Expansum Growth Research Articles

English

This study was conducted to investigate the effect of Candida ernobii strain Pg2-5 against postharvest disease of Penicillium expansum and to evaluate the possible mechanisms involved. Disc diffusion assay showed that Pg2-5 exhibited an inhibitory effect against P. expansum growth. An antifungal compound was isolated from the cell-free supernatant culture of Pg2-5 and identified as farnesol. The minimal inhibitory concentration (MIC) and minimum fungicidal concentrations (MFC) of farnesol for P. expansum were 15 and 100 μM, respectively. The addition of farnesol promoted reactive oxygen species (ROS) burst, and increased ultrastructure disintegration of mitochondria. These results indicate that antifungal compound production is one of the modes of action of C. ernobii Pg2-5 against postharvest pathogen of P. expansum. ROS may be an inducer, and mitochondria are potential target of the cytotoxicity process of farnesol. Key words: Postharvest, Candida ernobii, biocontrol, farnesol.

Interaction with Penicillium expansum enhances Botrytis cinerea growth in grape juice medium and prevents patulin accumulation in vitro

Interactions between fungi occur when they grow on the same host plant. This is the case of Botrytis cinerea and Penicillium expansum on grape. P.expansum is also responsible for production of the mycotoxin patulin. In this study, the influence of the interaction between both fungi on fungal growth parameters was studied as well as the effect on the accumulation of patulin by P.expansum. For that purpose, spores of B.cinerea and P.expansum were inoculated together (mixed inoculum), and the parameters growth rate, time for growth and patulin accumulation were assessed. The presence of P.expansum conidia shortened the time for growth of mixed inoculum colonies which, at the end of incubation, were B.cinerea-like. Although some P.expansum growth was observed in mixed inoculum colonies, very low levels of patulin were observed. In assays carried out in patulin-spiked medium, B.cinerea was capable to metabolize the mycotoxin. The capabilities of B.cinerea to shorten time for growth and prevent patulin accumulation are competing abilities that facilitate grape colonization.

English

  Antagonistic effect of Pseudomonas fluorescens and influence of gamma irradiation on the development of Penicillium expansum, the causal agent of postharvest disease on apple fruit was studied. P. fluorescens was originally isolated from rhizosphere of the apple trees. Suspension of P. fluorescens and P. expansumwere mixed in test tubes in proportions of 1:5, 2:5, 3:5 and 4:5 (V/V). The inhibitory effect depended on the proportion of the bacterium to the fungus in the mixture. The best inhibition was observed after 48 h at the ratio of 3:5. Gamma irradiation above 3 KGy completely inhibited mycelial growth, while the highest dose around 600 Gy mostly killed P. expansum spores. Our experiment demonstrates that the combination of gamma irradiation and P. fluorescens was more effective in reducing P. expansum growth, than either treatment alone and that the integration of irradiation and antagonist treatments can be more effective. The results of this study show that improved control by irradiation at labeled dose in combination with antagonist could allow direct incorporation of the biocontrol agent. Thus, the combination of the P. fluorescens with gamma irradiation showed an impressive effect on increasing applied range of irradiation for postharvest control by decreasing of dose rate.   Key words: Antagonist treatment, gamma irradiation, Penicillium expansum,Pseudomonas fluorescens, postharvest disease.

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.

Photocatalytic Activity against Penicillium Expansum of Ag-Doped TiO&lt;sub&gt;2&lt;/sub&gt;/SnO&lt;sub&gt;2&lt;/sub&gt;/SiO&lt;sub&gt;2&lt;/sub&gt;

The effect of 0.1-1 mol%Ag doping on crystallite size, morphology, photocatalytic and fungal growth suppression activities of TiO2/SnO2/SiO2 nano-composite powders were investigated. The powder was synthesized by sol-gel method and studied for physicochemical properties by XRD, spectrometric techniques, FTIR, SEM, EDX and BET. The anatase structure was formed after calcination at 500oC. Photocatalytic activities towards Penicillium expansum growth suppression correlated to the amount of sliver doped in TiO2/SnO2/SiO2 nano-composite powder under ultraviolet radiation. Ag 1.0mol% doped in TiO2/SiO2/SnO2 composite has a significant effect on antifungal behaviour. It could completely kill Penicillium expansum within one day of photacatalytic treatment under UV irradiation.

