Increasing maturity reduces wound response and lignification processes against Penicillium expansum (pathogen) and Penicillium digitatum (non-host pathogen) infection in apples

Penicillium expansum is the main postharvest pathogen of pome fruit and is a necrotrophic fungus that requires wounds to infect the fruit. Therefore, injuries caused during harvest and postharvest handling provide an optimal locus for infection. In this study, the effect of wound response in apples harvested at three different maturity stages and stored at two different temperatures (20 and 0 °C) infected with P. expansum (pathogen) and Penicillium digitatum (non-host pathogen) was evaluated. The effect of wounding and pathogen inoculation on lignin content was also quantified. At 20 °C, less decay incidence and severity were observed when time between wounding and inoculation increased, and these differences were more important in fruit from immature and commercial harvests. However, at 0 °C, wound response was too slow to prevent P. expansum infection. Lignin content was highest in fruit from the immature harvest. Our results indicated that maturity and storage temperature play an important role in apple wound response. This is the first report demonstrating that P. digitatum, a non-host pathogen, was able to develop rots in over-mature apples.     

Real time polymerase chain reaction for rapid and quantitative determination of Cystofilobasidium infirmominiatum on the surfaces of apple,pear, and sweet cherry fruit

The objectives of this study were to develop primers and a real time PCR protocol for the postharvest biocontrol yeast Cystofilobasidium infirmominiatum (Cim). The application of this technology was developed to quantify Cim on the surfaces of apple, two pear cultivars, and sweet cherry fruit treated over a range of concentrations. Statistically significant relationships were observed between Cim DNA on fruit surfaces, expressed as μg/m², and CFU/L of dip suspensions for apple, pear, and sweet cherry. In addition, the relationship for each fruit was significantly different from the other three fruits. Threshold values of concentrations of Cim DNA on the fruit surface were calculated based on regression equations and a dose of 2.0 × 10¹¹ CFU/L of dip suspension, the dose for optimum decay control, and were 4.8, 7.0, 16.5, and 25.2 μg/m² for Bosc pear, Lapins sweet cherry, d’Anjou pear, and Golden Delicious apple, respectively. Monitoring Cim DNA concentration on fruit surfaces will assure that Cim is being properly applied to fruit and that a sufficient number of cells are present for optimum decay control.     

Pathogen aggressiveness and postharvest biocontrol efficiency in Pantoea agglomerans

Pathogen aggressiveness on the host was studied as one of the influencing factors in the variability of the efficiency of biocontrol by Pantoea agglomerans. The effect of the relative dose of the pathogen and biocontrol agent (BCA) on efficacy of biocontrol was analyzed in six postharvest pathogens (Rhizopus stolonifer, Botrytis cinerea, Penicillium expansum, Monilinia laxa, Penicillium digitatum and Penicillium italicum), five fruit types (apple, pear, nectarine, strawberry, orange) and two strains of P. agglomerans. Median effective dose (ED₅₀) of the pathogens and of the BCAs was estimated by fitting data to a hyperbolic saturation model. The raw data required were either obtained from the literature or generated by the appropriate experiments. The ED₅₀ of the pathogens covered a range from 1 to 475 spores/wound and of the BCA strains ranged from 207 to 30,000 cfu/wound. The efficiency of the P. agglomerans strains was estimated as the ratio between the ED₅₀ for the BCA and the pathogen, and ranged from 7 to 25,000 cfu/spore. Low values indicate high efficiencies. A significant inverse relationship was observed between the efficiency of biocontrol and the ED₅₀ of the pathogen on the corresponding host, indicating that the higher the aggressiveness of the pathogen the lower the efficiency of the BCA. It is expected that this relationship can be extended to other postharvest biocontrol-pathogen systems.     

