Search

Filamentous fungi are prolific producers of secondary metabolites (SecMets), including compounds with antibiotic properties, like penicillin, that allows the producing fungus to combat competitors in a shared niche. However, the biological function of the majority of these small complex metabolites for the producing fungi remains unclear (Macheleidt et al., 2016). In an effort to address this lack of knowledge, we have chosen to study the microbial community of moldy apples in the hope of shedding more light on the role of SecMets for the dynamics of the microbial community. Penicillium expansum is one of the prevalent fungal species in this system, and in co-culture experiments with other apple fungal pathogens, we have observed up- and downregulation of several SecMets when compared to monocultures. However, molecular genetic dissection of the observed changes is challenging, and new methodologies for targeted genetic engineering in P. expansum are needed. In the current study, we have established a CRISPR-Cas9 dependent genetic engineering toolbox for the targeted genetic manipulation of P. expansum to allow for single-step construction of marker-free strains. The method and effect of different combinations of a Cas9-sgRNA expressing plasmids and repair template substrates in the NHEJ-proficient WT strain is tested by targeted deletion of melA, encoding a PKS responsible for pigment formation, which upon deletion resulted in white mutants. Co-transformation with a linear double-stranded DNA fragment consisting of two 2 kb homology arms flanking the PKS gene proved to be the most efficient strategy with 100% confirmed deletions by diagnostic PCR. Shorter homology arms (500-1000bp) resulted in 20-30% deletion efficiency. Furthermore, we demonstrate the application of the CRISPR-Cas9 method for targeted deletion of biosynthetic genes without a visible phenotype, insertion of a visual reporter-encoding gene (mRFP), and overexpression of bi

Patulin is a secondary metabolite produced primarily by the fungus Penicillium expansum, responsible for the blue mold disease on apples. It is found in apple products including apple cider when apple juice is added after fermentation. In the present study, two hundred and twenty-five cider-apples of the variety “Bedan”, cultivated in Brittany in France, were sampled from the orchard during harvesting until the storage step, right before processing. The patulin analysis on these samples reported a low contamination at the orchard and a significantly higher-level of contamination in the cider-apples starting from the transporting bin. The percentage of positive samples increased from 6% to 47% after 12 h in the harvesting bin before transporting and reached 95% after 24 h of transporting, decreasing then to 69% at the end of the storage. Penicillium expansum was quantified on the surface of apples using real-time PCR and was observed to be mostly consistent between the harvest and post-harvest steps. It was detected on average, on the surface of 85% of all sampled apples with a mean value around 2.35 × 106 Penicillium expansum DNA/g of apple. Moreover, the changes in the fungal and bacterial epiphytic microbiota in the different steps were studied using a metabarcoding approach. The alpha and beta diversity analysis revealed the presence of unique and more diverse bacterial and fungal communities on the surface of apples picked from the orchard compared to the rest of the sampling steps. Potential indigenous biological control agents were identified on the surface of sampled apples. Future perspective includes developing actions of prevention and control of the contamination by Penicillium expansum during the harvest and along the various critical post-harvest stages before transformation in a sustainable development concern.

PVC is one of the most widely used plastics today and the one that produces the most waste. In recent years, microorganisms capable of degrading it have been reported, most of which come from environments in which PVC is accumulated. The aim of this work was to study the degradation of PVC without plasticizer, from fungi isolated from a soil sample contaminated with PVC resin. The fungi were isolated and morphologically characterized, 30 morphospecies were obtained, 8 were chosen to undergo preliminary tests in a medium whose only carbon source was a PVC film. Growth curves of the two isolates with better results were made and their molecular identification showed that they corresponded to Penicillium sp. and Mucor sp., the latter gained biomass from PVC and in both cases, the films showed visible changes, which were supported by infrared spectra. Although the results shown in this article are preliminary, they open the door to new forms of PVC waste degradation that are very persistent., El PVC es uno de los plásticos más usados y de los que más residuos se producen. En los últimos años, se han reportado microorganismos capaces de degradarlo y que en su mayoría provienen de ambientes en los que se acumula. El objetivo de este trabajo fue estudiar la degradación del PVC sin plastificante, a partir de hongos aislados de una muestra de suelo contaminada con resina de PVC. Los hongos se aislaron y caracterizaron morfológicamente, obteniendo 30 aislamientos de los que se escogieron 8 para someterlos a pruebas preliminares, en un medio cuya única fuente de carbono era una película de PVC. Se hicieron curvas de crecimiento de los dos aislamientos con mejores resultados, y su identificación molecular mostró que correspondían a Penicillium sp. y Mucor sp.. Este último ganó biomasa a partir del PVC y en los dos casos, las películas mostraron cambios visibles, que fueron respaldados por espectros de infrarrojo. Aunque los resultados mostrados en este artículo son preliminares, abren la puerta a nuevas formas de degradación de los residuos de PVC, que son muy persistentes.

Blue mold, caused by Penicillium spp., is one of the most economically important postharvest diseases of pome fruits, globally. Pome fruits, in particular apple, is the most widely grown pome fruit in Serbia, and the distribution of Penicillium spp. responsible for postharvest decay is unknown. A two-year survey was conducted in 2014 and 2015, where four pome fruits (apple, pear, quince, and medlar) with blue mold symptoms were collected from 20 storage locations throughout Serbia. Detailed morphological characterization, analysis of virulence in three apple cultivars, and multilocus phylogeny revealed three main Penicillium spp. in order of abundance: P. expansum, P. crustosum, and P. solitum. Interestingly, P. expansum split into two distinct clades with strong statistical support that coincided with several morphological observations. Findings from this study are significant and showed previously undocumented diversity in blue mold fungi responsible for postharvest decay including the first finding of P. crustosum, and P. solitum as postharvest pathogens of quince and P. crustosum of medlar fruit in the world, and P. expansum of quince in Serbia. Data from this study provide timely information regarding phenotypic, morphological and genotypic plasticity in P. expansum that will impact the design of species-specific detection tools and guide the development of blue mold management strategies.

