Effects of TrPLDs on the pathogenicity of Trichothecium roseum infected apple fruit.

Phospholipase D plays a critical regulatory role in the pathogenicity of filamentous fungi. However, the molecular mechanism of PLD regulating the pathogenicity of filamentous fungi has not been reported. In this research, the previously constructed TrPLD1 and TrPLD2 (TrPLDs) mutants were used as test strains. Firstly, the function of TrPLDs in Trichothecium roseum was studied. Then, the effects of TrPLDs on the pathogenicity of T. roseum and the quality of the inoculated apples were verified. The results suggested that the deletion of TrPLD1 delayed the spore germination of ΔTrPLD1 and inhibited germ tube elongation by down-regulating the expressions of TrbrlA, TrabaA and TrwetA. By down-regulating the extracellular enzyme-coding gene expressions, ΔTrPLD1 inhibited the degradation of apple fruit cell wall and the change of fatty acid content during infection, reduced the cell membrane permeability and malondialdehyde (MDA) content of apple fruit, thereby maintaining the integrity of fruit cell membrane, and reduced the pathogenicity of ΔTrPLD1 to apple and kept the quality of apple. However, ΔTrPLD2 did not have a significant effect on the infection process of apple fruit by the pathogen.

Ozone Application Suppressed the Blue Mold Development and Maintained the Main Active Ingredients Content of Postharvest Fresh Codonopsis pilosula during Storage.

Penicillium expansum is the predominant causal agent causing blue mold in postharvest fresh Codonopsis pilosula during storage. The pathogen reduces the yield and affects the quality of C. pilosula and even generates patulin, threatening human health. In this study, postharvest fresh, healthy C. pilosula was sprayed with P. expansum, and the control effect of ozone on postharvest diseases of C. pilosula was studied, and the effect of ozone on the contents in the main active ingredients of C. pilosula was compared; finally, the effect of ozone on reactive oxygen species (ROS) metabolism in C. pilosula was analyzed. The results showed that 2 mg L-1 ozone application significantly inhibited the occurrence of postharvest blue mold caused by P. expansum, reduced weight loss rate, controlled the accumulation of patulin and maintained the contents of the main active components in C. pilosula. The study will provide a theoretical basis for ozone treatment to control the occurrence of postharvest diseases of C. pilosula.

ROS mediated by TrPLD3 of Trichothecium roseum participated cell membrane integrity of apple fruit by influencing phosphatidic acid metabolism.

Trichothecium roseum is a typical necrotrophic fungal pathogen that not only bring about postharvest disease, but contribute to trichothecenes contamination in fruit and vegetables. Phospholipase D (PLD), as an important membrane lipid degrading enzyme, can produce phosphatidic acid (PA) by hydrolyzing phosphatidylcholine (PC) and phosphatidylinositol (PI). PA can promote the production of reactive oxygen species (ROS) by activating the activity of NADPH oxidase (NOX), thereby increasing the pathogenicity to fruit. However, the ROS mediated by TrPLD3 how to influence T. roseum infection to fruit by modulating phosphatidic acid metabolism, which has not been reported. In this study, the knockout mutant and complement strain of TrPLD3 were constructed through homologous recombination, TrPLD3 was tested for its effect on the colony growth and pathogenicity of T. roseum. The experimental results showed that the knockout of TrPLD3 inhibited the colony growth of T. roseum, altered the mycelial morphology, completely inhibited the sporulation, and reduced the accumulation of T-2 toxin. Moreover, the knockout of TrPLD3 significantly decreased pathogenicity of T. roseum on apple fruit. Compared to inoculated apple fruit with the wide type (WT), the production of ROS in apple infected with ΔTrPLD3 was slowed down, the relative expression and enzymatic activity of NOX, and PA content decreased, and the enzymatic activity and gene expression of superoxide dismutase (SOD) increased. In addition, PLD, lipoxygenase (LOX) and lipase activities were considerably decreased in apple fruit infected with ΔTrPLD3, the changes of membrane lipid components were slowed down, the decrease of unsaturated fatty acid content was alleviated, and the accumulation of saturated fatty acid content was reduced, thereby maintaining the cell membrane integrity of the inoculated apple fruit. We speculated that the decreased PA accumulation in ΔTrPLD3-inoculated apple fruit further weakened the interaction between PA and NOX on fruit, resulting in the reduction of ROS accumulation of fruits, which decreased the damage to the cell membrane and maintained the cell membrane integrity, thus reducing the pathogenicity to apple. Therefore, TrPLD3-mediated ROS plays a critical regulatory role in reducing the pathogenicity of T. roseum on apple fruit by influencing phosphatidic acid metabolism.

