Abscisic acid triggers vitamin E accumulation by transient transcript activation of VTE5 and VTE6 in sweet cherry fruits.

Tocopherols are lipophilic antioxidants known as vitamin E and synthesized from the condensation of two metabolic pathways leading to the formation of homogentisate and phytyl diphosphate. While homogentisate is derived from tyrosine metabolism, phytyl diphosphate may be formed from geranylgeranyl diphosphate or phytol recycling from chlorophyll degradation. Here, we hypothesized that abscisic acid (ABA) could induce tocopherol biosynthesis in sweet cherries by modifying the expression of genes involved in vitamin E biosynthesis, including those from the phytol recycling pathway. Hence, the expression of key tocopherol biosynthesis genes was determined together with vitamin E and chlorophyll contents during the natural development of sweet cherries on the tree. Moreover, the effects of exogenously applied ABA on the expression of key tocopherol biosynthesis genes were also investigated during on-tree fruit development, and tocopherols and chlorophylls contents were analyzed. Results showed that the expression of tocopherol biosynthesis genes, including VTE5, VTE6, HPPD and HPT showed contrasting patterns of variation, but in all cases, increased by 2- and 3-fold over time during fruit de-greening. This was not the case for GGDR and VTE4, the first showing constitutive expression during fruit development and the second with marked down-regulation at ripening onset. Furthermore, exogenous ABA stimulated the production of both α- and γ-tocopherols by 60% and 30%, respectively, promoted chlorophyll degradation and significantly enhanced VTE5 and VTE6 expression, and also that of HPPD and VTE4, altogether increasing total tocopherol accumulation. In conclusion, ABA increases promote the transcription of phytol recycling enzymes, which may contribute to vitamin E biosynthesis during fruit development in stone fruits like sweet cherries.

Sweet cherry TCP gene family analysis reveals potential functions of PavTCP1, PavTCP2 and PavTCP3 in fruit light responses.

BACKGROUND: TCP proteins are plant specific transcription factors that play important roles in plant growth and development. Despite the known significance of these transcription factors in general plant development, their specific role in fruit growth remains largely uncharted. Therefore, this study explores the potential role of TCP transcription factors in the growth and development of sweet cherry fruits. RESULTS: Thirteen members of the PavTCP family were identified within the sweet cherry plant, with two, PavTCP1 and PavTCP4, found to contain potential target sites for Pav-miR159, Pav-miR139a, and Pav-miR139b-3p. Analyses of cis-acting elements and Arabidopsis homology prediction analyses that the PavTCP family comprises many light-responsive elements. Homologs of PavTCP1 and PavTCP3 in Arabidopsis TCP proteins were found to be crucial to light responses. Shading experiments showed distinct correlation patterns between PavTCP1, 2, and 3 and total anthocyanins, soluble sugars, and soluble solids in sweet cherry fruits. These observations suggest that these genes may contribute significantly to sweet cherry light responses. In particular, PavTCP1 could play a key role, potentially mediated through Pav-miR159, Pav-miR139a, and Pav-miR139b-3p. CONCLUSION: This study is the first to unveil the potential function of TCP transcription factors in the light responses of sweet cherry fruits, paving the way for future investigations into the role of this transcription factor family in plant fruit development.

Genome-Wide Identification and Expression Analysis of the Sweet Cherry Whirly Gene Family.

Sweet cherry (Prunus avium) is one of the economically valuable horticultural fruit trees and it is widely cultivated throughout the world. Whirly (WHY) genes are a unique gene family with few members and have important biological functions in plant growth, development, and response to abiotic stress. This study utilized whole-genome identification to conduct a comprehensive analysis of the WHY genes in sweet cherry and examined their transcription levels in different tissues and under abiotic stress to explore their functions. Two WHY genes were identified in the sweet cherry genome and named PavWHY1 and PavWHY2, respectively, based on their homology with those in Arabidopsis thaliana. Both genes have theoretical isoelectric points greater than seven and are hydrophilic proteins, suggesting that they may be localized in plastids. The two genes are evolutionarily classified into two categories, with large differences in gene structure, and highly similar protein tertiary structures, and both have conserved domains of WHY. PavWHY1 and PavWHY2 are collinear with AtWHY1 and AtWHY2, respectively. The promoter sequence contains cis-acting elements related to hormones and abiotic stress, which are differentially expressed during flower bud differentiation, fruit development, and cold accumulation. qRT-PCR showed that PavWHY1 and PavWHY2 were differentially expressed in flower and fruit development and responded to low temperature and exogenous ABA treatment. The recombinant plasmid pGreenII-0800-Luc with the promoters of these two genes can activate luciferase expression in tobacco. Protein interaction predictions indicate that these gene products may interact with other proteins. This study reveals the molecular features, evolutionary relationships, and expression patterns of sweet cherry WHY genes, and investigates the activities of their promoters, which lays the foundation for further exploration of their biological functions and provides new insights into the WHY gene family in Rosaceae.

