A fungal microRNA-like RNA regulated effector promotes pathogen infection by targeting a host defense-related transcription factor.

Effectors play important roles in facilitating the infection of plant pathogenic fungi. However, the gene expression regulatory mechanism of effector genes, in particular at the post-transcriptional level, is largely unknown. In this study, we uncovered the post-transcriptional regulation of an effector gene VmSP1 by a miRNA-like RNA (Vm-milR16) facilitating the infection of the apple tree Valsa canker pathogen Valsa mali. Genetic and molecular biological assays indicated that the expression of VmSP1 could be suppressed by Vm-milR16-mediated mRNA cleavage in a sequence-specific manner. During V. mali infection, Vm-milR16 was downregulated, whereas VmSP1 was upregulated, which further indicated the regulation relationship. VmSP1 was further demonstrated to be a secreted protein and could suppress plant immunity. Deletion of VmSP1 did not affect the vegetative growth but significantly reduced the virulence of V. mali. Further study indicated that VmSP1 could interact with the transcription factor MdbHLH189 of apple. Transiently overexpression of MdbHLH189 enhanced host resistance to V. mali by enhancing the expression of apple defense-related genes, together with the increased callose deposition. Silencing of MdbHLH189 compromised host resistance to V. mali. Our findings uncovered the novel epigenetic regulation mechanism of a virulence-associated effector gene mediated by a fungal milRNA at the post-transcriptional level, and the results enriched the understanding of the function and action mechanism of effectors in tree pathogenic fungi.

Identification of an SCF Ubiquitin Ligase Complex that Contributes to Resistance Against Valsa Canker in Apple.

E3 ubiquitin ligases play a critical role in plant disease resistance. Among them, the Skp1-Cullin-F-box protein (SCF) ubiquitin ligase complex is the largest family and regulates the ubiquitination of a wide range of proteins. Apple Valsa canker (AVC) is a fungal disease of apple trees caused by the fungus Valsa mali, which can lead to significant economic losses. However, the function of the SCF complex in apple resistance to this disease is still largely unknown. In this study, we identified an SCF ubiquitin ligase complex that can enhance resistance to Valsa canker in apple. Disease evaluation experiments demonstrated that MdSkp1 increased apple resistance to AVC. Furthermore, MdSkp1 interacted with an F-box protein, MdSKIP14, and interacted with a cullin-1 protein, MdCUL1, to form an SCF ubiquitin ligase complex. Additionally, we revealed both MdSKIP14 and MdCUL1 as positive regulators of AVC resistance. In conclusion, our results identified an SCF complex capable of contributing to apple resistance against AVC, providing a theoretical basis for apple disease resistance and the sustainable development of the industry. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Glucose uptake from the rhizosphere mediated by MdDOF3-MdHT1.2 regulates drought resistance in apple.

In plants under drought stress, sugar content in roots increases, which is important for drought resistance. However, the molecular mechanisms for controlling the sugar content in roots during response to drought remain elusive. Here, we found that the MdDOF3-MdHT1.2 module-mediated glucose influx into the root is essential for drought resistance in apple (Malus × domestica). Drought induced glucose uptake from the rhizosphere and up-regulated the transcription of hexose transporter MdHT1.2. Compared with the wild-type plants, overexpression of MdHT1.2 promoted glucose uptake from the rhizosphere, thereby facilitating sugar accumulation in root and enhancing drought resistance, whereas silenced plants showed the opposite phenotype. Furthermore, ATAC-seq, RNA-seq and biochemical analysis demonstrated that MdDOF3 directly bound to the promoter of MdHT1.2 and was strongly up-regulated under drought. Overexpression of MdDOF3 in roots improved MdHT1.2-mediated glucose transport capacity and enhanced plant resistance to drought, but MdDOF3-RNAihr apple plants showed the opposite phenotype. Moreover, overexpression of MdDOF3 in roots did not attenuate drought sensitivity in MdHT1.2-RNAi plants, which was correlated with a lower glucose uptake capacity and glucose content in root. Collectively, our findings deciphered the molecular mechanism through which glucose uptake from the rhizosphere is mediated by MdDOF3-MdHT1.2, which acts to modulate sugar content in root and promote drought resistance.

