FvemiR160-FveARF18A-FveAP1/FveFUL module regulates flowering time in woodland strawberry.

Flowering is an indicator of plant transformation from vegetative to reproductive growth. miR160 has been shown to have a significant effect on the growth and development of fruits, leaves, and roots of plants or their stress response to environment, but the participation of miR160 in regulating flowering time in plants is unclear. In this study, we found that two FvemiR160s (FvemiR160a/FvemiR160b) mature sequences in strawberry (Fragaria vesca) were consistent. It was displayed that the miR160 mature sequence is highly conserved in various species, and the miR160 mature sequence formed by the 5' arm of the MIR160 precursor was more conserved. Three FveARFs in woodland strawberry were negatively regulated by FvemiR160a, among which FveARF18A was the most significant. Phylogenetic analysis indicated that FvemiR160 is closely related to apple (Malus domestica), grape (Vitis vinifera), and Arabidopsis thaliana, while FveARF18A is closely related to RcARF18. Subsequently, we demonstrated that FvemiR160a can target cutting FveARF18A to negatively regulate its expression by RLM-5' RACE, cleavage site mutation, and GFP fluorescence assay. Moreover, we observed that FveMIR160a overexpressed plants have advanced flowering, while mFveARF18A overexpressed plants have delayed flowering. We also verified that FveARF18A negatively regulates the expression of FveAP1 and FveFUL by binding their promoters by yeast one-hybrid, LUC, and GUS assay, and FveAP1 and FveFUL transgenic Arabidopsis showed early flowering phenotype. In addition, the expression level of FvemiR160a was decreased obviously while that of FveARF18A was increased obviously by MeJA, GA and IAA. In conclusion, our study reveals the important role of the FvemiR160-FveARF18A-FveAP1/FveFUL module in the flowering process of woodland strawberry and provides a new pathway for studying flowering.

Abscisic acid controls sugar accumulation essential to strawberry fruit ripening via the FaRIPK1-FaTCP7-FaSTP13/FaSPT module.

Strawberry is considered as a model plant for studying the ripening of abscisic acid (ABA)-regulated non-climacteric fruits, a process in which sugar plays a fundamental role, while how ABA regulates sugar accumulation remains unclear. This study provides a direct line of physiological, biochemical, and molecular evidence that ABA signaling regulates sugar accumulation via the FaRIPK1-FaTCP7-FaSTP13/FaSPT signaling pathway. Herein, FaRIPK1, a red-initial protein kinase 1 previously identified in strawberry fruit, not only interacted with the transcription factor FaTCP7 (TEOSINTE BRANCHEN 1, CYCLOIDEA, and PCF) but also phosphorylated the critical Ser89 and Thr93 sites of FaTCP7, which negatively regulated strawberry fruit ripening, as evidenced by the transient overexpression (OE) and virus-induced gene silencing transgenic system. Furthermore, the DAP-seq experiments revealed that FvTCP7 bound the motif "GTGGNNCCCNC" in the promoters of two sugar transporter genes, FaSTP13 (sugar transport protein 13) and FaSPT (sugar phosphate/phosphate translocator), inhibiting their transcription activities as determined by the electrophoretic mobility shift assay, yeast one-hybrid, and dual-luciferase reporter assays. The downregulated FaSTP13 and FaSPT transcripts in the FaTCP7-OE fruit resulted in a reduction in soluble sugar content. Consistently, the yeast absorption test revealed that the two transporters had hexose transport activity. Especially, the phosphorylation-inhibited binding of FaTCP7 to the promoters of FaSTP13 and FaSPT could result in the release of their transcriptional activities. In addition, the phosphomimetic form FaTCP7S89D or FaTCP7T93D could rescue the phenotype of FaTCP7-OE fruits. Importantly, exogenous ABA treatment enhanced the FaRIPK1-FaTCP7 interaction. Overall, we found direct evidence that ABA signaling controls sugar accumulation during strawberry fruit ripening via the "FaRIPK1-FaTCP7-FaSTP13/FaSPT" module.

A clathrin-related protein FaRRP1/SCD2 integrates ABA trafficking and signaling to regulate strawberry fruit ripening.

