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Red grapes often suffer from postharvest diseases like blue mold and black mold caused by Penicillium expansum and Aspergillus niger. Biological control using beneficial yeasts and bacteria is an effective method to manage these diseases. Rhodotorula sp. and Bacillus sp. are effective microorganisms for the control of postharvest diseases of red grapes. This study combined two yeast strains (Rhodotorula graminis and Rhodotorula babjevae) and two bacterial strains (Bacillus licheniformis and Bacillus velezensis) to investigate their biological control effects on major postharvest diseases of red grapes and explore the underlying physiological mechanisms. Research showed that compound microorganism W3 outperformed the others; it reduced spore germination and germ tube growth of P. expansum and A. niger, while its volatiles further inhibited pathogen growth. Additionally, the treatment enhanced the antioxidant capacity of grapes and increased resistance to pathogens by boosting peroxidase activities, superoxide dismutase, catalase and ascorbate peroxidase, phenylalanine ammonolyase, and polyphenol oxidase. Furthermore, the combined treatment increased the activity and accumulation of antifungal compounds such as total phenols and flavonoids, thereby improving disease resistance and reducing decay. Therefore, composite microorganisms combining various antagonistic strains may offer a viable substitute for tackling postharvest diseases in red grapes.

Postharvest diseases, such as black spots caused by Alternaria alternata, have caused huge economic losses to the tomato industry and seriously restricted its development. In recent years, biological control has become a new method to control postharvest diseases of fruits and vegetables. Our research group screened W. anomalus, a yeast demonstrating a promising control effect on a postharvest black spot disease of tomatoes, and explored its physiological mechanism of prevention and control. Therefore, this study investigated the prevention and control effect of metabolites of W. anomalus on tomato black spot disease and the inhibition effect of main components on A. alternata. A GC-MS analysis found that isoamyl acetate was the main component of W. anomalus that played an inhibitory role. The results showed that isoamyl acetate could inhibit the growth of A. alternata and had a certain control effect on postharvest black spots in tomatoes. Our findings suggest that isoamyl acetate could be a promising alternative to fungicides for controlling postharvest black spots in tomatoes.

Cladosporium cladosporioides is an emerging pathogenic fungus that causes Cladosporium rot in postharvest table grapes (Vitis vinifera). However, studies investigating the infection process of C. cladosporioides are lacking. Therefore, this study aimed to elucidate the infection process by investigating the influence of microclimatic parameters (temperature, wetness and fungal age) in C. cladosporioides pathogenesis, activities of grape defense-related enzymes and grape quality during the infection. C. cladosporioides effectively infects grapes by developing distinct colonies on the artificial wounds and berry surfaces, completing its life cycle within 48 h. The C. cladosporioides disease incidence optimally occurred at 20 °C and 25 °C. Wetness played an influential role in the infectivity of C. cladosporioides and 7-day-old C. cladosporioides resulted in the most serious disease incidence of table grapes. As a result of infection, the quality of grapes was affected, including berry weight, pH, titratable acidity (TA), total soluble solids (TSS), and ascorbic acid level. This infection also induced defense-related enzymes, including polyphenoloxidase (PPO), peroxidase (POD), phenylalanine ammonialyase (PAL), and catalase (CAT), at certain times. The findings of this study demonstrated that Cladosporium rot development depended on the microclimatic parameters of grapes, significantly affected the grape quality and induced grape's defense-related enzymes.

Mechanical stress induced by compression during preharvest and postharvest handling of fresh fruit is a major factor influencing the quality of fresh fruit. The degree of compression primarily governs the extent of quality deterioration. However, research on the damage mechanisms of mechanical compression in table grapes remains scarce. This investigation evaluated the impact of varying compression levels (0%, 20%, 40%, 60%, and 80%) on the postharvest quality attributes of table grapes. Changes in postharvest physical properties (overall appearance and color), structural properties (firmness, springiness, cohesiveness, and chewiness), physiological qualities (total soluble solids, titratable acidity, ascorbic acid, malonaldehyde content, and relative electrical conductivity), and cell microstructure of the berries was determined at 4 °C during 15 d of storage. Mechanical compression contributed to the deterioration of the quality of table grapes during storage, resulting in increased weight loss, decay rate, malonaldehyde content, and relative electrical conductivity; and decreased total soluble solids, titratable acidity, and ascorbic acid content. Furthermore, as compared to the control group, mechanical compression resulted in substantial yellowing and diminished textural qualities of grapes. In particular, compression treatment caused significant deformation of grape cell microstructure. In conclusion, mechanical compression stress significantly affects the physical and physiological properties of postharvest table grapes, as well as the internal cellular organization. As compression levels increase, the quality of table grapes progressively deteriorates, leading to a substantial reduction in storage life and commercial value. This study offers essential information for devising damage prevention strategies in preharvest and postharvest handling of table grapes.

