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MicroRNAs (miRNAs) are a kind of short noncoding RNA (20–24 nt), playing versatile roles in plant growth and development. Strawberry generates leaves and flowers with unique features. However, few miRNAs have been functionally characterised in strawberry, especially for their developmental regulation. Here, we identified one ethyl methanesulfonate (EMS) mutant, deeply serrated (des), in the woodland strawberry Fragaria vesca that has wrinkled leaves with deeper serrations, serrated petals and deformed carpels. The causative mutation occurs in the 19th nucleotide of the FvemiR164a mature sequence. Overexpressing FveMIR164A rescued the phenotypes of des/fvemir164a except the petal serrations. Furthermore, we identified two allelic mutants of FveCUC2a, one target of FvemiR164a, which developed leaves with smooth margins and fused leaflets. Phenotypes of the double mutant fvemir164a fvecuc2a indicated that the two genes act linearly in leaf and carpel development, but synergistically in the development of other floral organs and inflorescence architecture. This work demonstrates the conserved and novel roles of the miR164-CUC2 module in leaf and flower development in different plant species, and reveals that the 19th nucleotide of FvemiR164a is important for its processing.

Antisense-mediated down-regulation of the fruit-specific polygalacturonase (PG) gene FaPG1 in strawberries (Fragaria×ananassa Duch.) has been previously demonstrated to reduce fruit softening and to extend post-harvest shelf life, despite the low PG activity detected in this fruit. The improved fruit traits were suggested to be attributable to a reduced cell wall disassembly due to FaPG1 silencing. This research provides empirical evidence that supports this assumption at the biochemical, cellular, and tissue levels. Cell wall modifications of two independent transgenic antisense lines that demonstrated a >90% reduction in FaPG1 transcript levels were analysed. Sequential extraction of cell wall fractions from control and ripe fruits exhibited a 42% decrease in pectin solubilization in transgenic fruits. A detailed chromatographic analysis of the gel filtration pectin profiles of the different cell wall fractions revealed a diminished depolymerization of the more tightly bound pectins in transgenic fruits, which were solubilized with both a chelating agent and sodium carbonate. The cell wall extracts from antisense FaPG1 fruits also displayed less severe in vitro swelling. A histological analysis revealed more extended cell–cell adhesion areas and an enhanced tissue integrity in transgenic ripe fruits. An immunohistological analysis of fruit sections using the JIM5 antibody against low methyl-esterified pectins demonstrated a higher labelling in transgenic fruit sections, whereas minor differences were observed with JIM7, an antibody that recognizes highly methyl-esterified pectins. These results support that the increased firmness of transgenic antisense FaPG1 strawberry fruits is predominantly due to a decrease in pectin solubilization and depolymerization that correlates with more tightly attached cell wall-bound pectins. This limited disassembly in the transgenic lines indicates that these pectin fractions could play a key role in tissue integrity maintenance that results in firmer ripe fruit.

The diploid woodland strawberry, Fragaria vesca, is being recognized as a model for the more complex octoploid commercial strawberry, Fragaria × ananassa. F. vesca exhibits a short seed to seed cycle, can be easily transformed by Agrobacteria, and a draft genome sequence has been published. These features, together with its similar flower structure, potentially make F. vesca a good model for studying the flower development of other members of the Rosaceae family, which contains many economically important fruit trees and ornamental plants. To propel F. vesca’s role in genetic and genomic research and to facilitate the study of its reproductive development, we have investigated in detail F. vesca flower and early fruit development using a seventh generation inbred diploid line, Yellow Wonder 5AF7. We present here standardized developmental staging and detailed descriptions of morphological changes associated with flower and early fruit development based on images of hand dissected flowers, histological sections, and scanning electron microscopy. In situ hybridization with the F. vesca AGAMOUS homolog, FvAG, showed expression in young stamen and carpel primordia. This work lays the essential groundwork and standardization for future molecular, genetic, and genomic studies of F. vesca.

Pectins are essential components of primary plant cell walls and middle lamellae, and are related to the consistency of the fruit and its textural changes during ripening. In fact, strawberries become soft as the middle lamellae of cortical parenchyma cells are extensively degraded during ripening, leading to the observed short post-harvest shelf life. Using a custom-made oligonucleotide-based strawberry microarray platform, a putative rhamnogalacturonate lyase gene (FaRGlyase1) was identified. Bioinformatic analysis of the FaRGlyase1 sequence allowed the identification of a conserved rhamnogalacturonate lyase domain, which was also present in other putative RGlyase sequences deposited in the databases. Expression of FaRGlyase1 occurred mainly in the receptacle, concurrently with ripening, and it was positively regulated by abscisic acid and negatively by auxins. FaRGLyase1 gene expression was transiently silenced by injecting live Agrobacterium cells harbouring RNA interference constructs into fruit receptacles. Light and electron microscopy analyses of these transiently silenced fruits revealed that this gene is involved in the degradation of pectins present in the middle lamella region between parenchymatic cells. In addition, genetic linkage association analyses in a strawberry-segregating population showed that FaRGLyase1 is linked to a quantitative trait loci linkage group related to fruit hardness and firmness. The results showed that FaRGlyase1 could play an important role in the fruit ripening-related softening process that reduces strawberry firmness and post-harvest life.

