Some factors influencing patulin production by Penicillium expansum in pome fruits

BACKGROUND: The objective of our study was to examine the effects of Penicillium expansum on patulin production in relation to isolates, species and cultivar type, incidence and severity of decay. In addition, patulin production at different incubation times and its diffusion were also investigated. These factors were evaluated in pome fruits inoculated with P. expansum and kept at 20 °C for short periods of time. RESULTS: The ability of five P. expansum isolates to grow and produce patulin in inoculated Golden Delicious apples varied among the strains from below the limit of quantification to 662 µg kg^?1. Variety and species of pome fruits influenced patulin production. P. expansum isolate PE97.IT produced a higher patulin content in apples than in pears. The highest patulin production was 386 µg kg^?1 in Golden Delicious. No blue mould symptom appeared in pears inoculated with P. expansum and no patulin was detected after 3 days at 20 °C. However, patulin increased with incubation time after 6 and 8 days. No patulin was detected in healthy pear tissue but it was high in the decayed area. CONCLUSION: Since patulin production is associated primarily with infected rotten tissue, patulin control is possible by using healthy fruits, sorting damaged and rotten fruits before processing. Copyright © 2010 Society of Chemical Industry     

Biocontrol ability and action mechanisms of Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 against Penicillium digitatum DSM2750 and Penicillium expansum DSM62841 causing postharvest diseases

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 10⁶ and 10⁸ cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 10⁸ cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.     

Inhibitory effect and mechanism of curcumin-based photodynamic inactivation on patulin secretion by Penicillium expansum

Patulin (PAT) contamination caused by Penicillium expansum growth in postharvest fruit brings safety problems. This study investigated the inhibitory effect and mechanism of curcumin (CUR)-based photodynamic inactivation (PDI) on PAT secreted by P. expansum in vitro using a multifunctional light source instrument. After being treated with photosensitive CUR, the growth of mycelium was completely inhibited on potato dextrose agar medium, and the maximum PAT reduction of 93.06% can be achieved (330.0 J/cm², 300 μM CUR) in liquid medium. The inherent mechanism was that PDI causes the reactive oxygen species accumulation in cells, which destroyed the antioxidant defense system and induced severe oxidative damage, resulting in the loss of cell integrity. Furthermore, mycelial apoptosis caused up-regulation of five genes, including PatB, PatE, PatG, PatI, and PatL, but down-regulated ten genes in the PAT biosynthetic pathway. These findings suggest that CUR-based PDI can inhibit fungal infection and toxin secretion.Industrial relevanceThis study found that photosensitized curcumin can significantly inhibit mycelial growth and the secretion of patulin by Penicillium expansum. Curcumin-based photodynamic inactivation has the feasibility of the application on fresh fruits and vegetables, such as apples or oranges, and can avoided excessive waste of resources in picking, transporting, storing, and selling.     

Control of <Emphasis Type="Italic">Penicillium Expansum</Emphasis> and <Emphasis Type="Italic">Botrytis Cinerea</Emphasis> on Apple Fruit by Mixtures of Bacteria and Yeast

Antagonistic activity of the mixtures Rahnella aquatilis–Rhodotorula glutinis and R. aquatilis–Cryptococcus laurentii was assessed against Penicillium expansum (cause of blue rot) and Botrytis cinerea (cause of grey rot) on apple fruit at 4 °C and 95% relative humidity (RH). Under these cold-storage conditions, the mixture R. aquatilis–R. glutinis inhibited the development of B. cinerea and P. expansum in apples stored for 40 days and reduced the incidence of disease produced by these moulds to nearly zero. The other mixture, R. aquatilis–C. laurentii, was less effective; the incidences of the grey rot and the blue rot were about 25% and 15%, respectively. Population dynamics of the mixtures showed that the growth of R. aquatilis was strongly stimulated by the presence of the yeast R. glutinis, but in the case of the mixture R. aquatilis–C. laurentii, the same effect could not be observed. In this study, it was demonstrated that the combination of two microorganisms with different requirements and antagonistic abilities resulted in a successful mixture against the two pathogen molds, B. cinerea and P. expansum. In addition, it was also proved that it is possible to improve the biocontrol of these pathogens without increasing the inoculum size of the antagonist (alone or in mixtures), which was always 10⁶cell/ml, despite the high concentration of the pathogen (10⁶ conidia/ml) utilized.     

