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ABSTRACT: Biofilm formation is usually affected by many environmental factors, including divalent cations. The purpose of the current work was to analyze how calcium (Ca2+) affects the biofilm formation of dairy Pseudomonas fluorescens isolates by investigating their growth, swarming motility, biofilm-forming capacity, extracellular polymeric substance production, and biofilm structures. Moreover, the regulation mechanism of Ca2+ involved in its biofilm formation was explored through RNA-sequencing analysis. This work revealed that supplementation of 5, 10, 15, and 20 mM Ca2+ significantly reduced the swarming motility of P. fluorescens strains (P.F2, P.F4, and P.F17), but the biofilm-forming ability and polysaccharide production were increased after the supplementation of 5 and 10 mM Ca2+. By the supplementation of Ca2+, complex structures with more cell clusters glued together in P. fluorescens P.F4 biofilms were confirmed by scanning electron microscopy, and increased biomass and coverage of P. fluorescens P.F4 biofilms were observed by confocal laser scanning microscopy. In addition, RNA-sequencing results showed that P. fluorescens P.F4 showed a transcriptional response to the supplementation of 10 mM Ca2+, and a total of 137 genes were significantly expressed. The differential genes were represented in 4 upregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways (nonribosomal peptide structures, quorum sensing, biosynthesis of siderophore group nonribosomal peptides, and phenylalanine metabolism), and 4 downregulated KEGG pathways (flagellar assembly, amino sugar and nucleotide sugar metabolism, nitrotoluene degradation, and cationic antimicrobial peptide resistance). The results indicate that Ca2+ might serve as an enhancer to substantially trigger the biofilm formation of dairy P. fluorescens isolates in the dairy industry.

A simple, rapid and novel method involving ultrahigh-performance liquid chromatography-electrospray ionization tandem triple quadrupole mass spectrometry (UHPLC-ESI–MS/MS) was developed to simultaneously detect erythromycin, its major metabolite and clarithromycin in chicken tissues (muscle, liver and kidney) and eggs (whole egg, albumen and yolk). Samples were extracted using acetonitrile–water (80:20, v/v), and a Cleanert MAS-Q cartridge was used to perform quick, easy, cheap, effective, rugged, and safe (QuEChERS) purification. The average recoveries were 87.78–104.22 %, and the corresponding intraday and interday relative standard deviations were less than 7.10 %. The decision limits and detection capabilities of the chicken tissues and eggs were 2.15–105.21 μg/kg and 2.26–110.42 μg/kg, respectively. For chicken tissues and eggs, the limits of detection and limits of quantification were 0.5 μg/kg and 2.0 μg/kg, respectively. The proposed method was successfully employed to analyse real samples, demonstrating its applicability.

The conducive conditions of warm and humid climates can facilitate mold proliferation and subsequent mycotoxin production during food processing and distribution, thereby posing a potential risk to consumer health. However, there exists a significant lack of research regarding the diversity of molds and the presence of ochratoxin A (OTA) and patulin (PAT) in food products available in the Yangzhou market. This study was conducted to assess OTA contamination levels and fungal presence in 57 cereal-based food samples, as well as PAT contamination levels and fungal presence in 50 types of foods, including apples, hawthorn berries, pears, and their derivatives. Ochratoxin A (OTA) was detected in 17 out of 57 cereal-based food samples, with concentrations ranging from 0.93 to 32.69 μg/kg. The contamination rate was determined to be 31.48%, and no samples exceeded the established regulatory limits. Furthermore, seven apple products were identified as contaminated with patulin (PAT), exhibiting concentrations between 26.85 and 192.78 μg/kg. Additionally, three food samples derived from hawthorn showed PAT contamination levels ranging from 29.83 to 88.56 μg/kg. Through purification on potato dextrose agar (PDA) medium, observation of colony morphology, and analysis of internal transcribed spacer (ITS) sequences, a total of 35 fungal strains belonging to 13 genera were identified in cereal-based foods. The predominant genera in cereals included Talaromyces, Fusarium, Aspergillus, and Penicillium. Additionally, twelve fungal strains from five genera (Penicillium, Cladosporium, Aureobasidium, Curvularia, and Alternaria) were isolated and identified in fruits and their derivatives. The findings indicate that OTA and PAT toxins are one of the important risk factors that threaten consumer health. Furthermore, the contamination of some other toxigenic strains is also a matter of substantial concern, with potential implications for consumer health.