Patulin is a secondary metabolite produced primarily by the fungus Penicillium expansum, responsible for the blue mold disease on apples. It is found in apple products including apple cider when apple juice is added after fermentation. In the present study, two hundred and twenty-five cider-apples of the variety “Bedan”, cultivated in Brittany in France, were sampled from the orchard during harvesting until the storage step, right before processing. The patulin analysis on these samples reported a low contamination at the orchard and a significantly higher-level of contamination in the cider-apples starting from the transporting bin. The percentage of positive samples increased from 6% to 47% after 12 h in the harvesting bin before transporting and reached 95% after 24 h of transporting, decreasing then to 69% at the end of the storage. Penicillium expansum was quantified on the surface of apples using real-time PCR and was observed to be mostly consistent between the harvest and post-harvest steps. It was detected on average, on the surface of 85% of all sampled apples with a mean value around 2.35 × 106 Penicillium expansum DNA/g of apple. Moreover, the changes in the fungal and bacterial epiphytic microbiota in the different steps were studied using a metabarcoding approach. The alpha and beta diversity analysis revealed the presence of unique and more diverse bacterial and fungal communities on the surface of apples picked from the orchard compared to the rest of the sampling steps. Potential indigenous biological control agents were identified on the surface of sampled apples. Future perspective includes developing actions of prevention and control of the contamination by Penicillium expansum during the harvest and along the various critical post-harvest stages before transformation in a sustainable development concern.

This thesis investigated the regulation of patulin production in Penicillium expansum in relation to fungal global regulators as well as the cluster-specific binuclear zinc transcription factor (BZTF) patL in silico. It also explored the state of BZTF research in filamentous ascomycetes fungi, and questioned if results in Saccharomyces cerevisiae can be freely translated to other taxonomic groups. Results indicate that a similar percentage of BZTFs bind as homodimers in filamentous ascomycetes as in S. cerevisiae, however, they also suggest that BZTFs are more prone to bind as monomers than previously thought, which could be beneficial in binding site prediction. However, the results are inconclusive if the presence of a coiled-coil domain can be used to predict the type of binding site. Global regulators are involved in a complex regulation system of patulin and 5'- CCBRAAGGAG -3' is identified as a putative binding motif for patL. A new generation of meta binding site prediction tools is proposed., Alla levande varelser har metabolism och då även metaboliter, som är ämnen som produceras och konsumeras under metabolismen. En del av dessa produceras konstant, som när vi bryter ner mat vi äter eller när vi ska röra på oss. Det finns också metaboliter som vi inte har, men finns i till exempel svampar. De kallar vi sekundära metaboliter. De är viktiga för hur svamparna beter sig och interagerar med sin omvärld. Vi har ett intresse av dessa ämnen då de kan vara användbara för oss i medicinskt syfte. Ett bra exempel på en sekundär metabolit är antibiotikan penicillin, som produceras av många svampar från släktet Penicillium. P. expansum är en mögelsvamp från släktet, men kan inte producera antibiotikan. Istället är den en utbredd patogen av äpplen. Den får äpplena att ruttna och producerar i dem det giftiga ämnet patulin. Patulin är ett annat exempel på en sekundär metabolit, och forskning pågår för att få nya sätt att bekämpa ämnet. Gener som är involverade i processen med att producera en viss kemisk förening grupperas i svampgenom i ett så kallat biosyntetiskt genkluster (BGC). Detta är användbart för svampar då de kan vara effektivare när de producerar dessa föreningar. Dessa kluster kan sedan styras av olika mekanismer, såsom de brett verksamma globala regulatoriska proteinerna eller mer specifika lokala transkriptionsfaktorer. Transkriptionsfaktorer är proteiner som kan användas för att kontrollera produktionen av en förening i en organism. I svampar kan dessa vara av klassen binukleära zink-transkriptionsfaktorer (BZTF). Dessa har en specifik egenskap och finns bara i svampar. De binder ofta i par när de modulerar metabolismen, eftersom de har en specifik proteinstruktur som gör att de kan hålla ihop. De binder sedan till palindromiska DNA-sekvenser. Detta examensarbete undersökte regleringen av produktion av patulin i P. expansum, både när det gäller de globala regulatoriska proteiner och för en specifik lokal transkriptionsfaktor som kallas patL, som är rela, All living things have metabolism and associated metabolites. Some of these are produced constantly, such as when we break down food, or need to move muscles. There are also metabolites called secondary metabolites (SecMets), which are compounds produced by fungi and have key roles relating to how the organism reacts and interacts with its environment. SecMets are of interest to mankind due to their potential use in the pharmaceutical industry, but also to understand fungal behaviour. A good example of a prominent secondary metabolite is the antibiotic penicillin, which is produced by many fungus from the genus Penicillium. P. expansum is a species of mold from the genus, but can't produce the antibiotic and is a wide-spread pathogen which infects apples. It causes the apples to rot and also produces the harmful mycotoxin patulin, which is in itself a secondary metabolite and is under a lot of research on ways to prevent it. Genes which are related to producing a certain compound are in fungi generally grouped together in the genome, in a so-called biosynthetic gene cluster (BGC). This is useful for fungi so that they can be more efficient when producing these compounds. These clusters can then be controlled by different mechanisms, such as widely-impactful global regulator proteins or specific local transcription factors. Transcription factors are proteins which can be used to control the production of a compound in an organism. In fungi these can be of the class binuclear zinc transcription factors (BZTFs). These have a specific characteristic and are only found in fungi. They may work in pairs when modulating metabolism, since they have a specific protein structure which allows them to stick together. They then bind to DNA sites which look palindromic. This thesis investigated the regulation of patulin production in P. expansum, both in regards to the global regulators but also for a specific local transcription factor called patL, which is known to be related to

Penicillium phytopathogenic species provoke severe postharvest disease and economic losses. Penicillium expansum is the main pome fruit phytopathogen while Penicillium digitatum and Penicillium italicum cause citrus green and blue mold, respectively. Control strategies rely on the use of synthetic fungicides, but the appearance of resistant strains and safety concerns have led to the search for new antifungals. Here, the potential application of different antifungal proteins (AFPs) including the three Penicillium chrysogenum proteins (PAF, PAFB and PAFC), as well as the Neosartorya fischeri NFAP2 protein to control Penicillium decay, has been evaluated. PAFB was the most potent AFP against P. digitatum, P. italicum and P. expansum, PAFC and NFAP2 showed moderate antifungal activity, whereas PAF was the least active protein. In fruit protection assays, PAFB provoked a reduction of the incidence of infections caused by P. digitatum and P. italicum in oranges and by P. expansum in apples. A combination of AFPs did not result in an increase in the efficacy of disease control. In conclusion, this study expands the antifungal inhibition spectrum of the AFPs evaluated, and demonstrates that AFPs act in a species-specific manner. PAFB is a promising alternative compound to control Penicillium postharvest fruit decay.