Overexpression of PavHIPP16 from Prunus avium enhances cold stress tolerance in transgenic tobacco.

BACKGROUND: The heavy metal-associated isoprenylated plant protein (HIPP) is an important regulatory element in response to abiotic stresses, especially playing a key role in low-temperature response. RESULTS: This study investigated the potential function of PavHIPP16 up-regulated in sweet cherry under cold stress by heterologous overexpression in tobacco. The results showed that the overexpression (OE) lines' growth state was better than wild type (WT), and the germination rate, root length, and fresh weight of OE lines were significantly higher than those of WT. In addition, the relative conductivity and malondialdehyde (MDA) content of the OE of tobacco under low-temperature treatment were substantially lower than those of WT. In contrast, peroxidase (POD), superoxide dismutase (SOD), catalase (CAT) activities, hydrogen peroxide (H2O2), proline, soluble protein, and soluble sugar contents were significantly higher than those of WT. Yeast two-hybrid assay (Y2H) and luciferase complementation assay verified the interactions between PavbHLH106 and PavHIPP16, suggesting that these two proteins co-regulated the cold tolerance mechanism in plants. The research results indicated that the transgenic lines could perform better under low-temperature stress by increasing the antioxidant enzyme activity and osmoregulatory substance content of the transgenic plants. CONCLUSIONS: This study provides genetic resources for analyzing the biological functions of PavHIPPs, which is important for elucidating the mechanisms of cold resistance in sweet cherry.

Effect of preharvest biofilm application regimes on cracking and fruit quality traits in '0900 Ziraat' sweet cherry cultivar.

BACKGROUND: Fruit cracking impacts the quality of sweet cherry, significantly affecting its marketability due to increased susceptibility to injury, aesthetic flaws, and susceptibility to pathogens. The effect of 1% biofilm (Parka™) application regimes on fruit cracking and other quality parameters in the '0900 Ziraat' cherry cultivar was investigated in this study. Fruit sprayed with water were served as control (U1). Fruit treated only once with biofilm three, two and one week before the commercial harvest were considered as U2, U3 and U4, respectively. Fruit treated with biofilm three, two, and one week before harvest were considered as U5; three and two week before harvest as U6; two and one week before harvest as U7; and fruit treated three and one week before harvest as U8. RESULTS: In both measurement periods, the lower cracking index was obtained in biofilm-treated sweet cherry fruit. However, the firmness of biofilm-treated fruit was higher than that of the control fruit. The lowest respiration rate was observed in U7, while the highest weight was recorded in U4 and U5 than the control. The biofilm application decreased fruit coloration. The biofilm application also increased the soluble solids content of the fruit. The U2, U3 and U4 applications at harvest showed higher titratable acidity than the control. In both measurement periods, the vitamin C content of the U2, U5, U6, U7 and U8 applications was found to be higher than that of the control. The total monomeric anthocyanin of the U3 and U8 applications was higher than that of the control. Furthermore, the antioxidant activity of the U2, U3 and U5 in the DPPH, and the U7 and U8 in FRAP were measured higher thanthat of the control. CONCLUSIONS: The application of biofilms has the potential to mitigate fruit cracking, prolong postharvest life of sweet cherries, and enhance fruit firmness.

A 5.2-kb insertion in the coding sequence of PavSCPL, a serine carboxypeptidase-like enhances fruit firmness in Prunus avium.