Commercial strawberries are mainly propagated using daughter plants produced on aerial runners because asexual propagation is faster than seed propagation, and daughter plants retain the characteristics of the mother plant. This study was conducted to investigate the effective factors for runner induction, as well as the molecular mechanisms behind the runner induction. An orthogonal test with 4 factors (photoperiod, temperature, gibberellin, and 6-benzyladenine), each with 3 levels was performed. Proteins were also extracted from the crowns with or without runners and separated by two-dimensional electrophoresis. The results of the orthogonal test showed that a long-day (LD) environment was the most influential factor for the runner formation, and 50 mg·L−1 of 6-BA significantly increased the number of runners. A proteomic analysis revealed that 32 proteins were differentially expressed (2-fold, p < 0.05) in the strawberry crowns with and without runners. A total of 16 spots were up-regulated in the crowns with runners induced by LD treatment. Identified proteins were classified into seven groups according to their biological roles. The most prominent groups were carbohydrate metabolism and photosynthesis, which indicated that the carbohydrate content may increase during runner formation. A further analysis demonstrated that the soluble sugar content was positively correlated with the number of runners. Thus, it is suggested that the photoperiod and 6-BA break the dormancy of the axillary buds and produce runners by increasing the soluble sugar content in strawberry.

Nanotechnology-based pesticide delivery systems provide controlled release of agrochemicals along with enhanced stability, target specificity, and reduced environmental impact. Strawberries ( Fragaria × ananassa ), valued for their flavor, nutritional content, and antioxidant properties, face a short shelf-life and significant postharvest losses due to Botrytis cinerea ( B. cinerea )-induced decay, posing a challenge to producers. This study reports the development of a biocompatible, botanical nano-formulated fungicide (BNF) to manage B. cinerea , as a resistant post-harvest pathogen. BNF, encapsulating citrus peel essential oil (CEO) within a biocompatible inorganic-organic matrix consisting of silica nanoporous and polyethylene glycol (PEG), was characterized using scanning electron microscopy (SEM), transmission electron microscopy (TEM), Thermogravimetric Analysis (TGA), Gas chromatography-mass spectrometry (GC-MS) analysis, and Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) methods. To evaluate the efficacy of our treatments against B. cinerea , Signum (BASF), a commercial fungicide, was used as a positive control. Its antifungal efficacy was assessed at four different concentrations (0, 5000, 7000, and 10000 mg L −1 , when BNF contains 10% of the active ingredient) and Signum (1000 mg L −1 ) against B. cinerea on strawberry flowers and postharvest fruits. Results indicate that BNF at 5000, 7000, and 10,000 mg L⁻¹ significantly inhibited the growth of B. cinerea ( p  ≤ 0.01) and reduced fruit spoilage. Notably, BNF at a concentration of 10,000 mg L⁻¹ achieved 74.03% inhibition, closely comparable to Signum (77.52% inhibition), while the water-treated control (BNF 0 ) showed only minimal inhibition (13.01%). These findings demonstrate BNF’s potential as an effective and sustainable alternative to conventional fungicides for managing B. cinerea in strawberry production, with a recommended dose of 10,000 mg L⁻¹ for optimal efficacy. This study demonstrates that BNF is a promising eco-friendly alternative to synthetic fungicides for controlling B. cinerea , enhancing strawberry shelf life, and reducing chemical residues, with potential utilization in other crops.

Xanthomonas phage RiverRider is a novel N4-like bacteriophage and the first phage isolated from the plant pathogen Xanthomonas fragariae. Electron microscopy revealed a Podoviridae morphology consisting of isometric heads and short noncontractile tails. The complete genome of RiverRider is 76,355 bp in length, with 90 open reading frames and seven tRNAs. The genome is characteristic of N4-like bacteriophages in both content and organization, having predicted proteins characterized into the functional groups of transcription, DNA metabolism, DNA replication, lysis, lysis inhibition, structure and DNA packaging. Amino acid sequence comparisons for proteins in these categories showed highest similarities to well-characterized N4-like bacteriophages isolated from Achromobacter xylosoxidans and Erwinia amylovora. However, the tail fiber proteins of RiverRider are clearly distinct from those of other N4-like phages. RiverRider was able to infect seven different strains of X. fragariae and none of the other species of Xanthomonas tested.