Characterization and Pathogenicity of Fungal Species Associated with Dieback of Apple Trees in Northern Italy.

Severe dieback symptoms were recently observed on apple (Malus × domestica) trees in Northern Italy, representing a growing concern for producers. Surveys were conducted over a 3-year period (2019 to 2021), and five apple orchards, from 5 to 12 years old, were monitored. A total of 33 fungal isolates isolated from symptomatic plants was selected for characterization. The species identification was achieved through multilocus phylogenetic analyses performed on sequences of three genomic loci (ITS, tub2, and tef1). Morphological features were assessed, and the average growth rate at different temperatures was determined. Seven species were identified in association with dieback of apple trees: Botryosphaeria dothidea, Cadophora luteo-olivacea, Diaporthe rudis, Diplodia seriata, Eutypa lata, Kalmusia longispora, and Paraconiothyrium brasiliense. All the species were pathogenic when inoculated on healthy apple plants. B. dothidea resulted in the most aggressive infections. This study provides an insight into the fungal species diversity associated with apple dieback and provides basis for further investigations to assess the phytosanitary status of plant materials to recommend and implement effective management strategies.

MdGH3.6 is targeted by MdMYB94 and plays a negative role in apple water-deficit stress tolerance.

Drought significantly limits apple fruit production and quality. Decoding the key genes involved in drought stress tolerance is important for breeding varieties with improved drought resistance. Here, we identified GRETCHEN HAGEN3.6 (GH3.6), an indole-3-acetic acid (IAA) conjugating enzyme, to be a negative regulator of water-deficit stress tolerance in apple. Overexpressing MdGH3.6 reduced IAA content, adventitious root number, root length and water-deficit stress tolerance, whereas knocking down MdGH3.6 and its close paralogs increased IAA content, adventitious root number, root length and water-deficit stress tolerance. Moreover, MdGH3.6 negatively regulated the expression of wax biosynthetic genes under water-deficit stress and thus negatively regulated cuticular wax content. Additionally, MdGH3.6 negatively regulated reactive oxygen species scavengers, including antioxidant enzymes and metabolites involved in the phenylpropanoid and flavonoid pathway in response to water-deficit stress. Further study revealed that the homolog of transcription factor AtMYB94, rather than AtMYB96, could bind to the MdGH3.6 promoter and negatively regulated its expression under water-deficit stress conditions in apple. Overall, our results identify a candidate gene for the improvement of drought resistance in fruit trees.

H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple.

Bud dormancy helps woody perennials survive winter and activate robust plant development in the spring. For apple (Malus × domestica), short-term chilling induces bud dormancy in autumn, then prolonged chilling leads to dormancy release and a shift to a quiescent state in winter, with subsequent warm periods promoting bud break in spring. Epigenetic regulation contributes to seasonal responses such as vernalization. However, how histone modifications integrate seasonal cues and internal signals during bud dormancy in woody perennials remains largely unknown. Here, we show that H3K4me3 plays a key role in establishing permissive chromatin states during bud dormancy and bud break in apple. The global changes in gene expression strongly correlated with changes in H3K4me3, but not H3K27me3. High expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes, key regulators of dormancy, in autumn was associated with high H3K4me3 levels. In addition, known DAM/SHORT VEGETATIVE PHASE (SVP) target genes significantly overlapped with H3K4me3-modified genes as bud dormancy progressed. These data suggest that H3K4me3 contributes to the central dormancy circuit, consisting of DAM/SVP and abscisic acid (ABA), in autumn. In winter, the lower expression and H3K4me3 levels at DAMs and gibberellin metabolism genes control chilling-induced release of dormancy. Warming conditions in spring facilitate the expression of genes related to phytohormones, the cell cycle, and cell wall modification by increasing H3K4me3 toward bud break. Our study also revealed that activation of auxin and repression of ABA sensitivity in spring are conditioned at least partly through temperature-mediated epigenetic regulation in winter.