Abstract Background Chlorophyll is a very important pigment involved in photosynthesis, while plant acyl-CoA biosynthesis is derived from plastid-localized fatty acids (FAs). Until now, the regulation of the acyl-CoA pathway for chlorophyll biosynthesis is still unknown. Results Here, we identified a long-chain acyl-CoA synthetase (LACS) gene BnLACS9 from Brassica napus. BnLACS9 complemented a LACS-deficient yeast strain YB525, which indicated that BnLACS9 has the LACS function. BnLACS9 was localized in the chloroplast envelope membrane, while mainly expressed in young leaves and flowers. Overexpression of BnLACS9 in Nicotiana benthamiana resulted in an increase in total CoA and MGDG content. In B. napus with overexpression of BnLACS9, the number of chloroplast grana lamellae and the chlorophyll content, as well as the MGDG and DGDG contents, increased compared to wild type. The net photosynthetic rate, dry weight of the entire plant and oil content of seeds increased significantly, accompanied by an increase in chlorophyll content. Transcriptome analysis revealed that overexpression of BnLACS9 improved the pathway of acyl-CoA biosynthesis and further improved the enzymes in the glycolipid synthesis pathway, while acyl-CoA was the substrate for glycolipid synthesis. The increased glycolipids, especially MGDG and DGDG, accelerated the formation of the chloroplast grana lamellae, which increased the number of chloroplast thylakoid grana lamella and further lead to increased chlorophyll content. Conclusions In the present study, we demonstrated that BnLACS9 played a crucial role in glycolipids and chlorophyll biosynthesis in B. napus. The results also provide a new direction and theoretical basis for the improvement of the agronomic traits of plants.

Antrodia cinnamomea is a valuable edible and medicinal mushroom with antitumor, hepatoprotective, and antiviral effects that play a role in intestinal flora regulation. Spore-inoculation submerged fermentation has become the most efficient and well-known artificial culture process for A. cinnamomea. In this study, a specific low-molecular compound named 1,8-cineole (cineole) from Cinnamomum kanehirae Hay was first reported to have remarkably promoted the asexual sporulation of A. cinnamomea in submerged fermentation (AcSmF). Then, RNA sequencing, real-time quantitative PCR, and a literature review were performed to predict the molecular regulatory mechanisms underlying the cineole-promoted sporulation of AcSmF. The available evidence supports the hypothesis that after receiving the signal of cineole through cell receptors Wsc1 and Mid2, Pkc1 promoted the expression levels of rlm1 and wetA and facilitated their transfer to the cell wall integrity (CWI) signal pathway, and wetA in turn promoted the sporulation of AcSmF. Moreover, cineole changed the membrane functional state of the A. cinnamomea cell and thus activated the heat stress response by the CWI pathway. Then, heat shock protein 90 and its chaperone Cdc37 promoted the expression of stuA and brlA, thus promoting sporulation of AcSmF. In addition, cineole promoted the expression of areA, flbA, and flbD through the transcription factor NCP1 and inhibited the expression of pkaA through the ammonium permease of MEP, finally promoting the sporulation of AcSmF. This study may improve the efficiency of the inoculum (spores) preparation of AcSmF and thereby enhance the production benefits of A. cinnamomea.

Foodborne pathogens cause many diseases and significantly impact human health and the economy. Foodborne pathogens mainly include Salmonella spp., Escherichia coli, Staphylococcus aureus, Shigella spp., Campylobacter spp. and Listeria monocytogenes, which are present in agricultural products, dairy products, animal-derived foods and the environment. Various pathogens in many different types of food and water can cause potentially life-threatening diseases and develop resistance to various types of antibiotics. The harm of foodborne pathogens is increasing, necessitating effective and efficient methods for early monitoring and detection. Traditional methods, such as real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and culture plate, are time-consuming, labour-intensive and expensive and cannot satisfy the demands of rapid food testing. Therefore, new fast detection methods are urgently needed. Electrochemical biosensors provide consumer-friendly methods to quickly detect foodborne pathogens in food and the environment and achieve extensive accuracy and reproducible results. In this paper, by focusing on various mechanisms of electrochemical transducers, we present a comprehensive overview of electrochemical biosensors for the detection of foodborne pathogens. Furthermore, the review introduces the hazards of foodborne pathogens, risk analysis methods and measures of control. Finally, the review also emphasizes the recent research progress and solutions regarding the use of electrochemical biosensors to detect foodborne pathogens in food and the environment, evaluates limitations and challenges experienced during the development of biosensors to detect foodborne pathogens and discusses future possibilities.

