Xiangfeng Zheng

Biological Control of Patulin by Antagonistic Yeast: A case study and possible model

Abstract The occurrence of patulin in fresh apples and apple products is a great burden from health, safety and economic perspectives. Attempts to prevent patulin accumulation in fruits might lead to the excessive use of fungicides. Therefore, guaranteeing the safety of apple foods is crucial for the international apple industry. Recently, literature revealed that application of antagonistic yeasts and other BCAs have been able to disrupt the process of fungal infection and patulin production in apples. Although, over the years the effect of interaction between BCAs and fungi on patulin production has been reported, the exact mechanism(s) of their action remain unclear. Here, the review focused on toxicology and occurrence of PAT; research advances made over the past few years on the interaction between antagonistic yeast, fruits and patulin-producing fungi; the prevalence of patulin in apple fruits and products and the implications of synthetic-fungicide applications. In addition, attention was focused on the mechanism(s) and the enhancement of the biocontrol efficacy of antagonistic for patulin control.

The Possible Mechanisms Involved in Degradation of Patulin by Pichia caribbica

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.

Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes

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.

Phytic Acid Enhances Biocontrol Activity of Rhodotorula mucilaginosa against Penicillium expansum Contamination and Patulin Production in Apples

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.

Investigating Proteome and Transcriptome Defense Response of Apples Induced by Yarrowia lipolytica

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. [Formula: see text]

Biocontrol agents increase the specific rate of patulin production by Penicillium expansum but decrease the disease and total patulin contamination of apples

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.

The mechanisms involved in ochratoxin A elimination by Yarrowia lipolytica Y-2: The mechanisms involved in ochratoxin A elimination

Biological control of mycotoxin in cereals, fruits and vegetables have emerged as a promising method. In a previous study, Yarrowia lipolytica Y‐2 isolated by our research team showed biocontrol effect on the post‐harvest decay of grapes and ochratoxin A (OTA) elimination in polytoma medium. The aim of this study was to elucidate the possible mechanisms of OTA elimination by Y. lipolytica Y‐2. The results indicated that OTA elimination by Y. lipolytica Y‐2 was attributed to the degradation action of intracellular enzymes but not extracellular enzymes. A degradation product was identified as ochratoxin alpha (OTα) by liquid chromatography‐tandem mass spectrometry. The intracellular enzymes precipitated with 65% saturation of ammonium sulphate degrade OTA the most quickly and 97.2% OTA was degraded within 4 h. Analysis of this fraction showed that two proteins of carboxypeptidase were expressed in Y. lipolytica Y‐2 but not in Y. lipolytica Polh without the ability to degrade OTA. The results of the protein identification combined with product identification indicated that OTA was degraded to OTα by Y. lipolytica Y‐2 through the hydrolysis activity of carboxypeptidases. Additionally, many proteins of Y. lipolytica Y‐2 involved in stress response and reactive O₂ species elimination also played essential role in OTA degradation.

Reply to Comment on “Screening and Identification of Novel Ochratoxin A-Producing Fungi from Grapes”. Toxins 2016, 8, 333”–in Reporting Ochratoxin A Production from Strains of Aspergillus, Penicillium and Talaromyces

n/a.

Screening of Deoxynivalenol Producing Strains and Elucidation of Possible Toxigenic Molecular Mechanism

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.

Proteomics profile of Hanseniaspora uvarum enhanced with trehalose involved in the biocontrol efficacy of grape berry

This present study tested the extent to which 2% w/v trehalose enhanced the proteins expression profile of Hanseniaspora uvarum Y3. Furthermore, it explored the relative gene expression of stilbene synthase (StSy), one of the vital defense-related genes found in the skin of grapes. The proteomics profile revealed that 29 proteins were differentially expressed out of which 26 were significantly up-regulated and 3 were download-regulated. The pathogenesis related (PR) and other protein spots were visible at 97.4 kDa and 14.4 kDa. Peroxiredoxin TSA1 and superoxide dismutase were the main proteins involved in defense response and both proteins were significantly up-regulated. The carbohydrate and energy metabolism proteins were also significantly up-regulated. The results revealed that the treatments were associated with substantial increase in peroxidase activity compared to the control. StSy relative gene expression level was observed to increase by 2.5-fold in grapes treated with the pre-enhanced H. uvarum compared to the control.