CCR -NB-LRR proteins MdRNL2 and MdRNL6 interact physically to confer broad-spectrum fungal resistance in apple (Malus × domestica).

Apple leaf spot, a disease caused by Alternaria alternata f. sp. mali and other fungal species, leads to severe defoliation and results in tremendous losses to the apple (Malus × domestica) industry in China. We previously identified three RPW8, nucleotide-binding, and leucine-rich repeat domain CCR -NB-LRR proteins (RNLs), named MdRNL1, MdRNL2, and MdRNL3, that contribute to Alternaria leaf spot (ALT1) resistance in apple. However, the role of NB-LRR proteins in resistance to fungal diseases in apple remains poorly understood. We therefore used MdRNL1/2/3 as baits to screen ALT1-inoculated leaves for interacting proteins and identified only MdRNL6 (another RNL) as an interactor of MdRNL2. Protein interaction assays demonstrated that MdRNL2 and MdRNL6 interact through their NB-ARC domains. Transient expression assays in apple indicated that complexes containing both MdRNL2 and MdRNL6 are necessary for resistance to Alternaria leaf spot. Intriguingly, the same complexes were also required to confer resistance to Glomerella leaf spot and Marssonina leaf spot in transient expression assays. Furthermore, stable transgenic apple plants with suppressed expression of MdRNL6 showed hypersensitivity to Alternaria leaf spot, Glomerella leaf spot, and Marssonina leaf spot; these effects were similar to the effects of suppressing MdRNL2 expression in transgenic apple plantlets. The identification of these novel broad-spectrum fungal resistance genes will facilitate breeding for fungal disease resistance in apple.

Genome-wide identification and stress response analysis of cyclophilin gene family in apple (Malus × domestica).

BACKGROUND: Cyclophilin (CYP) belongs to the immunophilin family and has peptidyl-prolyl cis-trans isomerase (PPIase) activity, which catalyzes the cis-trans isomerization process of proline residues. CYPs widely exist in eukaryotes and prokaryotes, and contain a conserved cyclophilin-like domain (CLD). Plant cyclophilins are widely involved in a range of biological processes including stress response, metabolic regulation, and growth and development. RESULT: In this study, 30 cyclophilin genes on 15 chromosomes were identified from the 'Golden Delicious' apple (M. domestica) genome. Phylogenetic analysis showed that the cyclophilin family genes can be divided into three clades in Malus. Collinear analysis showed that ten gene pairs were the result of segmental duplication. Analysis of gene and protein structure further supported the phylogenetic tree and collinearity analysis. The expression of MdCYPs in different organs was higher in leaves, flowers, and fruits. Ten and eight CYPs responded to drought and salt stress, respectively. MdCYP16, a nuclear-localized MD CYP, was screened from the intersection of the two expression profiling datasets and was highly sensitive to drought and salt stress. GUS staining of transgenic Arabidopsis indicated that MdCYP16 may be involved in the regulation of abiotic stress. CONCLUSION: This study systematically analyzed members of the apple cyclophilin family and confirmed the involvement of MdCYP16 as a nuclear-localized MD cyclophilin that acts in response to salt and drought stress in apple. Our work identifies members of the apple cyclophilin gene family, and provides an important theoretical basis for in-depth study of cyclophilin function. Additionally, the analysis provides candidate genes that may be involved in stress response in apple.

Relationships between trunk radial growth and fruit yield in apple and pear trees on size-controlling rootstocks.

