Constitutive expression of apple endo-POLYGALACTURONASE1 in fruit induces early maturation, alters skin structure and accelerates softening.

During fruit ripening, polygalacturonases (PGs) are key contributors to the softening process in many species. Apple is a crisp fruit that normally exhibits only minor changes to cell walls and limited fruit softening. Here, we explore the effects of PG overexpression during fruit development using transgenic apple lines overexpressing the ripening-related endo-POLYGALACTURONASE1 gene. MdPG1-overexpressing (PGox) fruit displayed early maturation/ripening with black seeds, conversion of starch to sugars and ethylene production occurring by 80 days after pollination (DAP). PGox fruit exhibited a striking, white-skinned phenotype that was evident from 60 DAP and most likely resulted from increased air spaces and separation of cells in the hypodermis due to degradation of the middle lamellae. Irregularities in the integrity of the epidermis and cuticle were also observed. By 120 DAP, PGox fruit cracked and showed lenticel-associated russeting. Increased cuticular permeability was associated with microcracks in the cuticle around lenticels and was correlated with reduced cortical firmness at all time points and extensive post-harvest water loss from the fruit, resulting in premature shrivelling. Transcriptomic analysis suggested that early maturation was associated with upregulation of genes involved in stress responses, and overexpression of MdPG1 also altered the expression of genes involved in cell wall metabolism (e.g. ß-galactosidase, MD15G1221000) and ethylene biosynthesis (e.g. ACC synthase, MD14G1111500). The results show that upregulation of PG not only has dramatic effects on the structure of the fruit outer cell layers, indirectly affecting water status and turgor, but also has unexpected consequences for fruit development.

The Ca2+-MdCRF4-MdWRKY9 module negatively affects apple fruit watercore formation by suppressing the transcription of MdSOT2.

Watercore is a common physiological disease of Rosaceae plants, such as apples (Malus domestica), usually occurring during fruit ripening. Apple fruit with watercore symptoms is prone to browning and rotting, thus losing commercial viability. Sorbitol and calcium ions are considered key factors affecting watercore occurrence in apples. However, the mechanism by which they affect the occurrence of watercore remains unclear. Here, we identified that the transcription factor MdWRKY9 directly binds to the promoter of MdSOT2, positively regulates the transcription of MdSOT2, increases sorbitol content in fruit, and promotes watercore occurrence. Additionally, MdCRF4 can directly bind to MdWRKY9 and MdSOT2 promoters, positively regulating their expression. Since calcium ions can induce the ubiquitination and degradation of the transcription factor MdCRF4, they can inhibit the transcription of MdWRKY9 and MdSOT2 by degrading MdCRF4, thereby reducing the sorbitol content in fruit and inhibiting the occurrence of fruit watercore disease. Our data sheds light on how calcium ions mitigate watercore in fruit, providing molecular-level insights to enhance fruit quality artificially.

The BTB-BACK-TAZ domain protein MdBT2 reduces drought resistance by weakening the positive regulatory effect of MdHDZ27 on apple drought tolerance via ubiquitination.

Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB-BACK-TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain-leucine zipper (HD-Zip) transcription factor, MdHDZ27, using a yeast two-hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance-related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2-MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants.

The MdCBF1/2-MdTST1/2 module regulates sugar accumulation in response to low temperature in apple.

