LaeA regulation of secondary metabolism modulates virulence in Penicillium expansum and is mediated by sucrose

Penicillium expansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxin patulin in colonized apple fruit tissue. Although patulin is produced by many Penicillium species, the factor(s) activating its biosynthesis are not clear. Sucrose, a key sugar component of apple fruit, was found to modulate patulin accumulation in a dose‐responsive pattern. An increase in sucrose culture amendment from 15 to 175 mm decreased both patulin accumulation and expression of the global regulator laeA by 175‐ and five‐fold, respectively, whilst increasing expression of the carbon catabolite repressor creA. LaeA was found to regulate several secondary metabolite genes, including the patulin gene cluster and concomitant patulin synthesis in vitro. Virulence studies of ΔlaeA mutants of two geographically distant P. expansum isolates (Pe‐21 from Israel and Pe‐T01 from China) showed differential reduction in disease severity in freshly harvested fruit, ranging from no reduction for Ch‐Pe‐T01 strains to 15%–25% reduction for both strains in mature fruit, with the ΔlaeA strains of Is‐Pe‐21 always showing a greater loss in virulence. The results suggest the importance of abiotic factors in LaeA regulation of patulin and other secondary metabolites that contribute to pathogenicity.     

Patulin is a cultivar‐dependent aggressiveness factor favouring the colonization of apples by Penicillium expansum

The blue mould decay of apples is caused by Penicillium expansum and is associated with contamination by patulin, a worldwide regulated mycotoxin. Recently, a cluster of 15 genes (patA–patO) involved in patulin biosynthesis was identified in P. expansum. blast analysis revealed that patL encodes a Cys₆ zinc finger regulatory factor. The deletion of patL caused a drastic decrease in the expression of all pat genes, leading to an absence of patulin production. Pathogenicity studies performed on 13 apple varieties indicated that the PeΔpatL strain could still infect apples, but the intensity of symptoms was weaker compared with the wild‐type strain. A lower growth rate was observed in the PeΔpatL strain when this strain was grown on nine of the 13 apple varieties tested. In the complemented PeΔpatL:patL strain, the ability to grow normally in apple and the production of patulin were restored. Our results clearly demonstrate that patulin is not indispensable in the initiation of the disease, but acts as a cultivar‐dependent aggressiveness factor for P. expansum. This conclusion was strengthened by the fact that the addition of patulin to apple infected by the PeΔpatL mutant restored the normal fungal colonization in apple.     

Dissection of patulin biosynthesis,spatial control and regulation mechanism in Penicillium expansum

The patulin biosynthesis is one of model pathways in an understanding of secondary metabolite biology and network novelties in fungi. However, molecular regulation mechanism of patulin biosynthesis and contribution of each gene related to the different catalytic enzymes in the biochemical steps of the pathway remain largely unknown in fungi. In this study, the genetic components of patulin biosynthetic pathway were systematically dissected in Penicillium expansum, which is an important fungal pathogen and patulin producer in harvested fruits and vegetables. Our results revealed that all the 15 genes in the cluster are involved in patulin biosynthesis. Proteins encoded by those genes are compartmentalized in various subcellular locations, including cytosol, nucleus, vacuole, endoplasmic reticulum, plasma membrane and cell wall. The subcellular localizations of some proteins, such as PatE and PatH, are required for the patulin production. Further, the functions of eight enzymes in the 10-step patulin biosynthetic pathway were verified in P. expansum. Moreover, velvet family proteins, VeA, VelB and VelC, were proved to be involved in the regulation of patulin biosynthesis, but not VosA. These findings provide a thorough understanding of the biosynthesis pathway, spatial control and regulation mechanism of patulin in fungi.     

Occurrence of<Emphasis Type="Italic">penicillium expansum</Emphasis> and patulin in apples as raw materials for processing of foods — case study

The contamination of apples Gloster variety withPenicillium expansum was investigated in the case study. The apples serve as a raw material in thefood industry for processing of baby foodstuffs and other apple products. The core of 3 – 5 % apple samples has been contaminated with spores and mycelium of untested microfungi. The surface tissue of stored apples has not been damaged. Penicillium expansum was isolated in all tested apple samples. Patulin has been found at levels tens to hundreds of üg/kg in tested apple samples. Patulin was primarily associated with core and areas of decomposing tissue and can penetrate up to approx. 1 cm of the surrounding healthy tissue. The production of patulin after the experimental contamination of apples was tested after 14 days of storage (21 °C, dark). The maximum value of patulin was found 110 mg/kg of rotten part of apple sample.     