Postharvest pigmentation in red Chinese sand pears (Pyrus pyrifolia Nakai) in response to optimum light and temperature

The development of red color in the peel of red Chinese sand pears (Pyrus pyrifolia Nakai) is influenced by temperature and light; however, the response patterns vary among different cultivars. In this study, we systematically investigated the influence of postharvest treatment with various temperatures (low, high, variant and constant) on detached mature fruit of red Chinese sand pear ‘Mantianhong’ and ‘Meirensu’. Fruit of red apple (Malus domestica Borkh.) ‘Royal Gala’ and red European pear (P. communis L.) ‘Cascade’ received the same treatments for comparison. Furthermore, the effects of light quality and irradiance level on ‘Mantianhong’ pears were evaluated at the optimum temperature for anthocyanin accumulation. Fruit firmness and concentrations of total soluble sugars and organic acids were measured to determine fruit quality. The effect of temperature on red Chinese sand pear fruit color was similar to that of apples, but not European pear. Moreover, low temperature more effectively induced red coloration in ‘Mantianhong’ and ‘Meirensu’ pears than high temperature; anthocyanin levels increased with increasing irradiance level from 0 to 532 μmol m⁻² s⁻¹, and UV-B and visible light synergistically improved the red color of the fruit. Therefore, a combination of low temperature and high intensity of UV-B/visible light could improve the postharvest coloration of red sand pear fruit. The results will contribute to an improved understanding of the mechanism responsible for the coloration of red Chinese sand pears and will aid development of new techniques to improve color in postharvest fruit.     

Sensitivity of Penicillium expansum to diphenylamine and thiabendazole and postharvest control of blue mold with fludioxonil in ‘McIntosh’ apples

Penicillium expansum is one of the most important pathogens that cause blue mold in stored apples. Due to the development of resistance to the postharvest fungicide, thiabendazole (TBZ), an increase in blue mold has been observed in apple storages. The sensitivity of three TBZ-sensitive and three TBZ-resistant isolates of P. expansum to diphenylamine (DPA), scald inhibitor, was tested in vitro. Of the 94 isolates, collected later in the storage season, 41% were found resistant to both DPA and TBZ. To manage the fungicide resistance, a reduced-risk fungicide, fludioxonil, was tested against blue mold caused by TBZ-sensitive and -resistant P. expansum on ‘McIntosh’ apples treated with or without 1000 μg ml⁻¹ of diphenylamine. Fruit were assessed for disease and scald incidence during storage. Diphenylamine controlled scald in treated fruit. Higher disease incidence of blue mold was observed in apples treated with diphenylamine and low concentrations of fludioxonil (3, 5, and 75 μg ml⁻¹). DPA neither positively nor negatively affected the control of blue mold when DPA was applied together with 150, 300 and 600 μg ml⁻¹ of fludioxonil during 12 weeks of storage at 4 °C.     

UV-C light inactivation kinetics of Penicillium expansum on pear surfaces: Influence on physicochemical and sensory quality during storage

UV-C inactivation kinetic data of Penicillium expansum on intact and wounded pear disks were determined. P. expansum conidia (0.5 mL, 1.6 × 10⁷ CFU/mL) were spot inoculated onto intact and wounded pear tissue with skin (excised disks), treated with UV-C doses ranging 0.101–3.06 kJ/m² at 23 °C and surviving conidia were enumerated. Changes in selected physicochemical parameters and sensory quality following UV-C treatment of whole pears were determined immediately after treatment, and 4 and 8 weeks of storage at 4 °C. A greater UV-C intensity was required for similar inactivation levels of P. expansum populations on wounded pear disks (3.1 kJ/m² for 2.7 log reduction) compared to intact pear disks (1.7 kJ/m² for 2.8 log reduction). No significant difference in % weight loss, or soluble solids content and texture was observed between UV-C treated and untreated pears. However, browning was observed on UV-C treated pear surfaces after 4 and 8 weeks along with changes in flavor and texture. An increase in consumer preference was noticed for the untreated control pears after 4 weeks storage.     

