Patulin Production Research Articles

PePalA/B/C are required for virulence and patulin biosynthesis by regulating the PePacC-processing proteolytic activity in Penicillium expansum

Abstract Background The Pal/Rim signaling pathway is crucial for fungal responses to ambient pH conditions. It comprises the transcription factor PacC/Rim101 and six upstream Pal proteins. While the role of PacC has been extensively studied, there is limited information on the functions of the upstream Pal proteins. Results In the genome of Penicillium expansum, genes encoding the endosomal membrane complex (PalA/B/C) were identified. Among these, only the expression of PePalB, not PePalA or PePalC, is pH-dependent. Subcellular localization and functional analyses showed that PePalA/B/C are localized to the cytosol and peripheral punctate structures. Deletion of PePalA/B/C resulted in reduced growth and conidiation of P. expansum across various pH conditions. The virulence of the ΔPePalA/B/C mutant was significantly reduced in pear and apple fruits. Additionally, the mutants exhibited a loss of patulin production under both acidic and alkaline conditions and the down-regulation of genes in the patulin biosynthetic cluster. pH shift experiments further demonstrated that PePalA/B/C are essential for both the PePacC expression and the pH-dependent proteolytic activation of PePacC. Conclusions These findings highlight the significant roles of PePalA/B/C in regulating growth, conidiation, virulence, and patulin production in P. expansum, thereby enhancing our understanding of the regulatory mechanisms underlying the Pal/Rim signaling pathway.

Molecular and chemical evaluation of patulin production of Aspergillus and Penicillium-like species isolated from Hungarian apples

Mycotoxins are secondary fungal metabolites harmful to humans and animals. Patulin (PAT) is a toxin found in different food products but especially in apples and their derivative products. The most common fungi producers of this compound are Aspergillus clavatus and Penicillium expansum. The production of patulin, as other mycotoxins, can be impacted by diverse phenomena such as water and nutrient availability, UV exposure, and the presence of antagonistic organisms. Consequently, gaining a comprehensive understanding of climate and environmental conditions is a crucial step in combating patulin contamination. In this study, moulds were isolated from 40 apple samples collected from seven locations across Hungary: Csenger, Damak, Pallag, Lövőpetri, Nagykálló, and Újfehértó. A total of 183 moulds were morphologically identified, with 67 isolates belonging to the Alternaria, 45 to the Aspergillus, and 13 to the Penicillium groups. The location possessed a higher influence than farming method on the distribution of mould genera. Despite the requirement of higher temperature, Aspergillus species dominated only for the region of Újfehértó with approximately 50% of the isolates belonging to the genus. Four of the seven locations assessed: Csenger, Debrecen-Pallag, Nyírtass and Nagykálló, were dominated by Alternaria species. All isolates belonging to the genera Aspergillus and Penicillium were tested for the presence of the isoepoxidone dehydrogenase (idh) gene, a key player in the patulin metabolic pathway. To guarantee patulin production, this ability was confirmed with TLC assays. The only Aspergillus strain that presented a positive result was the strain Aspergillus clavatus B9/6, originated from the apple cultivar Golden Reinders grown in Debrecen-Pallag by integrated farming. Of the Penicillium isolates only one strain, B10/6, presented a band of the right size (500-600 bp) for the idh gene. Further sequencing of the ITS gene showed that this strain should be classified as Talaromyces pinophilus. The TLC tests confirmed this microorganism as the only patulin producer under the studied conditions for its cluster.

Effects of temperature, pH, and relative humidity on the growth of Penicillium paneum OM1 isolated from pears and its patulin production