The study of bacteriophages is experiencing a resurgence owing to their antibacterial efficacy, lack of side effects, and low production cost. Nonetheless, the interactions between Staphylococcus aureus bacteriophages and their hosts remain unexplored. In this study, whole-genome sequences of 188 S. aureus bacteriophages—20 Podoviridae, 56 Herelleviridae, and 112 Siphoviridae—were obtained from the National Center for Biotechnology Information (NCBI, USA) genome database. A phylogenetic tree was constructed to estimate their genetic relatedness using single-nucleotide polymorphism analysis. Comparative analysis was performed to investigate the structural diversity and ortholog groups in the subdividing clusters. Mosaic structures and gene content were compared in relation to phylogeny. Phylogenetic analysis revealed that the bacteriophages could be distinguished into three lineages (I–III), including nine subdividing clusters and seven singletons. The subdividing clusters shared similar mosaic structures and core ortholog clusters, including the genes involved in bacteriophage morphogenesis and DNA packaging. Notably, several functional modules of bacteriophages 187 and 2368A shared more than 95% nucleotide sequence identity with prophages in the S. aureus strain RJ1267 and the Staphylococcus pseudintermedius strain SP_11306_4, whereas other modules exhibited little nucleotide sequence similarity. Moreover, the cluster phages shared similar types of holins, lysins, and DNA packaging genes and harbored diverse genes associated with DNA replication and virulence. The data suggested that the genetic diversity of S. aureus bacteriophages was likely due to gene replacement, acquisition, and loss among staphylococcal phages, which may have crossed species barriers. Moreover, frequent module exchanges likely occurred exclusively among the subdividing cluster phages. We hypothesize that during evolution, the S. aureus phages enhanced their DNA replication in host cells and the adaptive environment of their host.

A better understanding of the mode of action of postharvest biocontrol agents on fruit surfaces is critical for the advancement of successful implementation of postharvest biocontrol products. This is due to the increasing importance of biological control of postharvest diseases over chemical and other control methods. However, most of the mechanisms involved in biological control remain unknown and need to be explored. Yarrowia lipolytica significantly inhibited blue mold decay of apples caused by Penicillium expansum. The findings also demonstrated that Y. lipolytica stimulated the activities of polyphenoloxidase, peroxidase, chitinase, l-phenylalanine ammonia lyase involved in enhancing defense responses in apple fruit tissue. Proteomic and transcriptomic analysis revealed a total of 35 proteins identified as up- and down-regulated in response to the Y. lipolytica inducement. These proteins were related to defense, biotic stimulus, and stress responses, such as pathogenesis-related proteins and dehydrin. The analysis of the transcriptome results proved that the induced resistance was mediated by a crosstalk between salicylic acid (SA) and ethylene/jasmonate (ET/JA) pathways. Y. lipolytica treatment activated the expression of isochorismate synthase gene in the SA pathway, which up-regulates the expression of PR4 in apple. The expression of 1-aminocyclopropane-1-carboxylate oxidase gene and ET-responsive transcription factors 2 and 4, which are involved in the ET pathway, were also activated. In addition, cytochrome oxidase I, which plays an important role in JA signaling for resistance acquisition, was also activated. However, not all of the genes had a positive effect on the SA and ET/JA signal pathways. As transcriptional repressors in JA signaling, TIFY3B and TIFY11B were triggered by the yeast, but the gene expression levels were relatively low. Taken together, Y. lipolytica induced the SA and ET/JA signal mediating the defense pathways by stimulating defense response genes, such as peroxidase, thaumatin-like protein, and chitinase 4-like, which are involved in defense response in apple.[Graphic: see text] Copyright © 2017 The Author(s) This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Patulin (PAT) is a mycotoxin produced by some Penicillium, Aspergillus, and Byssochlamys species. Rhodotorula mucilaginosa is able to degrade PAT in vivo as well as in vitro, up till date, the process and molecular mechanism(s) involved patulin degradation still remains unknown. Protein lysine crotonylation (Kcr) plays an important role in regulating chromatin dynamics, gene expression, and metabolic pathways in mammals and eukaryotes. Investigation of the Kcr changes accompanying degradation of patulin in R. mucilaginosa were observed to investigate the mechanisms of patulin inhibition. Tandem mass tag (TMT) labeling and Kcro affinity enrichment, followed by high-resolution LC-MS/MS analysis, were used to perform quantitative lysine crotonylome analysis on R. mucilaginosa. Consequently, 1691 lysine crotonylation sites in 629 protein groups were identified, among which we quantified 1457 sites in 562 proteins. Among the quantified proteins, 79 and 46 crotonylated proteins were up-regulated and down-regulated, respectively. The differentially up expressed modified proteins were mainly involved in tricarboxylic acid cycle and gluconeogenic pathway. The differentially down expressed Kcr proteins were mainly classified to ribosome and carbohydrate transport and metabolism. Bioinformatic analyses were performed to annotate the quantifiable lysine crotonylated targets. Moreover, interaction networks and high confidence domain architectures of crotonylated proteins were investigated with the aid of bioinformatic tools, and these results showed that there was an increase in the number of yeasts with crotonylated proteins. The results also provided information on the various roles of crotonylation, which are involved in PAT degradation.

