A methyl esterase from Bifidobacterium longum subsp. longum reshapes the prebiotic properties of apple pectin by triggering differential modulatory capacity in faecal cultures.

Pectin structures have received increasing attention as emergent prebiotics due to their capacity to promote beneficial intestinal bacteria. Yet the collective activity of gut bacterial communities to cooperatively metabolize structural variants of this substrate remains largely unknown. Herein, the characterization of a pectin methylesterase, BpeM, from Bifidobacterium longum subsp. longum, is reported. The purified enzyme was able to remove methyl groups from highly methoxylated apple pectin, and the mathematical modelling of its activity enabled to tightly control the reaction conditions to achieve predefined final degrees of methyl-esterification in the resultant pectin. Demethylated pectin, generated by BpeM, exhibited differential fermentation patterns by gut microbial communities in in vitro mixed faecal cultures, promoting a stronger increase of bacterial genera associated with beneficial effects including Lactobacillus, Bifidobacterium and Collinsella. Our findings demonstrate that controlled pectin demethylation by the action of a B. longum esterase selectively modifies its prebiotic fermentation pattern, producing substrates that promote targeted bacterial groups more efficiently. This opens new possibilities to exploit biotechnological applications of enzymes from gut commensals to programme prebiotic properties.

Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by Wall-associated kinase gene MbWAK1.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.

Identification of Hsp20 gene family in Malus domestica and functional characterization of Hsp20 class I gene MdHsp18.2b.

Heat shock protein 20 (Hsp20) is a small molecule heat shock protein that plays an important role in plant growth, development, and stress resistance. Little is known about the function of Hsp20 family genes in apple (Malus domestica). Here, we performed a genome-wide analysis of the apple Hsp20 gene family, and a total of 49 Hsp20s genes were identified from the apple genome. Phylogenetic analysis revealed that the 49 genes were divided into 11 subfamilies, and MdHsp18.2b, a member located in the CI branch, was selected as a representative member for functional characterization. Treatment with NaCl and Botryosphaeria dothidea (B. dothidea), the causal agent of apple ring rot disease, significantly induced MdHsp18.2b transcription level. Further analysis revealed that overexpressing MdHsp18.2b reduced the resistance to salt stress but enhanced the resistance to B. dothidea infection in apple calli. Moreover, MdHsp18.2b positively regulated anthocyanin accumulation in apple calli. Physiology assays revealed that MdHsp18.2b promoted H2O2 production, even in the absence of stress factors, which might contribute to its functions in response to NaCl and B. dothidea infection. Hsps usually function as homo- or heterooligomers, and we found that MdHsp18.2b could form a heterodimer with MdHsp17.9a and MdHsp17.5, two members from the same branch with MdHsp18.2b in the phylogenetic tree. Therefore, we identified 49 Hsp20s genes from the apple genome and found that MdHsp18.2b was involved in regulating plant resistance to salt stress and B. dothidea infection, as well as in regulating anthocyanin accumulation in apple calli.

Molecular Mechanism of Resistance to Alternaria alternata Apple Pathotype in Apple by Alternative Splicing of Transcription Factor MdMYB6-like.

As a fruit tree with great economic value, apple is widely cultivated in China. However, apple leaf spot disease causes significant damage to apple quality and economic value. In our study, we found that MdMYB6-like is a transcription factor without auto-activation activity and with three alternative spliced variants. Among them, MdMYB6-like-ß responded positively to the pathogen infection. Overexpression of MdMYB6-like-ß increased the lignin content of leaves and improved the pathogenic resistance of apple flesh callus. In addition, all three alternative spliced variants of MdMYB6-like could bind to the promoter of MdBGLU H. Therefore, we believe that MdMYB6-like plays an important role in the infection process of the pathogen and lays a solid foundation for breeding disease-resistant cultivars of apple in the future.

Nitrogen-fixing bacterial communities differ between perennial agroecosystem crops.