Patulin is a mycotoxin produced by several species of Penicillium sp., Aspergillus sp., and Byssochlamys sp. on apples and pears. Most studies have been focused on Penicillium expansum, a common postharvest pathogen, but little is known about the characteristics of Penicillium paneum. In the present study, we evaluated the effects of temperature, pH, and relative humidity (RH) on the growth of P. paneum OM1, which was isolated from pears, and its patulin production. The fungal strain showed the highest growth rate at 25 °C and pH 4.5 on pear puree agar medium (PPAM) under 97 % RH, while it produced the highest amount of patulin at 20 °C and pH 4.5 on PPAM under 97 % RH. Moreover, RT-qPCR analysis of relative expression levels of 5 patulin biosynthetic genes (patA, patE, patK, patL, and patN) in P. paneum OM1 exhibited that the expression of the 4 patulin biosynthetic genes except patL was up-regulated in YES medium (patulin conducive), while it was not in PDB medium (patulin non-conducive). Our data demonstrated that the 3 major environmental parameters had significant impact on the growth of P. paneum OM1 and its patulin production. These results could be exploited to prevent patulin contamination by P. paneum OM1 during pear storage

Growth kinetics and patulin production by Penicillium setosum in pineapple juice under different temperatures and initial pH values

Penicillium setosum, a member of the Penicillium section Lanata-divaricata, is known for its ability to produce high levels of patulin. However, there is a notable lack of information regarding the influence of environmental factors on the growth and patulin production in fruit juice of this species. In this study, we investigated the impacts of temperature (25, 30, 35, or 40 °C), initial pH value (3, 5, or 7), and incubation time (2, 4, or 6 days) on the growth and patulin production in pineapple juice of the selected P. setosum strain HR9-5. The modified Gompertz model was applied to estimate the maximum growth rate and lag time prior to growth. This strain had the highest growth rate with the shortest lag time within the temperature range 30–35 °C, with an initial pH range of 3–5. Furthermore, it had the greatest biomass dry weight and patulin production at 35 °C with an initial pH of 3, and at 25 °C with an initial pH of 7, respectively. The growth rate and lag time prior to growth of P. setosum HR9-5 were significantly influenced by the initial pH, whereas the biomass dry weight was strongly affected by temperature and incubation time. Additionally, all factors and their interactions had a significant impact on patulin production. This study provided the first report on the effect of environmental factors on growth and patulin production by P. setosum in pineapple juice. These findings can contribute to formulating effective prevention strategies to control the growth of P. setosum and subsequent patulin production in pineapple and other fruit juices.

WSC1 Regulates the Growth, Development, Patulin Production, and Pathogenicity of Penicillium expansum Infecting Pear Fruits.

In this study, the role of WSC1 in the infection of pear fruit by Penicillium expansum was investigated. The WSC1 gene was knocked out and complemented by Agrobacterium-mediated homologous recombination technology. Then, the changes in growth, development, and pathogenic processes of the knockout mutant and the complement mutant were analyzed. The results indicated that deletion of WSC1 slowed the growth rate, reduced the mycelial and spore yield, and reduced the ability to produce toxins and pathogenicity of P. expansum in pear fruits. At the same time, the deletion of WSC1 reduced the tolerance of P. expansum to cell wall stress factors, enhanced antioxidant capacity, decreased hypertonic sensitivity, decreased salt stress resistance, and was more sensitive to most metal ions. Our results confirmed that WSC1 plays an important role in maintaining cell wall integrity and responding to stress, toxin production, and the pathogenicity of P. expansum.

Genetic identification, pathogenicity and patulin production of Penicillium species from apple blue mold in China

Abstract Objectives Blue mold is a common postharvest disease that leads to severe rot and patulin (PAT) contamination in fruits, which is a key factor endangering the storage and quality of apple products. However, the Penicillium species from apple blue mold in China and their pathogenicity and PAT production ability have not been well studied. Materials and Methods In this study, 62 strains of pathogenic Penicillium species were isolated from blue mold apples collected from different regions of China. The ITS, BenA, and CaM genes were sequenced for molecular identification. Results Penicillium strains were identified as three species, 57 P. expansum, 3 P. crustosum, and 2 P. polonicum. Twelve variants of the ITS sequence, 61 variants of BenA and 67 variants of CiaM genes were identified between Penicillium species. Fungal morphology was observed on potato dextrose agar (PDA). After 10 d of cultivation, P. expansum cultures were detected with PAT concentrations ranging from 457.98 to 1333.85 mg/kg, and two P. polonicum strains were 6.02 mg/kg and 6.30 mg/kg. The three P. crustosum strains could not produce PAT on PDA. In apple inoculation, P. expansum strains showed significantly faster infection rates than P. crustosum and P. polonicum strains. All P. expansum strains can produce PAT during infection, and the concentrations in the rot apple tissues were 2.31–88.67 mg/kg. The P. crustosum and P. polonicum strains could not produce PAT during infection. Conclusions This study provides data and information regarding the morphology, virulence and PAT production of Penicillium pathogens that will improve the understanding of apple blue mold and postharvest disease control.

