Intragenesis is an all-native engineering technology for crop improvement. Using an intragenic strategy to bring genes from wild species to cultivated strawberry could expand the genetic variability. A robust regeneration protocol was developed for the strawberry cv. ‘Shanghai Angel’ by optimizing the dose of Thidiazuron and identifying the most suitable explants. The expression cassette was assembled with all DNA fragments from F. vesca, harboring a sugar transporter gene FvSTP8 driven by a fruit-specific FvKnox promoter. Transformed strawberry was developed through an Agrobacterium-mediated strategy without any selectable markers. Other than PCR selection, probe-based duplex droplet digital PCR (ddPCR) was performed to determine the T-DNA insert. Four independent transformed shoots were obtained with a maximum of 5.3% efficiency. Two lines were confirmed to be chimeras, while the other two were complete transformants with six and 11 copies of the intragene, respectively. The presence of a vector backbone beyond the T-DNA in these transformants indicated that intragenic strawberries were not obtained. The current work optimized the procedures for producing transformed strawberry without antibiotic selection, and accurately determined the insertion copies by ddPCR in the strawberry genome for the first time. These strategies might be promising for the engineering of ‘Shanghai Angel’ and other cultivars to improve agronomic traits.

The Colletotrichum fructicola (C. fructicola) is a hemibiotrophic fungus, which causes devastating anthracnose in strawberry. At present, the resistance mechanism to C. fructicola remains poorly understood. Here, we used RNA-sequencing and liquid chromatography-mass spectrometry (LC-MS) metabolomics to excavate the molecular mechanism of strawberry resistance to C. fructicola. The differentially accumulated metabolites (DAMs) and differentially expressed genes (DEGs) were screened at different stages after C. fructicola infection in the susceptible ‘Benihoppe’ and resistant cultivar ‘Sweet Charlie’. The core common DEGs with high association of common DAMs were identified by multi-omics integration analysis, and showed convergence and divergence in the two strawberry cultivars. Strikingly, the phenylpropanoids biosynthesis was simultaneously enriched in a multi-level omics at different stages after C. fructicola infection in the resistant (R) and susceptible (S) strawberries. Furthermore, we constructed the DEGs-DAMs map of phenylpropanoid biosynthesis. More importantly, we showed that chloroplasts and starch and sugar metabolism related genes, such as chlorophyII A-B binding genes, glycosyl hydrolase (GH) family genes and so on, were differentially expressed. Taken together, our study revealed major changes in genes and metabolites expression associated with C. fructicola resistance, and identified the multi-level regulatory network based on phenylpropanoid biosynthesis, useful for further mechanistic excavation of resistance to C. fructicola in strawberries.

Strawberry anthracnose, caused by Colletotrichum spp., is a devastating disease that significantly reduces strawberry yield and quality. This study aimed to develop a simple diagnostic method to detect infection by the Colletotrichum gloeosporioides species complex (CGSC), the most predominant and virulent Colletotrichum species complex causing strawberry anthracnose in China. In this study, a Cas12aVIP diagnostic method was developed for the rapid detection of the CGSC in strawberry seedlings. This method targets the β-tubulin gene and combines recombinase polymerase amplification (RPA), the CRISPR/Cas12a system, and a cationic-conjugated polythiophene derivative [poly(3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride) (PMNT)] mixed with single-stranded DNA. This method shows high sensitivity (10 copies per reaction) and no cross-reactivity against related pathogens. The entire procedure, from sample to result, can be completed within 50 min, including simplified DNA extraction (15 min), RPA reaction (37°C for 20 min), CRISPR/Cas12a detection (37°C for 10 min), and visual detection by the naked eye (1 to 2 min). Furthermore, the Cas12aVIP assay successfully detected the CGSC in naturally infected strawberry seedling samples in field conditions. Asymptomatic infected plants and plant residues have been identified as primary inoculum sources for the CGSC. This method enables visible detection without the need for expensive equipment or specialized technical skills, thereby offering an efficient and straightforward approach for detecting the CGSC in strawberries. The newly developed detection method can be used to promote healthier strawberry production.