β-1,3-glucanase plays an important role in the biodegradation, reconstruction, and development of β-1,3-glucan. An endo-β-1,3-glucanase which was encoded by PeBgl1 was expressed, purified and characterized from Penicillium expansum for the first time. The PeBgl1 gene was amplified and transformed into the competent cells of E. coli Rosetta strain with the help of the pET-30a cloning vector. The recombinant protein PeBgl1 was expressed successfully at the induction conditions of 0.8 mmol/L IPTG at 16 °C for 16 h and then was purified by nickel ion affinity chromatography. The optimum reaction temperature of PeBgl1 was 55 °C and it had maximal activity at pH 6.0 according to the enzymatic analysis. Na2HPO4-NaH2PO4 buffer (pH 6.0) and NaCl have inhibitory and enhancing effects on the enzyme activities, respectively. SDS, TritonX-100 and some metal ions (Mg2+, Ca2+, Ba2+, Cu2+, and Zn2+) have an inhibitory effect on the enzyme activity. The results showed that PeBgl1 protein has good enzyme activity at 50–60 °C and at pH 5.0–9.0, and it is not a metal dependent enzyme, which makes it robust for storage and transportation, ultimately holding great promise in green biotechnology and biorefining.

Studies on the molecular mechanism of antagonistic yeasts to control apple postharvest diseases are not comprehensive enough. Our preliminary investigations screened the biocontrol effect of Hannaella sinensis, an antagonistic yeast, and discovered its control efficacy on apple blue mold decay. However, the molecular mechanism of H. sinensis-induced resistance in apple has not been studied. In this study, proteins from apple treated with H. sinensis and sterile saline were analyzed using TMT proteomics technology. It was found that H. sinensis treatment induced the expressions of apple resistance-related proteins. Among the proteins in H. sinensis-induced apple, proteins related to plant defense mechanisms, such as reactive oxygen species scavenging, improvement of plant resistance and synthesis of resistant substances, improvement of plant disease resistance, the degradation of the pathogen cell wall, cell signaling, antibacterial activity, transport of defense-related substances, and protein processing, were differentially regulated. The results of this study revealed the underlying molecular mechanisms of H. sinensis-induced apple resistance at the protein level; the results also provided a theoretical basis for the commercial application of H. sinensis.

Biological control utilizing antagonistic yeasts is an effective method for controlling postharvest diseases. Long non-coding RNAs (lncRNAs) have been found to be involved in a variety of plant growth and development processes, including those associated with plant disease resistance. In the present study, the yeast antagonist, Wickerhamomyces anomalus, was found to strongly inhibit postharvest blue mold (Penicillium expansum) and gray mold (Botrytis cinerea) decay of kiwifruit. Additionally, lncRNA high-throughput sequencing and bioinformatic analysis was used to identify lncRNAs in W. anomalus-treated wounds in kiwifruit and predict their function based on putative target genes. Our results indicate that lncRNAs may be involved in increasing ethylene (ET), jasmonic acid (JA), abscisic acid (ABA), and auxin (IAA) levels, as well as activating signal transduction pathways that regulate the expression of several transcription factors (WRKY72, WRKY53, JUB1AP2). These transcription factors (TFs) then mediate the expression of downstream, defense-related genes (ZAR1, PAD4, CCR4, NPR4) and the synthesis of secondary metabolites, thus, potentially enhancing disease resistance. Notably, by stimulating the accumulation of antifungal compounds, such as phenols and lignin, disease resistance in kiwifruit was enhanced. Our study provides new information on the mechanism underlying the induction of disease resistance in kiwifruit by W. anomalus, as well as a new disease resistance strategy that can be used to enhance the defense response of fruit to pathogenic fungi.