Patulin removal and biopreservation of apple by three lactic acid bacteria strains isolated from foods

ABSTRACTPenicillium expansum contamination and patulin production can occur at various stages of the food chain, leading to detrimental effects on the quality and shelf life of food. Consequently, it is imperative to develop safe and efficient methods to control fungal growth and prevent mycotoxin contamination. The use of lactic acid bacteria (LAB) for food biopreservation has become one of the most promising alternatives. In this study, we investigated the antifungal activity of three LAB strains, namely Lactiplantibacillus plantarum 4F, Lactiplantibacillus plantarum 4JC, and Lacticaseibacillus paracasei 3MI3 against Penicillium expansum, and we further evaluated their biopreservative potential using apple as a food model against the blue mould. Moreover, their ability to remove patulin (10 µg/mL) from MRS broth and apple juice was also examined. The mechanism involved in this decontamination was investigated using scanning electron microscopy with energy-dispersive X-ray spectroscopy (SEM-EDS) and fourier transform infrared spectroscopy (FTIR). It was found that in vitro antifungal activity of the three strains was validated in vivo. All strains were able to reduce patulin from MRS medium, notably L. plantarum4F exhibited the highest rate of reduction (88.18%). This particular strain eliminated patulin from juice, with a reduction of 87.26%. The C = O, C–H, and N–H were the main functional groups engaged in patulin adsorption indicated by FTIR and SEM-EDS. Furthermore, in terms of morphological changes, SEM analysis revealed that bacterial cell wall became irregular. This research provides evidence of control of blue Penicilium growth on apple and patulin removal using antifungal LAB strains.

The small GTPase Ypt7 of Penicillium expansum is required for growth, patulin biosynthesis and virulence

Ypt GTPases are the largest subfamily of small GTPases involved in membrane transport. Here, a PeYpt7 gene deletion mutant of P. expansum was constructed. The ΔPeYpt7 mutant showed reduced colony growth with abnormal mycelial growth, reduced conidiation, and insufficient spore development. The mutation rendered the pathogen susceptible to osmotic stress and cell wall stressors. In addition, the absence of PeYpt7 reduced patulin production in P. expansum and significantly limited gene expression (PatG, PatH, PatI, PatD, PatF, and PatL). In addition, the mutant showed attenuated virulence in infected fruit and reduced expression of pathogenic factors was (PMG, PG, PL, and GH1). Thus, PeYpt7 modulates the growth, morphology, patulin accumulation, and pathogenicity of P. expansum by limiting the expression of related genes.

Volatile organic compounds produced by Bacillus aryabhattai AYG1023 against Penicillium expansum causing blue mold on the Huangguan pear

Previous research on Bacillus aryabhattai has mainly focused on bioremediation, biosynthesis, and promotion of plant growth, whereas the function of B. aryabhattai on antifungal activity remains to be explored. In this study, we isolated a biocontrol bacterium with antagonistic activity against post-harvest pathogenic fungi by releasing volatile organic compounds (VOCs). We aimed to assess the effectiveness of VOCs produced by B. aryabhattai in prevention of the development of blue mold caused by Penicillium expansum in the Huangguan pear, and reveal the inhibitory mechanism against the pathogenic fungi. Using molecular methods, the biocontrol bacterium was identified as Bacillus aryabhattai AYG1023. 2-Nonanol was identified as the main VOC by solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC/MS). It showed strong inhibition of mycelial growth and conidial germination when treated at the minimum inhibitory concentration (MIC). Scanning and transmission electron microscopy showed that 2-nonanol caused abnormal changes in mycelial and conidial ultrastructure. 2-Nonanol also damaged the integrity of fungal cell membranes and reduced the ergosterol content to 44.77% of P. expansum. In addition, the production of secondary metabolites in P. expansum including patulin and citrinin was significantly reduced by 2-nonanol. Transcriptome analysis revealed that 2-nonanol modulated the expression of genes involved in development, and conidiation pathways, as well as secondary metabolite biosynthesis including steroid biosynthesis, citrinin production and patulin production. Furthermore, blue mold was completely inhibited by treatment with 0.04 μL mL−1 2-nonanol for 48 h on the Huangguan pear. In conclusion, Bacillus aryabhattai AYG1023 was identified as a promising and efficient agent for controlling post-harvest diseases via the release of VOCs, and the outcome of this study lays a theoretical foundation for future applications.