LOW OXYGEN EFFECT ON PENICILLIUM EXPANSUM MOULD IN STORED APPLE

Penicillium expansum causing blue mould is a serious postharvest pathogen in all growing pome fruit areas. Physical treatments, including storage atmosphere manipulation, have been developed to control postharvest fungal decay. The present study aimed to determine the influence of oxygen (O 2 ) levels on growth of P. expansum (P37 strain). Pathogen enzymatic production at low oxygen levels was also tested in in vitro trials by agarose diffusion assay. In in vitro trials, 100 μl of conidial suspension (10 3 conidia per ml) was spread on MEA dishes, stored for 30 days at 0°C with 0.7, 1.5, 3, 6 and 21% O 2 . The concentration of carbon dioxide was 1%. In in vivo trials, fruit were wounded and inoculated with 20 μl of a conidial suspension (10 3 conidia per ml) of P. expansum and stored with the same oxygen concentrations for five months at 0°C plus 3 days at 20°C. No significant differences were observed, within the different oxygen concentrations, on P. expansum growth in vitro. The percentage of infected fruit was significantly greater at low oxygen concentrations (0.7, 1.5, 3%) than at 6 or 21%. However, the dimensions of decay lesions, on apples exposed to 21 and 6% of O 2 , were significantly larger than those of apples stored at 0.7, 1.5 and 3% O 2 . In addition, O 2 levels greatly influenced the enzymatic activity. Polygalacturonase, protease and amylase activities were increased by low oxygen level (0.7%), whereas cellulases were reduced by the same oxygen concentration. More investigations are required to better elucidate the interactions between pathogen-fruit-oxygen level and the role of the enzymes in pathogenicity.

Fructooligosaccharide production by Penicillium expansum

Fructooligosaccharides (FOS) production by Penicillium expansum was evaluated. In a first stage, the best conditions for P. expansum growth and sporulation were established with potato/dextrose/agar being the most suitable medium at between 22 and 25 degrees C, giving good growth and good sporulation. The inocula from this medium were used for FOS production using shake-flask cultures, and yielded 0.58 g FOS/g sucrose (3.25 g FOS/l.h), demonstrating the potential of this strain for sucrose conversion to FOS.

Lentinula edodes enhances the biocontrol activity of Cryptococcus laurentii against Penicillium expansum contamination and patulin production in apple fruits

Penicillium expansum is a post-harvest pathogen of apples which can produce the hazardous mycotoxin patulin. The yeast Cryptococcus laurentii (LS28) is a biocontrol agent able to colonize highly oxidative environments such as wounds in apples. In this study culture filtrates of the basidiomycete Lentinula edodes (LF23) were used to enhance the biocontrol activity of LS28. In vitro L. edodes culture filtrates improved the growth of C. laurentii and the activity of its catalase, superoxide dismutase and glutathione peroxidase , which play a key role in oxidant scavenging. In addition, LF23 also delayed P. expansum conidia germination. The biocontrol effect of LS28 used together with LF23 in wounded apples improved the inhibition of P. expansum growth and patulin production in comparison with LS28 alone, under both experimental and semi-commercial conditions. The biocontrol effect was confirmed by a semi-quantitative PCR analysis set up for monitoring the growth of P. expansum.

Characterization of genes associated with induced resistance against Penicillium expansum in apple fruit treated with quercetin

Penicillium expansum causes blue mould, a serious postharvest disease of apples, and is the main producer of the mycotoxin patulin. Since control by synthetic fungicides is less accepted by consumers, the demand for alternative means is pressing. In a recent study, the flavonoid quercetin, although scarcely effective in in vitro assays against P. expansum growth, significantly reduced blue mould rots on ‘Golden Delicious’ apples, suggesting an enhancement of host disease resistance. To confirm or reject this hypothesis, genes differentially expressed in quercetin-treated ‘Golden Delicious’ apples were identified by suppression subtractive hybridization (SSH). A pool of 88 unique gene transcripts was obtained. Several sequences revealed high similarities with different classes of pathogenesis-related proteins (RNase-like PR10 and PR8), or with proteins expressed under stress conditions. Other transcripts had high similarity to genes of unknown function or genes coding for proteins having a role in pathogen recognition and in signalling pathways. SSH data were validated by analysing the expression of 14 genes by quantitative real-time PCR (qPCR). Eleven genes proved to be up-regulated at a medium-high level in freshly harvested apples. Among these, 5 genes selected for temporal expression profiling revealed the existence of a combined effect, particularly at 24 or 48 h, between wounding and phenolic treatment. These results provide evidence that quercetin induces resistance to P. expansum in apples, by acting on the transcription level of genes involved in several distinct metabolic processes.