Abscisic acid (ABA) is a critical regulator for nonclimacteric fruit ripening such as in the model plant of strawberry (Fragaria × ananassa). Although FaRRP1 is proposed to participate in clathrin-mediated endocytosis of ABA, its action molecular mechanisms in ABA signaling are not fully understood. Here, using our isolated FaRRP1 (ripening-regulation protein) and candidate ABA receptor FaPYL2 and FaABAR from strawberry fruit, a series of silico and molecular interaction analyses demonstrate that they all bind to ABA, and FaRRP1 binds both FaPYL2 and FaABAR; by contrast, the binding affinity of FaRRP1 to FaPYL2 is relatively higher. Interestingly, the binding of FaRRP1 to FaPYL2 and FaABAR affects the perception affinity to ABA. Furthermore, exogenous ABA application and FaRRP1 transgenic analyses confirm that FaRRP1 participates in clathrin-mediated endocytosis and vesicle transport. Importantly, FaRRP1, FaPYL2, and FaABAR all trigger the initiation of strawberry fruit ripening at physiological and molecular levels. In conclusion, FaRRP1 not only binds to ABA but also affects the binding affinity of FaPYL2 and FaABAR to ABA, thus promoting strawberry fruit ripening. Our findings provide novel insights into the role of FaRRP1 in ABA trafficking and signaling, at least in strawberry, a model plant for nonclimacteric fruit ripening.

FaMYB123 interacts with FabHLH3 to regulate the late steps of anthocyanin and flavonol biosynthesis during ripening.

In this work, we identified and functionally characterized the strawberry (Fragaria × ananassa) R2R3 MYB transcription factor FaMYB123. As in most genes associated with organoleptic properties of ripe fruit, FaMYB123 expression is ripening-related, receptacle-specific, and antagonistically regulated by ABA and auxin. Knockdown of FaMYB123 expression by RNAi in ripe strawberry fruit receptacles downregulated the expression of enzymes involved in the late steps of anthocyanin/flavonoid biosynthesis. Transgenic fruits showed a parallel decrease in the contents of total anthocyanin and flavonoid, especially malonyl derivatives of pelargonidin and cyanidins. The decrease was concomitant with accumulation of proanthocyanin, propelargonidins, and other condensed tannins associated mainly with green receptacles. Potential coregulation between FaMYB123 and FaMYB10, which may act on different sets of genes for the enzymes involved in anthocyanin production, was explored. FaMYB123 and FabHLH3 were found to interact and to be involved in the transcriptional activation of FaMT1, a gene responsible for the malonylation of anthocyanin components during ripening. Taken together, these results demonstrate that FaMYB123 regulates the late steps of the flavonoid pathway in a specific manner. In this study, a new function for an R2R3 MYB transcription factor, regulating the expression of a gene that encodes a malonyltransferase, has been elucidated.

Auxin biosynthesis gene FveYUC4 is critical for leaf and flower morphogenesis in woodland strawberry.

Auxin plays an essential role in plant growth and development, particularly in fruit development. The YUCCA (YUC) genes encode flavin monooxygenases that catalyze a rate-limiting step in auxin biosynthesis. Mutations that disrupt YUC gene function provide useful tools for dissecting general and specific functions of auxin during plant development. In woodland strawberry (Fragaria vesca), two ethyl methanesulfonate mutants, Y422 and Y1011, have been identified that exhibit severe defects in leaves and flowers. In particular, the width of the leaf blade is greatly reduced, and each leaflet in the mutants has fewer and deeper serrations. In addition, the number and shape of the floral organs are altered, resulting in smaller fruits. Mapping by sequencing revealed that both mutations reside in the FveYUC4 gene, and were therefore renamed as yuc4-1 and yuc4-2. Consistent with a role for FveYUC4 in auxin synthesis, free auxin and its metabolites are significantly reduced in the yuc4 leaves and flowers. This role of FveYUC4 in leaf and flower development is supported by its high and specific expression in young leaves and flower buds using GUS reporters. Furthermore, germline transformation of pYUC4::YUC4, which resulted in elevated expression of FveYUC4 in yuc4 mutants, not only rescued the leaf and flower defects but also produced parthenocarpic fruits. Taken together, our data demonstrate that FveYUC4 is essential for leaf and flower morphogenesis in woodland strawberry by providing auxin hormone at the proper time and in the right tissues.

Diversity of the volatilome and the fruit size and shape in European woodland strawberry (Fragaria vesca).

