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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Quantitative Trait Locus Mapping for Fire Blight Resistance in an F2 Population of Malus fusca MAL0045 Uncovers Novel Resistance Loci.

Several fire blight resistance loci in Malus genotypes map on different linkage groups (LGs) representing chromosomes of the domesticated apple. Prior genetics studies primarily focused on F1 populations. A strong resistance quantitative trait locus (QTL) explained up to 66% of phenotypic variance in an F1 progeny derived from crossing the highly resistant wild apple genotype Malus fusca MAL0045 and the highly susceptible apple cultivar 'Idared', which was previously mapped on LG10 (Mfu10) of MAL0045. Strains of the causative bacterial pathogen Erwinia amylovora, notably those that show a single nucleotide polymorphism in the avrRpt2EA effector protein sequence at position 156 (e.g., Ea3049), are more virulent and overcome some known fire blight resistance donors and their QTLs. However, MAL0045 is resistant to Ea3049 and Mfu10 is not overcome, but most of the F1 progeny were highly susceptible to this strain. This phenomenon led to the assumption that other putative resistance factors not segregating in the F1 progeny might be present in the genome of MAL0045. Here, we crossed F1 progeny together to obtain 135 F2 individuals. Facilitated by genotyping-by-sequencing and phenotypic assessments, we identified and mapped two novel resistance QTLs in these F2 individuals on LGs 4 and 15, which were not identified in the F1. To our knowledge, these are the first resistance QTLs mapped in F2 progeny in Malus. In addition, we report that neither MAL0045 nor Mfu10 is broken down by a highly aggressive U.S. strain, LA635, after analyses in the original F1 individuals. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Novel approach toward the understanding of genetic diversity based on the two types of amino acid repeats in Erwinia amylovora.

Erwinia amylovora is a notorious plant pathogenic bacterium of global concern that has devastated the apple and pear production industry worldwide. Nevertheless, the approaches available currently to understand the genetic diversity of E. amylovora remain unsatisfactory because of the lack of a trustworthy index and data covering the globally occurring E. amylovora strains; thus, their origin and distribution pattern remains ambiguous. Therefore, there is a growing need for robust approaches for obtaining this information via the comparison of the genomic structure of Amygdaloideae-infecting strains to understand their genetic diversity and distribution. Here, the whole-genome sequences of 245 E. amylovora strains available from the NCBI database were compared to identify intraspecific genes for use as an improved index for the simple classification of E. amylovora strains regarding their distribution. Finally, we discovered two kinds of strain-typing protein-encoding genes, i.e., the SAM-dependent methyltransferase and electron transport complex subunit RsxC. Interestingly, both of these proteins carried an amino acid repeat in these strains: SAM-dependent methyltransferase comprised a single-amino-acid repeat (asparagine), whereas RsxC carried a 40-amino-acid repeat, which was differentially distributed among the strains. These noteworthy findings and approaches may enable the exploration of the genetic diversity of E. amylovora from a global perspective.

Cold atmospheric plasma fumigation suppresses postharvest apple Botrytis cinerea by triggering intracellular reactive oxygen species and mitochondrial calcium.

Infection by Botrytis cinerea poses a great threat to the postharvest life of apple fruit. In this study, the effects of cold atmospheric plasma (CAP) fumigation on apple B. cinerea under different exposure times and intensities were investigated. The growth of B. cinerea in vitro and in vivo was significantly suppressed by the CAP fumigation at least 700 µL/L for 5 min. To reveal the possible mechanism of antifungal activity of CAP fumigation, the pathogen was exposed to 700 µL/L and 1000 µL/L for 5 min, respectively. The results indicated that the CAP-treated spores of the pathogen underwent shrinkage, cell membrane collapse and cytoplasmic vacuolation. The results obtained from the fluorescent probe assay and flow cytometry indicated that CAP caused the accumulation of reactive oxygen species (ROS), the elevation of mitochondrial and intracellular Ca2+ levels, and the decrease in mitochondrial membrane potential of the pathogen. Investigation on statues of cell life showed that typical hallmarks of apoptosis in the CAP-treated B. cinerea spores occurred, as indicted by a large degree of increased phosphatidylserine externalization, dysfunction of membrane permeability, DNA fragmentation, distortion of morphology, chromatin condensation, and metacaspase activation observed in B. cinerea spores after CAP fumigation. Overall, CAP fumigation triggered a metacaspase-dependent apoptosis of B. cinerea spores mediated by intracellular ROS burst and Ca2+ elevation via mitochondrial dysfunction and disruption, and therefore reduced the pathogenicity of B. cinerea and suppressed postharvest Botrytis rot of apple fruit. These results would provide an insight into the underlying mechanism of CAP fumigation acting on the pathogen. The CAP fumigation makes much convenient application of CAP in storage environment to deactivate microorganism.

Exogenous dsRNA trigger RNAi in Venturia inaequalis resulting in down regulation of target genes and growth reduction.