Wild yeasts isolated from the surface of apples were screened for antagonistic activity against Penicillium expansum, the main producer of the mycotoxin patulin. Three antagonistic yeasts (Y33, Y29 and Y24) from a total of 90 were found to inhibit P. expansum growth. Identification by ITS region sequence and characterization showed that three selected isolates of yeast should be different strains of Metschnikowia pulcherrima. Several concentrations of the selected yeasts were used to study their in vitro antifungal effectivity against P. expansum on Petri dishes (plates with 63.6 cm2 surface) whereas their potential activity on patulin reduction was studied in liquid medium. Finally, the BCA that had the best in vitro antifungal capacity against P. and the best patulin degradation capacity was selected to be assessed directly on apples. All the selected strains demonstrated antifungal activity in vitro but the most efficient was the strain Y29. Isolated strains were able to reduce patulin content in liquid medium, Y29 being the only strain that completely reduced patulin levels within 120 h. The application of Y29 as biocontrol agent on the surface of apples inoculated with P. expansum, inhibited fungal growth and patulin production during storage. Therefore, the results shown that this yeast strain could be used for the reduction of P. expansum and its mycotoxin in apples or apple-based products by adapting the procedure application.

[ES] En el presente trabajo se han estudiado diferentes formulaciones de recubrimientos elaborados a base de alcohol polivinílico (PVA) y almidón de patata (S) con la incorporación de carvacrol (C) que proporciona la acción antifúngica al recubrimiento. Se evaluó la retención de carvacrol por el recubrimiento y la densidad de sólidos en superficie. También se evaluó su efectividad antifúngica durante el almacenamiento poscosecha de manzanas Golden Delicious inoculadas con Penicillium expansum y la aceptación por los consumidores a través de catas sensoriales. Su aplicación fue muy efectiva contra el crecimiento de Penicillium expansum, al reducir significativamente la incidencia y severidad del hongo, especialmente aquellas formuladas con mayor proporción de almidón y carvacrol (50PVA:50S-C40 y 50PVA:50S-C80). Sin embargo, los resultados del análisis sensorial pusieron de manifiesto el impacto negativo del carvacrol sobre el aroma y sabor de las manzanas, incluso tras el lavado y el pelado de la fruta., [CA] Al present treball s'han estudiat diferents formulacions de recobriments elaborats amb base d'alcohol polivinílic (PVA) i midó de creïlla (S) amb l’incorporació de carvacrol (C) que proporciona l'acció antifúngica al recobriment. Es va avaluar la retenció de carvacrol pel recobriment i la densitat de sòlids en superfície i la seua l'efectivitat antifúngica sobre l'emmagatzemament postcollita de les pomes Golden Delicious inoculades amb Penicillium expansum. A mes, l'acceptació pels consumidors a través d’anàlisis sensorials també fou estudiada. Els resultats mostraren que la aplicació del recobriments amb carvacrol van ser molt efectives contra el 3 creixement de Penicillium expansum, al reduir significativament la incidència i severitat del fong, especialment en aquelles formulades amb major proporció de midó i carvacrol (50PVA:50S-C40 i 50PVA:50S-C80). No obstant això, els resultats de l'anàlisi sensorial mostraren un possible rebuig dels consumidors a estos recobriments, donada la baixa acceptació de l'aroma i el sabor del producte tractat, inclús després del llavat i el pelat de la fruita recoberta., [EN] In this work, different coatings based on polyvinyl alcohol (PVA) and potato starch (S) have been studied with the incorporation of carvacrol (C) that provides the antifungal action to the coating. The retention of carvacrol by the coating and the density of solids on the surface was evaluated together with the antifungal effectiveness on the post-harvest storage of Golden Delicious apples previously inoculated with Penicillium expansum. Moreover, a sensory analysis was also carried out to study the acceptability of the coated product. The carvacrol loaded coatings were highly effective against the growth of Penicillium expansum, significantly reducing the incidence and severity of the fungus, especially in those formulated with higher proportion of starch and carvacrol (50PVA:50S-C40 and 50PVA:50S-C80). However, the results of the sensory analysis showed the negative impact of these coatings on the aroma and taste of the coated apples, even after the washing and peeling of the fruit.

[ES] En el presente trabajo se han estudiado diferentes formulaciones de recubrimientos elaborados a base de alcohol polivinílico (PVA) y almidón de patata (S) con la incorporación de carvacrol (C) que proporciona la acción antifúngica al recubrimiento. Se evaluó la retención de carvacrol por el recubrimiento y la densidad de sólidos en superficie. También se evaluó su efectividad antifúngica durante el almacenamiento poscosecha de manzanas Golden Delicious inoculadas con Penicillium expansum y la aceptación por los consumidores a través de catas sensoriales. Su aplicación fue muy efectiva contra el crecimiento de Penicillium expansum, al reducir significativamente la incidencia y severidad del hongo, especialmente aquellas formuladas con mayor proporción de almidón y carvacrol (50PVA:50S-C40 y 50PVA:50S-C80). Sin embargo, los resultados del análisis sensorial pusieron de manifiesto el impacto negativo del carvacrol sobre el aroma y sabor de las manzanas, incluso tras el lavado y el pelado de la fruta., [CA] Al present treball s'han estudiat diferents formulacions de recobriments elaborats amb base d'alcohol polivinílic (PVA) i midó de creïlla (S) amb l’incorporació de carvacrol (C) que proporciona l'acció antifúngica al recobriment. Es va avaluar la retenció de carvacrol pel recobriment i la densitat de sòlids en superfície i la seua l'efectivitat antifúngica sobre l'emmagatzemament postcollita de les pomes Golden Delicious inoculades amb Penicillium expansum. A mes, l'acceptació pels consumidors a través d’anàlisis sensorials també fou estudiada. Els resultats mostraren que la aplicació del recobriments amb carvacrol van ser molt efectives contra el 3 creixement de Penicillium expansum, al reduir significativament la incidència i severitat del fong, especialment en aquelles formulades amb major proporció de midó i carvacrol (50PVA:50S-C40 i 50PVA:50S-C80). No obstant això, els resultats de l'anàlisi sensorial mostraren un possible rebuig dels consumidors a estos recobriments, donada la baixa acceptació de l'aroma i el sabor del producte tractat, inclús després del llavat i el pelat de la fruita recoberta., [EN] In this work, different coatings based on polyvinyl alcohol (PVA) and potato starch (S) have been studied with the incorporation of carvacrol (C) that provides the antifungal action to the coating. The retention of carvacrol by the coating and the density of solids on the surface was evaluated together with the antifungal effectiveness on the post-harvest storage of Golden Delicious apples previously inoculated with Penicillium expansum. Moreover, a sensory analysis was also carried out to study the acceptability of the coated product. The carvacrol loaded coatings were highly effective against the growth of Penicillium expansum, significantly reducing the incidence and severity of the fungus, especially in those formulated with higher proportion of starch and carvacrol (50PVA:50S-C40 and 50PVA:50S-C80). However, the results of the sensory analysis showed the negative impact of these coatings on the aroma and taste of the coated apples, even after the washing and peeling of the fruit.