Biocrusts, common in natural ecosystems, are specific assemblages of microorganisms at or on the soil surface with associated microorganisms extending into the top centimeter of soil. Agroecosystem biocrusts have similar rates of nitrogen (N) fixation as those in natural ecosystems, but it is unclear how agricultural management influences their composition and function. This study examined the total bacterial and diazotrophic communities of biocrusts in a citrus orchard and a vineyard that shared a similar climate and soil type but differed in management. To contrast climate and soil type, these biocrusts were also compared with those from an apple orchard. Unlike natural ecosystem biocrusts, these agroecosystem biocrusts were dominated by proteobacteria and had a lower abundance of cyanobacteria. All of the examined agroecosystem biocrust diazotroph communities were dominated by N-fixing cyanobacteria from the Nostocales order, similar to natural ecosystem cyanobacterial biocrusts. Lower irrigation and fertilizer in the vineyard compared with the citrus orchard could have contributed to biocrust microbial composition, whereas soil type and climate could have differentiated the apple orchard biocrust. Season did not influence the bacterial and diazotrophic community composition of any of these agroecosystem biocrusts. Overall, agricultural management and climatic and edaphic factors potentially influenced the community composition and function of these biocrusts.

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.

Biocontrol activity of an endophytic Alternaria alternata Aa-Lcht against apple Valsa canker.

Apple Valsa canker (AVC), caused by Valsa mali, is the most serious branch disease for apples in East Asia. Biocontrol constitutes a desirable alternative strategy to alleviate the problems of orchard environment pollution and pathogen resistance risk. It is particularly important to explore efficient biocontrol microorganism resources to develop new biocontrol technologies and products. In this study, an endophytic fungus, which results in the specific inhibition of the growth of V. mali, was isolated from the twig tissue of Malus micromalus with a good tolerance to AVC. The fungus was identified as Alternaria alternata, based on morphological observations and phylogenetic analysis, and was named Aa-Lcht. Aa-Lcht showed a strong preventive effect against AVC, as determined with an in vitro twig evaluation method. When V. mali was inhibited by Aa-Lcht, according to morphological and cytological observations, the hyphae was deformed and it had more branches, a degradation in protoplasm, breakages in cell walls, and then finally died completely due to mycelium cells. Transcriptome analysis indicated that Aa-Lcht could suppress the growth of V. mali by inhibiting the activity of various hydrolases, destroying carbohydrate metabolic processes, and damaging the pathogen membrane system. It was further demonstrated that Aa-Lcht could colonize apple twig tissues without damaging the tissue's integrity. More importantly, Aa-Lcht could also stimulate the up-regulated expression of defense-related genes in apples together with the accumulation of reactive oxygen species and callose deposition in apple leaf cells. Summarizing the above, one endophytic biocontrol resource was isolated, and it can colonize apple twig tissue and play a biocontrol role through both pathogen inhibition and resistance inducement.

Transcriptomic investigation of the interaction between a biocontrol yeast, Papiliotrema terrestris strain PT22AV, and the postharvest fungal pathogen Penicillium expansum on apple.

Biocontrol strategies offer a promising alternative to control plant pathogens achieving food safety and security. In this study we apply a RNAseq analysis during interaction between the biocontrol agent (BCA) Papiliotrema terrestris, the pathogen Penicillium expansum, and the host Malus domestica. Analysis of the BCA finds overall 802 upregulated DEGs (differentially expressed genes) when grown in apple tissue, with the majority being involved in nutrients uptake and oxidative stress response. This suggests that these processes are crucial for the BCA to colonize the fruit wounds and outcompete the pathogen. As to P. expansum analysis, 1017 DEGs are upregulated when grown in apple tissue, with the most represented GO categories being transcription, oxidation reduction process, and transmembrane transport. Analysis of the host M. domestica finds a higher number of DEGs in response to the pathogen compared to the BCA, with overexpression of genes involved in host defense signaling pathways in the presence of both of them, and a prevalence of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) only during interaction with P. expansum. This analysis contributes to advance the knowledge on the molecular mechanisms that underlie biocontrol activity and the tritrophic interaction of the BCA with the pathogen and the host.

Uncovering the antifungal activities of wild apple-associated bacteria against two canker-causing fungi, Cytospora mali and C. parasitica.