Fruit firmness is an important trait in sweet cherry breeding because it directly positively influences fruit transportability, storage and shelf life. However, the underlying genes responsible and the molecular mechanisms that control fruit firmness remain unknown. In this study, we identified a candidate gene, PavSCPL, encoding a serine carboxypeptidase-like protein with natural allelic variation, that controls fruit firmness in sweet cherry using map-based cloning and functionally characterized PavSCPL during sweet cherry fruit softening. Genetic analysis revealed that fruit firmness in the 'Rainier' × 'Summit' F1 population was controlled by a single dominant gene. Bulked segregant analysis combined with fine mapping narrowed the candidate gene to a 473-kb region (7418778-7 891 914 bp) on chromosome 6 which included 72 genes. The candidate gene PavSCPL, and a null allele harbouring a 5244-bp insertion in the second exon that completely inactivated PavSCPL expression and resulted in the extra-hard-flesh phenotype, were identified by RNA-sequencing analysis and gene cloning. Quantitative RT-PCR analysis revealed that the PavSCPL expression level was increased with fruit softening. Virus-induced gene silencing of PavSCPL enhanced fruit firmness and suppressed the activities of certain pectin-degrading enzymes in the fruit. In addition, we developed functional molecular markers for PavSCPL and the Pavscpl5.2-k allele that co-segregated with the fruit firmness trait. Overall, this research identified a crucial functional gene for fruit firmness. The results provide insights into the genetic control and molecular mechanism of the fruit firmness trait and present useful molecular markers for molecular-assisted breeding for fruit firmness in sweet cherry.

Adsorption of Organic Pollutants on Carbonaceous Adsorbents Prepared by Direct Activation of Sweet Cherry Stones.

The main objective of this study was to assess the usefulness of the sweet cherry stones for the production of carbonaceous adsorbents by means of direct physical activation method, using conventional and microwave variant of heating. The adsorbents were characterized in terms of textural parameters, acidic-basic character of the surface, electrokinetic properties and their suitability for drinking water purification. Adsorption tests were carried out against three organic compounds - Triton X-100 (surfactant), bovine serum albumin (protein) and methylene blue (synthetic dye). Depending on the variant of heating applied during activation procedure, the obtained activated biochars differed significantly in terms of the elemental composition, acidic-basic properties as well as degree of specific surface development and the type of porous structure generated. Adsorption tests have showed that the efficiency of organic pollutants removal from aqueous solutions depends significantly not only on the type of the adsorbent and adsorbate applied, but also on the temperature and pH of the system. The sample prepared by microwave-assisted direct activation proved to be very effective in terms of all tested organic pollutants adsorption. The maximum sorption capacity toward Triton X-100, bovine serum albumin and methylene blue reached the level of 86.5, 23.4 and 81.1 mg/g, respectively.

Unraveling Morphological, Physiological, and Transcriptomic Alterations Underlying the Formation of Little Leaves in Phytoplasma-Infected Sweet Cherry Trees.

Phytoplasmas are minute phytopathogenic bacteria that induce excessive vegetative growth, known as witches'-broom (WB), in many infected plant species during the later stages of infection. The WB structure is characterized by densely clustered little (small) leaves, which are frequently accompanied by chlorosis (yellowing). The mechanisms behind the formation of little leaves within WB structures (LL-WB) are poorly understood. To address this gap, the LL-WB formation was extensively studied using sweet cherry virescence (SCV) phytoplasma-infected sweet cherry plants. Based on morphological examinations, signs of premature leaf senescence were observed in LL-WB samples, including reduced leaf size, chlorosis, and alterations in shape. Subsequent physiological analyses indicated decreased sucrose and glucose levels and changes in hormone concentrations in LL-WB samples. Additionally, the transcriptomic analysis revealed impaired ribosome biogenesis and DNA replication. As an essential process in protein production, the compromised ribosome biogenesis and the inhibited DNA replication led to cell cycle arrest, thus affecting leaf morphogenesis and further plant development. Moreover, the expression of marker genes involved in premature leaf senescence was significantly altered. These results indicate a complicated interplay between the development of leaves, premature leaf senescence, and the pathogen-induced stress responses in SCV phytoplasma-infected sweet cherry trees. The results of this study provide insight into understanding the underlying molecular mechanisms driving the formation of little leaves and interactions between plants and pathogens. The findings might help control phytoplasma diseases in sweet cherry cultivation.

bZIP Transcription Factor PavbZIP6 Regulates Anthocyanin Accumulation by Increasing Abscisic Acid in Sweet Cherry.