[ES] En el presente trabajo se han estudiado diferentes formulaciones de recubrimientos elaborados a base de alcohol polivinílico (PVA) y almidón de patata (S) con la incorporación de carvacrol (C) que proporciona la acción antifúngica al recubrimiento. Se evaluó la retención de carvacrol por el recubrimiento y la densidad de sólidos en superficie. También se evaluó su efectividad antifúngica durante el almacenamiento poscosecha de manzanas Golden Delicious inoculadas con Penicillium expansum y la aceptación por los consumidores a través de catas sensoriales. Su aplicación fue muy efectiva contra el crecimiento de Penicillium expansum, al reducir significativamente la incidencia y severidad del hongo, especialmente aquellas formuladas con mayor proporción de almidón y carvacrol (50PVA:50S-C40 y 50PVA:50S-C80). Sin embargo, los resultados del análisis sensorial pusieron de manifiesto el impacto negativo del carvacrol sobre el aroma y sabor de las manzanas, incluso tras el lavado y el pelado de la fruta., [CA] Al present treball s'han estudiat diferents formulacions de recobriments elaborats amb base d'alcohol polivinílic (PVA) i midó de creïlla (S) amb l’incorporació de carvacrol (C) que proporciona l'acció antifúngica al recobriment. Es va avaluar la retenció de carvacrol pel recobriment i la densitat de sòlids en superfície i la seua l'efectivitat antifúngica sobre l'emmagatzemament postcollita de les pomes Golden Delicious inoculades amb Penicillium expansum. A mes, l'acceptació pels consumidors a través d’anàlisis sensorials també fou estudiada. Els resultats mostraren que la aplicació del recobriments amb carvacrol van ser molt efectives contra el 3 creixement de Penicillium expansum, al reduir significativament la incidència i severitat del fong, especialment en aquelles formulades amb major proporció de midó i carvacrol (50PVA:50S-C40 i 50PVA:50S-C80). No obstant això, els resultats de l'anàlisi sensorial mostraren un possible rebuig dels consumidors a estos recobriments, donada la baixa acceptació de l'aroma i el sabor del producte tractat, inclús després del llavat i el pelat de la fruita recoberta., [EN] In this work, different coatings based on polyvinyl alcohol (PVA) and potato starch (S) have been studied with the incorporation of carvacrol (C) that provides the antifungal action to the coating. The retention of carvacrol by the coating and the density of solids on the surface was evaluated together with the antifungal effectiveness on the post-harvest storage of Golden Delicious apples previously inoculated with Penicillium expansum. Moreover, a sensory analysis was also carried out to study the acceptability of the coated product. The carvacrol loaded coatings were highly effective against the growth of Penicillium expansum, significantly reducing the incidence and severity of the fungus, especially in those formulated with higher proportion of starch and carvacrol (50PVA:50S-C40 and 50PVA:50S-C80). However, the results of the sensory analysis showed the negative impact of these coatings on the aroma and taste of the coated apples, even after the washing and peeling of the fruit.

[ES] En el presente trabajo se han estudiado diferentes formulaciones de recubrimientos elaborados a base de alcohol polivinílico (PVA) y almidón de patata (S) con la incorporación de carvacrol (C) que proporciona la acción antifúngica al recubrimiento. Se evaluó la retención de carvacrol por el recubrimiento y la densidad de sólidos en superficie. También se evaluó su efectividad antifúngica durante el almacenamiento poscosecha de manzanas Golden Delicious inoculadas con Penicillium expansum y la aceptación por los consumidores a través de catas sensoriales. Su aplicación fue muy efectiva contra el crecimiento de Penicillium expansum, al reducir significativamente la incidencia y severidad del hongo, especialmente aquellas formuladas con mayor proporción de almidón y carvacrol (50PVA:50S-C40 y 50PVA:50S-C80). Sin embargo, los resultados del análisis sensorial pusieron de manifiesto el impacto negativo del carvacrol sobre el aroma y sabor de las manzanas, incluso tras el lavado y el pelado de la fruta., [CA] Al present treball s'han estudiat diferents formulacions de recobriments elaborats amb base d'alcohol polivinílic (PVA) i midó de creïlla (S) amb l’incorporació de carvacrol (C) que proporciona l'acció antifúngica al recobriment. Es va avaluar la retenció de carvacrol pel recobriment i la densitat de sòlids en superfície i la seua l'efectivitat antifúngica sobre l'emmagatzemament postcollita de les pomes Golden Delicious inoculades amb Penicillium expansum. A mes, l'acceptació pels consumidors a través d’anàlisis sensorials també fou estudiada. Els resultats mostraren que la aplicació del recobriments amb carvacrol van ser molt efectives contra el 3 creixement de Penicillium expansum, al reduir significativament la incidència i severitat del fong, especialment en aquelles formulades amb major proporció de midó i carvacrol (50PVA:50S-C40 i 50PVA:50S-C80). No obstant això, els resultats de l'anàlisi sensorial mostraren un possible rebuig dels consumidors a estos recobriments, donada la baixa acceptació de l'aroma i el sabor del producte tractat, inclús després del llavat i el pelat de la fruita recoberta., [EN] In this work, different coatings based on polyvinyl alcohol (PVA) and potato starch (S) have been studied with the incorporation of carvacrol (C) that provides the antifungal action to the coating. The retention of carvacrol by the coating and the density of solids on the surface was evaluated together with the antifungal effectiveness on the post-harvest storage of Golden Delicious apples previously inoculated with Penicillium expansum. Moreover, a sensory analysis was also carried out to study the acceptability of the coated product. The carvacrol loaded coatings were highly effective against the growth of Penicillium expansum, significantly reducing the incidence and severity of the fungus, especially in those formulated with higher proportion of starch and carvacrol (50PVA:50S-C40 and 50PVA:50S-C80). However, the results of the sensory analysis showed the negative impact of these coatings on the aroma and taste of the coated apples, even after the washing and peeling of the fruit.