BACKGROUND AND AIMS: Understanding the mutual co-ordination of vegetative and reproductive growth is important in both agricultural and ecological settings. A competitive relationship between vegetative growth and fruiting is often highlighted, resulting in an apparent trade-off between structural growth and fruit production. However, our understanding of factors driving this relationship is limited. METHODS: We used four scions grafted onto a series of size-controlling rootstocks to evaluate the relationships between the annual fruit yield and radial growth of trunks, branches and roots. To assess tree radial growth, we measured ring widths on extracted tree cores, which is an approach not frequently used in a horticultural setting. KEY RESULTS: We found that the yield and radial growth were negatively related when plotted in absolute terms or as detrended and normalized indices. The relationship was stronger in low vigour trees, but only after the age-related trend was removed. In contrast, when trunk radial growth was expressed as basal area increment, the negative relationship disappeared, suggesting that the relationship between trunk radial growth and fruit yield might not be a true trade-off related to the competition between the two sinks. The effect of low yield was associated with increased secondary growth not only in trunks but also in branches and roots. In trunks, we observed that overcropping was associated with reduced secondary growth in a subsequent year, possibly due to the depletion of reserves. CONCLUSIONS: Our results show that variation in annual fruit yield due to tree ageing, weather cueing and inherent alternate bearing behaviour is reflected in the magnitude of secondary growth of fruit trees. We found little support for the competition/architecture theory of rootstock-induced growth vigour control. More broadly, our study aimed at bridging the gap between forest ecology and horticulture.

The Apple Receptor-Like Kinase MdSRLK3 Positively Regulates Resistance Against Pathogenic Fungus Valsa mali by Affecting the Ca2+ Signaling Pathway.

Valsa mali is the main pathogenic fungus that causes the apple Valsa canker, a destructive disease severely threatening apple production in the world. However, the underlying key components involved in resistance against V. mali in apple trees remain largely unexplored. Here, we isolated and functionally characterized a G-type lectin S-receptor-like protein kinase MdSRLK3 from the cultivar Royal Gala derivative line GL-3. qRT-PCR showed that the relative expression of MdSRLK3 in apple branches reached its highest level at 24 h post V. mali inoculation, which was 13.42 times higher than without inoculation. Transient overexpression of MdSRLK3 enhanced apple resistance against V. mali, while transient silencing of MdSRLK3 reduced its resistance against the pathogen. More importantly, stable silencing of MdSRLK3 resulted in reduced resistance against this fungus. Furthermore, we demonstrated that MdSRLK3 positively regulated apple resistance by affecting the Ca2+ signaling pathway, and the regulation was also related to the H2O2 and callose signaling pathways. Overall, our data reveal that MdSRLK3 is a positive regulator of apple immunity.

No adverse dietary effect of a cisgenic fire blight resistant apple line on the non-target arthropods Drosophila melanogaster and Folsomia candida.

Genetic modification of apple cultivars through cisgenesis can introduce traits, such as disease resistance from wild relatives, quickly and without crossing. This approach was used to generate the cisgenic apple line C44.4.146, a 'Gala Galaxy' carrying the fire blight resistance gene FB_MR5. In contrast to traditionally bred apple cultivars, genetically modified (GM) plants need to undergo a regulatory risk assessment considering unintended effects before approval for commercial release. To determine potential unintended effects of C44.4.146, we assessed major leaf components and effects on the fitness of the decomposers Drosophila melanogaster (fruit fly) and Folsomia candida (collembolan), which were fed a diet amended with powdered apple leaf material. Leaf material of 'Gala Galaxy', several natural 'Gala' mutants, and the unrelated apple cultivar 'Ladina' were used for comparison. The genetic modification did not alter major leaf components and did not adversely affect survival, growth, or fecundity of the two decomposers. Consistent with previous studies with other GM crops, the differences between conventionally bred cultivars were greater than between the GM line and its non-GM wild type. These data provide a baseline for future risk assessments.

Transcriptome and metabolite profiling analyses provide insight into volatile compounds of the apple cultivar 'Ruixue' and its parents during fruit development.