Patulin (PAT), mainly produced by Penicillium expansum, is a potential threat to health. In recent years, PAT removal using antagonistic yeasts has become a hot research topic. Meyerozyma guilliermondii, isolated by our group, produced antagonistic effects against the postharvest diseases of pears and could degrade PAT in vivo or in vitro. However, the molecular responses of M. guilliermondii over PAT exposure and its detoxification enzymes are not apparent. In this study, transcriptomics is used to unveil the molecular responses of M. guilliermondii on PAT exposure and the enzymes involved in PAT degradation. The functional enrichment of differentially expressed genes indicated that the molecular response mainly includes the up-regulated expression of genes related to resistance and drug-resistance, intracellular transport, growth and reproduction, transcription, DNA damage repair, antioxidant stress to avoid cell damage, and PAT detoxification genes such as short-chain dehydrogenase/reductases. This study elucidates the possible molecular responses and PAT detoxification mechanism of M. guilliermondii, which could be helpful to further accelerate the commercial application of antagonistic yeast toward mycotoxin decontamination.

The composition of microbial communities can directly affect fruit quality, health status, and storability. The present study characterized the epiphytes and endophytes of “Hongyang” and “Cuiyu” kiwifruit at harvest under grown under open-field (OF) and rain-shelter (RS) cultivation systems. Disease incidence in kiwifruit was significantly lower (p < 0.05) under the RS system than it was under the OF system. High-throughput sequencing [16S V3-V4 ribosomal region and the fungal internal transcribed spacer (ITS2)] was conducted to compare the composition of the epiphytic and endophytic microbial community of kiwifruit under the two cultivation systems. Results indicated that the abundance of Actinobacteria, Bacteroidetes, Enterobacteriales, Acetobacterales, Sphingomonas, Pseudomonas, and Sphingobacterium was higher under the RS system, relative to the OF system, while the abundance of Capnodiales, Hypocreales, Vishniacozyma, and Plectosphaerella was also higher under the RS system. Some of these bacterial and fungal taxa have been reported to as act as biocontrol agents and reduce disease incidence. Notably, the α-diversity of the epiphytic bacterial and fungal communities on kiwifruit was higher under RS cultivation. In summary, RS cultivation reduced natural disease incidence in kiwifruit, which may be partially attributed to differences in the structure and composition of the microbial community present in and on kiwifruit.

To study the mechanism by which Pichia anomala induced with chitosan (1% w/v) controls blue mold disease in table grapes caused by Penicillium expansum, this study evaluated alterations in three yeast enzymatic activities. The changes in the five primary disease defense-related enzymes and two non-enzyme activities of table grapes were assayed. The results of the study showed that chitosan (1% w/v) significantly increased the yeast β-1,3-glucanase, catalase (CAT), and malondialdehyde (MDA) activities. Furthermore, P. anomala alone or induced with chitosan (1% w/v) significantly increased the table grapes enzymatic activities of Polyphenol oxidase (PPO), phenylalanine (PAL), peroxidase (POD), and catalase (CAT) compared to the control. The RT-qPCR results also confirmed that the genes of these major disease defense enzymes were up-regulated when the table grapes were treated with P. anomala. The highest results were recorded when the fruit was treated by yeast induced with chitosan (1% w/v). The phenolic compounds, in addition to their nutritional value, can also increase the antimicrobial properties of table grapes. The current experiment determined that the total phenol and flavonoid contents of table grapes showed the highest results for fruits treated by P. anomala induced with chitosan compared with the control. Generally, the increment of these fruit enzymatic and non-enzymatic activities shows improved table grape defense against the pathogenic fungus. The induction of the yeast with chitosan also increases its bio-control efficacy against the pathogen. This study will enable future detailed investigation in the yeast pathogen control mechanisms and the use of yeasts as bio-pesticides.

Gray mold decay is a widespread postharvest disease in tomato that results from infection by the pathogen Botrytis cinerea, leading to huge economic losses. The objective of this study was to select the most effective antagonistic yeast to control tomato gray mold from six potential biocontrol agents and to investigate the possible control mechanism. The results showed that the yeast Wickerhamomyces anomalus was the most effective in inhibiting B. cinerea among the six strains both in vivo and in vitro on tomato, with a colony diameter of 11 mm, a decay diameter of 20 mm, and the lowest decay incidence (53%)—values significantly smaller and lower than the values recorded for the control group and the other yeasts. The efficacy of the control depended on the increase in yeast concentration, and the decay incidence and lesion diameter were reduced to 31%, 28% and 7 mm, 6 mm, respectively, when treated with 1 × 108 and 1 × 109 cells/mL W. anomalus. In addition, W. anomalus was able to rapidly colonize and stably multiply in tomato, occupying the space to control pathogen infection. W. anomalus was also able to motivate the defense mechanism of tomato with stimulation of defense-related enzymes PPO, POD, APX, and SOD and promotion of the content of total phenols and flavonoid compounds. All these results suggest that W. anomalus exhibited exceptional ability to control gray mold in tomato.