Intraspecific variability of growth and patulin production of 79 Penicillium expansum isolates at two temperatures

Penicillium expansum is the main species responsible for patulin production in apples and pears. Generally, fruit is stored at suboptimal conditions for mould growth and this situation could influence on the intra-species variability in both capability for growth and mycotoxin production. The aim of this research was to assess the impact of suboptimal environmental conditions on the intra-specific variability of P. expansum growth and patulin production using seventy nine isolates of this mould. Petri dishes with Apple Concentrate Agar Medium (ACAM) were inoculated centrally and incubated at two temperatures, one near optimal (20 °C) and the other representative of suboptimal cold storage (1 °C). For each condition, 10 Petri dishes were inoculated, and colony growth and patulin production was measured over time. The Kruskal-Wallis test revealed significant differences among growth rate (μ) and lag phase (λ) within the seventy nine assayed isolates. Coefficients of variation revealed a wider dispersion of μ (mm/day) and λ (days) at 1 °C compared with 20 °C. There were significant differences (p < 0.05) among patulin levels (ng/mm²) for the different conditions, values being lower at the lower temperature. Coefficients of variation revealed a wider dispersion of mycotoxin production at 1 °C. In order to address the strain variability in growth initiation and prove the well-established notion of reducing patulin production in foods by preventing fungal growth, a greater number of strains should be included.     

Combined effects of potassium sorbate,hot water and thiabendazole against green mould of citrus fruit and residue levels

Postharvest treatments of potassium sorbate only controlled recently established infections of Penicillium digitatum on Femminello siracusano lemons but did not confer any persistent protection. The loss of efficacy of potassium sorbate to control green mould decay was related to its irregular deposition on the fruit surface, as revealed by environmental scanning electron microscopy of oranges, and to the brief persistence of potassium sorbate residues. When treatment was done at 53 °C, the co-application of potassium sorbate with thiabendazole reduced thiabendazole residues in Moro and Sanguinello oranges, compared to thiabendazole treatment alone. However, treatment efficacy against two isolates of P. digitatum (thiabendazole-sensitive and thiabendazole-resistant) notably improved, indicating that potassium sorbate and hot water potentiated thiabendazole activity. Potassium sorbate residues remarkably decreased during fruit storage and were not affected by the co-application of thiabendazole.     

Biological control of postharvest green mold decay of oranges by Rhodotorula glutinis

The biocontrol activity of Rhodotorula glutinis on green mold decay of oranges caused by Penicillium digitatum was investigated in vitro and in vivo. Significant control was achieved with a washed cell suspension and an unwashed cell culture mixture of R. glutinis. Treatment of wounds with autoclaved cell cultures or cell-free culture filtrate did not prevent decay. The protection provided by the washed yeast cells was dose-dependent. The higher the concentration of R. glutinis, the better the effect of the biocontrol capacity. At concentrations of yeast of 1×10⁹ colony-forming units per milliliter or higher and pathogen spore suspensions of 5×10⁴ spores per milliliter, green mold was almost inhibited after 4-days incubation at 20 °C. The interval between the pathogen inoculation and the antagonist application significantly influenced the biocontrol ability. The biocontrol efficacy of R. glutinis applied before the pathogen was better than that of applied after the pathogen. Surprisingly, R. glutinis was also effective in controlling green mold at low temperature (4 °C). Rapid colonization of the yeast in wounds was observed during the first 3 days at 20 °C, and remained stable after 5-days incubation. On fruits stored at 4 °C, even after 21 days, the population of R. glutinis in wounded fruits was more than 1,600-fold of what it was just prior to storage. In the test on potato dextrose agar plates, agar disks of R. glutinis nutrient yeast dextrose agar cultures placed on PDA plates seeded with pathogens did not inhibit the growth of P. digitatum. Spore germination of pathogens in potato dextrose broth was greatly controlled in the presence of living cell suspensions.     

Combination of Pichia membranifaciens and ammonium molybdate for controlling blue mould caused by Penicillium expansum in peach fruit

The potential enhancement of Pichia membranifaciens by ammonium molybdate (NH₄Mo) to control blue mould caused by Penicillium expansum on peach fruit was investigated. Combining P. membranifaciens at 1 × 10⁸ cell/ml with 1 mM NH₄Mo provided a more effective control of blue mould rot than applying the yeast or NH₄Mo alone. Addition of 1 mM NH₄Mo significantly increased the growth of P. membranifaciens in peach wounds, but did not affect the population in nutrient yeast dextrose broth medium. The in vitro experiment showed that the combined treatment inhibited spore germination and germ tube elongation of P. expansum in comparison with the treatment of P. membranifaciens or NH₄Mo alone. Moreover, P. membranifaciens, NH₄Mo, and the combination of them did not impair the quality parameters including fruit firmness and content of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of NH₄Mo is a useful approach to improve the efficacy of P. membranifaciens for postharvest disease control in peach fruit.     