The histone demethylase KdmB is part of a trimeric protein complex and mediates virulence and mycotoxin production in Penicillium expansum

Epigenetic modification of chromosome structure has increasingly been associated with alterations in secondary metabolism and sporulation defects in filamentous fungal pathogens. Recently, the epigenetic reader protein SntB was shown to govern virulence, spore production and mycotoxin synthesis in the fruit pathogen Penicillium expansum. Through immunoprecipitation-coupled mass spectrometry, we found that SntB is a member of a protein complex with KdmB, a histone demethylase and the essential protein RpdA, a histone deacetylase. Deletion of kdmB phenocopied some but not all characteristics of the ΔsntB mutant. KdmB deletion strains exhibited reduced lesion development on Golden Delicious apples and this was accompanied by decreased production of patulin and citrinin in host tissue. In addition, ΔkdmB mutants were sensitive to several cell wall stressors which possibly contributed to the decreased virulence observed on apples. Slight differences in spore production and germination rates of ΔkdmB mutants in vitro did not impact overall diameter growth in culture.

ArfA and SarA small GTPases involved in growth, patulin biosynthesis and virulence of Penicillium expansum

Although the Sar/Arf GTPase belonging to a small family of GTPases contributes to growth of fungi, its function in postharvest fungi has not been characterized. Here, ArfA and SarA gene deletion mutants were generated in Penicillium expansum. We found that ΔArfA and ΔSarA exhibited attenuated colony growth and rough colony morphology, as well as insufficient spore development. ΔArfA also increased hyphal branches. Stress experiments showed that knocking out the ArfA or SarA genes affected cell wall integrity of the P. expansum strain. In addition, ΔArfA and ΔSarA significantly downregulated the expression of genes controlling spore development (wetA, abaA and brlA) and MAPK pathway regulators (Fus3, Kss1, Hog1 and Mpk1). Absence of ArfA and SarA reduced patulin production, and the related gene expression was significantly limited. ΔArfA showed attenuated virulence in infected fruit and downregulated PG and PL gene expression. However, ΔSarA had no significant effect on fungal virulence. Taken together, both ArfA and SarA contribute to the growth, development and patulin biosynthesis in P. expansum. ArfA is also involved in the virulence of P. expansum in fruit.

Ena Proteins Respond to PacC-Mediated pH Signaling Pathway and Play a Crucial Role in Patulin Biosynthesis.

Penicillium expansum is a main producer of patulin that causes severe postharvest decay and food safety issues in the fruit industry. Development, pathogenicity, and patulin production of P. expansum are strongly influenced by the PacC-pH signaling pathway. Global transcription factor PacC regulates various fungal biological processes through a complicated molecular network. In the present study, three Ena family genes (PeEnas), PeEnaA, PeEnaB, and PeEnaC, as important downstream targets of PePacC, were identified in P. expansum. Deletion of PeEnaA, PeEnaB, and PeEnaC showed little effect on mycelial growth under alkaline or high salinity conditions, but double and triple deletion of these genes impaired the virulence of P. expansum on apple fruit. Notably, patulin biosynthesis of P. expansum was distinctly inhibited in the deletion mutants of PeEnas. PeEnas regulated expressions of the patulin gene cluster, AP1, CreA, Sge1, and Hog1 at the transcriptional level and played roles in maintaining membrane potential. Overexpression of PeEnaC in ΔPePacC restored the patulin production defect of ΔPePacC. Our results indicated that, as downstream targets of PePacC, the PeEna family proteins play a crucial role in patulin biosynthesis in P. expansum.