PATULIN IN APPLE FRUITS: II. EFFECT OF APPLE TREATMENTS WITH SOME CHEMICAL SANITIZERS ON PATULIN PRODUCTION BY Penicillium expanusm

Penicillium expansum is a wide spread fungi found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, basically patulin. This study was carried out to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing washing treatments that would inhibit P. expansum growth and subsequent patulin production on Anna apples destined for cider .Wash treatments with chemical sanitizers including NaOCl (100 to 200 ppm) , Potassium sorbate (0.1 to 0.5% ), SO2(100 to 300 ppm) and acetic acid (0.5 and 3% ) . The results showed that washing with NaOCl (200 ppm) delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solutions (2 to 3%) were the most efficient chemical against P. expansum and subsequent patulin production on apple juices. While no significant effect in patulin production was found by SO­­2 and potassium sorbate. The effect of pressing on molded Anna apple discs fruits was studied. The pressing resulted in a partially elimination of patulin in apple juices. Patulin in the final juices is dependent on initial the concentrations.

Some strains of Burkholderia gladioli are potential candidates for postharvest biocontrol of fungal rots in citrus and apple fruits.

SUMMARY Three Burkholderia gladioli strains (DISTEF), recovered from Strelitzia reginae, were biochemically and genomically characterized and evaluated as potential candidates for post harvest biocontrol of fungal rots in citrus and apple fruits. Cell and cell-free culture filtrates inhibited in vitro, to a different extent, the growth of Rhodotorula pilimanae, Penicillium digitatum, Penicillium expansum, Botrytis cinerea, Aspergillus flavus, Aspergillus ochraceus and Alternaria alternata. Conidial germination was also inhibited in liquid culture assays by bacterial cells, culture filtrates and concentrated culture filtrates. All strains completely inhibited or significantly reduced the incidence and severity of diseases caused by P. digitatum and P. expansum to orange, lemon and apple fruits after 4 days incubation at 20°C. B. gladioli strain DISTEF-G completely inhibited P. digitatum and P. expansum growth after 9 days incubation. A partial control of green and blue mould was obtained n citrus and apple wounds inoculated with culture filtrates. FAFLP analysis showed clear separation of DISTEF strains of B. gladioli from type strains of B. gladioli and B. cepacia. The results suggest that B. gladioli DISTEF- G would be an interesting microorganism for use as a biocontrol agent.

Control of Penicillium expansum and patulin accumulation on apples by quercetin and umbelliferone

The effectiveness of seven phenolic compounds (esculetin, ferulic acid, quercetin, resveratrol, scopoletin, scoparone and umbelliferone) in controlling Penicillium expansum growth and patulin accumulation was evaluated by both in vitro and in vivo studies. The in vitro screening showed that quercetin and umbelliferone were the most effective compounds in controlling Penicillium growth and patulin accumulation, respectively. Quercetin and umbelliferone, resulted to be effective also in the in vivo screening, were further tested (alone and in combination) on Granny Smith and Golden Delicious apples. The efficacy in controlling Penicillium growth was better expressed on Golden Delicious than on Granny Smith apples, with quercetin providing a better control of both incidence of decay and disease severity as compared to umbelliferone. Both compounds exerted a considerable control of patulin accumulation on the two apple cultivars. Quercetin and umbelliferone can be considered as natural compounds to be used as alternative strategy to chemical fungicides in postharvest control of P. expansum infections on apples.

Salicylic acid alleviated pathogen-induced oxidative stress in harvested sweet cherry fruit

The role of exogenous salicylic acid (SA) in regulating an antioxidative defense response of sweet cherry ( Prunus avium L. cv. Hongdeng) fruit inoculated with Penicillium expansum was investigated by immunodetection of carbonylated proteins. After inoculation with P. expansum, carbonylated proteins accumulated to a lesser extent in SA-treated fruit than in control fruit, ranging from molecular mass 29–45 kDa. Higher activities of catalase (CAT), glutathione peroxidase (GPX), chitinase and β-1,3-glucanase were observed in SA-treated fruit. Similarly, the expressions of CAT, GPX and β-1,3-glucanase genes were also stimulated by SA treatment. Moreover, 2 mM SA did not inhibit P. expansum growth in vitro. These results indicate that SA activated antioxidant defense responses of sweet cherry fruit, which may play a role in the resistance against P. expansum.