Woodland strawberry (Fragaria vesca subsp. vesca) is a wild relative of cultivated strawberry (F. × ananassa) producing small and typically conical fruits with an intense flavor and aroma. The wild strawberry species, F. vesca, is a rich resource of genetic and metabolic variability, but its diversity remains largely unexplored and unexploited. In this study, we aim for an in-depth characterization of the fruit complex volatilome by GC-MS as well as the fruit size and shape using a European germplasm collection that represents the continental diversity of the species. We report characteristic volatilome footprints and fruit phenotypes of specific geographical areas. Thus, this study uncovers phenotypic variation linked to geographical distribution that will be valuable for further genetic studies to identify candidate genes or develop markers linked to volatile compounds or fruit shape and size traits.

Secreted in Xylem 6 (SIX6) Mediates Fusarium oxysporum f. sp. fragariae Race 1 Avirulence on FW1-Resistant Strawberry Cultivars.

Fusarium oxysporum f. sp. fragariae (Fof) race 1 is avirulent on cultivars with the dominant resistance gene FW1, while Fof race 2 is virulent on FW1-resistant cultivars. We hypothesized there was a gene-for-gene interaction between a gene at the FW1 locus and an avirulence gene (AvrFW1) in Fof race 1. To identify a candidate AvrFW1, we compared genomes of 24 Fof race 1 and three Fof race 2 isolates. We found one candidate gene that was present in race 1, was absent in race 2, was highly expressed in planta, and was homologous to a known effector, secreted in xylem 6 (SIX6). We knocked out SIX6 in two Fof race 1 isolates by homologous recombination. All SIX6 knockout transformants (ΔSIX6) gained virulence on FW1/fw1 cultivars, whereas ectopic transformants and the wildtype isolates remained avirulent. ΔSIX6 isolates were quantitatively less virulent on FW1/fw1 cultivars Fronteras and San Andreas than fw1/fw1 cultivars. Seedlings from an FW1/fw1 × fw1/fw1 population were genotyped for FW1 and tested for susceptibility to a SIX6 knockout isolate. Results suggested that additional minor-effect quantitative resistance genes could be present at the FW1 locus. This work demonstrates that SIX6 acts as an avirulence factor interacting with a resistance gene at the FW1 locus. The identification of AvrFW1 enables surveillance for Fof race 2 and provides insight into the mechanisms of FW1-mediated resistance. [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.

Genome-wide analysis and functional validation reveal the role of late embryogenesis abundant genes in strawberry (Fragaria × ananassa) fruit ripening.

BACKGROUND: Late embryogenesis abundant (LEA) proteins play important roles in plant growth and development, as well as stresses responsiveness. Nowadays, it has been found that LEAs also have function in fruit ripening. However, the comprehensive analysis on a genome-wide basis of LEA family remains limited, and the role of LEA in fruit ripening has not been fully explored yet, especially in strawberry, an economic important plant and ideal material for studying fruit ripening. RESULTS: In this study, a total of 266 putative LEA proteins were identified and characterized in strawberry genome. Subcellular localization prediction indicated that they were mostly localized in chloroplast, cytoplasm and nucleus. Duplication events detection revealed that whole genome duplication or segmental was the main driver for the expansion of LEA family in strawberry. The phylogenetic analysis suggested that FaLEAs were classified into eight groups, among which, LEA2 was the largest subgroup with 179 members, followed by LEA3, dehydrin (DHN), LEA4 and SMP (seed maturation protein). The LEA1 and DHN groups were speculated to play dominant roles in strawberry fruit development and ripening, according to their larger proportion of members detected as differentially expressed genes during such process. Notably, the expression of FaLEA167 belonging to LEA1 group was altered by strawberry maturation, and inhibited by overexpression of negative regulators of ripening (a cytosolic/plastid glyceraldehyde-3-phosphate dehydrogenase, FaGAPC2 and a cytosolic pyruvate kinase, FaPKc2.2). Subsequently, overexpression of FaLEA167 significantly increased the percentage of fruit at green stage, while reduced the full red fruit proportion. In consistent, the anthocyanins content and the fruit skin color variable reflecting a range from greenness to redness (a* value) were significantly reduced. Whereas, FaLEA167 overexpression apparently up-regulated citric acid, soluble protein and malondialdehyde content, but had no obvious effects on total soluble solids, sugar, flavonoids, phenolics content and antioxidant capacity. CONCLUSIONS: These findings not only provided basic information of FaLEA family for further functional research, but also revealed the involvement of FaLEA167 in negatively regulating strawberry fruit ripening, giving new insights into understanding of FaLEA functions.