Blue LED light treatment inhibits virulence and patulin biosynthesis in Penicillium expansum

Blue light-emitting diode (LED) light has been considered to influence the growth, quality, and preservation of certain fruit and vegetable. However, the blue LED light impact on apple postharvest blue mold remains elusive. In this study, the influence of blue LED light on Penicillium expansum virulence and patulin secretion was investigated in vitro and in vivo, as well as on the apple quality. The growth of P. expansum was inhibited by blue LED light via changes in mycelium morphology changes, cell membrane integrity, and potential mitochondrial membrane destruction, thereby causing reactive oxygen species accumulation, oxidative stress, and mitochondrial dysfunction. Furthermore, the expression level of LaeA relevant to the pathogenicity of P. expansum, was down-regulated. Specifically, the patulin production of P. expansum was inhibited via expression down-regulation of the patulin biosynthesis-related genes PatA, PatE, and PatN rather than direct degradation by the blue LED light treatment. Additionally, no significant effects on the fruit quality were observed when the light intensity was lower than 120 μmol m-2 s-1. In summary, blue LED light treatment has the potential for a low-cost, safe, and non-chemical approach to control apple postharvest decay.

Aggressiveness and Patulin Production in Penicillium expansum Multidrug Resistant Strains with Different Expression Levels of MFS and ABC Transporters, in the Presence or Absence of Fludioxonil.

Penicillium expansum is the most common postharvest pathogen of apple fruit, causing blue mold disease. Due to the extensive use of fungicides, strains resistant to multiple chemical classes have been selected. A previous study by our group proposed that the overexpression of MFS (major facilitator superfamily) and ABC (ATP binding cassette) transporters constitute an alternative resistance mechanism in Multi Drug resistant (MDR) strains of this pathogen. This study was initiated to determine two main biological fitness parameters of MDR strains: aggressiveness against apple fruit and patulin production. In addition, the expression pattern of efflux transporters and hydroxylase-encoding genes that belong to the patulin biosynthesis pathway, in the presence or absence of fludioxonil and under in vitro and in vivo conditions were investigated. Results showed that the MDR strains produced higher concentrations of patulin but showed a lower pathogenicity compared to the wild-type isolates. Moreover, expression analysis of patC, patM and patH genes indicated that the higher expression levels do not correlate with the detected patulin concentration. The selection of MDR strains in P. expansum populations and the fact that they produce more patulin, constitutes a serious concern not only for successful disease control but also for human health. The above-mentioned data represent the first report of MDR in P. expansum associated with its patulin-production ability and the expression level of patulin biosynthesis pathway genes.

Transcriptome Analysis and Functional Characterization Reveal That Peclg Gene Contributes to the Virulence of Penicillium expansum on Apple Fruits.

Penicillium expansum is the causal agent of blue mold decay on apple fruits and is also known to be the major producer of patulin, a mycotoxin that represents serious hazard to human health. Several mechanisms have been suggested to explain the pathogenesis of P. expansum in host plants. Secreted effector proteins are vital for the pathogenicity of many fungal pathogens through manipulating their hosts for efficient colonization. In this study, we performed a RNA-Seq analysis followed by computational prediction of effector proteins from P. expansum during infection of the host apple fruits, and a total of 212 and 268 candidate effector protein genes were identified at 6 and 9 h after inoculation (hai), respectively. One of the candidate effector protein genes was identified as a concanavalin A-like lectin/glucanase (Peclg), which was dramatically induced during the pathogen-host interaction. Targeted knockout of Peclg resulted in significant reduction in conidial production and germination relative to the wild type. Further studies showed that in addition to salt stress, the mutant was much more sensitive to SDS and Congo red, suggesting a defect in cell wall integrity. Pathogenicity assays revealed that the ΔPeclg mutant showed significant decrease in virulence and infectious growth on apple fruits. All these results suggest that Peclg is required for fungal growth, stress response, and the virulence of P. expansum.