Isolation and Identification of Pathogens Causing Blue Mold of Lanzhou Lily during Postharvest Storage and Control of Disease and Mycotoxin Accumulation by Ozone Treatment.

Blue mold (penicilliosis) is a common disease of Lanzhou lily (Lilium davidii var. willmottiae) during postharvest storage, which not only seriously affects the appearance and reduces the quality of lily bulbs, but also leads to the accumulation of mycotoxins in rotten lily tissues, seriously endangering human health. Therefore, it is of great significance to clarify the main isolates causing postharvest blue mold of fresh Lanzhou lily and put forward effective measures to control the disease caused by these pathogens. In this study, pathogens were isolated and purified from the naturally diseased blue-mold tissue of Lanzhou lily, and then morphological and molecular biology techniques were applied to identify the isolates, verify the pathogenicity, determine the disease index and disease incidence, and finally, to analyze the control effect of ozone treatment on the blue mold of lily scale and mycotoxin accumulation. The results indicated that the main isolates causing postharvest blue mold of lily were Talaromyces adpressus, Penicillium gladioli, T. calidominioluteus, and P. polonicum. The pathogenicity test showed that P. gladioli and P. polonicum had a higher disease index than T. calidominioluteus and T. adpressus. Ozone treatment significantly reduced the incidence of disease caused by P. gladioli and P. polonicum, and effectively controlled the accumulation of patulin. This study characterized the main pathogens causing blue mold of postharvest Lanzhou lily during storage, and confirmed ozone application has a significant inhibitory effect on blue mold development and patulin accumulation in Lanzhou lily, which could be helpful in commercially controlling blue mold of postharvest Lanzhou lily during storage.

Pathogenicity, Mycotoxin Production, and Control of Potato Dry Rot Caused by Fusarium spp.: A Review.

Fusarium dry rot is one of the major potato diseases during storage after harvest, which not only results in quality degradation but also causes great economic losses. The disease can be elicited by some species of Fusarium, and the pathogenic fungi of Fusarium causing potato dry rot are considerably diverse in various countries and regions. The disease caused by Fusarium spp. is associated with mycotoxins accumulation, which has phytotoxic and mycotoxic effects on humans and animals. Chemical synthetic fungicide is considered the main control measure for the Fusarium dry rot of potato; nevertheless, it is unfortunate that persistent application inevitably results in the emergency of a resistant strain and environmental contamination. A comprehensive disease control strategy includes potato cultivar selection, appropriate cultural practices (crop rotation, cultivate pattern, fertilization, and irrigation), harvesting processes and postharvest treatments (harvesting, classification, packaging, wound healing), and storage conditions (environmental disinfection, temperature, humidity and gas composition) along with the application of fungicide pre-harvest or post-harvest. Recently, emerging studies have indicated that eco-friendly strategies include physical control, chemical methods (such as the application of generally-recognised-as-safe (GRAS) compounds or chemical (elicitors) and biological control have been introduced to combat the Fusarium dry rot of potato.

Effects of Aspergillus niger Infection on the Quality of Jujube and Ochratoxin A Cumulative Effect.

The jujube is one of the most popular fruits in China because of its delicious taste and high nutritional value. It has a long history of usage as an important food or traditional medicine. However, the jujube is easily infected by fungi, which causes economic losses and threatens human health. When the jujube was infected by Aspergillus niger (H1), the changes in nutritional qualities were determined, such as the content of total acid, vitamin C, reducing sugar, etc. In addition, the ability of A. niger (H1) to produce ochratoxin A (OTA) in different inoculation times and culture media was evaluated, and the content of OTA in jujubes was also analyzed. After jujubes were infected by A. niger (H1), the total acid, and vitamin C contents increased, while the total phenol content decreased, and the reducing sugar content increased after an initial decrease. Although A. niger (H1) infection caused the jujubes to rot and affected its quality, OTA had not been detected. This research provides a theoretical foundation for maximizing edible safety and evaluating the losses caused by fungal disease in jujubes.