Basic leucine zipper (bZIP) transcription factors (TFs) play a crucial role in anthocyanin accumulation in plants. In addition to bZIP TFs, abscisic acid (ABA) increases anthocyanin biosynthesis. Therefore, this study aimed to investigate whether bZIP TFs are involved in ABA-induced anthocyanin accumulation in sweet cherry and elucidate the underlying molecular mechanisms. Specifically, the BLAST method was used to identify bZIP genes in sweet cherry. Additionally, we examined the expression of ABA- and anthocyanin-related genes in sweet cherry following the overexpression or knockdown of a bZIP candidate gene. In total, we identified 54 bZIP-encoding genes in the sweet cherry genome. Basic leucine zipper 6 (bZIP6) showed significantly increased expression, along with increased anthocyanin accumulation in sweet cherry. Additionally, yeast one-hybrid and dual-luciferase assays indicated that PavbZIP6 enhanced the expression of anthocyanin biosynthetic genes (PavDFR, PavANS, and PavUFGT), thereby increasing anthocyanin accumulation. Moreover, PavbZIP6 interacted directly with the PavBBX6 promoter, thereby regulating PavNCED1 to promote abscisic acid (ABA) synthesis and enhance anthocyanin accumulation in sweet cherry fruit. Conclusively, this study reveals a novel mechanism by which PavbZIP6 mediates anthocyanin biosynthesis in response to ABA and contributes to our understanding of the mechanism of bZIP genes in the regulation of anthocyanin biosynthesis in sweet cherry.

Identification of PavHB16 gene in Prunus avium and validation of its function in Arabidopsis thaliana.

Sweet cherry (Prunus avium L.) is one of the most economically important fruits in the world. However, severe fruit abscission has brought significant challenges to the cherry industry. To better understand the molecular regulation mechanisms underlying excessive fruit abscission in sweet cherry, the fruit abscission characteristics, the anatomical characteristics of the abscission zone (AZ), as well as a homeodomain-Leucine Zipper gene family member PavHB16 function were analyzed. The results showed that the sweet cherry exhibited two fruit abscission peak stages, with the "Brooks" cultivar demonstrating the highest fruit-dropping rate (97.14%). During these two fruit abscission peak stages, both the retention pedicel and the abscising pedicel formed AZs. but the AZ in the abscising pedicel was more pronounced. In addition, a transcription factor, PavHB16, was identified from sweet cherry. The evolutionary analysis showed that there was high homology between PavHB16 and AtHB12 in Arabidopsis. Moreover, the PavHB16 protein was localized in the nucleus. Overexpression of PavHB16 in Arabidopsis accelerated petal shedding. In the PavHB16-overexpressed lines, the AZ cells in the pedicel became smaller and denser, and the expression of genes involved in cell wall remodeling, such as cellulase 3 gene (AtCEL3), polygalacturonase 1 (AtPG1), and expandin 24(AtEXPA24) were upregulated. The results suggest that PavHB16 may promote the expression of genes related to cell wall remodeling, ultimately facilitating fruit abscission. In summary, this study cloned the sweet cherry PavHB16 gene and confirmed its function in regulating sweet cherry fruit abscission, which provided new data for further study on the fruit abscission mechanism. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01443-8.

Genome-wide identification and expression analysis of the ALDH gene family and functional analysis of PaALDH17 in Prunus avium.

ALDH (Aldehyde dehydrogenase), as an enzyme that encodes the dehydroxidization of aldehydes into corresponding carboxylic acids, played an important role inregulating gene expression in response to many kinds of biotic and abiotic stress, including saline-alkali stress. Saline-alkali stress was a common stress that seriously affected plant growth and productivity. Saline-alkali soil contained the characteristics of high salinity and high pH value, which could cause comprehensive damage such as osmotic stress, ion toxicity, high pH, and HCO3-/CO32- stress. In our study, 18 PaALDH genes were identified in sweet cherry genome, and their gene structures, phylogenetic analysis, chromosome localization, and promoter cis-acting elements were analyzed. Quantitative real-time PCR confirmed that PaALDH17 exhibited the highest expression compared to other members under saline-alkali stress. Subsequently, it was isolated from Prunus avium, and transgenic A. thaliana was successfully obtained. Compared with wild type, transgenic PaALDH17 plants grew better under saline-alkali stress and showed higher chlorophyll content, Superoxide dismutase (SOD), Peroxidase (POD) and Catalase (CAT) enzyme activities, which indicated that they had strong resistance to stress. These results indicated that PaALDH17 improved the resistance of sweet cherries to saline-alkali stress, which in turn improved quality and yields. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01444-7.

The perennial fruit tree proteogenomics atlas: a spatial map of the sweet cherry proteome and transcriptome.