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.

Antagonistic yeasts can inhibit fungal growth. In our previous research, Meyerozyma guilliermondii, one of the antagonistic yeasts, exhibited antagonistic activity against Penicillium expansum. However, the mechanisms, especially the molecular mechanisms of inhibiting activity of M. guilliermondii, are not clear. In this study, the protein expression profile and transcriptome characterization of P. expansum induced by M. guilliermondii were investigated. In P. expansum induced by M. guilliermondii, 66 proteins were identified as differentially expressed, among them six proteins were upregulated and 60 proteins were downregulated, which were associated with oxidative phosphorylation, ATP synthesis, basal metabolism, and response regulation. Simultaneously, a transcriptomic approach based on RNA-Seq was applied to annotate the genome of P. expansum and then studied the changes of gene expression in P. expansum treated with M. guilliermondii. The results showed that differentially expressed genes such as HEAT, Phosphoesterase, Polyketide synthase, ATPase, and Ras-association were significantly downregulated, in contrast to Cytochromes P450, Phosphatidate cytidylyltransferase, and Glutathione S-transferase, which were significantly upregulated. Interestingly, the downregulated differentially expressed proteins and genes have a corresponding relationship; these results revealed that these proteins and genes were important in the growth of P. expansum treated with M. guilliermondii.

Transcriptome analysis (TA) was conducted to characterize the transcriptome changes in postharvest disease-related genes of table grapes following treatment with Pichia anomala induced with chitosan (1% w/v). In the current study, the difference in the gene expression of table grapes after treatment with P. anomala induced with chitosan and that of a control group was compared 72 h post-inoculation. The study revealed that postharvest treatment of table grapes with P. anomala induced with chitosan could up-regulate genes that have a pivotal role in the fruit’s disease defense. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) results also confirmed that GO terms and the KEGG pathways, which have pivotal roles in plant disease resistance, were significantly enriched. The up-regulated genes of the treatment group have a unique function in the fruit’s disease resistance compared to the control group. Generally, most genes in the plant–pathogen interaction pathway; the plant Mitogen-activated protein kinase (MAPK) signaling pathway; the plant hormone signal transduction pathway; the pathway of glutathione metabolism; the pathway of phenylalanine, tyrosine, and tryptophan biosynthesis; and the pathway of flavonoid biosynthesis were all up-regulated. These up-regulations help the fruit to synthesize disease-resistant substances, regulate the reactive oxygen species (ROS), enhance the fruit cell wall, and enrich hormone signal transduction during the pathogen’s attack. This study is useful to overcome the lags in applying transcriptomics technology in postharvest pathology, and will provide insight towards developing other alternative methods to using bio-pesticides to control postharvest diseases of perishables.

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.

Green mold disease, a common citrus post-harvest disease caused by Penicillium digitatum, has an unresolved initial infection mechanism. Understanding the infection mechanism leads to the development of potential controls and preventive measures against the disease. The present study aimed to delineate the infection mechanism by investigating spore germination, changes of organic molecules and enzyme activity, and differential expression of genes in the P. digitatum infection. P. digitatum spore germination was observed by a pathology section scanner and it was found that in vivo germination was 3 h behind the in vitro germination. In addition, cell wall degrading enzymes and soluble sugar and titratable acid content during the infection process measured dynamically. The level of pectinase reached its maximum of 6067 U/g before 48 hpi, while cellulase increased rapidly after 48 hpi. The soluble sugar and organic acid content increased considerably with the progression of the infection. The transcriptomic profile of P. digitatum before and after infection was analyzed by RNA-seq. The genes related to cell wall degrading enzymes were significantly up-regulated and annotated to participate in two major carbon source synthesis pathways. The study delineated the initial infection mechanism of P. digitatum which eventually opened the gate way for the development of new control strategies in the future.

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.