Erg4 Is Involved in Ergosterol Biosynthesis, Conidiation and Stress Response in Penicillium expansum.

erg4 is a key gene for ergosterol biosynthesis in filamentous fungi, but its function in Penicillium expansum remains unknown. Our results showed that P. expansum contains three erg4 genes, including erg4A, erg4B and erg4C. The expression levels of the three genes showed differences in the wild-type (WT) strain, and the expression level of erg4B was the highest, followed by erg4C. Deletion of erg4A, erg4B or erg4C in the WT strain revealed functional redundancy between them. Compared to the WT strain, erg4A, erg4B or erg4C knockout mutants reduced ergosterol levels, with erg4B deletion having the greatest effect. Furthermore, deletion of the three genes reduced sporulation of the strain, and Δerg4B and Δerg4C mutants showed defective spore morphology. In addition, Δerg4B and Δerg4C mutants were found to be more sensitive to cell wall integrity and oxidative stress. However, deletion of erg4A, erg4B or erg4C had no significant effect on colony diameter, spore germination rate, conidiophore structure of P. expansum or pathogenicity to apple fruit. Taken together, erg4A, erg4B and erg4C have redundant functions and are all involved in ergosterol synthesis and sporulation in P. expansum. In addition, erg4B and erg4C contribute to spore morphogenesis, cell wall integrity and response to oxidative stress in P. expansum.

Isolation of Main Pathogens Causing Postharvest Disease in Fresh Codonopsis pilosula during Different Storage Stages and Ozone Control against Disease and Mycotoxin Accumulation.

Codonopsis pilosula is an important Chinese herbal medicine. However, fresh C. pilosula is prone to decay during storage due to microorganism infections, seriously affecting the medicinal value and even causing mycotoxin accumulation. Therefore, it is necessary to study the pathogens present and develop efficient control strategies to mitigate their detrimental effects on the herbs during storage. In this study, fresh C. pilosula was collected from Min County in Gansu Province, China. The natural disease symptoms were observed during different storage stages, and the pathogens causing C. pilosula postharvest decay were isolated from the infected fresh C. pilosula. Morphological and molecular identification were performed, and pathogenicity was tested using Koch's postulates. In addition, the control of ozone was examined against the isolates and mycotoxin accumulation. The results indicated that the naturally occurring symptom increased progressively with the extension of storage time. The mucor rot caused by Mucor was first observed on day 7, followed by root rot caused by Fusarium on day 14. Blue mold disease caused by Penicillum expansum was detected as the most serious postharvest disease on day 28. Pink rot disease caused by Trichothecium roseum was observed on day 56. Moreover, ozone treatment significantly decreased the development of postharvest disease and inhibited the accumulations of patulin, deoxynivalenol, 15-Acetyl-deoxynivalenol, and HT-2 toxin.

Isolation of the Main Pathogens Causing Postharvest Disease in Fresh Angelica sinensis during Different Storage Stages and Impacts of Ozone Treatment on Disease Development and Mycotoxin Production.

Angelica sinensis, a Chinese herbal medicine, is susceptible to molds during storage, reducing its quality, and even generating mycotoxins with toxic effects on human health. Fresh A. sinensis was harvested from Min County of Gansu Province in China and kept at room temperature. Naturally occurring symptoms were observed during different storage stages. Molds were isolated and identified from the diseased A. sinensis using morphological and molecular biology methods. The impact of ozone treatment on postharvest disease development and mycotoxin production was investigated. The results indicated that A. sinensis decay began on day 7 of storage and progressed thereafter. Nine mold species were isolated and characterized: day 7, two Mucormycetes; day 14, Clonostachys rosea; day 21, two Penicillium species and Aspergillus versicolor; day 28, Alternaria alternata and Trichoderma atroviride; and day 49, Fusarium solani. Ozone treatment markedly inhibited the development of postharvest disease and the mycotoxin production (such as, patulin, 15-acetyl-deoxynivalenol, and sterigmatocystin) in the rotten tissue of A. sinensis inoculated with the nine isolates.