To develop new agents against strawberry grey mould and to aid in the development of biological pesticides, we investigated the inhibitory effect of a natural compound, phenazine-1-carboxamide (PCN), against Botrytis cinerea using a growth rate assay. Additionally, indoor toxicity and the in vitro control effect of PCN were further studied to determine its potential mechanisms of action on B. cinerea. PCN was inhibitory against B. cinerea with a 50% effective concentration (EC50) of 108.12 μg/mL; the toxicity of PCN was equivalent to that of carbendazim (CBM). The best in vitro control effect of PCN against grey mould in strawberry (fruit) reached 75.32%, which was slightly higher than that of CBM. The field control effect of PCN against grey mould reached a maximum of 72.31% at a PCN concentration of 700 μg/mL, which was 1.02 times higher than that of CBM. Fungistatic activity was observed at low concentrations of PCN, while high concentrations of PCN resulted in fungicidal activity against B. cinerea. This natural compound strongly inhibited both spore and sclerotium germination of B. cinerea, with the best relative inhibition rates of 77.03% and 82.11%, respectively. The inhibitory effect of PCN on mycelial growth of B. cinerea was significant and reached levels of 87.32%. Scanning electron microscopy observations revealed that after 48 h of PCN treatment, the mycelia appeared loose, locally twisted, and folded, with exudation of contents; the mycelia was withered and twisted, with edge burrs, deformations, ruptures and a sheet-like structure. Transmission electron microscopy observations revealed that after 48 h of PCN treatment, the structure of the cell nucleus was unclear and the vacuoles had ruptured; additionally, various organelles exhibited disordered structures, there were substantial non-membrane transparent inclusions, the cells were plasmolysed, the cell walls were collapsed in some cases, and the hyphal tissue was essentially necrotic. A PCN dosage of 35-140 μg/mL had no effect on the cell membrane permeability of the mycelia, while a PCN dosage of 700 μg/mL resulted in significant permeability. PCN inhibited B. cinerea toxin; the mycotoxin level was approximately 0.41 of the value recorded for the control at a PCN dosage of 700 μg/mL. PCN affected the activity of pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β-glucosidase (BG); the lowest activities of PMG, PG, BG and Cx reached 0.3 U/mg, 0.62 U/mg, 0.64 U/mg, and 0.79 U/mg, respectively, after treatment with 700 μg/mL PCN.

Two biocontrol agents, a yeast (Pichia guilermondii) and a bacterium (Bacillus mycoides), were tested separately and together for suppression of Botrytis cinerea on strawberry leaves and plants. Scanning electron microscopy revealed significant inhibition of Botrytis cinerea conidial germination in the presence of Pichia guilermondii, whereas Bacillus mycoides caused breakage and destruction of conidia. When both biocontrol agents were applied in a mixture, conidial destruction was more severe. The modes of action of each of the biocontrol agents were elucidated and the relative quantitative contribution of each mechanism to suppression of Botrytis cinerea was estimated using multiple regression with dummy variables. The improvement in control efficacy achieved by introducing one or more mechanisms at a time was calculated. Pichia guilermondii competed with Botrytis cinerea for glucose, sucrose, adenine, histidine, and folic acid. Viability of the yeast cells played a crucial role in suppression of Botrytis cinerea and they secreted an inhibitory compound that had an acropetal effect and was not volatile. Bacillus mycoides did not compete for any of the sugars, amino acids, or vitamins examined at a level that would affect Botrytis cinerea development. Viable cells and the compounds secreted by them contributed similarly to Botrytis cinerea suppression. The bacteria secreted volatile and non-volatile inhibitory compounds and activated the defense systems of the host. The nonvolatile compounds had both acropetal and basipetal effects. Mixture of Pichia guilermondii and Bacillus mycoides resulted in additive activity compared with their separate application. The combined activity was due to the summation of biocontrol mechanisms of both agents. This work provides a theoretical explanation for our previous findings of reduced disease control variability with a mixture of Pichia guilermondii and Bacillus mycoides.

A strawberry (Fragaria x ananassa cv. Chandler) fruit cDNA (Fahyprp-cDNA) and its corresponding gene (Fahyprp) showing sequence homology to higher plant hyprp genes have been isolated. The cDNA contains an open reading frame encoding a 16 kDa protein with 156 amino acids. The peptide has an amino-terminal signal sequence, a repetitive proline-rich sequence, and a cysteine-rich carboxy-terminal region homologous to other HyPRP proteins. Northern blot and QRT-PCR analysis have shown that the strawberry transcript is specifically expressed in fruit, not being detected in other plant tissues. \"In situ\" hybridization and immunolocalization studies have indicated that the Fahyprp gene is strongly expressed in achene sclerenchyma cells, in the vascular and receptacle cells of immature green fruit and in the vascular cells of mature red fruits. The achenes removal from unripe green fruits induced the expression of this Fahyprp gene. This induction was reverted by treatment of deachened fruit with the auxin NAA, supporting the idea that Fahyprp gene expression is regulated by auxins. Furthermore, the HyPRP protein has been localized in parenchymatic cells of immature fruits associated to structures containing condensed tannins. The results are discussed supporting a putative role of this protein in the anchoring of polymeric polyphenols in the strawberry fruit during growth and ripening.