Soluble sugar content is a key component in controlling fruit flavor, and its accumulation in fruit is largely determined by sugar metabolism and transportation. When the diurnal temperature range is greater, the fleshy fruits accumulated more soluble sugars and become more sweeter. However, the molecular mechanism underlying this response remains largely unknown. In this study, we verified that low-temperature treatment promoted soluble sugar accumulation in apple fruit and found that this was due to the upregulation of the Tonoplast Sugar Transporter genes MdTST1/2. A combined strategy using assay for transposase-accessible chromatin (ATAC) sequencing and gene expression and cis-acting elements analyses, we identified two C-repeat Binding Factors, MdCBF1 and MdCBF2, that were induced by low temperature and that might be upstream transcription factors of MdTST1/2. Further studies established that MdCBF1/2 could bind to the promoters of MdTST1/2 and activate their expression. Overexpression of MdCBF1 or MdCBF2 in apple calli and fruit significantly upregulated MdTST1/2 expression and increased the concentrations of glucose, fructose, and sucrose. Suppression of MdTST1 and/or MdTST2 in an MdCBF1/2-overexpression background abolished the positive effect of MdCBF1/2 on sugar accumulation. In addition, simultaneous silencing of MdCBF1/2 downregulated MdTST1/2 expression and apple fruits failed to accumulate more sugars under low-temperature conditions, indicating that MdCBF1/2-mediated sugar accumulation was dependent on MdTST1/2 expression. Hence, we concluded that the MdCBF1/2-MdTST1/2 module is crucial for sugar accumulation in apples in response to low temperatures. Our findings provide mechanistic components coordinating the relationship between low temperature and sugar accumulation as well as new avenues to improve fruit quality.

Fungal invasion-induced accumulation of salicylic acid promotes anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple fruits.

In the field, necrosis area induced by pathogens is usually surrounded by a red circle in apple fruits. However, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we demonstrated that accumulated salicylic acid (SA) induced by fungal infection promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module in apple (Malus domestica). Inoculating apple fruits with Valsa mali or Botryosphaeria dothidea induced a red circle surrounding the necrosis area, which mimicked the phenotype observed in the field. The red circle accumulated a high level of anthocyanins, which was positively correlated with SA accumulation stimulated by fungal invasion. Further analysis showed that SA promoted anthocyanin biosynthesis in a dose-dependent manner in both apple calli and fruits. We next demonstrated that MdNPR1, a master regulator of SA signaling, positively regulated anthocyanin biosynthesis in both apple and Arabidopsis. Moreover, MdNPR1 functioned as a co-activator to interact with and enhance the transactivation activity of MdTGA2.2, which could directly bind to the promoters of anthocyanin biosynthetic and regulatory genes to promote their transcription. Suppressing expression of either MdNPR1 or MdTGA2.2 inhibited coloration of apple fruits, while overexpressing either of them significantly promoted fruit coloration. Finally, we revealed that silencing either MdNPR1 or MdTGA2.2 in apple fruits repressed SA-induced fruit coloration. Therefore, our data determined that fungal-induced SA promoted anthocyanin biosynthesis through MdNPR1-MdTGA2.2 module, resulting in a red circle surrounding the necrosis area in apple fruits.

Strigolactone regulates adventitious root formation via the MdSMXL7-MdWRKY6-MdBRC1 signaling cascade in apple.

Propagation through stem cuttings is a popular method worldwide for species such as fruit tree rootstocks and forest trees. Adventitious root (AR) formation from stem cuttings is crucial for effective and successful clonal propagation of apple rootstocks. Strigolactones (SLs) are newly identified hormones involved in AR formation. However, the regulatory mechanisms underpinning this process remain elusive. In the present study, weighted gene co-expression network analysis, as well as rooting assays using stable transgenic apple materials, revealed that MdBRC1 served as a key gene in the inhibition of AR formation by SLs. We have demonstrated that MdSMXL7 and MdWRKY6 synergistically regulated MdBRC1 expression, depending on the interactions of MdSMXL7 and MdWRKY6 at the protein level downstream of SLs as well as the direct promoter binding on MdBRC1 by MdWRKY6. Furthermore, biochemical studies and genetic analysis revealed that MdBRC1 inhibited AR formation by triggering the expression of MdGH3.1 in a transcriptional activation pathway. Finally, the present study not only proposes a component, MdWRKY6, that enables MdSMXL7 to regulate MdBRC1 during the process of SL-controlled AR formation in apple, but also provides prospective target genes to enhance AR formation capacity using CRISPR (i.e. clustered regularly interspaced short palindromic repeats) technology, particularly in woody plants.

CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.

Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.

Transcriptional repression of MdMa1 by MdMYB21 in Ma locus decreases malic acid content in apple fruit.

Malic acid is a major organic acid component of apples and a crucial determinant of fruit organoleptic quality. A candidate gene for malic acid content, designated MdMa1, was previously identified in the Ma locus, which is a major quantitative trait locus (QTL) for apple fruit acidity located on the linkage group 16. Region-based association mapping to detect candidate genes in the Ma locus identified MdMa1 and an additional MdMYB21 gene putatively associated with malic acid. MdMYB21 was significantly associated with fruit malic acid content, accounting for ~7.48% of the observed phenotypic variation in the apple germplasm collection. Analyses of transgenic apple calli, fruits and tomatoes demonstrated that MdMYB21 negatively regulated malic acid accumulation. The apple fruit acidity-related MdMa1 and its tomato ortholog, SlALMT9, exhibited lower expression profiles in apple calli, mature fruits and tomatoes in which MdMYB21 was overexpressed, compared with their corresponding wild-type variety. MdMYB21 directly binds to the MdMa1 promoter and represses its expression. Interestingly, a 2-bp variation in the MdMYB21 promoter region altered its expression and regulation of its target gene, MdMa1, expression. Our findings not only demonstrate the efficiency of integrating QTL and association mapping in the identification of candidate genes controlling complex traits in apples, but also provide insights into the complex regulatory mechanism of fruit malic acid accumulation.

Mdm-miR160-MdARF17-MdWRKY33 module mediates freezing tolerance in apple.

Apple (Malus domestica) trees are vulnerable to freezing temperatures. Cold resistance in woody perennial plants can be improved through biotechnological approaches. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the tree's response to cold. In this study, we demonstrated that the Mdm-miR160-MdARF17-MdWRKY33 module is crucial for apple freezing tolerance. Mdm-miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm-miR160, is a positive regulator of apple freezing tolerance. RNA sequencing analysis revealed that in apple, MdARF17 mediates the cold response by influencing the expression of cold-responsive genes. EMSA and ChIP-qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpression of MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm-miR160-MdARF17-MdWRKY33 module regulates cold tolerance in apple by regulating reactive oxygen species (ROS) scavenging, as revealed by (i) increased H2 O2 levels and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants and (ii) decreased H2 O2 levels and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm-miR160-MdARF17-MdWRKY33 module in freezing tolerance in apple, thus providing support for breeding of cold-tolerant apple cultivars.

MdHB7-like positively modulates apple salt tolerance by promoting autophagic activity and Na+ efflux.

Salt stress adversely affects the yield and quality of crops and limits their geographical distribution. Studying the functions and regulatory mechanisms of key genes in the salt stress response is important for breeding crops with enhanced stress resistance. Autophagy plays an important role in modulating the tolerance of plants to various types of abiotic stressors. However, the mechanisms underlying salt-induced autophagy are largely unknown. Cation/Ca2+ exchanger proteins enhance apple salt tolerance by inhibiting Na+ accumulation but the mechanism underlying the response to salt stress remains unclear. Here, we show that the autophagy-related gene MdATG18a modulated apple salt tolerance. Under salt stress, the autophagic activity, proline content, and antioxidant enzyme activities were higher and Na+ accumulation was lower in MdATG18a-overexpressing transgenic plants than in control plants. The use of an autophagy inhibitor during the salt treatment demonstrated that the regulatory function of MdATG18a depended on autophagy. The yeast-one-hybrid assay revealed that the homeodomain-leucine zipper (HD-Zip) transcription factor MdHB7-like directly bound to the MdATG18a promoter. Transcriptional regulation and genetic analyses showed that MdHB7-like enhanced salt-induced autophagic activity by promoting MdATG18a expression. The analysis of Na+ efflux rate in transgenic yeast indicated that MdCCX1 expression significantly promoted Na+ efflux. Promoter binding, transcriptional regulation, and genetic analyses showed that MdHB7-like promoted Na+ efflux and apple salt tolerance by directly promoting MdCCX1 expression, which was independent of the autophagy pathway. Overall, our findings provide insight into the mechanism underlying MdHB7-like-mediated salt tolerance in apple through the MdHB7-like-MdATG18a and MdHB7-like-MdCCX1 modules. These results will aid future studies on the mechanisms underlying stress-induced autophagy and the regulation of stress tolerance in plants.