Genome-wide transcriptome analysis provides systems-level insights into plant biology. Due to the limited depth of quantitative proteomics our understanding of gene-protein-complex stoichiometry is largely unknown in plants. Recently, the complexity of the proteome and its cell-/tissue-specific distribution have boosted the research community to the integration of transcriptomics and proteomics landscapes in a proteogenomic approach. Herein, we generated a quantitative proteome and transcriptome abundance atlas of 15 major sweet cherry (Prunus avium L., cv 'Tragana Edessis') tissues represented by 29 247 genes and 7584 proteins. Additionally, 199 984 alternative splicing events, particularly exon skipping and alternative 3' splicing, were identified in 23 383 transcribed regions of the analyzed tissues. Common signatures as well as differences between mRNA and protein quantities, including genes encoding transcription factors and allergens, within and across the different tissues are reported. Using our integrated dataset, we identified key putative regulators of fruit development, notably genes involved in the biosynthesis of anthocyanins and flavonoids. We also provide proteogenomic-based evidence for the involvement of ethylene signaling and pectin degradation in cherry fruit ripening. Moreover, clusters of genes and proteins with similar and different expression and suppression trends across diverse tissues and developmental stages revealed a relatively low RNA abundance-to-protein correlation. The present proteogenomic analysis allows us to identify 17 novel sweet cherry proteins without prior protein-level annotation evidenced in the currently available databases. To facilitate use by the community, we also developed the Sweet Cherry Atlas Database (https://grcherrydb.com/) for viewing and data mining these resources. This work provides new insights into the proteogenomics workflow in plants and a rich knowledge resource for future investigation of gene and protein functions in Prunus species.

Overexpression of PavbHLH28 from Prunus avium enhances tolerance to cold stress in transgenic Arabidopsis.

BACKGROUND: The basic helix-loop-helix (bHLH) gene family is one of plants' largest transcription factor families. It plays an important role in regulating plant growth and abiotic stress response. RESULTS: In this study, we determined that the PavbHLH28 gene participated in cold resistance. The PavbHLH28 gene was located in the nucleus and could be induced by low temperature. Under the treatment of ABA, PEG, and GA3, the transcript level of PavbHLH28 was affected. At low temperature, overexpression of the PavbHLH28 gene enhanced the cold resistance of plants with higher proline content, lower electrolyte leakage (EL) and malondialdehyde (MDA) content. Compared with the WT plants, the transgenic plants accumulated fewer reactive oxygen species (ROS), and the activity and expression levels of antioxidant enzymes were significantly increased. The expression of proline synthesis enzyme genes was up-regulated, and the transcripts levels of degradation genes were significantly down-regulated. The transcripts abundance of the cold stressed-related genes in the C-repeat binding factor (CBF) pathway was not significantly different between WT plants and transgenic plants after cold stress. Moreover, the PavbHLH28 could directly bind to the POD2 gene promoter and promote its gene expression. CONCLUSIONS: Overall, PavbHLH28 enhanced the cold resistance of transgenic plants through a CBF-independent pathway, which may be partly related to ROS scavenging.

Sweet cherry AP2/ERF transcription factor, PavRAV2, negatively modulates fruit size by directly repressing PavKLUH expression.

For sweet cherry, fruit size is one of the main targets in breeding programs owing to the high market value of larger fruits. KLUH/CYP78A5 is an important regulator of seed/fruit size in several plant species, but its molecular mechanism is largely unknown. In this study, we characterized the function of PavKLUH in the regulation of sweet cherry fruit size. The ectopic overexpression of PavKLUH in Arabidopsis increased the size of its siliques and seeds, whereas virus-induced gene silencing of PavKLUH in sweet cherry significantly decreased fruit size by restricting mesocarp cell expansion. We screened out an AP2/ERF transcription factor containing a B3-like domain, designated as PavRAV2, which was able to physically interact with PavKLUH promoter in a yeast one-hybrid (Y1H) system. In Y1H assays, electrophoretic mobility shift assays, and dual-luciferase reporter analyses, PavRAV2 directly bound to the promoter of PavKLUH in vitro and in vivo, and suppressed PavKLUH expression. Silencing of PavRAV2 resulted in enlarged fruit as a result of enhanced mesocarp cell expansion. Together, our results provide new insights into signaling pathways related to fruit size, and outline a possible mechanism for how the RAV transcription factor directly regulates CYP78A family members to influence fruit size and development.

Functional analysis of sweet cherry PavbHLH106 in the regulation of cold stress.