BACKGROUND: Aroma is one the most crucial inherent quality attributes of fruit. 'Ruixue' apples were selected from a cross between 'Pink Lady' and 'Fuji', a later ripening yellow new cultivar. However, there is little known about the content and composition of aroma compounds in 'Ruixue' apples or the genetic characters of 'Ruixue' and its parents. In addition, the metabolic pathways for biosynthesis of aroma volatiles and aroma-related genes remain poorly understood. RESULTS: Volatile aroma compounds were putatively identified using gas chromatography-mass spectrometry (GC-MS). Our results show that the profile of volatile compounds changes with ripening. Aldehydes were the dominant volatile compounds in early fruit development, with alcohols and esters increasing dramatically during maturation. On the basis of a heatmap dendrogram, these aroma compounds clustered into seven groups. In ripe fruit, esters and terpenoids were the main aroma volatiles in ripening fruit of 'Pink Lady' and 'Fuji' apples, and they included butyl 2-methylbutanoate; propanoic acid, hexyl ester; propanoic acid, hexyl ester; hexanoic acid, hexyl ester; acetic acid, hexyl ester and (Z, E)-α-farnesene. Interestingly, aldehydes and terpenoids were the dominant volatile aroma compounds in ripening fruit of 'Ruixue', and they mainly included hexanal; 2-hexenal; octanal; (E)-2-octenal; nonanal and (Z, E)-α-farnesene. By comparing the transcriptome profiles of 'Ruixue' and its parents fruits during development, we identified a large number of aroma-related genes related to the fatty acid, isoleucine and sesquiterpenoid metabolism pathways and transcription factors that may volatile regulate biosynthesis. CONCLUSIONS: Our initial study facilitates a better understanding of the volatile compounds that affect fruit flavour as well as the mechanisms underlying differences in flavour between 'Ruixue' and its parents.

Integrating genomics and multiplatform metabolomics enables metabolite quantitative trait loci detection in breeding-relevant apple germplasm.

Apple (Malus × domestica) has commercial and nutritional value, but breeding constraints of tree crops limit varietal improvement. Marker-assisted selection minimises these drawbacks, but breeders lack applications for targeting fruit phytochemicals. To understand genotype-phytochemical associations in apples, we have developed a high-throughput integration strategy for genomic and multiplatform metabolomics data. Here, 124 apple genotypes, including members of three pedigree-connected breeding families alongside diverse cultivars and wild selections, were genotyped and phenotyped. Metabolite genome-wide association studies (mGWAS) were conducted with c. 10 000 single nucleotide polymorphisms and phenotypic data acquired via LC-MS and 1 H NMR untargeted metabolomics. Putative metabolite quantitative trait loci (mQTL) were then validated via pedigree-based analyses (PBA). Using our developed method, 519, 726 and 177 putative mQTL were detected in LC-MS positive and negative ionisation modes, and NMR, respectively. mQTL were indicated on each chromosome, with hotspots on linkage groups 16 and 17. A chlorogenic acid mQTL was discovered on chromosome 17 via mGWAS and validated with a two-step PBA, enabling discovery of novel candidate gene-metabolite relationships. Complementary data from three metabolomics approaches and dual genomics analyses increased confidence in validity of compound annotation and mQTL detection. Our platform demonstrates the utility of multiomic integration to advance data-driven, phytochemical-based plant breeding.

Investigating the Distribution of Strains of Erwinia amylovora and Streptomycin Resistance in Apple Orchards in New York Using Clustered Regularly Interspaced Short Palindromic Repeat Profiles: A 6-Year Follow-Up.

Fire blight, caused by the bacterium Erwinia amylovora, is one of the most important diseases of apple. The antibiotic streptomycin is routinely used in the commercial apple industries of New York (NY) and New England to manage the disease. In 2002 and again, from 2011 to 2014, outbreaks of streptomycin resistance (SmR) were reported and investigated in NY. Motivated by new grower reports of control failures, we conducted a follow-up investigation of the distribution of SmR and E. amylovora strains for major apple production regions of NY over the last 6 years (2015 to 2020). Characterization of clustered regularly interspaced short palindromic repeat (CRISPR) profiles revealed that a few "cosmopolitan" strains were widely prevalent across regions, whereas many other "resident" strains were confined to one location. In addition, we uncovered novel CRISPR profile diversity in all investigated regions. SmR E. amylovora was detected only in a small area spanning two counties from 2017 to 2020 and was always associated with one CRISPR profile (41:23:38), which matched the profile of SmR E. amylovora, discovered in 2002. This suggests the original SmR E. amylovora was never fully eradicated and went undetected because of several seasons of low disease pressure in this region. Investigation of several representative isolates under controlled greenhouse conditions indicated significant differences in aggressiveness on 'Gala' apples. Potential implications of strain differences include the propensity of strains to become distributed across wide geographic regions and associated resistance management practices. Results from this work will directly influence sustainable fire blight management recommendations for commercial apple industries in NY state and other regions.