Comparative study of flavonoid and scoparone accumulation in different Citrus species and their susceptibility to Penicillium digitatum

Fungi of the genus Penicillium are responsible for substantial post-harvest losses in Citrus fruits. The results obtained following artificial inoculation of Citrus fruits with Penicillium digitatum showed that the degree of fungal development depended on the Citrus species. Thus, the mature fruit of Citrus paradisi were more susceptible to this fungus than the mature fruit of Citrus limon, Citrus sinensis,Citrus clementina Hort. ex Tan., and Citrus unshiu (mak) Marc. The results point to an inverse correlation between the degree of susceptibility of Citrus species to this fungus and the flavanone content – hesperidin in C. sinensis, C. clementina Hort. ex Tan. and C. unshiu (mak) Marc.; naringin in C. paradisi and the flavanone hesperidin and the flavone diosmin in C. limon. Thus, in C. sinensis,C. clementina Hort. ex Tan and C. unshiu (mak) Marc. the highest levels of the polymethoxyflavones, sinensetin, tangeretin, heptamethoxyflavone and nobiletin, were observed in the least susceptible varieties and viceversa. In the case of C. paradisi, no significant differences were detected in the polymethoxyflavone levels between varieties, while in C. limon, polymethoxyflavones were hardly detectable. The production of scoparone was observed in all the species and varieties studied after inoculation with the fungus, especially in C. limon fruits. Based on the evidence, it seems that flavanones, flavones, polymethoxyflavones (phytoanticipins) and scoparone (a phytoalexin) may well be involved in the defence mechanisms of Citrus fruits against P. digitatum. Depending on the Citrus species in question, the relative participation of one group of secondary compounds or another may vary.     

Efficacy of killer yeasts in the biological control of Penicillium digitatum on Tarocco orange fruits (Citrus sinensis)

Killer Saccharomyces cerevisiae and Wickerhamomyces anomalus yeast strains were tested as biocontrol agents against Penicillium digitatum, one the most important causes of postharvest decay in orange fruits.     

Use of High-Intensity Ultrasound to Increase the Efficiency of Imazalil in Postharvest Storage of Citrus Fruits

Imazalil (IMZ) plays a key role in the storage and marketing of Citrus fruits. It represents a very important tool in the fight against postharvest pathogens and is an efficient system for reducing economic losses due to the development of rots from Penicillium digitatum and Penicillium italicum. However, the current technologies employed to apply the fungicide by drenching, spraying, or dipping are ever much less suitable and progressive restrictions by law limit the residue level on the fruit and in the waste-water. A critical analysis of the factors affecting imazalil efficacy in postharvest preservation of Citrus fruit suggests that ultrasound could be useful to overcome these drawbacks. Data obtained confirmed a faster and deeper uptake of the fungicide in mandarins and lemons when the fruits were dipped in sonicated imazalil mixtures compared to the un-sonicated ones, allowing reduction of the dose of application and duration of the treatment. Indeed dipping for 1 min in 500 mg?L^?1 IMZ sonicated mixture produced in mandarins the same residue level of dipping for 5 min in 1,000 mg?L^?1 IMZ mixture without ultrasound. Moreover, imazalil concentration in fruit after sonication did not exceed the maximum residue limit allowed by the law. Sonication did not affect the chemical parameters, weight loss, and rind color of the fruit. No damage on the fruit surface was observed after scanning electron microscopy analyses of the rind of sonicated fruit. The data obtained open new interesting perspectives in the development of new postharvest treatment technologies for Citrus fruit.     

Original article: Antifungal activities of cinnamon oil against Rhizopus nigricans,Aspergillus flavus and Penicillium expansum in vitro and in vivo fruit test

The postharvest pathogens such as R. nigricans, A. flavas and P. expansum are the causal agents of jujube or orange fruit, therefore, in vitro and in vivo antifungal activities of cinnamon oil to inactivate these fungi were investigated. Cinnamaldehyde is the main constituent of cinnamon oil. The minimum inhibitory concentrations of cinnamon oil against Rhizopus nigricans, Aspergillus flavus and Penicillium expansum were 0.64% (v/v), 0.16% (v/v) and 0.16% (v/v), respectively. The antifungal activity of cinnamon oil against A. flavus and P. expansum was stronger than that against R. nigricans and the activity was improved with increasing its concentration. In an in vivo study, cinnamon oil with concentrations of 2.0% (v/v) and 3.0% (v/v) showed complete control the growth of fungi in wound‐inoculated Lingwu Long Jujube and Sand Sugar Orange fruits. These results revealed that cinnamon oil has a good potential to be as a natural antifungal agent for fruit applications.     