Brazilian Table Olives: A Source of Lactic Acid Bacteria with Antimycotoxigenic and Antifungal Activity.

Food and feed contamination by fungi, especially by toxigenic ones, is a global concern because it can pose serious health problems when the production of mycotoxins is involved. Lactic acid bacteria (LAB), well-known for fermenting foods, have been gaining attention for their antifungal and anti-mycotoxin properties. This work tested 14 LAB strains isolated from naturally fermented Brazilian table olives for growth inhibition of Aspergillus flavus, Aspergillus carbonarius, Penicillium nordicum, and Penicillium expansum. The strains Lacticaseibacillus paracasei subsp. paracasei CCMA 1764, Levilactobacillus brevis CCMA 1762, and Lactiplantibacillus pentosus CCMA 1768 showed the strongest antifungal activity, being more active against P. expansum. Aflatoxin B1 (AFB1), ochratoxin A (OTA), and patulin (PAT) production was reduced essentially by mycelia growth inhibition. The main organic acids detected in the cell free supernatant (CFS) were lactic and acetic acids. Tested LAB exhibited adsorption capacity against AFB1 (48-51%), OTA (28-33%), and PAT (23-24%). AFB1 was converted into aflatoxin B2a (AFB2a) by lactic and acetic acids produced by the strain CCMA 1764. A similar conversion was observed in solutions of these organic acids (0.1 M). These findings demonstrate the potential of isolated LAB strains as natural agents to control toxigenic fungi and their mycotoxins in fermented products, such as table olives.

Histone H3K4 Methyltransferase PeSet1 Regulates Colonization, Patulin Biosynthesis, and Stress Responses of Penicillium expansum.

Fruit blue mold disease and patulin contamination caused by Penicillium expansum lead to huge economic losses and food safety concerns worldwide. Many genes have been proven to be involved in the regulation of pathogenic and toxigenic processes of P. expansum. Histone H3 lysine 4 (H3K4) methylation is well recognized for its association with chromatin regulation and gene transcription. However, it is not clear whether H3K4 methylation is related to infection and patulin biosynthesis in Penicillium. Here, we characterized PeSet1, which is responsible for H3K4me1/me2/me3 in P. expansum. The deletion of PeSet1 caused severe defects in hyphal growth, conidiation, colonization, patulin biosynthesis, and stress responses. Moreover, we demonstrated that PeSet1 is involved in the regulation of patulin biosynthesis by mediating the expression of patulin cluster genes and crucial global regulatory factors. Likewise, PeSet1 positively regulated key genes in β-1,3-glucan biosynthesis and the reactive oxygen species scavenging process to modulate cell wall integrity and oxidative stress responses, respectively. Collectively, we have proven for the first time the function of Set1 in patulin biosynthesis and the crucial role of Set1 in colonization and stress responses in P. expansum. IMPORTANCE Penicillium expansum is one of the most important plant fungal pathogens, which not only causes blue mold rot in various fruits, leading to huge decay losses, but also produces mycotoxin patulin, posing a threat to human health. Both pathogenesis and patulin biosynthesis in P. expansum are regulated by complex and sophisticated networks. We focused on the epigenetic modification and identified a conserved histone H3K4 methyltransferase PeSet1 in P. expansum. Our work revealed the important role of PeSet1 in growth, development, colonization, patulin production, and stress responses of P. expansum. In particular, we originally described the regulation of Set1 on patulin biosynthetic pathway. These findings will provide new targets for the prevention and control of blue mold disease and patulin contamination.

Small GTPases RasA and RasB regulate development, patulin production, and virulence of Penicillium expansum

Ras GTPases belong to one of the small G proteins, which regulate morphology and growth of filamentous fungi. However, whether Ras GTPases are involved in the biological process of Penicillium expansum remains unclear. In this study, RasA and RasB gene knockout mutants of P. expansum were constructed. ΔPeRasA and ΔPeRasB showed defective growth, insufficient spore development and malformed mycelium. Loss of PeRasA and PeRasB resulted in a markedly reduction of patulin content and a down-regulation genes expression involved in patulin biosynthesis. Deletion of PeRasA and PeRasB significantly down-regulated the expression of MAPK and cAMP signal regulators. Additionally, ΔPeRasA and ΔPeRasB exhibited attenuated virulence on apple and pear fruit and lower gene expression of cell wall degrading enzymes. In summary, PeRasA and PeRasB were involved in the development, patulin synthesis and virulence of P. expansum by controlling MAPK and cAMP signaling pathways.