Induction of H2O2-metabolizing enzymes and total protein synthesis by antagonistic yeast and salicylic acid in harvested sweet cherry fruit

The immersion of sweet cherry fruit in Pichia membranefaciens at a concentration of 5 × 10⁷ cells ml⁻¹ or in salicyclic acid (SA) at 0.5 mM for 10 min reduced the incidence of decay and lesion size caused by Penicillium expansum. Without pathogen inoculation, peroxidase (POD) activity was enhanced in yeast-treated fruit, but activities of catalase (CAT) and superoxide dismutase (SOD) showed a decrease in the same fruit. SA-treatment significantly inhibited CAT activity, but stimulated SOD and POD activities. After inoculation with P. expansum, CAT activity decreased and SOD activity increased in both yeast- and SA-treated fruit. No obvious difference was found in POD activity between treatments and water control. Treatments with yeast and SA changed the expression of POD isozymes. In addition, yeast and SA treatment increased total protein content of sweet cherry and up-regulated 33 and 47 kDa protein bands shown by SDS-PAGE. These results indicated that yeast- and SA-treatments induced synthesis of anti-oxidant enzymes and specific proteins, which may play a role in the resistance against postharvest blue mold.     

Wound response in orange as a resistance mechanism against Penicillium digitatum (pathogen) and P. expansum (non-host pathogen)

Penicillium digitatum is the most devastating postharvest pathogen of citrus. In addition, Penicillium expansum is the main pathogen of pome fruit, although recent studies have demonstrated its ability to infect oranges under some conditions. In this study, we evaluated wound response in ‘Valencia’ oranges harvested at three different maturity stages and the effect of wound response on the establishment of both pathogens when fruit were stored at two different temperatures (20 and 4 °C). The effect of wounding and pathogen inoculation on lignin content, was also quantified. Lastly, the expression of several phenylpropanoid pathway-related genes was also analyzed by semi-quantitative RT-PCR. Results indicated that, in general, P. digitatum exhibited lower decay incidence and severity as time between wounding and inoculation increased. Decay incidence and severity were higher in fruit from the over-mature harvest than in fruit from immature and commercial harvests. P. expansum was able to infect fruit at 20 °C but lesions were small compared to lesion size of fruit stored at 4 °C. Lignin content in wounded fruit (control) and in samples wounded and inoculated with P. expansum was highest in fruit from the immature harvest at 7 d post-wounding and inoculation. Wounded fruit had higher expression of pal1, comt1 and pox1 genes at 48 h than at 24 h. However, samples inoculated with P. digitatum showed lower expression at 48 h than at 24 h. Our results indicated that maturity and storage temperature play an important role in orange wound response.     

Mechanisms of action and efficacy of four isolates of the yeast Metschnikowia pulcherrima active against postharvest pathogens on apples

The mechanisms of action and efficacy of four isolates (GS37, GS88, GA102, and BIO126) of the yeast Metschnikowia pulcherrima against Botrytis cinerea, Penicillium expansum, Alternaria sp., and Monilia sp., all postharvest pathogens of apple fruit, were studied in vitro and on apples, in controlled and semi-commercial conditions. An application of a cell suspension (10⁸ cells per ml) of the antagonists in artificial wounds of apples reduced growth of B. cinerea and P. expansum after storage at 23 °C. A complete suppression of the pathogen was obtained against Monilia sp., stored at 23 °C, and against B. cinerea and P. expansum, stored at 4 °C. The results against Alternaria sp. were more variable. Applications of culture filtrates and autoclaved cells of the isolates were ineffective in reducing the diameter of the lesions on the fruit, supporting the hypothesis that living cells are necessary for biocontrol. In experiments of antagonism in vitro, on different solid substrates, a reduction in the mycelium growth of the pathogens resulted, so that, at least in vitro, the antagonists could produce some diffusible toxic metabolites. In co-cultivation in vitro on a synthetic medium, B. cinerea spore (10⁵ ml⁻¹) germination was completely inhibited by the presence of 10⁸ cells of the antagonists, while culture filtrates and autoclaved suspensions were not able to reduce germination. Dipping boxes of apples cv. Golden delicious in a suspension of 10⁷ antagonist cells per ml and storing for 8 months in controlled atmosphere at 1 °C, showed levels of control against B. cinerea and P. expansum similar to those from thiabendazole.     