Contamination, Detection and Control of Mycotoxins in Fruits and Vegetables.

Mycotoxins are secondary metabolites produced by pathogenic fungi that colonize fruits and vegetables either during harvesting or during storage. Mycotoxin contamination in fruits and vegetables has been a major problem worldwide, which poses a serious threat to human and animal health through the food chain. This review systematically describes the major mycotoxigenic fungi and the produced mycotoxins in fruits and vegetables, analyzes recent mycotoxin detection technologies including chromatography coupled with detector (i.e., mass, ultraviolet, fluorescence, etc.) technology, electrochemical biosensors technology and immunological techniques, as well as summarizes the degradation and detoxification technologies of mycotoxins in fruits and vegetables, including physical, chemical and biological methods. The future prospect is also proposed to provide an overview and suggestions for future mycotoxin research directions.

Electrostatic adsorption and removal mechanism of ochratoxin A in wine via a positively charged nano-MgO microporous ceramic membrane.

Ochratoxin A (OTA) is a very important mycotoxin. However, there are few studies on the removal of OTA in wine because of the great influence on product quality and difficulty in practical application. A nano-MgO-modified diatomite ceramic membrane (MCM) with a high positive charge was prepared and applied to remove OTA in wine. The isotherm adsorption between the positively charged membrane and OTA was in accordance with the Langmuir model, with a maximum adsorption capacity of 806 ng/g at 25 °C. All of the changes in adsorption enthalpy (ΔH), adsorption free energy (ΔG) and adsorption entropy (ΔS) were negative, which indicated that the combination of nano-MgO MCM and OTA was a spontaneous exothermic and nonspecific physical adsorption process. The concentrations of OTA in adsorption-treated wines were lower than 2 µg/kg, and the removal rates exceeded 92%. After OTA removal, the composition of wines was preserved to some extent.

Effect of Ambient pH on Growth, Pathogenicity, and Patulin Production of Penicillium expansum.

Penicillium expansum is an important postharvest pathogen of pomaceous fruit and a causal agent of blue mold or soft rot. In this study, we investigated the effect of ambient pH on growth, ultrastructure alteration, and pathogenicity of P. expansum, as well as accumulation of patulin and expression of genes involved in patulin biosynthesis. Under different pH, the fungus was routinely cultured and collected for growth, pathogenicity, patulin production, and gene expression studies using transmission electron microscopy, apple inoculation, HPLC, and RT-qPCR methods. Different ambient pH had significant impact on expression of genes and growth factors involved in patulin biosynthesis. Under same range of pH, gene expression profile, growth factors, and patulin accumulation (in vivo and in vitro) all showed similar changing trends. A well-developed cell was observed in addition to upregulation of genes at pH between pH 5.0 and 7.0, while the opposite was observed when pH was too basic (8.5) or too acid (2.5). Additionally, ambient pH had direct or indirect influence on expression of PecreaA, PelaeA, and PepacC. These findings will help in understanding the effect of ambient pH on growth, pathogenicity, and patulin production and support the development of successful methods for combating P. expansum infection on apple fruits.

Modification performance and electrochemical characteristics of different groups of modified aptamers applied for label-free electrochemical impedimetric sensors.

Amino and thiolated aptamers are the main aptamers used to construct label-free electrochemical impedimetric aptasensors. In this study, the modification performance and electrochemical properties of amino aptamers and thiolated aptamers were studied in the construction of label-free impedimetric sensors. The results showed that the initial modification density of amino aptamers was higher than that of thiol aptamers. Aptamers can recognize and bind OTA to generate electrical signals. The higher the density of aptamer modification was, the better the electric signals were. If only considering the initial modification density, amino aptamers were more suitable for the preparation of aptasensors than thiolated aptamers. However, the modification density of the amino aptamer decreased with the prolonged immersion time in 1 mM HCl solution, which suggests that the stability of this sensor was poor. However, the thiolated aptamer maintained relatively constant density and could be reused. Thus, the thiolated aptasensor had a wide range and good reproducibility and stability for the determination of ochratoxin A (OTA). In addition, this study proved that gold nanoparticles play an important role in signal amplification by increasing the effective gold surface to fix more aptamers in the process of sensor preparation.

The effect of benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) treatment on regulation of reactive oxygen species metabolism involved in wound healing of potato tubers during postharvest.

Benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) can improve wound healing of potato tubers; however, how the chemical regulates reactive oxygen species (ROS) generation and scavenging during wound healing is not completely understood. BTH at 100 mg·L-1 regulated changes in ROS generation and scavenging in healing tissues of potato tubers. A higher H2O2 content was presented in healing tissues of potato tubers, while cell membrane permeability and malondialdehyde content declined due to BTH treatment. Additionally, the activities and transcript level of enzymes related with ROS generation, including NADPH oxidase, peroxidase and polyamine oxidase, as well as enzymes involved in ROS scavenging, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, were significantly enhanced by BTH treatment. It is suggested that ROS metabolism might play a crucial role in wound healing of potato tubers mediated by BTH during postharvest.

Genome-wide in silico identification of LysM-RLK genes in potato (Solanum tuberosum L.).

The receptor like kinases (RLKs) belong to the RLK/Pelle superfamily, one of the largest gene families in plants. RLKs play an important role in plant development, as well as in response to biotic and abiotic stresses. The lysine motif receptor like kinases (LysM-RLKs) are a subfamily of RLKs containing at least one lysine motif (LysM) that are involved in the perception of elicitors or pathogen-associated molecular patterns (PAMPs). In the present study, 77 putative RLKs genes and three receptor like proteins were identified in potato (Solanum tuberosum) genome, following a genome-wide search. The 77 potato RLK proteins are classified into two major phylogenetic groups based on their kinase domain amino acid sequence similarities. Out of 77 RLKs, 10 proteins had at least one LysM. Among them three RLP proteins were found in potato genome with either 2 or three tandem LysM but these lacked a cytoplasmic kinase domain. Expression analyses of a potato LysM-RLKs (StLysM-RLK05) was carried out by a Real time RT-PCR, following inoculation of potato leaves and immature tubers with late blight and common scab pathogens, respectively. The expression was significantly higher in resistant than in susceptible following S. scabies inoculation. The StLysM-RLK05 sequence was verified and it was polymorphic in scab susceptible cultivar. The present study provides an overview of the StLysM-RLKs gene family in potato genome. This information is helpful for future functional analysis of such an important protein family, in Solanaceae species.

Acetylsalicylic acid treatment reduce Fusarium rot development and neosolaniol accumulation in muskmelon fruit.

Fusarium rot of muskmelon is a common and frequently-occurring postharvest disease, which leads to quality deterioration and neosolaniol (NEO) contamination. New strategies to control postharvest decay and reduce NEO contamination are of paramount importance. The effects of acetylsalicylic acid (ASA) treatment on the growth of Fusarium sulphureum in vitro, and Fusarium rot development and NEO accumulation in fruits inoculated with F. sulphureum in vivo were investigated. The results showed that ASA inhibited the growth of F. sulphureum, evident morphological and major cellular changes were observed under the microscope. In vivo testing showed that 3.2 mg/mL ASA significantly suppressed Fusarium rot development and NEO accumulation after 6 and 8 d of pathogen inoculation. Meanwhile, Tri gene expressions involved in NEO biosynthesis were down-regulated after treatment. Taken together, ASA treatment not only reduced Fusarium rot development by inhibiting the growth of F. sulphureum, but decreased NEO accumulation by suppressing NEO biosynthesis pathway.

Sodium silicate prime defense responses in harvested muskmelon by regulating mitochondrial energy metabolism and reactive oxygen species production.

The effects of postharvest treatment with sodium silicate (Si) (100 mM) on mitochondrial ROS production and energy metabolism of the muskmelon fruits (cv. Yujinxiang) on development of defense responses to Trichothecium roseum were studied. Si treatment decreased decay severity of inoculated muskmelons, enhanced the activities of energy metabolism of key enzymes and kept the intracellular ATP at a higher level; meanwhile, Si also induced the mtROS accumulation such as H2O2 and superoxide anion. TMT-based quantitative proteomics analysis revealed that a total of 24 proteins with significant differences in abundance involved in energy metabolism, defense and stress responses, glycolytic and TCA cycle, and oxidation-reduction process. It is suggested by our study that melon fruit mitochondria, when induced by Si treatments, play a key role in priming of host resistance against T. roseum infection through the regulation of energy metabolism and ROS production in the pathogen infected muskmelon fruits.