KEY MESSAGE: Sweet cherry PavbHLH106 was up-regulated under cold induction and overexpressed to enhance the cold resistance in tobacco by mediating the scavenging of ROS through increasing of antioxidant enzyme activity. Sweet cherry (Prunus avium L.) is an economically important fruit. Chilling requirements are critical during dormancy, but abnormally low temperatures unfavorably affect fruit growth and development. Differences were found in the transcript level of PavbHLH106 under salt, dehydration, and low-temperature treatments, especially in response to cold stress, suggesting that this gene is involved in the regulation of different abiotic stresses. PavbHLH106 is homologous to Arabidopsis thaliana AtbHLH106 with a conserved bHLH domain, and transient expression in tobacco suggests that the protein is localized in the nucleus and has transcriptional activity in yeast. The PavbHLH106 overexpression in tobacco resulted in weaker electrolyte leakages, lower malondialdehyde, and higher proline content than the wild type at low-temperature treatment. Reactive oxygen species accumulation was significantly reduced in the overexpressed lines, negatively correlated with the antioxidant enzyme activity. In addition, overexpression of PavbHLH106 delayed the germination of tobacco seeds and promoted plant growth. Resistance-related genes were expressed more in the overexpressed plants compared to the wild type. PavbHLH106 bound to the PavACO promoter in yeast and potentially interacted with a bHLH162-like transcription factor. These results indicate that PavbHLH106 has various functions and is particularly active in controlling low-temperature stress.

First Report of Brown Rot Caused by Monilia yunnanensis on Sweet Cherry in China.

Chinese cherry industry has developed rapidly over the past few years, with the planting acreage continuously expanding, from Shandong province to Liaoning, Shaanxi, Hebei, Sichuan etc. Monilia spp. are the most important causal agents of brown rot of cherry, to date, M. fructicola, M. mumecola, and M. fructigena were reported to cause brown rot of cherry in China (Chen et al. 2013; Yin et al. 2014; Liu et al. 2012). In May 2023, fruit of sweet cherry cultivar 'Hongdeng' (Prunus avium L.) with symptoms resembling brown rot were collected from Tongchuan City, Shaanxi Province. Conidia on diseased tissues were spread on a water agar (WA, 1.5% agar and distilled water) medium and isolated with a glass needle under a professional single spore separation microscope (Wuhan Heipu Science and Technology Ltd., Wuhan, China). If no conidia were present, fruit pieces (5 × 5 mm) at the intersection of healthy and diseased tissues were surface sterilized with a sodium hypochlorite solution (1%) for 30 s and washed three times in sterilized water, followed by 75% ethanol for 30 s, then washed three times in sterilized water. After the tissue pieces were dried, they were placed on potato dextrose agar (PDA; 200 g of potato, 20 g of dextrose, and agar at 20 g/L) and incubated at 22 °C for about twenty days to produce spores and then single spore isolation was carried out. Thirty single-spore isolates were obtained and all were morphologically similar. The isolates produced white-gray colonies with even margins and concentric rings of sporogenous mycelium after 3 days incubation, and abundant black-colored stromata on the PDA medium after 15 days of incubation at 22°C. Conidia were one-celled, hyaline, ellipsoid to lemon shape (14.12 × 10.37 µm), with 1-2 germs which is similar to M. yunnanensis on peach. The genomic DNA of the isolates was extracted as described previously (Chi et al. 2009). The pathogen identity was confirmed by multiplex PCR which resulted in a 237bp amplicon, which is diagnostic of M. yunnanensis (Hu et al. 2011). Further sequencing of the internal transcribed spacer (ITS) region 1 and 2 and 5.8S gene (accession number: OR192774) indicated 100% identity with that of M. yunnanensis isolates (accession numbers: MW355895, ON024742). The average daily growth of mycelium on PDA at 22°C was 11.44 mm. Koch's postulates were fulfilled by inoculating 20 mature sweet cherry fruits of cv. 'Van' with mycelial plugs in a drilled hole. After 3 days of incubation at 22℃ in an airtight plastic tray with wet paper, the inoculated fruit developed typical brown rot symptoms. The developing spores on inoculated fruit were confirmed to be M. yunnanensis based on ITS sequence. All control fruit inoculated with a PDA plug remained healthy. M. yunnanensis was first reported as the causal agent of brown rot of peach in China (Hu et al. 2011). Later studies demonstrated that it is also pathogen on other fruits, e.g. hawthorn (Zhao et al. 2013), plum (Yin et al. 2015), apricot (Yin et al. 2017), apple, and pear in China (Zhu et al. 2016). To our knowledge, this is the first report of cherry brown fruit rot caused by M. yunnanensis, indicating the high risk of this species to cherry production, and effective strategies must be taken to prevent the possible control failure in practice.

First report of 'Candidatus Phytoplasma pruni' associated with X-disease on sweet cherry (Prunus avium L.) in Canada.