Synthetic fungicides are commonly employed for the control of postharvest diseases of fruits. However, due to health concerns about the use of these chemicals, alternative control methods including biocontrol based on antagonistic yeasts are gaining in popularity. In this study, we investigated the effects of two biocontrol yeasts, Rhodotorula mucilaginosa strain 3617 and Rhodotorula kratochvilovae strain LS11, on blue mold and patulin (PAT) contamination caused by Penicillium expansum strains PY and FS7 in artificially inoculated Fuji apples stored at 20°C for 9 days. To correlate the development of the P. expansum strains in yeast-treated and untreated apples with PAT production, we quantified their biomass in the infected fruits using a recently published quantitative real-time polymerase chain reaction method based on specific primers for patF, a gene from P. expansum that is involved in PAT biosynthesis. Both yeasts significantly reduced the disease incidence caused by the two strains of P. expansum up to 5–7 days of incubation, and lowered their biomass and the progression of symptoms up to 9 days. Interestingly, both yeasts strains increased the rate of PAT production (expressed as ng patulin/μg fungal DNA) by the two pathogenic strains. Nevertheless, both biocontrol agents reduced the total PAT contamination, especially in the case of P. expansum strain FS7, the higher PAT producer of the two tested P. expansum strains. Comparing between the yeast strains, R. kratochvilovae LS11 was more effective than R. mucilaginosa 3617 for the control of P. expansum.

Blue mold, caused by Penicillium expansum, is an important postharvest disease of apple, and can result in significant economic losses. The present study investigated the interaction between P. expansum and wounded apple fruit tissues during the early stages of the infection. Spores of P. expansum became activated one hour post-inoculation (hpi), exhibited swelling at 3 hpi, and the germ tubes were found entering into apple tissues at 6 hpi. RNA-seq was performed on samples of P. expansum and apple fruit tissue collected at 1, 3, and 6 hpi. The main differentially expressed genes (DEGs) that were identified in P. expansum were related to interaction, cell wall degradation enzymes, anti-oxidative stress, pH regulation, and effectors. Apple tissues responded to the presence of P. expansum by activating pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) at 1 hpi, then activated effector-triggered immunity (ETI) at 3 hpi. This research provides new information on the interaction between P. expansum and apple fruit tissue at an early stage of the infection process.

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Ochratoxin A (OTA) contamination has been established as a world-wide problem. In this study, the strains with the ability of OTA production were screened by analyzing the green fluorescence of the isolates colonies from the grapes in Zhenjiang with 365 nm UV light and confirmed by HPLC with fluorescent detection (HPLC-FLD). The results showed that seven isolates acquired the characteristic of the fluorescence, of which only five showed the ability of OTA production as confirmed by HPLC-FLD analysis. The five OTA-producing strains were identified based on comparative sequence analysis of three conserved genes (ITS, BenA and RPB2) of the strains, and they are Talaromyces rugulosus (O1 and Q3), Penicillium commune (V5-1), Penicillium rubens (MQ-5) and Aspergillus aculeatus (MB1-1). There are two Penicillium species of the five OTA-producing strains and our study is the first to report that P. rubens, T. rugulosus and A. aculeatus can produce OTA. This work would contribute to comprehensively understanding the fungi with an OTA-producing ability in grapes before harvest and then take effective measures to prevent OTA production.