Penicillium expansum-infected apple fruit resulted in a disrupted balance of primary metabolites and impacted the ascorbate-glutathione cycle. The initial decline in fructose, malic acid, shikimic acid and total ascorbate content (6 hpi) was followed by a significant increase in malic acid (24 hpi) and total glutathione (3 dpi). Significant increase in sucrose was accompanied by a decline in glucose and ascorbate content (5 dpi). Total glutathione and shikimic acid gradually deteriorated with symptom development. A negative correlation was found between sucrose and ascorbate-glutathione pool. Other correlations were strong amongst other metabolites and lesion size indicating that they may be involved or important for apple fruit decay.

Penicillium expansum-infected apple fruit resulted in a disrupted balance of primary metabolites and impacted the ascorbate-glutathione cycle. The initial decline in fructose, malic acid, shikimic acid and total ascorbate content (6 hpi) was followed by a significant increase in malic acid (24 hpi) and total glutathione (3 dpi). Significant increase in sucrose was accompanied by a decline in glucose and ascorbate content (5 dpi). Total glutathione and shikimic acid gradually deteriorated with symptom development. A negative correlation was found between sucrose and ascorbate-glutathione pool. Other correlations were strong amongst other metabolites and lesion size indicating that they may be involved or important for apple fruit decay.

Antifungal proteins of fungal origin (AFPs) are small, secreted, cationic, and cysteine-rich proteins. Filamentous fungi encode a wide repertoire of AFPs belonging to different phylogenetic classes, which offer a great potential to develop new antifungals for the control of pathogenic fungi. The fungus Penicillium expansum is one of the few reported to encode three AFPs each belonging to a different phylogenetic class (A, B, and C). In this work, the production of the putative AFPs from P. expansum was evaluated, but only the representative of class A, PeAfpA, was identified in culture supernatants of the native fungus. The biotechnological production of PeAfpB and PeAfpC was achieved in Penicillium chrysogenum with the P. chrysogenum-based expression cassette, which had been proved to work efficiently for the production of other related AFPs in filamentous fungi. Western blot analyses confirmed that P. expansum only produces PeAfpA naturally, whereas PeAfpB and PeAfpC could not be detected. From the three AFPs from P. expansum, PeAfpA showed the highest antifungal activity against all fungi tested, including plant and human pathogens. P. expansum was also sensitive to its self-AFPs PeAfpA and PeAfpB. PeAfpB showed moderate antifungal activity against filamentous fungi, whereas no activity could be attributed to PeAfpC at the conditions tested. Importantly, none of the PeAFPs showed hemolytic activity. Finally, PeAfpA was demonstrated to efficiently protect against fungal infections caused by Botrytis cinerea in tomato leaves and Penicillium digitatum in oranges. The strong antifungal potency of PeAfpA, together with the lack of cytotoxicity, and significant in vivo protection against phytopathogenic fungi that cause postharvest decay and plant diseases, make PeAfpA a promising alternative compound for application in agriculture, but also in medicine or food preservation.

Patulin er et naturligt giftstof, der bl.a. produceres af svampen Penicillium expansum i lagrede æbler. Artiklen gennemgår de forhold, der påvirker indholdet af patulin i æblemost, bl.a. høstmetoder, lagringsforhold, sortering og håndtering i mostprocessen. Der gives en vejledning i, hvordan man minimerer risikoen for patulin-udvikling i æblemost.

Malus sieversii from Central Asia is a progenitor of the modern domesticated apple (Malus × domestica). Several accessions of M. sieversii are highly resistant to the postharvest pathogen Penicillium expansum. A previous study identified the qM–Pe3.1 QTL on LG3 for resistance to P. expansum in the mapping population GMAL4593, developed using the resistant accession, M. sieversii –PI613981, and the susceptible cultivar “Royal Gala” (RG) (M. domestica), as parents. The goal of the present study was to characterize the transcriptomic response of susceptible RG and resistant PI613981 apple fruit to wounding and inoculation with P. expansum using RNA–Seq. Transcriptomic analyses 0–48 h post inoculation suggest a higher basal level of resistance and a more rapid and intense defense response to wounding and wounding plus inoculation with P. expansum in M. sieversii –PI613981 than in RG. Functional analysis showed that ethylene–related genes and genes involved in “jasmonate” and “MYB–domain transcription factor family” were over–represented in the resistant genotype. It is suggested that the more rapid response in the resistant genotype (Malus sieversii–PI613981) plays a major role in the resistance response. At least twenty DEGs were mapped to the qM–Pe3.1 QTL (M × d v.1: 26,848,396–28,424,055) on LG3, and represent potential candidate genes responsible for the observed resistance QTL in M. sieversii–PI613981. RT–qPCR of several of these genes was used to validate the RNA–Seq data and to confirm their higher expression in MS0.

Patulin er et naturligt giftstof, der bl.a. produceres af svampen Penicillium expansum i lagrede æbler. Artiklen gennemgår de forhold, der påvirker indholdet af patulin i æblemost, bl.a. høstmetoder, lagringsforhold, sortering og håndtering i mostprocessen. Der gives en vejledning i, hvordan man minimerer risikoen for patulin-udvikling i æblemost.