Cytospora canker has become a devastating disease of apple species worldwide, and in severe cases, it may cause dieback of entire trees. The aim of this study was to characterize the diversity of cultivable bacteria from the wild apple microbiota and to determine their antifungal ability against the canker-causing pathogenic fungi Cytospora mali and C. parasitica. Five bacterial strains belonging to the species Bacillus amyloliquefaciens, B. atrophaeus, B. methylotrophicus, B. mojavensis, and Pseudomonas synxantha showed strong antagonistic effects against pathogenic fungi. Therefore, since the abovementioned Bacillus species produce known antifungal compounds, we characterized the antifungal compounds produced by Ps. synxantha. Bacteria grown on nutritional liquid medium were dehydrated, and the active compound from the crude extract was isolated and analysed via a range of chromatographic processes. High-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance analyses revealed a bioactive antifungal compound, phenazine-1-carboxylic acid (PCA). The minimum inhibitory concentration (MIC) demonstrated that PCA inhibited mycelial growth, with a MIC of 10 mg mL-1. The results suggested that PCA could be used as a potential compound to control C. mali and C. malicola, and it is a potential alternative for postharvest control of canker disease.

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.

Host-induced gene silencing in wild apple germplasm Malus hupehensis confers resistance to the fungal pathogen Botryosphaeria dothidea.

Host-induced gene silencing (HIGS) is an inherent mechanism of plant resistance to fungal pathogens, resulting from cross-kingdom RNA interference (RNAi) mediated by small RNAs (sRNAs) delivered from plants into invading fungi. Introducing artificial sRNA precursors into crops can trigger HIGS of selected fungal genes, and thus has potential applications in agricultural disease control. To investigate the HIGS of apple (Malus sp.) during the interaction with Botryosphaeria dothidea, the pathogenic fungus causing apple ring rot disease, we evaluated whether apple miRNAs can be transported into and target genes in B. dothidea. Indeed, miR159a from Malus hupehensis, a wild apple germplasm with B. dothidea resistance, silenced the fungal sugar transporter gene BdSTP. The accumulation of miR159a in extracellular vesicles (EVs) of both infected M. hupehensis and invading B. dothidea suggests that this miRNA of the host is transported into the fungus via the EV pathway. Knockout of BdSTP caused defects in fungal growth and proliferation, whereas knockin of a miR159a-insensitive version of BdSTP resulted in increased pathogenicity. Inhibition of miR159a in M. hupehensis substantially enhanced plant sensitivity to B. dothidea, indicating miR159a-mediated HIGS against BdSTP being integral to apple immunity. Introducing artificial sRNA precursors targeting BdSTP and BdALS, an acetolactate synthase gene, into M. hupehensis revealed that double-stranded RNAs were more potent than engineered MIRNAs in triggering HIGS alternative to those natural of apple and inhibiting infection. These results provide preliminary evidence for cross-kingdom RNAi in the apple-B. dothidea interaction and establish HIGS as a potential disease control strategy in apple.

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Listeria monocytogenes during chlorine and peroxyacetic acid interventions in simulated apple dump tank water.

Sanitizers are widely incorporated in commercial apple dump tank systems to mitigate the cross-contamination of foodborne pathogens. This study validated the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Listeria monocytogenes during sanitizer interventions in dump tank water systems. E. faecium NRRL B-2354 inoculated on apples exhibited statistically equivalent susceptibility to L. monocytogenes when exposed to chlorine-based sanitizers (25-100 ppm free chlorine (FC)) and peroxyacetic acid (PAA, 20-80 ppm) in simulated dump tank water (SDTW) with 1000 ppm chemical oxygen demand (COD), resulting in 0.2-0.9 and 1.1-1.7 log CFU/apple reduction, respectively. Increasing the contact time did not affect sanitizer efficacies against E. faecium NRRL B-2354 and L. monocytogenes on apples. Chlorine and PAA interventions demonstrated statistically similar efficacies against both bacteria inoculated in SDTW. Chlorine at 25 and 100 ppm FC for 0.5-5 min contact yielded ~37.68-78.25 % and > 99.85 % inactivation, respectively, in water with 1000-4000 ppm COD, while ~51.55-99.86 % and > 99.97 % inactivation was observed for PAA at 20 and 80 ppm, respectively. No statistically significant difference was observed between the transference of E. faecium NRRL B-2354 and L. monocytogenes from inoculated apples to uninoculated apples and water, and from water to uninoculated apples during chlorine- or PAA-treated SDTW exposure. The data suggest E. faecium NRRL B-2354 is a viable surrogate for L. monocytogenes in dump tank washing systems, which could be used to predict the anti-Listeria efficacy of chlorine and PAA interventions during commercial apple processing. Further investigations are recommended to assess the suitability of E. faecium NRRL B-2354 as a surrogate for L. monocytogenes, when using different sanitizers and different types of produce to ensure reliable and comprehensive results.