In this work, we examined the mechanisms involved in the degradation of patulin by Pichia caribbica. Our results indicate that cell-free filtrate of P. caribbica reduced patutlin content. The heat-killed cells could not degrade patulin. However, the live cells significantly reduced the concentration of the patulin. In furtherance to this, it was observed that patulin was not detected in the broken yeast cells and cell wall. The addition of cycloheximide to the P. caribbica cells decreased the capacity of degradation of patulin. Proteomics analyses revealed that patulin treatment resulted in an upregulated protein which was involved in metabolism and stress response processes. Our results suggested that the mechanism of degradation of patulin by P. caribbica was not absorption; the presence of patulin can induce P. caribbica to produce associated intracellular and extracellular enzymes, both of which have the ability to degrade patulin. The result provides a new possible method that used the enzymes produced by yeast to detoxify patulin in food and feed.

In this study, seven strains of Fusarium graminearum were isolated from wheat, of which six were identified to produce deoxynivalenol and the production of deoxynivalenol was assessed. F. graminearum strain Fg1 was noted to produce 1.0 μg/g deoxynivalenol during the incubation period in the Czapek yeast broth, while none was detected in F. graminearum strain Fg2. Hence, the differences in proteomes and transcriptomes of Fg1 and Fg2 were compared to analyze the mechanism underlying deoxynivalenol production. Among the 66 significantly differentially expressed proteins in Fg1, 39 and 27 were more or less abundant expressed. Functional analysis suggested that the enzymes involved in the methylerythritol 4-phosphate and mevalonate pathways, which provide a substrate for biosynthesis of farnesyl pyrophosphate, a precursor of DON, were activated in Fg1. The transcriptomics data demonstrated that the expression level of a majority of genes, including trichothecene biosynthetic genes, protein kinases, and transcription factors, involved in trichothecene biosynthesis was higher in Fg1 than in Fg2. The results also revealed differential expression profiles of deoxynivalenol biosynthesis genes in strains Fg1 and Fg2, which emphasized their deoxynivalenol producing ability and the underlying mechanism. [ABSTRACT FROM AUTHOR]

Recently a species in the genus Talaromyces, a uniseriate species of Aspergillus section Nigri and an isolate each of two widespread species, Penicillium rubens and P. commune, were reported to produce ochratoxin A. This claim was based on insufficient biological and chemical data. We propose a list of criteria that need to be met before an unexpected mycotoxin producer is reported. There have only been convincing data on ochratoxin A production for Penicillium verrucosum, P. nordicum, P. thymicola, all from Penicillium series Verrucosa, and from species in three sections of Aspergillus: section Circumdati, section Nigri and section Flavi. [ABSTRACT FROM AUTHOR]

The effect of Rhodotorula mucilaginosa in combination with phytic acid (PA) on blue mold decay and patulin contamination of apples was investigated. Results from this study show that different concentrations of PA were effective in reducing the disease incidence of apples and that PA at concentration of 4 µmol/mL, decreased the incidence of blue mold decay in apples from 86.1 to 62.5%, and showed higher control efficacy compared to untreated, control fruit during storage at 20?C. However, R. mucilaginosa combined with PA (4 μmol/mL) showed better control efficacy of blue mold decay than R. mucilaginosa used as single treatment, the disease incidence was reduced to 62.5% and lesion diameter on apples was reduced to 16.59 cm. In in vitro experiments, the addition of PA enhanced the biocontrol effect of R. mucilaginosa against the growth of Penicillium expansum and reduced patulin level when compared with either R. mucilaginosa or PA used separately. R. mucilaginosa together with PA, improved the inhibition of patulin production in wounded apples, decreasing the content of patulin by 89.6% compared to the control, under experimental conditions. Both R. mucilaginosa and R. mucilaginosa in combination with PA degraded patulin in vitro. In conclusion, the appropriate combination of R. mucilaginosa and PA may provide an effective biocontrol method for reducing postharvest decay of apples. [ABSTRACT FROM AUTHOR]