BACKGROUND: Venturia inaequalis is an apple scab causing fungal pathogen. It is a highly contagious and destructive pathogen which rapidly spreads infection in the surrounding orchards if not managed. The management and control of disease require multiple fungicides to be sprayed at different development stages of the apple. Persistent applications of fungicides also raises environmental concerns. Here, we demonstrate the potential of using spray induced gene silencing (SIGS) by developing target specific gene constructs for the synthesis of corresponding double-stranded RNA (dsRNA). METHODS AND RESULTS: The exogenous application of dsRNAs was found to reduce mycelial growth and spore formation of V. inaequalis on culture plates. Four genes of V. inaequalis viz. CIN1, CE5, VICE12 and VICE16 which get upregulated during infection, were selected as targets for the development of gene construct expressing the corresponding dsRNA. The effect of exogenously supplied in vitro synthesized dsRNA on V. inaequalis was assessed in culture bioassay experiments with respect to growth, and spore formation. The expression level of the target genes in treated and control fungus was evaluated using quantitative PCR. Fungus treated with VICE12 targeted dsRNA showed maximum reduction in colony size (~ 55%), conidia formation (~ 93%) and expression level of the corresponding gene (2.2 fold), which was followed by CIN1-dsRNA. VICE16-dsRNA treatment was least effective with 32% reduction in growth, the non-significant effect of conidial spore formation and 1.13 fold down regulation of corresponding target gene expression level. CONCLUSION: The result of this investigation validates the hypothesis that RNAi is evoked in V. inaequalis by exogenously supplied dsRNA and spray induced gene silencing (SIGS) based solutions may reduce burden of fungicide usage on apple crop against apple scab disease in future.

The cytochrome P450 gene, MdCYP716B1, is involved in regulating plant growth and anthracnose resistance in apple.

Apple is one of the main cultivated fruit trees worldwide. Both biotic and abiotic stresses, especially fungal diseases, have serious effects on the growth and fruit quality of apples. Cytochrome P450, the largest protein family in plants, is critical for plant growth and stress responses. However, the function of apple P450 remains poorly understood. In our previous study, 'Hanfu' autotetraploid showed dwarfism and fungal resistance phenotypes compared to 'Hanfu' diploid. Digital gene expression sequencing analysis revealed that the transcript level of MdCYP716B1 was significantly downregulated in the autotetraploid apple cultivar 'Hanfu'. In this study, we identified and cloned the MdCYP716B1 gene from 'Hanfu' apples. The MdCYP716B1 protein fused to a green fluorescent protein was localized in the cytoplasm. We constructed the plant overexpression vector and RNAi vector of MdCYP716B1, and the apple 'GL-3' was transformed by Agrobacterium-mediated transformation to obtain transgenic plants. Overexpressing and RNAi silencing transgenic plants exhibited an increase and decrease in plant height to 'GL-3', respectively. RNAi silencing transgenic plants displayed increased resistance to Colletotrichum gloeosporioides, whereas overexpression transgenic plants were more sensitive to C. gloeosporioides. According to transcriptome analysis, the transcript levels of gibberellin biosynthesis genes were upregulated in MdCYP716B1-overexpression plants. In contrast with 'GL-3', GA3 accumulation was rose in MdCYP716B1-OE lines and impaired in MdCYP716B1-RNAi lines. Collectively, our data indicate that MdCYP716B1 regulates plant growth and resistance to fungal stress.

Applications of Germicidal Ultraviolet Light as a Tool for Fire Blight Management (Erwinia amylovora) in Apple Plantings.

Nighttime applications of germicidal UV light (UV-C) have been used to suppress several fungal diseases of plants, but less is known of UV-C's potential to suppress bacterial plant pathogens. Fire blight of apple and pear, caused by the bacterium Erwinia amylovora, is difficult to suppress using cultural practices, antibiotics, and host resistance. We therefore investigated the potential of UV-C as an additional means to manage the disease. Laboratory assays confirmed that in vitro exposure of cultures E. amylovora to UV-C at doses ranging from 0 to 400 J/m2 in the absence of visible light was more than 200% as effective as cultures exposed to visible light after the same UV-C treatments. In a 2-year orchard study, we demonstrated that with only two nighttime applications of UV-C at 200 J/m2 made at bloom resulted in an incidence of blossom blight and shoot blight equivalent to the results viewed when antibiotic and biopesticide commercial standards were applied. In vitro dose-response studies indicated consistency in pathogen response to suppressive UV-C doses, including pathogen isolates that were resistant to streptomycin. Based on these results, UV-C may be useful in managing bacterial populations with antibiotic resistance. Concurrent measurements of host growth after UV-C applications indicated that the dose required to suppress E. amylovora had no significant (P > 0.05) effects on foliar growth, shoot extension, internode length, or fruit finish but substantially reduced epiphytic populations of E. amylovora on host tissues.