Effect of Air Temperature and Velocity on Listeria monocytogenes Inactivation During Drying of Apple Slices.

A wide range of drying parameters and methods are used by industry to produce dried apples. To ensure end-product safety and regulatory compliance, it is essential to evaluate the effectiveness of such industrial practices on microbial inactivation. Therefore, the objective of this study was to evaluate the effects of drying air temperature and velocity on Listeria monocytogenes inactivation during drying of apple slices. Apples (cv. Gala) were cored, sliced as rings (∼6 mm thick), and surface-inoculated with broth-grown culture of an 8-strain cocktail of L. monocytogenes to achieve an inoculation level of 8.6 ± 0.3 log CFU/g. Apple rings were dried in batches using dry air in a pilot-scale impingement oven at 60 or 80 °C air temperature and 0.7 or 2.1 m/s air velocity, and sampled every 30 min for bacterial enumeration, water activity (aw), and moisture content analysis. L. monocytogenes reduction increased (P < 0.05) with higher air velocity or higher drying air temperature. By the end of drying, in which the standard moisture content for dried apple slices of <24% wet basis was reached, L. monocytogenes was reduced by 1.8 ± 0.3 and 2.8 ± 0.7 log CFU/g at 0.7 and 2.1 m/s air velocity, respectively, after 180 min at 60 °C. When using 80 °C drying temperature, L. monocytogenes reduction was 5.2 ± 0.5 log CFU/g at both air velocities after 150 min. Therefore, process conditions should be considered in the validation of fruit drying processes, instead of solely relying on product endpoint properties, such as moisture content.

The Influence of Long-Term Storage on the Epiphytic Microbiome of Postharvest Apples and on Penicillium expansum Occurrence and Patulin Accumulation.

Patulin is a secondary metabolite primarily synthesized by the fungus Penicillium expansum, which is responsible for blue mold disease on apples. The latter are highly susceptible to fungal infection in the postharvest stages. Apples destined to produce compotes are processed throughout the year, which implies that long periods of storage are required under controlled atmospheres. P. expansum is capable of infecting apples throughout the whole process, and patulin can be detected in the end-product. In the present study, 455 apples (organically and conventionally grown), destined to produce compotes, of the variety "Golden Delicious" were sampled at multiple postharvest steps. The apple samples were analyzed for their patulin content and P. expansum was quantified using real-time PCR. The patulin results showed no significant differences between the two cultivation techniques; however, two critical control points were identified: the long-term storage and the deck storage of apples at ambient temperature before transport. Additionally, alterations in the epiphytic microbiota of both fungi and bacteria throughout various steps were investigated through the application of a metabarcoding approach. The alpha and beta diversity analysis highlighted the effect of long-term storage, causing an increase in the bacterial and fungal diversity on apples, and showed significant differences in the microbial communities during the different postharvest steps. The different network analyses demonstrated intra-species relationships. Multiple pairs of fungal and bacterial competitive relationships were observed. Positive interactions were also observed between P. expansum and multiple fungal and bacterial species. These network analyses provide a basis for further fungal and bacterial interaction analyses for fruit disease biocontrol.

Inter-species interactions between two bacterial flower commensals and a floral pathogen reduce disease incidence and alter pathogen activity.