Assessing and Minimizing the Development and Spread of Fire Blight Following Mechanical Thinning and Pruning in Apple Orchards.

The adoption of mechanical thinning and pruning in commercial apple orchards has been limited largely by the risk of development and spread of fire blight. This devastating disease, caused by the bacterial pathogen Erwinia amylovora, may be transmitted by mechanical injury such as pruning, especially under warm, moist conditions conducive to bacterial growth, infection, and disease development. However, risk may be mitigated by avoiding highest-risk times and applying a bactericide, such as streptomycin, after mechanical thinning or pruning. In 'Gala' and 'Idared' orchards, we evaluated the risk of fire blight development and spread after mechanical thinning early in bloom (20% bloom), when seasonal temperatures are cooler and there are few open flowers available for infection. In both orchards, we also evaluated the spread and development of fire blight by mechanical pruning in July and in August, before and after terminal bud set, when shoot growth is slowed and the tree is less susceptible to infection. We also assessed the potential efficacy of a streptomycin or Bacillus subtilis biopesticide application after mechanical thinning and pruning to mitigate the spread of fire blight. In the 'Gala' orchard, disease never developed beyond the inoculated tree after thinning or pruning, which was unexpected for this highly susceptible cultivar. In the 'Idared' orchard, incidence of blossom or shoot blight from the point source, represented as relative area under the disease progress curve, was rarely different for trees that underwent mechanical thinning or mechanical pruning compared with untreated trees, and it was often reduced or eliminated when the antibiotic streptomycin or the B. subtilis biopesticide was applied within 24 h of mechanical thinning or pruning. For both thinning and pruning, incidence of fire blight dropped off quickly beyond the inoculated tree in the 'Idared' orchard and generally was not observed in trees beyond 10 to 15 m from the inoculated point source or predicted beyond 10 m by exponential and power law models fit to the disease progress curves. The results of this work demonstrate the low risk for fire blight development and spread by mechanical thinning and pruning when practiced under low-risk conditions-early in bloom for mechanical thinning and after terminal bud set (in August) for mechanical pruning-especially when paired with a subsequent bactericide application. This study demonstrates the safe use of mechanical thinning and pruning in commercial apple production, corroborated by anecdotal evidence from apple growers in western New York State.

Apple Autotetraploids with Enhanced Resistance to Apple Scab (Venturia inaequalis) Due to Genome Duplication-Phenotypic and Genetic Evaluation.

Among the fungal diseases of apple trees, serious yield losses are due to an apple scab caused by Venturia inaequalis. Protection against this disease is based mainly on chemical treatments, which are currently very limited. Therefore, it is extremely important to introduce cultivars with reduced susceptibility to this pathogen. One of the important sources of variability for breeding is the process of polyploidization. Newly obtained polyploids may acquire new features, including increased resistance to diseases. In our earlier studies, numerous tetraploids have been obtained for several apple cultivars with 'Free Redstar' tetraploids manifesting enhanced resistance to apple scab. In the present study, tetraploids of 'Free Redstar' were assessed in terms of phenotype and genotype with particular emphasis on the genetic background of their increased resistance to apple scab. Compared to diploid plants, tetraploids (own-rooted plants) were characterized with poor growth, especially during first growing season. They had considerably shorter shoots, fewer branches, smaller stem diameter, and reshaped leaves. In contrast to own-rooted plants, in M9-grafted three-year old trees, no significant differences between diplo- and tetraploids were observed, either in morphological or physiological parameters, with the exceptions of the increased leaf thickness and chlorophyll content recorded in tetraploids. Significant differences between sibling tetraploid clones were recorded, particularly in leaf shape and some physiological parameters. The amplified fragment length polymorphism (AFLP) analysis confirmed genetic polymorphism of tetraploid clones. Methylation-sensitive amplification polymorphism (MSAP) analysis showed that the level of DNA methylation was twice as high in young tetraploid plants as in a diploid donor tree, which may explain the weaker vigour of neotetraploids in the early period of their growth in the juvenile phase. Molecular analysis showed that 'Free Redstar' cultivar and their tetraploids bear six Rvi genes (Rvi5, Rvi6, Rvi8, Rvi11, Rvi14 and Rvi17). Transcriptome analysis confirmed enhanced resistance to apple scab of 'Free Redstar' tetraploids since the expression levels of genes related to resistance were strongly enhanced in tetraploids compared to their diploid counterparts.