Recombination Variation Shapes Phylogeny and Introgression in Wild Diploid Strawberries.

Introgressive hybridization is widespread in wild plants and has important consequences. However, frequent hybridization between species makes the estimation of the species' phylogeny challenging, and little is known about the genomic landscape of introgression as it results from complex interactions of multiple evolutionary processes. Here, we reconstructed the phylogeny of ten wild diploid strawberries with whole genome resequencing data and then investigated the influence of recombination rate variation on phylogeny and introgression. We found that genomic regions with low recombination showed reduced levels of incomplete lineage sorting and introgression, and concentrated phylogenetic signals, thus contributing to the most likely species tree of wild diploid strawberries. We revealed complex and widespread introgression across the genus Fragaria, with an average proportion of approximately 4.1% of the extant genome. Introgression tends to be retained in the regions with high recombination rates and low gene density. Furthermore, we identified four SLF genes under selective sweeps that may play potential roles in the possible regain of self-incompatibility by ancient introgression. Altogether, our study yielded novel insights into the evolutionary history and genomic characteristics of introgression in wild diploid strawberries and provides evidence for the role of introgression in plant mating system transitions.

Genomic analyses provide insights into sex differentiation of tetraploid strawberry (Fragaria moupinensis).

The strawberry genus, Fragaria, exhibits a wide range of sexual systems and natural ploidy variation. Nearly, all polyploid strawberry species exhibit separate sexes (dioecy). Research has identified the sex-determining sequences as roughly conserved but with repeatedly changed genomic locations across octoploid strawberries. However, it remains unclear whether tetraploid wild strawberries evolved dioecy independently or shared a common origin with octoploid strawberries. In this study, we investigated the sex determinants of F. moupinensis, a dioecious plant with heterogametic females (ZW). Utilizing a combination of haplotype-resolved genome sequencing of the female F. moupinensis, k-mer-based and coverage-based genome-wide association studies (GWAS), and transcriptomic analysis, we discovered a non-recombining, approximately 33.6 kb W-specific region on chromosome 2a. Within this region, only one candidate sex-determining gene (FmoAFT) was identified. Furthermore, an extensive resequencing of the entire Fragaria genus indicated that the W-specific region displays conservative female specificity across all tetraploid species. This observation suggests that dioecy evolved independently in tetraploid and octoploid strawberries. Moreover, employing virus-induced gene silencing (VIGS), we knocked down the expression of the FmoAFT homologue transcript in cultivated strawberries, revealing its potential role in promoting female functions during early carpel development. We also applied DNA affinity purification sequencing (DAP-seq) and yeast one-hybrid assays to identify potential direct targets of FmoAFT. These insights shed new light on the genetic basis and evolutionary history of sex determination in strawberries, thereby facilitating the formulation of strategies to manipulate sex determination in breeding programs.

FveDREB1B improves cold tolerance of woodland strawberry by positively regulating FveSCL23 and FveCHS.

Cold stress has seriously inhibited the growth and development of strawberry during production. CBF/DREB1 is a key central transcription factor regulating plant cold tolerance, but its regulatory mechanisms are varied in different plants. Especially in strawberry, the molecular mechanism of CBF/DREB1 regulating cold tolerance is still unclear. In this study, we found that FveDREB1B was most significantly induced by cold stress in CBF/DREB1 family of diploid woodland strawberry. FveDREB1B was localized to the nucleus, and DREB1B sequences were highly conserved in diploid and octoploid strawberry, and even similar in Rosaceae. And FveDREB1B overexpressed strawberry plants showed delayed flowering and increased cold tolerance, while FveDREB1B silenced plants showed early flowering and decreased cold tolerance. Under cold stress, FveDREB1B activated FveSCL23 expression by directly binding to its promoter. Meanwhile, FveDREB1B and FveSCL23 interacted with FveDELLA, respectively. In addition, we also found that FveDREB1B promoted anthocyanin accumulation in strawberry leaves by directly activating FveCHS expression after cold treatment and recovery to 25°C. DREB1B genes were also detected to be highly expressed in cold-tolerant strawberry resources 'Fragaria mandschurica' and 'Fragaria nipponica'. In conclusion, our study reveals the molecular mechanism of FveDREB1B-FveSCL23-FveDELLA module and FveDREB1B-FveCHS module to enhance the cold tolerance of woodland strawberry. It provides a new idea for improving the cold tolerance of cultivated strawberry and evaluating the cold tolerance of strawberry germplasm resources.

