conducive to Penicillium rots and a low level of disease incidence is normal. Moreover, it is well known that ‘Valencia late’ fruit are tough, ripening in a dry period, and therefore not prone to postharvest decay; clementines are more susceptible to decay but not as winter ripening oranges. In addition, with naturally occurring infection it is possible to test the efficacy of the control methods on every kind of infection, namely latent, quiescent, and incipient infections, not only on wound infections. Assessing the efficacy of candidate control means on natural infections is necessary and important when researchers are looking for a commercial application (Smilanick et al., 1997). Finally, to dispel all uncertainties it has to be considered that preharvest and double applications of some
K. Youssef et al. / Postharvest Biology and Technology 72 (2012) 57–63
8 Yeasts and yeast-like
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salts gave the same results as postharvest application of chemical fungicide, i.e. complete suppression of Penicillium rot. On the whole, for both citrus species, our results demonstrated that preharvest and combined applications of salts provided a significant reduction of the disease. In particular, some of them (sodium bicarbonate, sodium carbonate, and potassium carbonate) achieved the same protection level as with the standard chemical control (IMZ) applied after harvest. Also calcium chelate, potassium sorbate, and sodium silicate were effective when applied once (before harvest) or twice (before and after harvest). The efficacy of preharvest salts applications was very high, and from a statistical point of view it was not improved by further applications after harvest, except for calcium chloride. Salts applied in the field prior to harvest had a longer time to interact with the pathogen/fruit, as compared to the salts applied after harvest, thus affecting the pathogen inoculum density on fruit surface, the environment in the wound niche, and probably tissue resistance. Regarding the inoculum density, the filamentous fungal population on fruit treated
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Fig. 3. Populations of yeast and yeast-like fungi and filamentous fungi on the surface of ‘Valencia late’ oranges preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 4 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Population was evaluated at the end of storage. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
Fig. 4. Populations of yeast and yeast-like fungi and filamentous fungi on the surfaces of ‘Comune’ clementines preharvest (A), pre/postharvest (B), and postharvest (C) treated with salts, stored for two months at 6 ± 1 ◦ C followed by one week at 20 ± 2 ◦ C. Imazalil- and water-treated fruit were used as controls. Population was evaluated at the end of storage. Columns marked with the same letters are not statistically different by Fisher’s protected least significant difference test (P ≤ 0.05).
once in the field and with the double treatment had significantly lower values as compared to the water control, whereas no differences were observed for both species when salts were applied after harvest. This behavior can be ascribed to a direct activity of salts, as confirmed by our in vitro trials, except for those containing calcium. In these trials a MIC of 0.25% (w/v) was recorded for almost all salts, 10 times higher than the one recorded for IMZ, but equally interesting considering that salts are much cheaper and eco-friendly. The antifungal activity of carbonate, bicarbonate, sorbate, and silicate is well known (Palou et al., 2002; Smilanick et al., 2005; Ippolito et al., 2005; Nigro et al., 2006; Guo et al., 2007; Gregori et al., 2008; Youssef et al., 2010b), as well as the direct activity of carbonates and bicarbonates in inhibiting spore germination, germ tube elongation, and production of pectinolytic enzymes in several pathogens (Hervieux et al., 2002; Smilanick et al., 2005). Moreover, results reported herein seem to confirm that pH of salt solutions did not have a dominant role in the observed activity, as
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previously reported by Nigro et al. (2006). In fact, high disease reduction was obtained with pre- and postharvest applications of PC and SC (pH 11.4), but also with PB and SB (pH 8.4), and CCh (pH 7.7). The osmotic stress caused by the presence of salts in field applications may have contributed to the decrease in fungal populations (Elad and Kirshner, 1993; Ippolito et al., 2005). Osmotic stress should play a minor role in the case of postharvest application, where the RH is generally near the saturation point. The low influence of most of the tested salts on yeast and yeast-like populations is reported in the literature (Schena et al., 2003; Ippolito et al., 2005) and can be considered as an advantage since they are not pathogenic and may act as biocontrol agents (Janisiewicz and Korsten, 2002). In addition to the direct activity on the epiphytic fungal population, salts may have entered the wounds during infliction, leading to an interaction between salt residues and constituents of citrus rind, thus creating unfavorable environmental conditions for the pathogen, and possibly increasing tissue resistance. Indeed, specific experiments conducted by Youssef