Effect of biocontrol agents Candida sake and Pantoea agglomerans on Penicillium expansum growth and patulin accumulation in apples

Penicillium expansum is the major responsible of fruit pome decaying in cold storage. Apples spoiled by P. expansum are expected to contain patulin, a mycotoxin which is proven to affect human health. The use of chemicals is the most common procedure to prevent rots in postharvest but legislation is becoming more and more restrictive. The use of biocontrol agents (BCA) as an alternative tool is currently being proposed. The aim of this study was to evaluate the effect of two BCA ( Candida sake CPA-2 and Pantoea agglomerans CPA-1) on P. expansum growth and patulin accumulation in cold storage and further deck (ambient) storage. Wounded apples were inoculated with a cell suspension of either C. sake or P. agglomerans and with a P. expansum conidial suspension. Apples were cold stored at 1 °C until lesion diameter reached 2 or 4 cm. Half the apples of each treatment were further stored at 20 °C for three days before patulin analyses. Both BCA tested controlled blue rot and patulin accumulation during cold storage. The control of P. expansum growth was enhanced in C. sake treated apples. On the other side, control of patulin accumulation in P. agglomerans treated apples seemed to be more efficient. BCA treatment could not control blue rot and patulin accumulation during further storage at room temperature and in some cases, an increase in P. expansum aggressiveness was observed.

Inoculum size and intraspecific interactions affects Penicillium expansum growth and patulin accumulation in apples

Penicillium expansum is the most important fungal species causing spoilage in cold stored apples. P. expansum produces patulin, a toxic secondary metabolite. The aim of this work was to study the changes in growth parameters and patulin accumulation that may occur when two different isolates of P. expansum germinate and develop in the same wound. The effect of the inoculum size was also studied. For that purpose, apples were point inoculated with two different isolates of P. expansum at two different conidia concentrations. Both isolates were also inoculated together. Apples were cold stored at 1 °C or incubated at 20 °C until lesion diameter reached 2.5 cm. Patulin from decayed tissue was analysed at the end of cold storage/incubation. It seems that intraspecific interactions occur. The interactions resulted in slower growth in which patulin content was lower. A competition for resources followed by either a “combat-like” interaction may be involved in this. Higher inoculum size led to shorter lag phase, which reflects the importance of the hygiene in packinghouses facilities. The inoculum size also affected growth rate and patulin accumulation, which suggests that interactions between individuals of the same strain may occur. Competition for resources and a combat-like interaction may take place. In vitro studies should be carried out in order to assess the patterns of the interactions observed.

Penicillium expansum versus antagonist yeasts and patulin degradation in vitro

Taking into account the preliminary antagonistic/biodegradation property showed by Pichia membranifaciens and Sporobolomyces roseus, which decreased the initial patulin concentration of 588.4 to 290.0 µg/mL, ability of P. ohmeri 158 in biocontrol against Penicillium expansum and patulin decrease in vitro was performed. The culture supernatant of P. ohmeri 158 was effective against 66.17% micelial growth, indicating antibiosis related with the killer phenomenon. The initial patulin concentration of 223 µg in the presence of P. ohmeri 158 cells was decreased over 83% of the original concentration, when incubated at 25ºC/2 days and &gt; 99% after 5 days incubation time, with undetectable patulin level after 15 days. The initial pH 4.0 decreased to pH 3.3 along 15 days experiment, suggesting that patulin decrease was an active process and a consequence of yeast metabolism. The results suggested that P. ohmeri 158 could be a promising alternative for the inhibition of P. expansum growth and patulin degradation.

Patulin accumulation in apples during postharvest: Effect of controlled atmosphere storage and fungicide treatments

The aim of this study was to assess the opportunities of Penicillium expansum to develop and produce patulin in apples under two different CA storage methods (LOW and U-LOW) at 1 °C. Differences in lesion diameter and patulin accumulation depending on CO 2 and O 2 partial pressure were studied. Further apple rot and patulin production during a three days post-storing stage at 20 °C was also monitored so that effect of further storage at room temperature could be assesed. Two lots of apples of Golden variety with different ripeness degrees were used. Half of each lot was fungicide treated. Apples were inoculated with patulin producer P. expansum strains and stored at 1 °C for either two month or 2.5 months at both LOW and U-LOW conditions. The extent of lesions and patulin accumulation both at the end of CA cold storage and after three days at 20 °C were assessed. CA storage conditions had strong significance in P. expansum growth on apples and factors such as fruit ripeness, fungicide treatment and time at the storage room had significant influence. In general, bigger lesions were observed under U-LOW than under LOW conditions, lesions being similar or bigger when increasing the storage time from 2 to 2.5 months. P. expansum grew faster in riper apples, although fungicide application was clearly more effective for ripe rather than for underripe apples. Although lesions were evident after both storage conditions, no patulin was detected. Increase of lesion when fruits were subsequently stored at 20 °C was evident in all cases and patulin was detected at this moment. No differences in patulin content were found at this stage between LOW and U-LOW stored apples.