Cold tolerance of woodland strawberry (Fragaria vesca) is linked to Cold Box Factor 4 and the dehydrin Xero2.

Domesticated strawberry is susceptible to sudden frost episodes, limiting the productivity of this cash crop in regions, where they are grown during early spring. In contrast, the ancestral woodland strawberry (Fragaria vesca) has successfully colonised many habitats of the Northern Hemisphere. Thus, this species seems to harbour genetic factors promoting cold tolerance. Screening a germplasm established in frame of the German Gene Bank for Crop Wild Relatives we identified, among 70 wild accessions, a pair contrasting with respect to cold tolerance. By following the physiological, biochemical, molecular, and metabolic responses of this contrasting pair, we identified the transcription factor Cold Box Factor 4 and the dehydrin Xero-2 as molecular markers associated with superior tolerance to cold stress. Overexpression of GFP fusions with Xero-2 in tobacco BY-2 cells conferred cold tolerance to these recipient cells. A detailed analysis of the metabolome for the two contrasting genotypes allows to define metabolic signatures correlated with cold tolerance versus cold stress. This work provides a proof-of-concept for the value of crop wild relatives as genetic resources to identify genetic factors suitable to increase the stress resilience of crop plants.

Protein kinase FaSnRK2.6 phosphorylates transcription factor FabHLH3 to regulate anthocyanin homeostasis during strawberry fruit ripening.

Strawberry (Fragaria×ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA). However, the signaling of ABA in the regulation of fruit coloration is not fully understood. Here, a transcription factor FabHLH3 key to fruit coloration is identified by yeast two hybrid library screening using FaSnRK2.6 as a bait, an ABA core signaling component negative to ripening. Indeed, this interaction is also confirmed by firefly luciferase complementation assay and pull-down assay. RT-qPCR and Western blotting analysis confirm FabHLH3 is expressed ubiquitously in strawberry and stably during fruit development. Manipulating both FabHLH3 and FaSnRK2.6 expression by overexpression and interference demonstrates that FabHLH3 and FaSnRK2.6 promote and inhibit strawberry fruit coloration, respectively, using the marker gene FaUFGT, key to anthocyanin biosynthesis. FaSnRK2.6 can phosphorylate FabHLH3, which promotes FaUFGT expression by the directly binding to its promoter. The phosphorylation inhibits the binding of FabHLH3 to FaUFGT promoter, consequently suppressing FaUFGT expression. Altogether, FaSnRK2.6, a negative kinase in ripening, interacts with and phosphorylates FabHLH3 to suppress FaUFGT expression. With the increase of ABA content in strawberry fruit ripening, the expression of FaSnRK2.6 decreased, which released FabHLH3 transcription activity and enhanced FaUFGT expression, finally promoting the coloration. Thus, our findings fill a gap how FaSnRK2.6 negatively regulates strawberry fruit coloration and ripening by FabHLH3.

Strawberry phenotypic plasticity in flowering time is driven by interaction between genetic loci and temperature.

The flowering time (FT), which determines when fruits or seeds can be harvested, is subject to phenotypic plasticity, i.e. the ability of a genotype to display different phenotypes in response to environmental variations. Here, we investigated how the environment affects the genetic architecture of FT in cultivated strawberry (Fragaria ×ananassa) and modifies its QTL effects. To this end, we used a bi-parental segregating population grown for two years at widely divergent latitudes (5 European countries) and combined climatic variables with genomic data (Affymetrix® SNP array). Examination, using different phenological models, of the response of FT to photoperiod, temperature and global radiation, indicated that temperature is the main driver of FT in strawberry. We next characterized in the segregating population the phenotypic plasticity of FT by using three statistical approaches that generated plasticity parameters including reaction norm parameters. We detected 25 FT QTL summarized into 10 unique QTL. Mean values and plasticity parameters QTL were co-localized in three of them, including the major 6D_M QTL whose effect is strongly modulated by temperature. The design and validation of a genetic marker for the 6D_M QTL offers great potential for breeding programs, for example for selecting early-flowering strawberry varieties well adapted to different environmental conditions.

Pseudomonas fragariae sp. nov., a novel bacterial species causing leaf spots on strawberry (Fragaria×ananassa).