Coating citrus (Murcott tangor) fruit with low molecular weight chitosan increases postharvest quality and shelf life

This study investigates the effects of coating with low molecular weight chitosan (LMWC, Mw = 15 kDa) and high molecular weight chitosan (HMWC, Mw = 357 kDa) on the decay of Murcott tangor and the maintenance of its quality. A 0.1% LMWC coating substantially slowed the decay of Murcott tangor stored at 15 °C in relation to a control sample and reduced decay by over 20% as compared to the fungicide TBZ. A concentration of 0.2% LMWC was more effective in controlling the growth of fungus on citrus fruits caused by Penicillium digitatum and Penicillium italicum, exhibiting effective antifungal activity. LMWC coating improved firmness, titratable acidity, ascorbic acidity and the water content for Murcott tangor stored at 15 °C for 56 days. Consequently, Murcott tangor coated with LMWC exhibited greater antifungal resistance than TBZ and its quality was maintained for longer.     

Biocontrol of postharvest gray and blue mold decay of apples with Rhodotorula mucilaginosa and possible mechanisms of action

The efficacy of Rhodotorula mucilaginosa against postharvest gray mold, blue mold and natural decay development of apples and the possible mechanisms involved were investigated. The decay incidence and lesion diameter of gray mold and blue mold of apples treated by R. mucilaginosa were significantly reduced compared with the control fruits, and the higher concentration of R. mucilaginosa, the better the efficacy of the biocontrol. R. mucilaginosa also significantly reduced the natural decay development of apples following storage at 20 °C for 35 days or at 4 °C for 45 days followed by 20 °C for 15 days. Germination and survival of spores of Penicillium expansum and Botrytis cinerea were markedly inhibited by R. mucilaginosa in an in vitro test. Rapid colonization of the yeast in apple wounds was observed whether stored at 20 °C or 4 °C. In apples, the activities of peroxidase (POD) and polyphenoloxidase (PPO) were significantly induced and lipid peroxidation (malondialdehyde (MDA) content) was highly inhibited by R. mucilaginosa treatment compared with those of the control fruits. All these results indicated that R. mucilaginosa has great potential for development of commercial formulations to control postharvest pathogens on fruits. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, PPO and inhibition of lipid peroxidation (MDA content) of apples, so as to enhance the resistance and delay the ripening and senescence of apples.     

Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against Penicillium digitatum

Citrus peel is rich in flavanone glycosides and polymethoxyflavones. In view of their importance for industrial application as well as for their pharmacological properties, their content was analyzed in the mature fruits of several Citrus paradisi (grapefruit) and Citrus sinensis (orange) varieties, with a view to select the most interesting for isolation. The results shows that the Star Ruby grapefruit and the Sanguinelli orange stand out for their high contents of naringin and hesperidin, respectively. The presence of the polymethoxyflavones nobiletin, heptamethoxyflavone and tangeretin, could be ascertained in all the grapefruit varieties analysed. Higher polymethoxyflavone levels were recorded in orange, with Valencia Late showing the greatest nobiletin, sinensetin and tangeretin contents and Navelate the highest heptamethoxyflavone levels. An in vitro study revealed that these compounds acted as antifungal agents against Penicillium digitatum, the polymethoxyflavones being more active than the flavanones in this respect. The possible participation of these phenolic compounds in the defence mechanism of Citrus against P. digitatum is discussed.     

Inactivation of Penicillium expansum spores in apple juice by contact glow discharge electrolysis and its related mechanism

Penicillium expansum spores in apple juice were treated with contact glow discharge electrolysis (CGDE), and the effect of different parameters on the efficiency of spore inactivation and the related mechanism were investigated. We found that CGDE effectively inactivated spores of P. expansum in the clarified apple juice. CGDE achieved a 3.71 log inactivation of spores in the juice with 1 cm electrode gap, at 520 V, and discharge time for 15 min. CGDE produced hydrogen peroxide in the juice, and the concentration reached 58.1 mg/L after discharged for 30 min. CGDE destroyed the integrity of the cell membrane in the spores, and reduced the activities of superoxide dismutase, catalase, and peroxidase by 68.1%, 81.8%, and 68.0% after discharged for 30 min. In addition, CGDE inhibited mycelial growth, reduced colony diameter by 18.9%, and decreased the lesion diameter by 41.4% in apples surface-inoculated with P. expansum. Taken together, CGDE could inactivate the spores of P. expansum in apple juice by inducing reactive oxygen species accumulation and disrupting the integrity of cell membrane of spores.Industrial relevanceCGDE can effectively inactivate the spores of P. expansum in apple juice. The device of CGDE is composed of a high voltage DC power supply, electrodes and a reaction vessel, which is connected easily with material pipeline, and suitable for handling liquids. Therefore, CGDE can be applicated in treating liquids contaminated with fungal spores in the food industry.     