[EN] This work aimed to control the fungal deterioration of strawberry jams. The antifungal activity of the clove, cinnamon leaf, lemon and mandarin essential oils and their effectiveness in oil-in-water emulsions were evaluated. According to the results obtained, only clove and cinnamon leaf oils were selected to prepare emulsions. All the tested emulsions were stable, independently the amount of polymer and essential oil used. Essential oil loss was affected by the amount of polymer employed to prepare the emulsions. The oil-in-water emulsions with 5.0 mg/g xanthan gum, and with 0.55 mg/g clove or 0.65 mg/g cinnamon leaf essential oil, were used for the in vivo tests. The jams prepared with the oil-in-water emulsions showed a lower fungal decay compared with jams without emulsion. The present work demonstrated that emulsions can be employed to prevent strawberry jam mould spoilage.

[EN] This work aimed to control the fungal deterioration of strawberry jams. The antifungal activity of the clove, cinnamon leaf, lemon and mandarin essential oils and their effectiveness in oil-in-water emulsions were evaluated. According to the results obtained, only clove and cinnamon leaf oils were selected to prepare emulsions. All the tested emulsions were stable, independently the amount of polymer and essential oil used. Essential oil loss was affected by the amount of polymer employed to prepare the emulsions. The oil-in-water emulsions with 5.0 mg/g xanthan gum, and with 0.55 mg/g clove or 0.65 mg/g cinnamon leaf essential oil, were used for the in vivo tests. The jams prepared with the oil-in-water emulsions showed a lower fungal decay compared with jams without emulsion. The present work demonstrated that emulsions can be employed to prevent strawberry jam mould spoilage.

Patulin is a mycotoxin that can be found in many apple products all over the world. It has been well-known for many decades and is nowadays one of the most regulated mycotoxins in food. Today when fruit and vegetables are traded internationally more than ever before, the quality of the goods might fall victim for careless storage during logistics. Still, with natural resources becoming scarce, we need to take care of what we got in our reach. Maybe at some point in the future we might have to find a usage for a shipment of apple juice contaminated with patulin. In this study, we focus on finding ways to decrease the amount of patulin in apple juice. By inoculation apples with the mold Penicillium expansum, 3 grades of decay were produced in the fruits. Diameters of lesion that the infection reached were sorted into 25 mm, 35 mm and 45 mm. Using a household fruit juicer these 3 grades of infected apples were juiced separately, to produce 3 juices containing different amounts of patulin. Having these 3 juices, using 5 treatments found in commercial juicing businesses (unfermented, pasteurized, fermented with commercial yeast strains Safale S-04 or Safale US-05, or spontaneously fermented by wild Saccharomyces cerevisiae) could prove if there were any process that would reduce the patulin concentration. Using a common extraction method and a HPLC analysis with a UV diode array detector at 276 nm, it was concluded that both the commercial yeasts lowered the concentration significantly if compared to the unfermented and the pasteurized. A significant difference in patulin concentration was concluded between the 3 degrees of lesion, where the highest grade of decay (45 mm lesion diameter) had the highest levels of patulin in its juice and the lowest grade of decay (25 mm lesion diameter) resulted in the lowest patulin concentration.

Penicillium expansum was identified as a major contaminant in indoor air, settled dust and materials of several buildings connected to indoor air related health complaints. This fungus emitted large quantities of exudates when cultivated on laboratory media. The exudates proved toxic towards four different mammalian test cells up to 10000 fold dilution. Toxins identified by LC-MS/MS were communesins and chaetoglobosin. Air dispersal of the toxic exudates was investigated with an experimental set-up where natural convection was generated by temperature gradient. It was found that the exudate with the contained toxins became airborne transported from the warmer surface to the colder surface. The results thus demonstrate transportation of microbially produced toxic substances across the air space. The role of liquid emissions from indoor molds represents a novel mechanism for human exposure in mold contaminated buildings. In this paper we report that vapor condensed from the indoor air of building affected with molds Aspergillus versicolor, Aspergillus calidoustus and Penicillium expansum contained substances that were acutely toxic when exposed to mammalian cells in vitro. The results encourage further study of condensed indoor water vapor as a tool to assess the presence of airborne substances with possible adverse health effects.

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples.

Although the plant cell wall is an extremely effective physical barrier against attack by pathogens, most phytopathogenic microorganisms produce enzymes that are capable of degrading cell wall polymers such as pectin and hemicelluloses. In this work we investigate the possible implication of the cell wall enzyme XET in the host-pathogen interaction as well as the XTH gene regulation during apple fruit infection by Penicillium expansum Link. A. We characterized the time course of fungal infection in apple fruit, and the XET activity and the expression of eleven MdXTHs. The results showed an important decrease in XET specific activity in the fruits after 24, 48 and 72 h of fruit infection (42.9 %, 73.1 % and 95.1 % respectively as compared with the controls). The expression analysis of the MdXTHs showed an important decrease in expression, as the infection progressed, in MdXTH2 and MdXTH10, as well as in MdXTH3, MdXTH4 and MdXTH5, but mainly after 72 h of fruit infection; and in MdXTH1, and MdXTH9 after 24 and 48 h of infection. These results suggested that the decrease in XET activity during infection could be mainly attributed to the decrease in expression of MdXTH2 and MdXTH10, since those were the MdXTHs that showed the highest expression levels during ripening, particularly MdXTH10. These results support the suggestion that inhibition of MdXTHs expression, and consequently the decrease in XET specific activity, could represent a pathogenicity factor, since the potential reconstructing role of the enzyme also decreased and the infection could progress.