British Columbia (BC) is the lead producer of sweet cherries in Canada with more than 2,000 ha in production and a farm gate value of over CAD$100 million annually. Since 2010, an outbreak of little cherry disease caused by Little cherry virus 1 (LChV1) and Little cherry virus 2 (LChV2), as well as X-disease (XD) caused by 'Candidatus Phytoplasma pruni' has caused significant economic losses in neighboring Washington State (WA), USA. LChV1 and LChV2 have long been known to occur in BC (Theilmann et al. 2002); however, 'Ca. P. pruni' has not yet been reported in BC. Due to its geographical proximity to WA State, the BC cherry industry expressed significant concerns about the possible presence of the phytoplasma in cherry orchards. Accordingly, the main objective of this study was to survey cherry orchards to determine whether 'Ca. P. pruni' was present in symptomatic trees in BC. A total of 118 samples of leaves and fruit stems from individual symptomatic trees were collected prior to harvest from nine cherry orchards and one nectarine orchard in the Okanagan and Similkameen Valleys in BC. Characteristic symptoms included small and misshapen fruit with poor color development. Samples were submitted to AGNEMA, LLC (Pasco, WA) for testing using qPCR TaqMan assays for LChV1 (Katsiani et al. 2018), LChV2 (Shires et al. 2022) and 'Ca. P. pruni' (Kogej et al. 2020). Test results showed 21 samples (17.8%) from three cherry orchards positive for LChV2 and 2 samples (1.7%) from one cherry orchard positive for 'Ca. P. pruni'. In order to confirm the identification of 'Ca. P. pruni', part of the 16S ribosomal RNA gene was amplified by nested PCR using the P1/P7 followed by R16F2n/R2 primer sets (Gundersen and Lee 1996) and Sanger sequenced. BC-XD-Pa-1 (GenBank Acc. No. OR539920) and BC-XD-Pa-2 (OR537699) were identical to one another and showed 99.92% identity to the 'Ca. P. pruni' reference strain CX-95 (JQ044397). Analysis using iPhyClassifier (Zhou et al. 2009) indicated that they were 16SrIII-A strains. Interestingly, the two partial 16S sequences showed 100% nucleotide identity to strain 10324 (MH810016) and others from WA. For additional confirmation, partial secA (Hodgetts et al. 2008) and secY (Lee et al. 2010) translocases were amplified and sequenced. As with the 16S sequences, secY sequences (OR542980, OR542981) showed 99.92% nucleotide identity to strain CX-95 (JQ268249), and 100% to strain 10324 (MH810035). The secA sequences (OR542978, OR542979) had nucleotide identities of 99.77% to strain CX (MW547067), and 100% to the Green Valley strain from California (EU168733). Accordingly, 'Ca. P. Pruni' was confirmed to be present in sweet cherry samples from BC. 'Ca. P. Pruni'-related strains have been previously reported to occur in Canada in commercial poinsettias (Euphorbia pulcherrima) (Arocha-Rosete et al. 2021). To our knowledge, this is the first report of 'Ca. P. Pruni' in sweet cherry in Canada. Due to the important economic value of sweet cherries in BC, these findings are highly significant and represent the first steps towards the development of a surveillance system for early detection of XD, and consequent implementation of management strategies, including vector control. As required by federal and provincial regulations, cherry trees infected with LChV2 and 'Ca. P. Pruni' found in the survey were removed by the growers.

First report of Epicoccum nigrum causing brown leaf spot of sweet cherry (Prunus avium) in China.