Flowers are colonized by a diverse community of microorganisms that can alter plant health and interact with floral pathogens. Erwinia amylovora is a flower-inhabiting bacterium and a pathogen that infects different plant species, including Malus × domestica (apple). Previously, we showed that the co-inoculation of two bacterial strains, members of the genera Pseudomonas and Pantoea, isolated from apple flowers, reduced disease incidence caused by this floral pathogen. Here, we decipher the ecological interactions between the two flower-associated bacteria and E. amylovora in field experimentation and in vitro co-cultures. The two flower commensal strains did not competitively exclude E. amylovora from the stigma habitat, as both bacteria and the pathogen co-existed on the stigma of apple flowers and in vitro. This suggests that plant protection might be mediated by other mechanisms than competitive niche exclusion. Using a synthetic stigma exudation medium, ternary co-culture of the bacterial strains led to a substantial alteration of gene expression in both the pathogen and the two microbiota members. Importantly, the gene expression profiles for the ternary co-culture were not just additive from binary co-cultures, suggesting that some functions only emerged in multipartite co-culture. Additionally, the ternary co-culture of the strains resulted in a stronger acidification of the growth milieu than mono- or binary co-cultures, pointing to another emergent property of co-inoculation. Our study emphasizes the critical role of emergent properties mediated by inter-species interactions within the plant holobiont and their potential impact on plant health and pathogen behavior. IMPORTANCE: Fire blight, caused by Erwinia amylovora, is one of the most important plant diseases of pome fruits. Previous work largely suggested plant microbiota commensals suppressed disease by antagonizing pathogen growth. However, inter-species interactions of multiple flower commensals and their influence on pathogen activity and behavior have not been well studied. Here, we show that co-inoculating two bacterial strains that naturally colonize the apple flowers reduces disease incidence. We further demonstrate that the interactions between these two microbiota commensals and the floral pathogen led to the emergence of new gene expression patterns and a strong alteration of the external pH, factors that may modify the pathogen's behavior. Our findings emphasize the critical role of emergent properties mediated by inter-species interactions between plant microbiota and plant pathogens and their impact on plant health.

Biocontrol of Diplodia bulgarica, the causal agent of apple canker, using Trichoderma zelobreve.

Apple (Malus domestica Borkh) is one of the most consumed and nutritious fruits. Iran is one of the main producers of the apple in the world. Diplodia bulgarica is the major causal agent of apple tree decline in Iran. Biological control is a nature-friendly approach to plant disease management. Trichoderma zelobreve was isolated from apple trees infected with Diplodia bulgarica in West Azarbaijan province of Iran. The results showed that T. zelobreve strongly inhibited the colony growth of D. bulgarica. In vivo assay on detached branches of apple tree cv. Golden Delicious using T. zelobreve mycelial plug showed that canker length/stem length (CL/SL) and canker perimeter/stem perimeter (CP/SP) indices decreased by 76 and 69%, respectively, 21 days after inoculation. Additionally, wettable powder formulation (WPF) containing the antagonistic fungus "T. zelobreve" decreased CL and CP/SP by 75 and 67%, respectively, 6 months after inoculation. Moreover, canker progress curves and the area under the disease progress curve (AUDPC) supported these findings. The growth temperatures of the antagonist and pathogen were similar, indicating the adaptation of T. zelobreve for biocontrol of apple canker caused by D. bulgarica. The results also showed that T. zelobreve-based WPF stored at 25 °C assure excellent shelf life at least 4 months, allowing the bioproduct to be stored at room temperature, which is a great advantage and cost-effective option.

Valsa mali effector Vm_04797 interacts with adaptor protein MdAP-2ß to manipulate host autophagy.

Apple Valsa canker, caused by the ascomycete fungus Valsa mali, employs virulence effectors to disturb host immunity and poses a substantial threat to the apple industry. However, our understanding of how V. mali effectors regulate host defense responses remains limited. Here, we identified the V. mali effector Vm_04797, which was upregulated during the early infection stage. Vm_04797, a secreted protein, suppressed Inverted formin 1 (INF1)-triggered cell death in Nicotiana benthamiana and performed virulence functions inside plant cells. Vm_04797 deletion mutants showed substantially reduced virulence toward apple. The adaptor protein MdAP-2ß positively regulated apple Valsa canker resistance and was targeted and degraded by Vm_04797 via the ubiquitination pathway. The in vitro analysis suggested that Vm_04797 possesses E3 ubiquitin ligase activity. Further analysis revealed that MdAP-2ß is involved in autophagy by interacting with Malus domestica autophagy protein 16 MdATG16 and promoting its accumulation. By degrading MdAP-2ß, Vm_04797 inhibited autophagic flux, thereby disrupting the defense response mediated by autophagy. Our findings provide insights into the molecular mechanisms employed by the effectors of E3 ubiquitin ligase activity in ascomycete fungi to regulate host immunity.

Inactivation activity and mechanism of pulsed light against Alicyclobacillus acidoterrestris vegetative cells and spores in concentrated apple juice.