Field study of the fire-blight-resistant cisgenic apple line C44.4.146.

Cisgenesis, the genetic modification of a plant with genes from a sexually compatible plant, was used to confer fire blight resistance to the cultivar 'Gala Galaxy' by amendment of the resistance gene FB_MR5, resulting in the line C44.4.146. To verify whether cisgenesis changed other tree-, flower- or fruit-related traits, a 5-year field trial was conducted with trees of C44.4.146 and multiple control genotypes, including members of the 'Gala' sports group. None of the 44 investigated tree-, flower- or fruit-related traits significantly differed between C44.4.146 and at least one of the control genotypes in all observation years. However, fruits of C44.4.146 and its wild-type 'Gala Galaxy' from tissue culture were paler in color than fruits of 'Gala Galaxy' that had not undergone tissue culture. There was no significant and consistently detected difference in the fruit flesh and peel metabolome of C44.4.146 compared with the control genotypes. Finally, the disease resistance of C44.4.146 was confirmed also when the fire blight pathogen was inoculated through the flowers. We conclude that the use of cisgenesis to confer fire blight resistance to 'Gala Galaxy' in C44.4.146 did not have unintended effects, and that the in vitro establishment of 'Gala Galaxy' had a greater effect on C44.4.146 properties than its generation applying cisgenesis.

The identification and analysis of meristematic mutations within the apple tree that developed the RubyMac sport mutation.

BACKGROUND: Understanding the molecular basis of sport mutations in fruit trees has the potential to accelerate generation of improved cultivars. RESULTS: For this, we analyzed the genome of the apple tree that developed the RubyMac phenotype through a sport mutation that led to the characteristic fruit coloring of this variety. Overall, we found 46 somatic mutations that distinguished the mutant and wild-type branches of the tree. In addition, we found 54 somatic gene conversions (i.e., loss-of-heterozygosity mutations) that also distinguished the two parts of the tree. Approximately 20% of the mutations were specific to individual cell lineages, suggesting that they originated from the corresponding meristematic layers. Interestingly, the de novo mutations were enriched for GC = > AT transitions while the gene conversions showed the opposite bias for AT = > GC transitions, suggesting that GC-biased gene conversions have the potential to counteract the AT-bias of de novo mutations. By comparing the gene expression patterns in fruit skins from mutant and wild-type branches, we found 56 differentially expressed genes including 18 involved in anthocyanin biosynthesis. While none of the differently expressed genes harbored a somatic mutation, we found that some of them in regions of the genome that were recently associated with natural variation in fruit coloration. CONCLUSION: Our analysis revealed insights in the characteristics of somatic change, which not only included de novo mutations but also gene conversions. Some of these somatic changes displayed strong candidate mutations for the change in fruit coloration in RubyMac.

Self-(in)compatibility in Tunisian apple accessions [Malus domestica. Borkh]: S-genotypes identification and pollen tube growth analysis.