Pre-storage hypobaric treatments delay fungal decay of strawberries

Fungal decay is a major cause of postharvest losses in strawberries. The traditional approach for controlling fungal decay is the use of fungicides. However, the use of fungicides has been questioned as a sustainable and safe method, and is also prohibited in many countries. One potential physical method for reducing fungal decay is application of a short-term hypobaric treatment prior to storage. In this study efficacy of postharvest hypobaric treatments to control natural rot development in strawberries was evaluated. Strawberries were treated with hypobaric pressures (25 kPa_a, 50 kPa_a and 75 kPa_a) for 4 h at 20 °C and subsequently stored at 20 °C or 5 °C. A 50 kPa_a treatment consistently delayed rot development in samples stored at either temperature confirming that the technique has potential as a non-chemical treatment. Moreover 50 kPa_a treatments did not affect weight loss and firmness at either 20 °C or 5 °C. An initial increase in respiration rate was observed in 50 kPa_a treated samples potentially indicating mild stress due to hypobaric treatment. An in vitro fungal study found that 50 kPa_a treatment for 4 h did not affect the rate of radial growth of colonies of Botrytis cinerea and Rhizopus stolonifer, providing further evidence that the potential mechanism of hypobaric treatment is induction of the defence system within the fruit rather than a direct effect on fungal viability. Further molecular and biochemical research is required to evaluate the possible stimulation of resistance in fruit through short-term hypobaric treatments.     

Methyl jasmonate induces resistance against Penicillium citrinum in Chinese bayberry by priming of defense responses

Methyl jasmonate (MeJA) can act as an activator of defense responses in plants against pathogenic infection. However, the mechanisms involved in the postharvest induction of resistance by MeJA in fruit are largely unknown. Thus, we investigated the effect of a postharvest MeJA treatment on disease resistance against Penicillium citrinum infection in Chinese bayberries and the possible mechanisms. The results indicated that treatment with 10 μmol L⁻¹ of MeJA significantly inhibited green mould rot caused by P. citrinum, with the decay incidence being 66.2% lower than that of the control fruit after storage at 1 °C for 8 d. Moreover, it is clear that MeJA triggers a priming mechanism in Chinese bayberries, since only the MeJA-treated fruit showed an enhanced capacity to augment defense responses upon challenge with the pathogen. These augmented responses included an H₂O₂ burst, enhanced protein levels of phenylalanine ammonia-lyase and chitinase, and accumulation of phenolic compounds, lignin and phytoalexin. Therefore, our results suggest that a postharvest MeJA treatment induces disease resistance against P. citrinum in Chinese bayberries by priming of defense responses.     

UV-B irradiation retards chlorophyll degradation in lime (Citrus latifolia Tan.) fruit

Peel yellowing is a major postharvest problem of lime fruit. Research was conducted to control peel yellowing by UV-B irradiation. Mature green lime fruit were irradiated with UV-B doses at 0 (control), 8.8, and 13.2 kJ m^?2 and then stored at 25 °C in darkness. UV-B treatment at 8.8 kJ m^?2 efficiently delayed the decrease of chlorophyll content. A high level of chlorophyllide a accumulated in mature green fruit and then gradually decreased with the progress of peel yellowing. The chlorophyllide a level was higher in 8.8 kJ m^?2 UV-B-treated fruit than it was in the controls. The pheophorbide a level declined in lime fruit treated with 8.8 kJ m^?2 UV-B, especially during the development of yellowing. In addition, the pheophytin a level increased by 8.8 kJ m^?2 UV-B treatment at the late period of storage. We concluded that UV-B treatment effectively suppressed chlorophyll degradation in mature green lime during storage, which suggests that UV-B irradiation is a usable method for prolonging the postharvest life of lime fruit.     