Early Defense Responses Involved in Mitochondrial Energy Metabolism and Reactive Oxygen Species Accumulation in Harvested Muskmelons Infected by Trichothecium roseum.

Mitochondria play an essential part in fighting against pathogen infection in the defense responses of fruits. In this study, we investigated the reactive oxygen species (ROS) production, energy metabolism, and changes of mitochondrial proteins in harvested muskmelon fruits ( Cucumis melo cv. Yujinxiang) inoculated with Trichothecium roseum. The results indicated that the fungal infection obviously induced the H2O2 accumulation in mitochondria. Enzyme activities were inhibited in the first 6 h postinoculation (hpi), including succinic dehydrogenase, cytochrome c oxidase, H+-ATPase, and Ca2+-ATPase. However, the activities of Ca2+-ATPase and H+-ATPase and the contents of intracellular adenosine triphosphate (ATP) were improved to a higher level at 12 hpi. A total of 42 differentially expressed proteins were identified through tandem mass tags-based proteomic analyses, which are mainly involved in energy metabolism, stress responses and redox homeostasis, glycolysis and tricarboxylic acid cycle, and transporter and mitochondria dysfunction. Taken together, our results suggest that mitochondria play crucial roles in the early defense responses of muskmelons against T. roseum infection through regulation of ROS production and energy metabolism.

Rapid Determination of Ochratoxin A in Grape and Its Commodities Based on a Label-Free Impedimetric Aptasensor Constructed by Layer-by-Layer Self-Assembly.

A simple and sensitive label-free impedimetric aptasensor for rapid determination of ochratoxin A (OTA) has been developed, which was based on the combination between thiolated aptamer and gold nanoparticles by layer-by-layer self-assembly. Because of the interaction between aptamer and OTA, the relative normalized electron-transfer resistance (ΔRct) values obtained by electrochemical impedance spectroscopy (EIS) was proportional to the concentration of OTA and showed a good linear relationship from 0.1 to 10.0 ng/mL, with a lower detection limit (0.030 ng/mL) than one-step thiolated DNA aptasensor. The established method was successfully applied to detect and analyze OTA in table wine and grape juice, and the recovery was 90.56%⁻104.21% when PVP effective removed of phenolic substances. The label-free impedimetric aptasensor was used for rapid detection and quantitation of OTA in the inoculated grapes with the Aspergillus Nigri (H1), and the production of OTA (62.4 µg/kg, 20 µg/kg) far exceeded the maximum levels of 2 µg/kg after inoculation for three days. The developed method exhibited a good specificity, high sensitivity, time-efficient, and it could be applied to detect the OTA concentration in grape and its commodities.

Damage to Trichothecium roseum caused by sodium silicate is independent from pH.

Trichothecium roseum is one of the most important postharvest pathogens in arid and semiarid regions. Sodium silicate (NaSi) and environmental pH have significant inhibitory effects on fungal growth. However, no study has addressed the relationship of NaSi and pH in combination and the effects on T. roseum. In this work, we showed that spore germination, germ tube elongation, and mycelial growth of T. roseum were significantly inhibited by various NaSi concentrations, which had corresponding increasing pHs. Furthermore, these NaSi solutions showed a much greater impact than did pH treatments alone. The pathogenicity of NaSi-treated conidia on a model assay (conidia-inoculated apple fruit) was dramatically reduced, whereas no changes of pathogenicity were evident for the corresponding pH (various sodium hydroxide (NaOH) solutions) treatments. Fluorescent microscopy, using propidium iodide staining, showed damage of the plasma membranes of T. roseum conidia treated with both NaSi and NaOH, although the damage was more severe with NaSi. Leakage of proteins and sugars was significantly higher in NaSi-treated and NaOH-treated conidia than in untreated controls. In addition, serious damage was observed in the conidia exposed to NaSi for longer periods of time. Ultrastructural observations showed that treatment with either NaSi or NaOH caused a plasmolysis state and disorganized organelles. Taken together the results show that NaSi has inhibitory effects on T. roseum and that the inherent higher pH of NaSi solutions of higher concentrations simply acts as an enhancer of the inhibitory effects of NaSi.