Sweet cherry (Prunus avium L.) has become an important economic fruit in China, mainly produced in Shandong Province. In recent years, the planting area of Aba Prefecture in Sichuan Province has increased. In June 2022, sweet cherry brown leaf spot was found in a cherry plantation (100ha) in Wenchuan County (30°54'50.21″N, 103°24'49.10″E), with an incidence of 50 - 70%. The symptoms appeared as brown circular spots on the leaf, gradually expanding until multiple lesions coalesced to form large irregular brown spots; eventually entire leaves were killed. To isolate the causal pathogens, 10 diseased trees were randomly selected from an orchard, one diseased leaf was taken from each tree, and samples (4×4 mm2) were cut from the border between diseased and healthy tissues of 10 diseased leaves, surface sterilized with 75% ethanol for 30 sec, washed three times with sterilized water, dried on sterilized filter paper and placed on potato dextrose agar (PDA). After 5d at 25℃, five morphologically similar colonies were obtained, colony appears yellow fluffy and released a large amount of red-orangepigment. Microscopy revealed circular to ovoid, verrucose, and multicellular conidia measuring 20×25 µm diameter (n = 30) were produced on the mycelia. The morphological characteristics were consistent with the description of Epicoccum nigrum (Lima et al 2011). To further identify the strains, the internal transcribed spacer (ITS), ß-tubulin, and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4 , Bt2a/Bt2b, and 5f2/7cr (White et al. 1990; Glass and Donaldson 1995; Sung et al. 2007), respectively. BLAST analysis revealed that the ITS, ß-tubulin, and RPB2 sequences were 99.2%, 100% and 99.6% homologous, with those of E. nigrum (KU204750.1, OL782123.1, and MW602294.1), respectively. The sequences of the five isolates were identical; and those of representative strain TY3 were deposited in GenBank (ITS, OP410968; ß-tubulin, OR502448; RPB2, OP484927). Maximum likelihood phylogenetic analyses were performed for the combined data set with ITS , ß-tubulin and RPB2 using MEGA6 under the Tamura-Nei model (Tamura et al. 1993). Isolate TY3 clustered with E. nigrum type strain CBS 505.85. The pathogenicity of TY3 was tested on 10 sweet cherry trees aged 3 years (there were about 50 leaves per plant). Five plants were sprayed with 50 mL of spore suspension (1×105 spores/mL), while the controls (Five plants) were sprayed with 50 mL of sterile water. All plants were in closed plastic bags to maintain high humidity, placed in a greenhouse, and incubated at 25℃with a 12-h photoperiod. Twelve days after inoculation, 35% of the inoculated leaves showed lesions; that were consistent with those observed in the field, and the control group was asymptomatic. To confirm Koch´s postulates, two isolates were taken from the margins of leaf lesions and both were confirmed to be E. nigrum based on morphological observations and molecular identification using ITS ß-tubulin, and RPB2 sequences. This is the first report of brown leaf spot caused by E. nigrum on P. avium in China. This discovery needs to be considered in developing and implementing disease management programs in sweet cherry production.

Diagnostic and Historical Surveys of Sweet Cherry (Prunus avium) Virus and Virus-Like Diseases in Oregon.

There are over 35 known virus and virus-like diseases of sweet cherry (Prunus avium), some with potential to cause severe economic impact by reducing vegetative growth, vigor, or fruit quality. Oregon is the second-ranked state for sweet cherry production in the United States. Statewide surveys were conducted in Oregon sweet cherry orchards for virus and virus-like diversity and distribution. Orchards in key production regions with suspected virus disease symptoms were sampled. Virus-specific enzyme-linked immunosorbent assay, isothermal amplification, or quantitative real-time PCR were used to test for the presence of common or economically important sweet cherry pathogens, including cherry leaf roll virus (CLRV), little cherry virus 2 (LChV2), prune dwarf virus (PDV), prunus necrotic ringspot virus (PNRSV), tomato ringspot virus (ToRSV), and 'Candidatus Phytoplasma pruni'. CLRV, a new virus of sweet cherry in Oregon, was found associated with enation and dieback symptoms in The Dalles. Some viruses were found in new regions, which included Hood River (PDV, PNRSV, and ToRSV) and the Umpqua Valley (PDV and PNRSV). A subsequent survey was conducted in the Mid-Columbia production region for the presence of little cherry symptoms associated with little cherry and X-Diseases. All symptomatic samples from The Dalles and Mosier, OR, or Dallesport, WA, tested positive for 'Ca. P. pruni' but not LChV2. These findings provide a foundation for the current understanding and management of virus and virus-like diseases of sweet cherry in Oregon and context for further studies into these pathogens and their vectors.

Genome-Wide Identification of the SQUAMOSA Promoter-Binding Protein-like (SPL) Transcription Factor Family in Sweet Cherry Fruit.

Plant-specific SQUAMOSA promoter-binding protein-like (SPL) transcription factors play important regulatory roles during plant growth and development, fruit ripening, inflorescence branching, and biotic and abiotic stresses. However, there have been no identification or systematic studies of the SPL gene family in the sweet cherry. In this study, 12 SPL genes were identified in the sweet cherry reference genome, which were distributed over 6 chromosomes and classified into six groups according to phylogenetic relationships with other SPL gene families. Nine PavSPLs were highly expressed at green fruit stages and dramatically decreased at the onset of fruit ripening, which implied that they were important regulators during fruit development and ripening. The expression patterns of PavSPL genes under ABA, GA, and MeJA treatments showed that the PavSPLs were involved in the process of fruit ripening. A subcellular localization experiment proved that PavSPL4 and PavSPL7 proteins were localized in the nucleus. The genome-wide identification of the SPL gene family provided new insights while establishing an important foundation for sweet cherry studies.