Overexpression of an apple LysM-containing protein gene, MdCERK1-2, confers improved resistance to the pathogenic fungus, Alternaria alternata, in Nicotiana benthamiana.

BACKGROUND: Lysin motif (LysM)-containing proteins are involved in the recognition of fungal and bacterial pathogens. However, few studies have reported on their roles in the defense responses of woody plants against pathogens. A previous study reported that the apple MdCERK1 gene was induced by chitin and Rhizoctonia solani, and its protein can bind to chitin. However, its effect on defense responses has not been investigated. RESULTS: In this study, a new apple CERK gene, designated as MdCERK1-2, was identified. It encodes a protein that shares high sequence identity with the previously reported MdCERK1 and AtCERK1. Its chitin binding ability and subcellular location are similar to MdCERK1 and AtCERK1, suggesting that MdCERK1-2 may play a role in apple immune defense responses as a pattern recognition receptor (PRR). MdCERK1-2 expression in apple was induced by 2 fungal pathogens, Botryosphaeria dothidea and Glomerella cingulate, but not by the bacterial pathogen, Erwinia amylovora, indicating that MdCERK1-2 is involved in apple anti-fungal defense responses. Further functional analysis by heterologous overexpression (OE) in Nicotiana benthamiana (Nb) demonstrated that MdCERK1-2 OE improved Nb resistance to the pathogenic fungus, Alternaria alternata. H2O2 accumulation and callose deposition increased after A. alternata infection in MdCERK1-2 OE plants compared to wild type (WT) and empty vector (EV)-transformed plants. The induced expression of NbPAL4 by A. alternata significantly (p < 0.01, n = 4) increased in MdCERK1-2 OE plants. Other tested genes, including NbNPR1, NbPR1a, NbERF1, and NbLOX1, did not exhibit significant changes after A. alternata infection in OE plants compared to EV or WT plants. OE plants also accumulated more polyphenols after A. alternata infection. CONCLUSIONS: Heterologous MdCERK1-2 OE affects multiple defense responses in Nb plants and increased their resistance to fungal pathogens. This result also suggests that MdCERK1-2 is involved in apple defense responses against pathogenic fungi.

Technical benefit on apple fruit of controlled atmosphere influenced by 1-MCP at molecular levels.

The apple is a highly perishable fruit after harvesting and, therefore, several storage technologies have been studied to provide the consumer market with a quality product with a longer shelf life. However, little is known about the apple genome that is submitted to the storage, and even less with the application of ripening inhibitors. Due to these factors, this study sought to elucidate the transcriptional profile of apple cultivate Gala stored in a controlled atmosphere (AC) treated and not treated with 1-methyl cyclopropene (1-MCP). Through the genetic mapping of the apple, applying the microarray technique, it was possible to verify the action of treatments on transcripts related to photosynthesis, carbohydrate metabolism, response to hormonal stimuli, nucleic acid metabolism, reduction of oxidation, regulation of transcription and metabolism of cell wall and lipids. The results showed that the transcriptional profile in the entire genome of the fruit showed significant differences in the relative expression of the gene, this in response to CA in the presence and absence of 1-MCP. It should be noted that the transcription genes involved in the anabolic pathway were only maintained after six months in fruits treated with 1-MCP. The data in this work suggests that the apple in the absence of 1-MCP begins to prepare its metabolism to mature, even during the storage period in AC. Meanwhile, in the presence of the inhibitor, the transcriptional profile of the fruit is similar to that at the time of harvest. It was also found that a set of genes that code for ethylene receptors, auxin homeostasis, MADS Box, and NAC transcription factors may be involved in the regulation of post-harvest ripening after storage and in the absence of 1-MCP.