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity. [Int Microbiol 2014; 17(2):81-90]Keywords: Pantoea agglomerans · Erwinia amylovora · Meloidogyne javanica · Penicillium expansum · Caenorhabditis elegans · biocontrol · biosafety · toxicity · hemolytic activity · Ames test

Although the plant cell wall is an extremely effective physical barrier against attack by pathogens, most phytopathogenic microorganisms produce enzymes that are capable of degrading cell wall polymers such as pectin and hemicelluloses. In this work we investigate the possible implication of the cell wall enzyme XET in the host-pathogen interaction as well as the XTH gene regulation during apple fruit infection by Penicillium expansum Link. A. We characterized the time course of fungal infection in apple fruit, and the XET activity and the expression of eleven MdXTHs. The results showed an important decrease in XET specific activity in the fruits after 24, 48 and 72 h of fruit infection (42.9 %, 73.1 % and 95.1 % respectively as compared with the controls). The expression analysis of the MdXTHs showed an important decrease in expression, as the infection progressed, in MdXTH2 and MdXTH10, as well as in MdXTH3, MdXTH4 and MdXTH5, but mainly after 72 h of fruit infection; and in MdXTH1, and MdXTH9 after 24 and 48 h of infection. These results suggested that the decrease in XET activity during infection could be mainly attributed to the decrease in expression of MdXTH2 and MdXTH10, since those were the MdXTHs that showed the highest expression levels during ripening, particularly MdXTH10. These results support the suggestion that inhibition of MdXTHs expression, and consequently the decrease in XET specific activity, could represent a pathogenicity factor, since the potential reconstructing role of the enzyme also decreased and the infection could progress.

Certain strains of Pantoea are used as biocontrol agents for the suppression of plant diseases. However, their commercial registration is hampered in some countries because of biosafety concerns. This study compares clinical and plant-beneficial strains of P. agglomerans and related species using a phenotypic analysis approach in which plant-beneficial effects, adverse effects in nematode models, and toxicity were evaluated. Plant-beneficial effects were determined as the inhibition of apple fruit infection by Penicillium expansum and apple flower infection by Erwinia amylovora. Clinical strains had no general inhibitory activity against infection by the fungal or bacterial plant pathogens, as only one clinical strain inhibited P. expansum and three inhibited E. amylovora. By contrast, all biocontrol strains showed activity against at least one of the phytopathogens, and three strains were active against both. The adverse effects in animals were evaluated in the plant-parasitic nematode Meloidogyne javanica and the bacterial-feeding nematode Caenorhabditis elegans. Both models indicated adverse effects of the two clinical strains but not of any of the plant-beneficial strains. Toxicity was evaluated by means of hemolytic activity in blood, and genotoxicity with the Ames test. None of the strains, whether clinical or plant-beneficial, showed any evidence of toxicity. [Int Microbiol 2014; 17(2):81-90]Keywords: Pantoea agglomerans · Erwinia amylovora · Meloidogyne javanica · Penicillium expansum · Caenorhabditis elegans · biocontrol · biosafety · toxicity · hemolytic activity · Ames test

The pine weevil Hylobius abietis (L.) is a severe pest of conifer seedlings in reforested areas of Europe and Asia. To identify minimally toxic and ecologically sustainable compounds for protecting newly planted seedlings, we evaluated the volatile metabolites produced by microbes isolated from H. abietis feces and frass. Female weevils deposit feces and chew bark at oviposition sites, presumably thus protecting eggs from feeding conspecifics. We hypothesize that microbes present in feces/frass are responsible for producing compounds that deter weevils. Here, we describe the isolation of a fungus from feces and frass of H. abietis and the biological activity of its volatile metabolites. The fungus was identified by morphological and molecular methods as Penicillium expansum Link ex. Thom. It was cultured on sterilized H. abietis frass medium in glass flasks, and volatiles were collected by SPME and analyzed by GC-MS. The major volatiles of the fungus were styrene and 3-methylanisole. The nutrient conditions for maximum production of styrene and 3-methylanisole were examined. Large quantities of styrene were produced when the fungus was cultured on grated pine bark with yeast extract. In a multi-choice arena test, styrene significantly reduced male and female pine weevils' attraction to cut pieces of Scots pine twigs, whereas 3-methylanisole only reduced male weevil attraction to pine twigs. These studies suggest that metabolites produced by microbes may be useful as compounds for controlling insects, and could serve as sustainable alternatives to synthetic insecticides., QC 20130306

A strain of Penicillium expansum was studied for the production of styrene using forest waste biomass as a feeding substrate. The fungal strain was cultivated on bark of various trees supplemented with yeast extract and the volatiles produced were collected on Tenax TA and analyzed by gas chromatography-mass spectrometry. Fungus cultured on grated soft bark of pine (Pinus sylvestris) stems (GPB) and mature bark of oak (Quercus robur) supplemented with yeast extract produced relatively the highest amounts of styrene. The maximum styrene production rate was 52.5 mu g/h, 41 mu g/h and 27 mu g/h from fungus cultivated on 50 mL liquid media with 10 g GPB or mature bark of oak and potato dextrose broth respectively. These promising results suggest that the fungal strain could be used to produce "green" styrene plastics using renewable forest waste biomass., QC 20131017. Updated from submitted to published.