Implication of VelB in the development, pathogenicity, and secondary metabolism of Penicillium expansum

Penicillium expansum is the main cause of blue mold disease and the main producer of patulin in apple-derived products. This fungus also produces a wide variety of secondary metabolites, including citrinin and chaetoglobosin A. Secondary metabolism is regulated by global transcription factors that are affected by environmental signals. The light-dependent global transcription factor VelB, a member of the velvet complex, plays an important role in the regulation of secondary metabolism and fungal development in many fungal species. However, the specific roles of VelB in P. expansum remain unknown. A Pe∆velB mutant and a complemented Pe∆velB:velB strain were generated to investigate the role of VelB in pathogenicity and secondary metabolism. The Pe∆velB mutant colonized apples, but at a lower rate than the wild type and complemented strains. Conidiation was significantly reduced in the Pe∆velB strain. Under light conditions, the Pe∆velB strain showed a reduced level of spore viability. Deletion of the velB gene strongly inhibited the production of patulin, citrinin, and chaetoglobosin A on synthetic media or in planta, but increased the production of fumarylalanine; these changes were associated with down-regulation or upregulation of the genes involved in their respective biosynthesis. Analysis of the global secondary metabolism of the Pe∆velB strain revealed that 22 backbone genes were modulated. In addition, genes involved in siderophore biosynthesis, ergosterol biosynthesis, and nitrate assimilation were also upregulated in the PeΔvelB strain. Taken together, our results demonstrate that VelB is involved in the development, pathogenicity, and secondary metabolism of P. expansum.

Modulation of extracellular Penicillium expansum-driven acidification by Papiliotrema terrestris affects biosynthesis of patulin and has a possible role in biocontrol activity.

The active regulation of extracellular pH is critical for the virulence of fungal pathogens. Penicillium expansum is the causal agent of green-blue mold on stored pome fruits and during its infection process acidifies the host tissues by secreting organic acids. P. expansum is also the main producer of patulin (PAT), a mycotoxin found in pome fruit-based products and that represents a serious health hazard for its potential carcinogenicity. While it is known that PAT biosynthesis in P. expansum is regulated by nutritional factors such as carbon and nitrogen and by the pH, the mechanistic effects of biocontrol on PAT production by P. expansum are not known. In this work, we assessed how optimal and suboptimal concentrations of the biocontrol agent (BCA) Papiliotrema terrestris LS28 affect both extracellular pH and PAT biosynthesis in P. expansum. In wounded apples, the optimal and suboptimal concentrations of the BCA provided almost complete and partial protection from P. expansum infection, respectively, and reduced PAT contamination in both cases. However, the suboptimal concentration of the BCA increased the specific mycotoxigenic activity by P. expansum. In vitro, the rate of PAT biosynthesis was strictly related to the extracellular pH, with the highest amount of PAT detected in the pH range 4–7, whereas only traces were detectable at pH 3. Moreover, both in vitro and in apple wounds the BCA counteracted the extracellular P. expansum-driven acidification maintaining extracellular pH around 4, which is within the pH range that is optimal for PAT biosynthesis. Conversely, in the absence of LS28 the pathogen-driven acidification led to rapidly achieving acidic pH values (<3) that lie outside of the optimal pH range for PAT biosynthesis. Taken together, these results suggest that pH modulation by LS28 is important to counteract the host tissue acidification and, therefore, the virulence of P. expansum. On the other hand, the buffering of P. expansum-driven acidification provided by the BCA increases the specific rate of PAT biosynthesis through the extension of the time interval at which the pH value lies within the optimal range for PAT biosynthesis. Nevertheless, the antagonistic effect provided by the BCA greatly reduced the total amount of PAT.