Alicyclobacillus acidoterrestris has received much attention due to its unique thermo-acidophilic property and implication in the spoilage of pasteurized juices. The objective of this study was to evaluate the sterilization characteristics and mechanisms of pulsed light (PL) against A. acidoterrestris vegetative cells and spores in apple juice. The results indicated that bacteria cells in apple juice (8-20°Brix) can be completely inactivated within the fluence range of 20.25-47.25 J/cm2, which mainly depended on the soluble solids content (SSC) of juice, and the spores in apple juice (12°Brix) can be completely inactivated by PL with the fluence of 54.00 J/cm2. The PL treatment can significantly increase the leakage of reactive oxygen species (ROS) and proteins from cells and spores. Fluorescence studies of bacterial adenosine triphosphate (ATP) indicated that the loss of ATP was evident. Scanning electron microscopy and confocal laser scanning microscope presented that PL-treated cells or spores had serious morphological damage, which reduced the integrity of cell membrane and led to intracellular electrolyte leakage. In addition, there were no significant negative effects on total sugars, total acids, total phenols, pH value, SSC and soluble sugars, and organic acid content decreased slightly during the PL treatment. The contents of esters and acids in aroma components had a certain loss, while that of alcohols, aldehydes and ketones were increased. These results demonstrated that PL treatment can effectively inactivate the bacteria cells and spores in apple juice with little effect on its quality. This study provides an efficient method for the inactivation of A. acidoterrestris in fruit juice.

Patulin contamination of hard apple cider by Paecilomyces niveus and other postharvest apple pathogens: Assessing risk factors.

Hard apple cider is considered to be a low-risk product for food spoilage and mycotoxin contamination due to its alcoholic nature and associated food sanitation measures. However, the thermotolerant mycotoxin-producing fungus Paecilomyces niveus may pose a significant threat to hard cider producers. P. niveus is known to infect apples (Malus xdomestica), and previous research indicates that it can survive thermal processing and contaminate finished apple juice with the mycotoxin patulin. To determine if hard apple cider is susceptible to a similar spoilage phenomenon, cider apples were infected with P. niveus or one of three patulin-producing Penicillium species and the infected fruits underwent benchtop fermentation. Cider was made with lab inoculated Dabinett and Medaille d'Or apple cultivars, and patulin was quantified before and after fermentation. Results show that all four fungi can infect cider apples and produce patulin, some of which is lost during fermentation. Only P. niveus was able to actively grow throughout the fermentation process. To determine if apple cider can be treated to hinder P. niveus growth, selected industry-grade sanitation measures were tested, including chemical preservatives and pasteurization. High concentrations of preservatives inhibited P. niveus growth, but apple cider flash pasteurization was not found to significantly impact spore germination. This study confirms that hard apple cider is susceptible to fungal-mediated spoilage and patulin contamination. P. niveus is an important concern for hard apple cider producers due to its demonstrated thermotolerance, survival in fermentative environments, and resistance to sanitation measures.

Infection of postharvest pear by Penicillium expansum is facilitated by the glycoside hydrolase (eglB) gene.

The primary reason for postharvest loss is blue mold disease which is mainly caused by Penicillium expansum. Strategies for disease control greatly depend on the understanding of mechanisms of pathogen-fruit interaction. A member of the glycoside hydrolase family, ß-glucosidase 1b (eglB), in P. expansum was significantly upregulated during postharvest pear infection. Glycoside hydrolases are a large group of enzymes that can degrade plant cell wall polymers. High homology was found between the glycoside hydrolase superfamily in P. expansum. Functional characterization and analysis of eglB were performed via gene knockout and complementation analysis. Although eglB deletion had no notable effect on P. expansum colony shape or microscopic morphology, it did reduce the production of fungal hyphae, thereby reducing P. expansum's sporulation and patulin (PAT) accumulation. Moreover, the deletion of eglB (ΔeglB) reduced P. expansum pathogenicity in pears. The growth, conidia production, PAT accumulation, and pathogenicity abilities of ΔeglB were restored to that of wild-type P. expansum by complementation of eglB (ΔeglB-C). These findings indicate that eglB contributes to P. expansum's development and pathogenicity. This research is a contribution to the identification of key effectors of fungal pathogenicity for use as targets in fruit safety strategies.