Antifungal activity of TiO 2 photocatalysis against Penicillium expansum in vitro and in fruit tests

The antifungal activity of TiO 2 photocatalytic reaction in the form of TiO 2 powder and TiO 2 coated on a plastic film against Penicillium expansum was investigated in vitro and in fruit tests. The mixture of P. expansum conidial suspension and TiO 2 powder was added to potato dextrose agar (PDA) plates for vitro test. The TiO 2 photocatalytic reaction reduced conidial germination of the fungal pathogen. It was found that the ability of the TiO 2 photocatalytic reaction to suppress P. expansum growth correlated to the amount of TiO 2 added. Lower numbers of viable colonies of P. expansum were observed with increasing amount of TiO 2. Regardless of the kind of selected fruit inoculated with P. expansum, both TiO 2 powder and TiO 2-coated film exhibited antifungal activity to control fruit rot. Development of Penicillium rot in apple was significantly ( P = 0.05) retarded by the TiO 2 photocatalytic reaction. Similarly the TiO 2 photocatalytic reaction was the only treatment where no tomato fruit rot was noticeable after 1 week of storage. TiO 2-coated film also decreased brown lesions and Penicillium rot infection in lemons. The mean severity fruit rot scores (browning and softening flesh) were 3.2 and 1.9 for uncoated and TiO 2-coated film, respectively. Our findings suggest that “TiO 2 photocatalytic reaction” shows antifungal activity against P. expansum which may have potential for postharvest disease control.

Editorial

Penicillium expansum growth and patulin production occur mainly at post-harvest stage during the long-term storage of apples. Low temperature in combination with reduced oxygen concentrations is commonly applied as a control strategy to extend apple shelf life and supply the market throughout the year. Our in vitro study investigated the effect of temperature and atmosphere on expression of the idh gene in relation to the patulin production by P. expansum. The idh gene encodes the isoepoxydon dehydrogenase enzyme, a key enzyme in the patulin biosynthesis pathway. First, a reverse transcription real-time PCR (RT-qPCR) method was optimized to measure accurately the P. expansum idh mRNA levels relative to the mRNA levels of three reference genes (18S, β-tubulin, calmodulin), taking into account important parameters such as PCR inhibition and multiple reference gene stability. Subsequently, two P. expansum field isolates and one reference strain were grown on apple puree agar medium (APAM) under three conditions of temperature and atmosphere: 20 °C — air, 4 °C — air and 4 °C — controlled atmosphere (CA; 3% O2). When P. expansum strains reached a 0.5 and 2.0 cm colony diameter, idh expression and patulin concentrations were determined by means of the developed RT-qPCR and an HPLC-UV method, respectively. The in vitro study showed a clear reduction in patulin production and down-regulation of the idh gene expression when P. expansum was grown under 4 °C — CA. The results suggest that stress (low temperature and oxygen level) caused a delay of the fungal metabolism rather than a complete inhibition of toxin biosynthesis. A good correlation was found between the idh expression and patulin production, corroborating that temperature and atmosphere affected patulin production by acting at the transcriptional level of the idh gene. Finally, a reliable RT-qPCR can be considered as an alternative tool to investigate the effect of control strategies on the toxin formation in food.

Survival of Penicillium expansum and Patulin Production on Stored Apples after Wash Treatments

ABSTRACT: Penicillium expansum is a widespread fungus found on apples that causes fruit decay and may lead to production of a toxic secondary metabolite, patulin. This study was undertaken to evaluate the effectiveness of several chemical sanitizers against P. expansum NRRL 2304 and to establish sanitizing wash treatments that would inhibit P. expansum growth and subsequent patulin production on Empire apples destined for cider. Wash treatments included 200 ppm NaOCl, 1% StorOx®, 0.5% potassium sorbate, 300 ppm SO2, and 0% to 5% acetic acid. Spores of P. expansum or inoculated apple slices were dipped in sanitizing wash solution for 5 min, and mold growth and patulin production was monitored on subsequent storage. It was found that 0.5% potassium sorbate and 300 ppm SO2 did not affect mold survival or patulin production; 1% StorOx® was effective against mold spores in solution (4 log Most Probable Number destruction of spores), but there was no significant reduction in spore count when the same solution was used to sanitize mold‐inoculated apple discs. Washing with 200 ppm NaOCl delayed growth of P. expansum on inoculated apple discs but failed to completely inhibit patulin production. Acetic acid solution (2% to 5%) was the most efficient chemical against P. expansum. A wash treatment with ≥2% acetic acid for more than 1 min is recommended to completely inhibit growth of P. expansum and subsequent patulin production on apples destined for cider.