Control of blue mold of apple by combining controlled atmosphere,an antagonist mixture,and sodium bicarbonate

‘Golden Delicious’ apples were wound-inoculated with Penicillium expansum and treated with various combinations of sodium bicarbonate and two antagonists (Metschnikowia pulcherrima, Cryptococcus laurentii), and then stored in air or controlled atmosphere (CA = 1.4 kPa O₂ and 3 kPa CO₂) for 2 or 4 months at 1 °C. The antagonists survived and their populations increased during both air and CA storage. The antagonists alone reduced blue mold but were more effective when combined. Sodium bicarbonate tended to reduce lesion size when used with these antagonist, either when they were used alone or combined. Storage under CA conditions also increased the effectiveness of both antagonist, when used alone or in combination. The only treatment that completely eliminated P. expansum-incited decay was the combination of the two antagonists and sodium bicarbonate on fruit stored under CA conditions. The proper combination of alternative control measures can provide commercially acceptable long-term control of fruit decay and could help reduce our dependency on fungicides.     

NIR spectroscopy for the optimization of postharvest apple management

Apples can be stored for long time under controlled temperature and atmosphere conditions, and therefore, non-destructive and rapid tools are required to assess fruit quality and to monitor changes during the postharvest period. The aim of this study was to evaluate the feasibility of NIR spectroscopy to optimize postharvest apple management and to follow changes in fruit quality during storage. An FT-NIR system operating in diffuse reflectance in the range 12,500–3600 cm⁻¹ was used to evaluate the physico-chemical (dry matter, soluble solids, colour and firmness) and some nutraceutical characteristics (total phenolics, total flavonoids and antioxidant activity) of ‘Golden Delicious’ apples, which were stored for about six months at 1 °C in controlled atmosphere, over two subsequent years. Spectral data were elaborated by PLS regression and LDA classification techniques. Good correlation models between spectral data and chemical and physical parameters were obtained for soluble solids, a* colour coordinate and firmness (0.81 < R² < 0.90 in calibration and 0.79 < R² < 0.89 in cross validation). Even higher correlation values (0.89 < R² < 0.95 in calibration and 0.86 < R² < 0.92 in cross validation) were obtained for indexes correlated to the antioxidant capacity of apples. The classification technique Linear Discriminant Analysis was applied to spectral data, in order to discriminate apples on the basis of storage time. Average correct classification was higher than 93% in validation and close to 100% in calibration, indicating high potential of NIR spectroscopy for the estimation of storage time of apple lots.     

Integration of pre- and postharvest treatments for the control of black spot caused by Alternaria alternata in stored persimmon fruit

In Israel, black spot caused by Alternaria alternata is the main postharvest factor that impairs the quality and reduces the storability of persimmon fruit (Diospyros kaki cv. Triumph). The fungus infects the fruit in the orchard and remains quiescent until harvest. After harvest, the pathogen slowly colonizes the fruit during storage at 0 °C, which elicits black spot symptom development 2–3 months after storage entry. A commercial postharvest dip treatment in chlorine at 500 mg L^?1, released from sodium troclosene tablets, effectively controlled black spot in fruit stored for up to 2 months. However, decay incidence increased as the length of storage was extended beyond 2.5 months. The long incubation period that precedes black spot symptom development after harvest enabled the development of a series of integrative approaches for application at the pre- and postharvest stages, in combination with the commercial chlorine dip treatment, to improve the control of black spot disease. Preharvest treatments included treatment with the cytokinin-like N₁-(2-chloro-4-pyridyl)-N₃-phenylurea (CPPU) 30 d after fruit set, or a single spray with the curative fungicide polyoxin B 14 d before harvest, and when one of these was applied in combination with the postharvest chlorine dip treatment, the black spot infected area was reduced by 3 and 60%, respectively, compared with the chlorine dip alone. At the postharvest stage, fogging during storage, or post-storage on-line spraying with sodium troclosene, when applied in combination with the postharvest chlorine dip, improved the percentage of marketable fruit by 2 or 10%, respectively, compared with the chlorine dip alone. The results indicate that postharvest pathogens that show a slow colonization pattern might enable the integration of pre- and postharvest disease control methods to improve quality and reduce postharvest disease development.     