Propolis has been used for millennia for its antimicrobial and pharmaceutical properties, whereas its use as an agricultural antifungal agent has only recently been assessed. The chemical characteristics of six ethanolic extracts of Chilean propolis (EEP-1 to EEP-6) were evaluated. The total polyphenols ranged between 7.8 and 42.3 mg mL-1 equivalents of a 2:1 pinocembrin: galangin standard, and the total flavonoid contents ranged between 4.0 and 19.7 mg mL-1 equivalents of quercetin. A high-performance liquid chromatographic (HPLC) analysis allowed the identification of caffeic acid, myricetin, quercetin, kaempferol, apigenin, pinocembrin, galangin, caffeic acid phenethyl ester (CAPE) and rutin. The EEP-3 extract was selected for additional studies based on the higher total polyphenols and flavonoids contents (42.3 and 19.7 mg mL-1, respectively). The antifungal effects of four different concentrations (0.5, 1.0, 2.5 and 5.0 %) of EEP-3 on Alternaria alternata, Fusarium sp., Ulocladium sp., Botrytis cinerea, Penicillium expansum and Trichoderma reesei were evaluated in vitro using the agar dilution method. Although significant differences occurred among the extract concentrations, the results indicated that EEP-3 inhibited the mycelial growth of the six fungi evaluated., El propóleo ha sido utilizado por el hombre desde hace milenios por sus propiedades antimicrobianas y farmacéutica. Sin embargo, su uso como un agente antifúngico agrícola sólo recientemente ha sido evaluado. Con el objetivo de estudiar su capacidad antifúngica se caracterizaron químicamente seis extractos etanólicos (EEP1-EEP6), comercializados en la Región de La Araucanía. Se evaluó el contenido de polifenoles y flavonoides totales presentes en los EEP, estos presentaron concentraciones de polifenoles totales en un rango de 7,8 a 42,3 mg mL-1 equivalentes de la mezcla pinocembrina: galangina 2:1, y de flavonoides totales en un rango que varió entre 4,0 y 19,7 mg mL-1 equivalentes de quercetina. Análisis por cromatografía líquida de alta resolución (HPLC) permitió la identificación del ácido cafeico, miricetina, quercetina, kaempferol, apigenina, pinocembrina, galangina, éster del ácido cafeico (CAPE) y rutina. Se seleccionó el EEP-3, pues presentó las mayores concentraciones de polifenoles y flavonoides totales 42,3 y 19,7 mg mL-1, respectivamente. El efecto antifúngico de cuatro diferentes concentraciones (0,5; 1,0; 2,5 y 5,0%) del EEP-3 sobre los hongos Alternaria alternata, Fusarium sp., Ulocladium sp., Botrytis cinerea, Penicillium expansum y Trichoderma reesei, fue evaluado in vitro mediante el método de dilución en agar. Se presentaron diferencias significativas entre las concentraciones de extracto ensayadas. Los resultados indican que el EEP-3 inhibió el crecimiento micelial en los seis hongos evaluados.

Propolis has been used for millennia for its antimicrobial and pharmaceutical properties, whereas its use as an agricultural antifungal agent has only recently been assessed. The chemical characteristics of six ethanolic extracts of Chilean propolis (EEP-1 to EEP-6) were evaluated. The total polyphenols ranged between 7.8 and 42.3 mg mL-1 equivalents of a 2:1 pinocembrin: galangin standard, and the total flavonoid contents ranged between 4.0 and 19.7 mg mL-1 equivalents of quercetin. A high-performance liquid chromatographic (HPLC) analysis allowed the identification of caffeic acid, myricetin, quercetin, kaempferol, apigenin, pinocembrin, galangin, caffeic acid phenethyl ester (CAPE) and rutin. The EEP-3 extract was selected for additional studies based on the higher total polyphenols and flavonoids contents (42.3 and 19.7 mg mL-1, respectively). The antifungal effects of four different concentrations (0.5, 1.0, 2.5 and 5.0 %) of EEP-3 on Alternaria alternata, Fusarium sp., Ulocladium sp., Botrytis cinerea, Penicillium expansum and Trichoderma reesei were evaluated in vitro using the agar dilution method. Although significant differences occurred among the extract concentrations, the results indicated that EEP-3 inhibited the mycelial growth of the six fungi evaluated., El propóleo ha sido utilizado por el hombre desde hace milenios por sus propiedades antimicrobianas y farmacéutica. Sin embargo, su uso como un agente antifúngico agrícola sólo recientemente ha sido evaluado. Con el objetivo de estudiar su capacidad antifúngica se caracterizaron químicamente seis extractos etanólicos (EEP1-EEP6), comercializados en la Región de La Araucanía. Se evaluó el contenido de polifenoles y flavonoides totales presentes en los EEP, estos presentaron concentraciones de polifenoles totales en un rango de 7,8 a 42,3 mg mL-1 equivalentes de la mezcla pinocembrina: galangina 2:1, y de flavonoides totales en un rango que varió entre 4,0 y 19,7 mg mL-1 equivalentes de quercetina. Análisis por cromatografía líquida de alta resolución (HPLC) permitió la identificación del ácido cafeico, miricetina, quercetina, kaempferol, apigenina, pinocembrina, galangina, éster del ácido cafeico (CAPE) y rutina. Se seleccionó el EEP-3, pues presentó las mayores concentraciones de polifenoles y flavonoides totales 42,3 y 19,7 mg mL-1, respectivamente. El efecto antifúngico de cuatro diferentes concentraciones (0,5; 1,0; 2,5 y 5,0%) del EEP-3 sobre los hongos Alternaria alternata, Fusarium sp., Ulocladium sp., Botrytis cinerea, Penicillium expansum y Trichoderma reesei, fue evaluado in vitro mediante el método de dilución en agar. Se presentaron diferencias significativas entre las concentraciones de extracto ensayadas. Los resultados indican que el EEP-3 inhibió el crecimiento micelial en los seis hongos evaluados.

The aim of the study was to evaluate the efficacy of lactic acid bacteria isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 lactic acid bacteria strains was tested in vitro, and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also tested for inhibition of infection of P. expansum in wounds of Golden Delicious apples. Four strains TC97, AC318, TM319 and FF441 reduced the fungal rot diameter of apples by 20%, and only, Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred inhibition mechanism but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against Erwinia carotovora. These results support the potentiality of lactic acid bacteria as biocontrol agents against postharvest rot. Previous works reporting of antifungal activity by lactic acid bacteria are scarce.

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.

The aim of the study was to evaluate the efficacy of lactic acid bacteria isolated from fresh fruits and vegetables as biocontrol agents against the phytopathogenic and spoilage bacteria and fungi, Xanthomonas campestris, Erwinia carotovora, Penicillium expansum, Monilinia laxa, and Botrytis cinerea. The antagonistic activity of 496 lactic acid bacteria strains was tested in vitro, and all tested microorganisms except P. expansum were inhibited by at least one isolate. The 496 isolates were also tested for inhibition of infection of P. expansum in wounds of Golden Delicious apples. Four strains TC97, AC318, TM319 and FF441 reduced the fungal rot diameter of apples by 20%, and only, Weissella cibaria strain TM128 decreased infection levels by 50%. Cell-free supernatants of selected antagonistic bacteria were studied to determine the nature of the antimicrobial compounds produced. Organic acids were the preferred inhibition mechanism but hydrogen peroxide was also detected when strains BC48, TM128, PM141 and FF441 were tested against Erwinia carotovora. These results support the potentiality of lactic acid bacteria as biocontrol agents against postharvest rot. Previous works reporting of antifungal activity by lactic acid bacteria are scarce.