MAIN CONCLUSION: Self-incompatibility studies have revealed a potential use of Tunisian apple resources for crop improvement and modern breeding programs and a likely correlation between the pollen tube growth and flowering period. Apples [Malus domestica. Borkh] exhibit an S-RNase-based gametophytic self-incompatibility (GSI) system. Four primer combinations were used to S-genotype eighteen Tunisian local apple accessions and twelve introduced accessions that served as references. Within the Tunisian local accessions, S2, S3, S7, and S28 S-alleles were the most frequent and were assigned to 14 S-genotypes; among them, S7S28, S3S7, S2S5, and S2S3 were the most abundant. PCA plot showed that population structuring was affected by the S-alleles frequencies and revealed a modern origin of the Tunisian varieties rather than being ancient ones. Nonetheless, the results obtained with 17 SSR markers showed a separate grouping of local Tunisian accessions that calls into question the hypothesis discussed. Pollination experiments showed that the pollen started to germinate within 24 h of pollination but 48 h after pollination in the "El Fessi" accession. The first pollen tubes arrived in the styles within 36 h of pollination in two early flowering accessions known as "Arbi" and "Bokri", and after 72 h of pollination in late flowering "El Fessi" and 48 h after pollination in remaining accessions. The first pollen tube arrests were observed in accessions "Arbi" and "Bokri" within 84 h of pollination, within 108 h of pollination in "El Fessi" and within 108 h of pollination in remaining accessions. In the apple accession called "Boutabgaya," the pollen tubes reached the base of the style within 120 h of pollination without being aborted. Nevertheless, the self-compatible nature of "Boutabgaya" needs more studies to be confirmed. However, our results revealed the malfunction of the female component of the GSI in this accession. To conclude, this work paved the path for further studies to enhance the insight (i) into the relation between the flowering period and the pollen tube growth, (ii) self-compatible nature of "Boutabgaya", and (iii) the origin of the Tunisian apple.

Competition for nutrient niches within the apple blossom microbiota antagonizes the initiation of fire blight infection.

Changes in the plant microbiota composition are intimately associated with the health of the plant, but factors controlling the microbial community in flowers are poorly understood. In this study, we used apple flowers and fire blight as a model system to investigate the effects of floral microbiota and microbial competition on disease development and suppression. To compare changes in microbial flora with the RNA expression patterns of plants, the flower samples were collected in three different flowering stages (Bud, Popcorn, and Full-bloom). Using advanced sequencing technology, we analyzed the data and conducted both in vitro and in vivo experiments to validate our findings. Our results show that the Erwinia amylovora use arabinogalactan, which is secreted on the flowers, for early colonization of apple flowers. Pantoea agglomerans was more competitive for arabinogalactan than E. amylovora. Additionally, P. agglomerans suppressed the expression of virulence factors of E. amylovora by using arabinose, which is a major component of arabinogalactan, which induces virulence gene expression. The present data provide new insights into developing control strategies for diverse plant diseases, including fire blight, by highlighting the importance of nutrients in disease development or suppression.

The apple transcription factor MdZF-HD11 regulates fruit softening by promoting Mdß-GAL18 expression.

Fruit ripening and the associated softening are major determinants of fruit quality and post-harvest shelf life. Although the mechanisms underlying fruit softening have been intensively studied, there are limited reports on the regulation of fruit softening in apples (Malus domestica). Here, we identified a zinc finger homeodomain transcription factor MdZF-HD11that trans-activates the promoter of Mdß-GAL18, which encodes a pectin-degradation enzyme associated with cell wall metabolism. Both MdZF-HD11 and Mdß-GAL18 genes were up-regulated by exogenous ethylene treatment and repressed by 1-methylcyclopropene treatment. Further experiments revealed that MdZF-HD11 binds directly to the Mdß-GAL18 promoter and up-regulates its transcription. Moreover, using transgenic apple fruit calli, we found that overexpression of Mdß-GAL18 or MdZF-HD11 significantly enhanced ß-galactosidase activity, and overexpression of MdZF-HD11 induced the expression of Mdß-GAL18. We also discovered that transient overexpression of Mdß-GAL18 or MdZF-HD11 in 'Golden Delicious' apple significantly increased the release of ethylene, reduced fruit firmness, promoted the transformation of skin color from green to yellow, and accelerated ripening and softening of the fruit. Finally, the overexpression of MdZF-HD11 in tomato also promoted fruit softening. Collectively, these results indicate that ethylene-induced MdZF-HD11 interacts with Mdß-GAL18 to promote the post-harvest softening of apple.