Low occurrence of patulin‐ and citrinin‐producing species isolated from grapes

Aims: To assess the ability of fungi isolated from grapes to produce patulin and citrinin. Methods and Results: A total of 446 Aspergillus isolates belonging to 20 species and 101 Penicillium isolates were inoculated in Czapek yeast extract agar and yeast extract sucrose agar and incubated for 7 days at 25°C. Extracts were analysed for patulin and citrinin by thin‐layer chromatography. None of the isolates of Aspergillus spp. produced either patulin or citrinin. Patulin was produced by three isolates of Penicillium expansum and two of Penicillium griseofulvum. Citrinin was produced by five isolates of P. expansum, two of Penicillium citrinum and one of Penicillium verrucosum. Conclusions: Our results show that the Aspergillus and Penicillium species commonly isolated from grapes are not a source of the mycotoxins, patulin and citrinin. Significance and Impact of the Study: The possibility of co‐occurrence of patulin and citrinin with ochratoxin A in grapes and grape products remain low, owing to the low frequency of isolation of potentially producing species.     

Lactic acid bacteria with potential to eliminate fungal spoilage in foods

Aims: To investigate antifungal activity produced by lactic acid bacteria (LAB) isolated from malted cereals and to determine if such LAB have the capacity to prevent fungal growth in a particular food model system. Methods and Results: The effect of pH, temperature and carbon source on production of antifungal activity by four LAB was determined. Pediococcus pentosaceus was used to conduct a trial to determine if it is feasible to eliminate Penicillium expansum, the mould responsible for apple rot, using an apple model. Penicillium expansum was incapable of growth during the trial on apple‐based agar plates inoculated with the antifungal‐producing culture, whereas the mould did grow on apple plates inoculated with an LAB possessing no antifungal activity. Conclusion: Partial characterization of the antifungal compounds indicates that their activity is likely to be because of production of antifungal peptides. The trial conducted showed that the antifungal culture has the ability to prevent growth of the mould involved in apple spoilage, using apples as a model. Significance and Impact of the study: The ability of an LAB to prevent growth of Pen. expansum using the apple model suggests that these antifungal LAB have potential applications in the food industry to prevent fungal spoilage of food.     

Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation

Aspergillus flavus, A. niger, Penicillium expansum and Rhizopus stolonifer were the most frequently isolated fungi from healthy apple fruits. Alternaria alternata was the most common organism of rotten apple fruits, followed by A. niger, A. flavus, P. expansum and R. stolonifer. The prevalent type of decay, brown rot lesion, is caused by R. stolonifer followed by A. flavus, A. niger, A. alternata and P. expansum. Sodium hypochlorite had good curative properties against fruit rots. The main natural mycotoxins produced in rotten apple were patulin and aflatoxins. The optimum temperature for patulin production by P. expansum was 15 °C after 15 days. Complete inhibition of patulin formation was attained using 0.2% lemon oil and > 90% inhibition using 0.05% lemon and 0.2% orange oils. Also significant inhibition (> 90%) of aflatoxin production was observed with 0.2% lemon oil. This revised version was published online in July 2006 with corrections to the Cover Date.     

The isoepoxydon dehydrogenase gene of the patulin metabolic pathway differs for Penicillium griseofulvum and Penicillium expansum

Purified DNA from isolates of Penicillium griseofulvum and P. expansum was used as a template to amplify a 600-bp fragment of the isoepoxydon dehydrogenase (idh) gene of the patulin biosynthetic pathway. Primer pairs designed from the P. griseofulvum gene (GenBank accession AF006680) to amplify specific regions of the idh gene yielded similar-sized bands for all strains. Asymmetrical amplification produced DNA products for sequencing and DNA sequences were translated to produce the corresponding amino acid sequences. After removal of two introns present in the region sequenced, amino acid sequences were compared. There were 12 amino acid differences between P. expansum and P. griseofulvum in the coding region. The differences correlated with the amount of patulin previously produced in culture, with strains of P. griseofulvum producing the greatest amounts of patulin.     