In Florida, angular leaf spot, caused by Xanthomonas fragariae, was the only known bacterial disease in strawberry, which is sporadic and affects the foliage and calyx. However, from the 2019-2020 to 2023-2024 Florida strawberry seasons, unusual bacterial-like symptoms were observed in commercial farms, with reports of up to 30 % disease incidence. Typical lesions were water-soaked and angular in early stages that later became necrotic with a circular-ellipsoidal purple halo, and consistently yielded colonies resembling Pseudomonas on culture media. Strains were pathogenic on strawberry, fluorescent, oxidase- and arginine-dihydrolase-negative, elicited a hypersensitive reaction on tobacco, and lacked pectolytic activity. Although phenotypic assays, such as fatty acid methyl profiles and Biolog protocols, placed the strains into the Pseudomonas group, there was a low similarity at the species level. Further analysis using 16S rRNA genes, housekeeping genes, and whole genome sequencing showed that the strains cluster into the Pseudomonas group but do not share more than 95 % average nucleotide identity compared to representative members. Therefore, the genomic and phenotypic analysis confirm that the strains causing bacterial spot in strawberry represent a new plant pathogenic bacterial species for which we propose the name Pseudomonas fragariae sp. nov. with 20-417T (17T=LMG 32456T=DSM 113340 T) as the type strain, in relation to Fragaria×ananassa, the plant species from which the pathogen was first isolated. Future work is needed to assess the epidemiology, cultivar susceptibility, chemical sensitivity, and disease management of this possible new emerging strawberry pathogen.

Rapid immunochemical methods for the analysis of proquinazid in strawberry QuEChERS extracts.

Proquinazid is a new-generation fungicide authorized in the EU for combating powdery mildew infections in high-value crops. Due to the perishable nature of fruits, alternative analytical methods are necessary to protect consumer's health from pesticide residues. Currently, immunoassays are a well-established approach for rapidly monitoring chemical contaminants. However, the production of high-quality immunoreagents, such as antibodies and bioconjugates, is essential. This study presents a newly designed hapten that maintains the characteristic moieties of proquinazid unmodified. The linear aliphatic substituents of this molecule were used to introduce the spacer arm. A three-step synthesis strategy was optimized to prepare a hapten that displays the entire 6-iodoquinazolin-4(3H)-one moiety with excellent yields. The N-hydroxysuccimidyl ester of the hapten was activated and purified to prepare a protein conjugate with high hapten density, which was used as an immunogen. Antibodies were raised and competitive enzyme-linked immunosorbent assays were developed. To enhance the assay's sensitivity, two additional heterologous haptens were prepared by modifying the halogenated substituent at C-6. The optimized assays demonstrated low limits of detection in buffer, approximately 0.05 µg/L. When applied to the analysis of proquinazid in QuEChERS extracts of strawberry samples, the immunoassays produced precise and accurate results, particularly in the 10-1000 µg/kg range.

Fractionation of metals in soil for strawberry cultivation: Effect on metal migration in food chain and application in risk assessment.

Although trace metals in strawberry production system have attracted growing attention, little is known about metal fractionation in soil for strawberry cultivation. We hypothesized that the metal fractions in soil influenced by strawberry production had significant effect on food chain transport of metals and their risk in soil. Here, samples of strawberries and soil were gathered in the Yangtze River Delta, China to verify the hypothesis. Results showed that the acid-soluble Cr, Cd, and Ni in soil for strawberry cultivation were 21.5%-88.3% higher than those in open field soil, which enhanced uptake and bioaccessible levels of these metals in strawberries. Overall, the ecological, mobility, and health risks of Pb, Zn, Ni, and Cu in soil were at a low level. However, the ecological risk of bioavailable Cd, mobility risk of Cd, and cancer risk of bioavailable Cr in over 70% of the soil samples were at moderate, high, and acceptable levels, respectively. Since the increased acid-soluble Cr and Ni in soil were related to soil acidification induced by strawberry production, nitrogen fertilizer application should be optimized to prevent soil acidification and reduce transfer of Cr and Ni. Additionally, as Cd and organic matter accumulated in soil, the acid-soluble Cd and the ecological and mobility risks of Cd in soil were enhanced. To decrease transfer and risk of Cd in soil, organic fertilizer application should be optimized to mitigate Cd accumulation, alter organic matter composition, and subsequently promote the transformation of bioavailable Cd into residual Cd in soil.