et al. (2010c) pointed out an indirect action of some salts mediated by tissue-induced resistance; in these trials, SB, SC, PC, and PB applied in wounds 5 mm apart from the pathogen-inoculated ones induced a significant reduction of disease severity (diameter of the lesion) as compared to the water control. Further trials have shown also an increase in the phytoalexins scoparone, scopoletin, and umbelliferone in SC and SB treated orange tissues (Youssef et al., 2011). Similarly, Venditti et al. (2005) reported that SC induced structural changes in cell wall, production of scoparone and an increase in pH of citrus rind tissues. Regarding the sodium silicate mode of action, a specific study showed that the activity of peroxidase and PAL was enhanced in treated melon tissues (Guo et al., 2007). In contrast, with regard to potassium sorbate, to the best of our knowledge, it seems that no specific studies have been conducted to verify its activity in enhancing citrus natural resistance. Our data showed that treatment with calcium chloride was effective against decay only when applied twice (before and after harvest), whereas, when applied once, either before or after harvest, it did not influence decay incidence as compared to the water control. The reduced efficacy of CC in preharvest applications was unexpected since its field application is effective against a number of pathogens on different hosts, including kiwifruit, cactus, pears, table grapes, and sweet cherries (Gerasopoulos et al., 1996; Schirra et al., 1999; Ippolito et al., 2005; Nigro et al., 2006). It should be considered that CC has a limited direct action on the pathogen (Nigro et al., 2006; Latifa et al., 2011) and its activity is mainly related to the stabilization of the host cell wall and middle lamella (Gerasopoulos et al., 1996; Miceli et al., 1999). It can be hypothesized that CC applied once on ripening fruit did not accumulate enough in the citrus peel to exert its activity. In the case of calcium chelate, which was effective for both species when applied before harvest and in double application, its particular chemical structure (chelate) may have allowed an accumulation in rind tissue sufficient to enhance its natural resistance against the pathogen (Lester and Grusak, 2004). In conclusion, our results have demonstrated that the incidence of postharvest decays on citrus fruit can be reduced by applying, a few days before harvest, a number of salts which are safe for consumers and the environment. This strategy may be a way of overcoming one of the main limitations of most of the alternative control means, i.e. the lack of curative effects (Spadaro and Gullino, 2004; Palou et al., 2008). In addition, their preventive effect can be of interest for packinghouses, avoiding heavy decay when harvested fruit are kept at room temperature before sorting and packaging. Salts can be applied using the same equipment as for synthetic fungicides, and being common food additives marketed at a convenient price, their use should be easily accepted by
operators and consumers. Less contaminated/infected fruit on packing lines should also reduce the demand for sanitizers and water during washing procedures. Practical preharvest application of salts needs to be further optimized as the obtainable level of protection is affected by various factors, including the chemical composition of the salt, citrus variety, environmental conditions prevailing during their application, etc. Regarding the last aspect, the slight phytotoxicity on clementine fruit treated in the field with sodium and potassium carbonate, observed only when heavy dew followed salt application, is difficult to explain. Probably the phytotoxicity should be ascribed to the prolonged contact and imbibitions of rind tissues with solutions having a high pH. Long interaction times and/or osmotic stress associated with preharvest treatments should have affected weight loss and fruit rind quality but the high RH during storage and the short shelf life have probably masked these effects. Finally, although our experiments clearly demonstrate the advantage of preharvest application of alternative control means, generally, farmers are not interested in the storage life of products once fruit are sold on the plant. In this regard, more than in the past, an integrated control approach along the entire production chain is necessary to obtain a high quality product. Acknowledgments The research has been realized with the contribution of the Emilia-Romagna Region (leader partner) within the interregional project “Frutticoltura post-raccolta” (L. 499/99) coordinated by CRPV. Thanks are expressed to the “Azienda Agricola Sperimentale Pantanello, ALSIA” Metaponto – (MT), Italy, directed by Dr. C. Mennone, in which the trials were conducted. References Cerioni, L., Rodriguez-Montelongo, L., Ramallo, J., Prado, F.E., Rapisarda, V.A., Volentini, S.I., 2012. Control of lemon green mold by a sequential oxidative treatment and sodium bicarbonate. Postharvest Biology and Technology 63, 33–39. Cunningham, N.M., 2010. Combinations of treatments to replace the use of conventional fungicides for the commercial control of postharvest diseases of citrus fruit. Stewart Postharvest Review 1, 1–8. Eckert, J.W., Eaks, I.L., 1989. Postharvest disorders and diseases of citrus fruits. In: Reuter, W., Calavan, E.C., Carman, G.E. (Eds.), The Citrus Industry, vol. 5. Univ. California Press, Berkeley, pp. 179–260. Elad, Y., Kirshner, B., 1993. Survival in the phylloplane of an introduced biocontrol agent (Trichoderma harzianum) and populations of the plant pathogen Botrytis cinerea as modified by biotic conditions. Phytoparasitica 21, 303–313. El-Ghaouth, A., Smilanick, J.L., Brown, G.E., Ippolito, A., Wilson, C.L., 2001. Control of decay of apple and citrus fruits in semicommercial tests with Candida saitoana and 2-deoxy-d-glucose. Biological Control 20, 96–101. Gerasopoulos, D., Chouliaras, V.E., Lionikas, S., 1996. Effects of preharvest calcium chloride sprays on maturity and storability of Hayward kiwifruit. Postharvest Biology and Technology 7, 65–72. Gregori, R., Borsetti, F., Neri, F., Mari, M., Bertolini, P., 2008. Effects of potassium sorbate on postharvest brown rot of stone fruit. Journal of Food Protection 71, 1626–1631. Guo, Y., Liu, L., Zhao, J., Bi, Y., 2007. Use of silicon oxide and sodium silicate for controlling Trichothecium roseum postharvest rot in Chinese cantaloupe (Cucumis melo L.). International Journal of Food Science and Technology 42, 1012–1018. Hervieux, V., Yaganza, E.S., Arul, J., Tweddell, R.J., 2002. Effect of organic and inorganic salts on the development of Helminthosporium solani, the causal agent of potato silver scurf. Plant Diseases 86, 1014–1018. Ippolito, A., Nigro, F., 2000. Impact of preharvest application of biocontrol agents on postharvest diseases of fresh fruit and vegetables. Crop Protection 19, 723–725. Ippolito, A., Schena, L., Pentimone, I., Nigro, F., 2005. Control of postharvest rots of sweet cherries by pre- and postharvest applications of Aureobasidium pullulans in combination with calcium chloride or sodium bicarbonate. Postharvest Biology and Technology 363, 245–252. Janisiewicz, W.J., Korsten, L., 2002. Biological control of postharvest diseases of fruits. Annual Review of Phytopathology 40, 411–441. Janisiewicz, W.J., Conway, W.S., 2010. Combining biological control with physical and chemical treatments to control fruit decay after harvest. Stewart Postharvest Review 6, 1–16. Larrigaudiere, C., Pons, J., Torres, R., Usall, J., 2002. Storage performance of clementines treated with hot water, sodium carbonate, and sodium bicarbonate dips. Journal of Horticultural Science & Biotechnology 77, 314–319.
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Smilanick, J.L., Mansour, M.F., Mlikota Gabler, F., Sorenson, D., 2008. Control of citrus postharvest green mold and sour rot by potassium sorbate combined with heat and fungicides. Postharvest Biology and Technology 47, 226–238. Smilanick, J.L., 2011. Integrated approaches to postharvest disease management in California citrus packinghouses. Acta Horticulturae 905, 145–148. Snowdon, A.L., 1990. A Colour Atlas of Post-Harvest Diseases and Disorders of Fruits and Vegetables, vol. 1. Wolfe Scientific, London, pp. 102–103. Spadaro, D., Gullino, M.L., 2004. State of the art and future prospects of the biological control of postharvest fruit diseases. International Journal of Food Microbiology 91, 185–194. ˜ I., 2010. Preharvest strateTeixidó, N., Usall, J., Nunes, C., Torres, R., Abadias, M., Vinas, gies to control postharvest diseases in fruits. In: Prusky, D., Gullino, M.L. (Eds.), Postharvest Pathology, Plant Pathology in the 21st Century, vol. 2. Springer, New York, pp. 89–106. Venditti, T., Molinu, M.G., Dore, A., Agabbio, M., D’hallewin, G., 2005. Sodium carbonate treatment induces scoparone accumulation, structural changes, and alkalinization in the albedo of wounded citrus fruits. Journal of Agricultural and Food Chemistry 53, 3510–3518. Youssef, K., Ligorio, A., Pentimone, I., Sanzani, S., Donghia, A.M., Nigro, F., Ippolito, A., 2008a. Studies on application strategy of salts and chitosan for control Penicillium rot of Hernandina clementine. In: Kaiser, H.K., Kirner, R. (Eds.), Proc. Junior Scientist Conference. Vienna, Austria, pp. 189–190. Youssef, K., Ligorio, A., Pentimone, I., Nigro, F., Ippolito, A., Yaseen, T., 2008b. Studies on application strategy of salts for controlling Penicillium rot of Valencia late oranges. In: Proc. 11th International Citrus Congress, vol. 2, Wuhan, China, pp. 1276–1280. Youssef, K., Ahmed, Y., Ligorio, A., D’Onghia, A.M., Nigro, F., Ippolito, A., 2010a. First report of Penicillium ulaiense as a new postharvest pathogen of citrus fruit in Egypt. Plant Pathology 59, 1174. Youssef, K., Di Primo, P., Coniglione, M., Ippolito, A., 2010b. Evaluation of antifungal activity of Bioprotege, a mixture of sodium bicarbonate and silicium dioxide, against major postharvest fungal pathogens of fruit and vegetables. Journal of Plant Pathology 92, 104. Youssef, K., Ligorio, A., Sanzani, S.M., Nigro, F., Ippolito, A., 2010c. Evaluation of direct activity and induction of resistance of some common food additives against green mould of citrus fruit. Journal of Plant Pathology 92, 67. Youssef, K., Sanzani, S.M., Ligorio, A., Terry, L.A., Ippolito, A., 2011. Biochemical changes associated with induced resistance on citrus fruit. Journal of Plant Pathology 93, 22. Youssef, K., Ligorio, A., Nigro, F., Ippolito, A., 2012. Activity of salts incorporated in wax in controlling postharvest diseases of citrus fruit. Postharvest Biology and Technology 65, 39–43.