Management of Fire Blight Using Pre-bloom Application of Prohexadione-Calcium.

Fire blight, a bacterial disease of rosaceous plants caused by Erwinia amylovora, is one of the most important diseases affecting commercial apple production worldwide. Antibiotics, applied at bloom to protect against blossom infection, are the most effective means of management but raise concern due to the potential for antibiotic resistance in both the pathogen population and nontarget organisms. In addition, most fire blight outbreaks in New York State often emerge in late June to July as shoot blight, calling into question the role of blossom infections and the antibiotic applications made to manage them. Prohexadione-calcium (PhCa) is a gibberellic acid inhibitor used post-bloom to control shoot vigor and to manage shoot blight. However, the magnitude of shoot blight management is directly related to the suppression of shoot growth, which is undesirable, especially in young orchards during establishment years. PhCa is believed to control shoot blight by thickening cell walls in cortical parenchyma, preventing invasion of host tissues by E. amylovora. We hypothesize that PhCa applied pre-bloom could similarly prevent invasion of blossom pedicels following infection, leading to reduced disease incidence. We evaluated novel pre-bloom PhCa programs for their effects on disease management (blossom and shoot blight) as well as their impact on shoot growth for three years in a mature 'Gala' orchard in New York. In all three years of the study, all PhCa programs resulted in less than 27% incidence (71% control) of blossom blight and less than 13% incidence (77% control) of shoot blight with minimal effect on tree growth. Inclusion of a biopesticide during bloom further reduced the incidence of blossom blight in one year of three. Using light microscopy, we found that cell walls in the cortical parenchyma of fruitlet pedicels on trees receiving pre-bloom PhCa applications were significantly thicker than those of untreated trees 40 days after full bloom and inoculation. Overall, we found that pre-bloom applications of PhCa had utility in reducing blossom blight and shoot blight with minimal impacts on tree growth. These pre-bloom programs would fit with standard production practices and may contribute toward the development of fire blight management programs without the use of antibiotics.

Development of a Sensitive TaqMan qPCR Assay for Detection and Quantification of Venturia inaequalis in Apple Leaves and Fruit and in Air Samples.

A TaqMan quantitative PCR (qPCR) assay based on the translation elongation factor 1-α gene was developed for the quantification of Venturia inaequalis in leaves and fruits of Malus × domestica and in spore trap samples. The designed primers and hydrolysis probe amplified a specific 86-bp fragment for V. inaequalis. The specificity of the assay was tested using 35 strains of V. inaequalis and 20 different fungal species, including common pathogens of apple and other species of Venturia. The limit of detection was 20 fg, which is lower than a single genome of V. inaequalis. The selectivity of the assay was tested using DNA from three cultivars of Malus × domestica, and no influence on pathogen amplification was found. The assay was also validated for repeatability and reproducibility. With this assay, it was possible to detect and quantify V. inaequalis in four cultivars (Ambrosia, Florina, Golden Delicious, and Mondial Gala) in both symptomatic and asymptomatic leaves and in symptomatic Golden Delicious apple fruit stored for 2 months. Furthermore, the assay was successfully tested on spore trap samples originating from apple orchards. The quantification of the molecular assay when compared with the estimated number of V. inaequalis cells, using an optical microscope, showed a correlation coefficient of 0.8186. The developed technique could be used to detect V. inaequalis in asymptomatic samples without any cross-reaction with other fungal species. Furthermore, to improve the efficacy of disease management with a timely application of fungicides, this assay could be used for the analysis of spore trap samples by using an implemented extraction method.