Potential of patulin production by Penicillium expansum strains on various fruits

In this study, we investigated the pathogenicity and patulin production by ten strains of Penicillium expansum on various fruits (apples, apricots, kiwis, plums and peaches) at two (4°C and 25°C) different temperature regimes. All strains caused the infectious rots on all fruits at 4 and 25°C except one strain (PEX 09) at 4°C. Two strains (PEX 20 and PEX 12) out of ten produced the highest amounts of patulin on all fruits tested. The patulin production by P. expansum is high at 25°C compared to 4°C. All strains of P. expansum accumulated patulin ranging from 100–13,200 μg/kg and nine strains ranging from 100–12,100 μg/kg in all fruits at 25°C and 4°C, respectively. Among ten strains of P. expansum, strain PEX 20 produced the greatest amount of patulin on apricots (13,200 μg/kg of rotten fruit) and on apples (12,500 μg/kg) at 25°C after 9 days of incubation. At 4°C, this strain produced 12,100, 12,000, 2,100 and 1,200 μg/kg of patulin on apricots, apples, plums and peaches, respectively, after 45 days of incubation. Strain PEX 12 produced the highest amount of patulin on kiwis (10,700 μg/kg) at 25°C and 10,300 μg/kg at 4°C. Patulin production by P. expansum on peaches and plums at both temperatures were lower than other fruits. The results of this study showed that careful removal of rotten fruits is essential to produce patulin-free fruit juice, since high patulin levels in apricots, apples and kiwis could result in a level greater than 50 μg/kg of this mycotoxin in finished fruit juices, when one contaminated fruit occurs in 264, 250 and 214 fruits, respectively. So, the fruit processors should take care in not using rotten fruits for juice production to avoid the patulin problem worldwide, since this study proved that most important fruits being used for juice production and direct human consumption are susceptible to P. expansum and subsequent patulin production even at low temperatures. This is the first comprehensive report regarding patulin production by different strains of P. expansum on various fruits from Italy at different temperature regimes.     

Carbon,nitrogen and pH regulate the production and activity of a polygalacturonase isozyme produced by Penicillium expansum

The influence of carbon, nitrogen and pH on polygalacturonase (PG) activity produced by Penicillium expansum were investigated. P. expansum mycelial growth was greatest on lyophilized lyophilised fruit tissue and the highest PG activity occurred in apple pectin medium. Nitrogen source influenced PG activity and was highest with ammonia while the greatest mycelial mass was supported by glutamate or glutamine. PG activity and mycelial mass peaked 5 five days after inoculation as polyuronide content decreased and the pH and ammonium levels increased in apple pectin medium. A single active PG isozyme with an isoelectric point of ～7.6 was produced in apple pectin medium and a partial cDNA clone was obtained that was most homologous to the pggII gene from Penicillium. griseoroseum. The results from this study indicate that P. expansum can modulate the activity of PG in response to nutrient sources and ambient pH through signalling pathways that modulate nutrient acquisition, uptake and metabolism.     

Carbon regulation of environmental pH by secreted small molecules that modulate pathogenicity in phytopathogenic fungi

Fruit pathogens can contribute to the acidification or alkalinization of the host environment. This capability has been used to divide fungal pathogens into acidifying and/or alkalinizing classes. Here, we show that diverse classes of fungal pathogens—Colletotrichum gloeosporioides, Penicillium expansum, Aspergillus nidulans and Fusarium oxysporum—secrete small pH‐affecting molecules. These molecules modify the environmental pH, which dictates acidic or alkaline colonizing strategies, and induce the expression of PACC‐dependent genes. We show that, in many organisms, acidification is induced under carbon excess, i.e. 175 mm sucrose (the most abundant sugar in fruits). In contrast, alkalinization occurs under conditions of carbon deprivation, i.e. less than 15 mm sucrose. The carbon source is metabolized by glucose oxidase (gox2) to gluconic acid, contributing to medium acidification, whereas catalysed deamination of non‐preferred carbon sources, such as the amino acid glutamate, by glutamate dehydrogenase 2 (gdh2), results in the secretion of ammonia. Functional analyses of Δgdh2 mutants showed reduced alkalinization and pathogenicity during growth under carbon deprivation, but not in high‐carbon medium or on fruit rich in sugar, whereas analysis of Δgox2 mutants showed reduced acidification and pathogencity under conditions of excess carbon. The induction pattern of gdh2 was negatively correlated with the expression of the zinc finger global carbon catabolite repressor creA. The present results indicate that differential pH modulation by fruit fungal pathogens is a host‐dependent mechanism, affected by host sugar content, that modulates environmental pH to enhance fruit colonization.