Combination of hot water,Bacillus amyloliquefaciens HF-01 and sodium bicarbonate treatments to control postharvest decay of mandarin fruit

An antagonistic isolate Bacillus amyloliquefaciens HF-01, sodium bicarbonate (SBC) and hot water treatment (HW) were investigated individually and in combination against green and blue mold and sour rot caused by Penicillium digitatum, P. italicum and Geotrichum citri-aurantii respectively, in mandarin fruit. Populations of antagonists were stable in the presence of 1% or 2% SBC treatment, and spore germination of pathogens in potato dextrose broth was greatly controlled by the hot water treatment of 45 °C for 2 min. Individual application of sodium bicarbonate at low rates and hot water treatment, although reducing disease incidence after 8 weeks or 4 weeks of storage at 6 °C or 25 °C respectively, was not as effective as the fungicide treatment. The treatment comprising B. amyloliquefaciens combined with 2% SBC or/and HW (45 °C for 2 min) was as effective as the fungicide treatment and reduced decay to less than 80% compared to the control. B. amyloliquefaciens HF-01 alone or in combination with 2% SBC or/and HW significantly reduced postharvest decay without impairing fruit quality after storage at 25 °C for 4 weeks or at 6 °C for 8 weeks. These results suggest that the combination of B. amyloliquefaciens HF-01, SBC and HW could be a promising method for the control of postharvest decay on citrus while maintaining fruit quality after harvest.     

Essential oil vapours suppress the development of anthracnose and enhance defence related and antioxidant enzyme activities in avocado fruit

Anthracnose caused by Colletotrichum gloeosporioides is a major postharvest disease in avocados that causes significant losses during transportation and storage. Complete inhibition of the radial mycelia growth of C. gloeosporioides in vitro was observed with citronella or peppermint oils at 8 μL plate⁻¹ and thyme oil at 5 μL plate⁻¹. Thyme oil at 66.7 μL L⁻¹ significantly reduced anthracnose from 100% (untreated control) to 8.3% after 4 days, and to 13.9% after 6 days in artificially wounded and inoculated ‘Fuerte’ and ‘Hass’ fruit with C. gloeosporioides. GC/MS analysis revealed thymol (53.19% RA), menthol (41.62% RA) and citronellal (23.54% RA) as the dominant compounds in thyme, peppermint and citronella oils respectively. The activities of defence enzymes including chitinase, 1, 3-β-glucanase, phenylalanine ammonia-lyase and peroxidase were enhanced by thyme oil (66.7 μL L⁻¹) treatment and the level of total phenolics in thyme oil treated fruit was higher than that in untreated (control) fruit. In addition, the thyme oil (66.7 μL L⁻¹) treatment enhanced the antioxidant enzymes such as superoxide dismutase and catalase. These observations suggest that the effects of thyme oil on anthracnose in the avocado fruit are due to the elicitation of biochemical defence responses in the fruit and inducing the activities of antioxidant enzymes. Thus postharvest thyme oil treatment has positive effects on reducing anthracnose in avocados.     

Evaluation of postharvest treatments with chemical resistance inducers to control green and blue molds on orange fruit

Preventive and curative activities of postharvest treatments with selected chemical resistance inducers to control postharvest green (GM) and blue (BM) molds on oranges (cvs. ‘Valencia’ or ‘Lanelate’) artificially inoculated with Penicillium digitatum and Penicillium italicum, respectively, were evaluated. In vivo primary screenings to select the most effective chemicals and concentrations were performed with benzothiadiazole (BTH), β-aminobutyric acid (BABA), 2,6-dichloroisonicotinic acid (INA), sodium silicate (SSi), salicylic acid (SA), acetylsalicylic acid (ASA) and harpin. INA at 0.03 mM, SA at 0.25 mM, BABA at 0.3 mM and BTH at 0.9 mM were selected and tested afterwards as dips at 20 °C for 60 or 150 s with oranges artificially inoculated before or after the treatment and incubated for 7 d at 20 °C. Although it was an effective treatment, SSi at 1000 mM was discarded because of potential phytotoxicity to the fruit rind. Preventive or curative postharvest dips at room temperature had no effect or only reduced the development of GM and BM very slightly. Therefore, these treatments cannot be recommended for inclusion in postharvest decay management programs for citrus packinghouses.