VmSom1 is essential for growth, development, maintenance of cell wall integrity and virulence in Valsa mali.

Apple Valsa canker disease, caused by Valsa mali Miyabe et Yamada, severely endangers the healthy growth of apple trees. The Som1, located downstream of the cyclic AMP-dependent protein kinase A (cAMP-PKA) pathway, plays crucial roles in the growth, development, morphological differentiation, and virulence of filamentous fungi. In this study, we identify and functionally characterize VmSom1, a homolog of Som1, in Valsa mali. The VmSom1 gene is located on chromosome 12, encoding an 824 amino acid protein. Phylogenetic analysis reveals VmSom1 as a fungal Som1 homolog. The VmSom1 deletion mutants exhibit slower growth rates and fail to produce pycnidia. Additionally, their hyphal growth is significantly inhibited on media containing Calcofluor White, Congo Red, NaCl, and sorbitol. The growth rate of VmSom1 deletion mutants is reduced on maltose, lactose, sucrose and fructose media but increases on glucose medium. Moreover, the mycelial growth rate of the VmSom1 deletion mutant is significantly lower than that of the wild-type strain in peptone, NH4SO4, NaNO3, and no nitrogen. Notably, the distances between the septa increase, and chitin concentration shifts to the hyphal tip in the VmSom1 deletion mutant. Furthermore, compared with the wild-type strain, the VmSom1 deletion mutant exhibits fewer diseased spots on apple fruit and branches. Overall, our findings demonstrate that VmSom1 is involved in regulating the growth and development, colony surface hydrophobicity, osmotic stress, cell wall integrity maintenance, carbon and nitrogen source utilization, septa formation, and virulence of V. mali.

Effects of apples (Malus domestica) and their derivatives on metabolic conditions related to inflammation and oxidative stress and an overview of by-products use in food processing.

Apple (Malus domestica) is the third most produced fruit worldwide. It is a well-known source of bioactive compounds mainly represented by hydroxycinnamic acids, flavan-3-ols, dihydrochalcones, dehydroascorbic acid, carotenoids, chlorogenic acid, epicatechin, and phloridzin. Due to the lack of a recent evaluation of the clinical trials associated with apple consumption, this review investigated the effects of this fruit on metabolic conditions related to inflammation and oxidative stress and reviewed the applications of apple waste on food products. Thirty-three studies showed that apples or its derivatives exhibit anti-inflammatory and antioxidant actions, improve blood pressure, body fat, insulin resistance, dyslipidemia, and reduce cardiovascular risks. Apples have a great economic impact due to its several applications in the food industry and as a food supplement since it has impressive nutritional value. Dietary fiber from the fruit pomace can be used as a substitute for fat in food products or as an improver of fiber content in meat products. It can also be used in bakery and confectionary products or be fermented to produce alcohol. Pomace phytocompounds can also be isolated and applied as antioxidants in food products. The potential for the use of apples and by-products in the food industry can reduce environmental damage.

Investigating the spatiotemporal dynamics of apple tree phyllosphere bacterial and fungal communities across cultivars in orchards.