Development of a Large-Scale Soil DNA Extraction Method for Molecular Quantification of Fusarium oxysporum f. sp. fragariae in Soil.

The most common soilborne diseases affecting the strawberry industry in California include Verticillium wilt due to Verticillium dahliae, charcoal root rot due to Macrophomina phaseolina, and Fusarium wilt due to Fusarium oxysporum f. sp. fragariae. Detection of these pathogens in soil is an important facet of disease management and fumigation recommendations. Whereas the soil populations of both M. phaseolina and V. dahliae can be readily quantified with quantitative PCR (qPCR) assays using DNA extractions with 500 mg of soil, the single-cell nature of the F. oxysporum chlamydospore does not provide enough pathogen DNA from 500-mg extractions to be reliably quantified. Here, we describe an improved DNA extraction protocol from 10 to 15 g of soil that allows for the quantification of F. oxysporum f. sp. fragariae populations below 10 CFU/g. The relationship between results from the TaqMan qPCR assay and pathogen population density in soil was determined by using this extraction method in pathogen-free soils artificially infested with a hygromycin-resistant strain of F. oxysporum f. sp. fragariae to facilitate accurate colony counts when plated on a selective medium. Although the protocol was developed for F. oxysporum f. sp. fragariae, it is applicable for detection and quantification of other soilborne pathogens.

RNA interference-based strategies to control Botrytis cinerea infection in cultivated strawberry.

KEY MESSAGE: Gene silencing of BcDCL genes improves gray mold disease control in the cultivated strawberry. Gene silencing technology offers new opportunities to develop new formulations or new pathogen-resistant plants for reducing impacts of agricultural systems. Recent studies offered the proof of concept that the symptoms of gray mold can be reduced by downregulating Dicer-like 1 (DCL1) and 2 (DCL2) genes of Botrytis cinerea. In this study, we demonstrate that both solutions based on dsRNA topical treatment and in planta expression targeting BcDCL1 and BcDCL2 genes can be used to control the strawberry gray mold, the most harmful disease for different fruit crops. 50, 70 and 100 ng µL-1 of naked BcDCL1/2 dsRNA, sprayed on plants of Fragaria x ananassa cultivar Romina in the greenhouse, displayed significant reduction of susceptibility, compared to the negative controls, but to a lesser extent than the chemical fungicide. Three independent lines of Romina cultivar were confirmed for their stable expression of the hairpin gene construct that targets the Bc-DCL1 and 2 sequences (hp-Bc-DCL1/2), and for the production of hp construct-derived siRNAs, by qRT-PCR and Northern blot analyses. In vitro and in vivo detached leaves, and fruits from the hp-Bc-DCL1/2 lines showed significantly enhanced tolerance to this fungal pathogen compared to the control. This decreased susceptibility was correlated to the reduced fungal biomass and the downregulation of the Bc-DCL1 and 2 genes in B. cinerea. These results confirm the potential of both RNAi-based products and plants for protecting the cultivated strawberry from B. cinerea infection, reducing the impact of chemical pesticides on the environment and the health of consumers.

A multidisciplinary approach to the antioxidant and hepatoprotective activities of Arbutus pavarii Pampan fruit; in vitro and in Vivo biological evaluations, and in silico investigations.

The Libyan Strawberry, Arbutus pavarii Pampan (ARB), is an endemic Jebel Akhdar plant used for traditional medicine. This study presents the antioxidant and hepatoprotective properties of ARB fruit-extract. ARB phytochemical analysis indicated the presence of 354.54 GAE and 36.2 RE of the phenolics and flavonoids. LC-MS analysis identified 35 compounds belonging to phenolic acids, procyanidins, and flavonoid glycosides. Gallic acid, procyanidin dimer B3, ß-type procyanidin trimer C, and quercetin-3-O-glucoside were the major constituents of the plant extract. ARB administration to paracetamol (PAR)-intoxicated rats reduced serum ALT, AST, bilirubin, hepatic tissue MDA and proinflammatory markers; TNF-α and IL-6 with an increase in tissue GSH level and SOD activity. Histological and immunohistochemical studies revealed that ARB restored the liver histology and significantly reduced the tissue expression of caspase 3, IL-1B, and NF-KB in PAR-induced liver damage. Docking analysis disclosed good binding affinities of some compounds with XO, COX-1, 5-LOX, and PI3K.