Control of citrus green and blue molds by Chinese propolis

Green and blue molds, caused by Penicillium digitatum and Penicillium italicum, respectively, are economically important postharvest diseases of citrus fruits. In this study, Chinese propolis ethyl acetate extract (PEAE) was evaluated to control P. digitatum and P. italicum on postharvest citrus fruits. The results indicated PEAE strongly inhibited mycelia growth and induced hyphae prominent abnormal morphological alterations. Also, PEAE had strong detrimental effect on spore germination of the tested pathogens in a concentrationdependent manner. For in vivo tests, PEAE could both reduce decay caused by P. digitatum and P. italicum respectively in wound-inoculated fruit and naturally infected fruit; meanwhile, no negative influences on the overall quality of the citrus fruits were observed with PEAE treatment. Therefore, PEAE could be used as a natural antifungal agent to control citrus blue and green mold.     

The effects of the combination of Pichia membranefaciens and BTH on controlling of blue mould decay caused by Penicillium expansum in peach fruit

The feasibility of 0.2 g l⁻¹ benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mould decay in peach fruit was investigated. Our results showed that biocontrol activity of P. membranefaciens against blue mould caused by Penicillium expansum in peach fruit could be enhanced by addition of 0.2 g l⁻¹ BTH. The combination of P. membranefaciens and BTH resulted in a more effective control of blue mould than individual treatment of P. membranefaciens or BTH alone. The combined treatment had a synergistic effect on the induction of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities, which induced stronger disease resistance in fruit than BTH or yeast alone, and resulted in a lower lesion diameter and disease incidence of blue mould decay in peaches. Furthermore, the combined treatment did not impair the quality parameters including fruit firmness and contents of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of BTH may be an effective method to improve the biological activity of P. membranefaciens.     

Modeling Penicillium Expansum Growth Response to Thyme Essential oil at Selected Water Activities and pH Values Using Surface Response Methodology

Our objective was to evaluate, using a full factorial design, the effects of selected water activities (0.990, 0.945, or 0.900), pHs (5, 4, or 3), and thyme essential concentration (TEO, 0, 25, 50, or 100ppm) oil on Penicillium expansum lag time (λ) and radial growth rate (μ_m) obtained by modeling mold response using Gompertz equation, and corresponding polynomial quadratic models. Potato-dextrose agar formulated with every studied factor combination was inoculated with 10³ spores/ml, and incubated at 25°C up to 30 days. Mold colony diameter was periodically measured during incubation and adjusted with Gompertz equation to determine λ and μ_m. Decreasing a_w and pH, and increasing TEO concentration decreased μ_m and increased λ. At low a_w and pH, the increase in TEO concentration had a dramatic effect on P. expansum response since 25ppm of TEO inhibited its growth for 30 days at 25°C. Gompertz parameters exhibited that P. expansum was sensitive to the evaluated combined factors, allowing us to construct a secondary predictive growth model. TEO in combination with a_w and pH reduction effectively inhibited P. expansum growth.     

Electronic nose application for determination of Penicillium digitatum in Valencia oranges

BACKGROUND: Penicillium digitatum and Penicillium italicum are responsible for one the most serious diseases occurring during storage of citrus fruits. Its early detection allows a relevant increase in shelf life, and in situ monitoring of fungal infections represents a very efficient tool to improve storage quality. In the case of metabolic alterations due to physiological or fungal pathologies, olfactometric analysis allows the detection of specific volatile biomarkers, thus providing an effective tool for postharvest quality control of fruits and vegetables. RESULTS: A total of 300 Valencia oranges were analysed with an electronic nose and results were screened by a multivariate classification technique, partial least squares discriminant analysis, in order to investigate whether the electronic nose could distinguish between Penicillium‐infected and non‐infected samples and to evaluate the efficiency of the group classifications. High percentages of correct classification were obtained at low levels of infection (100% for 2–5% infection in an independent test). CONCLUSION: The electronic nose may be successfully applied as a reliable, non‐destructive and non‐contact indirect technology for the identification of fungal strains in storage rooms, especially when the infection occurs in small percentages that are not easily identifiable by classic methodologies of inspection. Copyright © 2012 Society of Chemical Industry