The phyllosphere, a reservoir of diverse microbial life associated with plant health, harbors microbial communities that are subject to various complex ecological processes acting at multiple scales. In this study, we investigated the determinants of the spatiotemporal variation in bacterial and fungal communities within the apple tree phyllosphere, employing 16S and ITS amplicon sequencing. Our research assessed the impact of key factors-plant compartment, site, time, and cultivar-on the composition and diversity of leaf and flower microbial communities. Our analyses, based on samples collected from three cultivars in three orchards in 2022, revealed that site and time are the strongest drivers of apple tree phyllosphere microbial communities. Conversely, plant compartment and cultivar exhibited minor roles in explaining community composition and diversity. Predominantly, bacterial communities comprised Hymenobacter (25%) and Sphingomonas (10%), while the most relatively abundant fungal genera included Aureobasidium (27%) and Sporobolomyces (10%). Additionally, our results show a gradual decrease in alpha-diversity throughout the growth season. These findings emphasize the necessity to consider local microbial ecology dynamics in orchards, especially as many groups worldwide aim for the development of biocontrol strategies (e.g., by manipulating plant-microbe interactions). More research is needed to improve our understanding of the determinants of time and site-specific disparities within apple tree phyllosphere microbial communities across multiple years, locations, and cultivars.

Bioactivities evaluation of an endophytic bacterial strain Bacillus tequilensis QNF2 inhibiting apple ring rot caused by Botryosphaeria dothidea on postharvest apple fruits.

Apple ring rot, one of the most common apple postharvest diseases during storage, is caused by Botryosphaeria dothidea. Presently, the disease management is primarily dependent on chemical fungicide application. Here we demonstrated an endophyte bacterium Bacillus tequilensis QNF2, isolated from Chinese leek (Allium tuberosum) roots considerably suppressed B. dothidea mycelial growth, with the highest suppression of 73.56 % and 99.5 % in the PDA and PDB medium, respectively in vitro confront experiments. In in vivo experiments, B. tequilensis QNF2 exhibited a control efficacy of 88.52 % and 100 % on ring rot disease on postharvest apple fruits inoculated with B. dothidea disc and dipped into B. dothidea culture, respectively. In addition, B. tequilensis QNF2 volatile organic compounds (VOCs) also manifested markedly inhibition against B. dothidea mycelial growth and the ring rot on postharvest apple fruits. Moreover, B. tequilensis QNF2 severely damaged the mycelial morphology of B. dothidea. Finally, B. tequilensis QNF2 significantly repressed the expression of six pathogenicity-related genes, such as adh, aldh, aldh3, galm, pdc1, pdc2, involved in glycolysis/gluconeogenesis of B. dothidea. The findings of the study proved that B. tequilensis QNF2 was a promising alternative for controlling apple ring rot of postharvest apple fruit.

Apple Bitter Rot and Glomerella Leaf Spot: A Comprehensive Review of Causal Species and Their Biology, Fungicide Sensitivities, and Management Strategies.

Bitter rot and Glomerella leaf spot (GLS) are two distinct diseases of apple fruit and foliage caused by members of the ascomycete fungal genus Colletotrichum. Although GLS is restricted to subtropical and, in some areas, to temperate climates, bitter rot is responsible for significant yield loss worldwide, particularly during the postharvest period. Initially thought to be caused by just two species of Colletotrichum, C. acutatum, and C. gloeosporioides, advances in molecular biology and sequencing techniques enabled the identification of 25 different species capable of causing bitter rot and/or GLS of apple belonging to the C. gloeosporioides species complex (CGSC), C. acutatum species complex (CASC), and C. boninense species complex (CBSC). Three species (C. gloeosporioides, C. fructicola, and C. chrysophilum) of CGSC cause both bitter rot and GLS, 18 species (6 of CGSC and 12 of CASC) only cause bitter rot, and 4 species (C. aenigma and C. asianum of CGSC, C. limetticola of CASC, and C. karsti of CBSC) only cause GLS. These species were found to differ in their geographical distribution, environmental and host tissue preference, pathogenicity, and fungicide sensitivities. In this review, we summarize the distribution, life cycle, and pathogenicity mechanisms of all currently known Colletotrichum species responsible for bitter rot and GLS of apple. Furthermore, we describe known apple defense mechanisms and management strategies for the control of these economically significant pathogens and identify gaps in our present understanding for future research.