Apple canker disease, also named as apple Valsa canker, is one of the most destructive diseases for apples (Malus domestica Borkh.). Cytospora/Valsa spp. are the dominant causal agent of this disease, but many studies have revealed that fungi from some other genus can also cause typical apple canker symptoms. In this study, we performed fungal pathogen isolation from cankered ‘Fuji’ apple barks. Six representative morphologically different fungi (Strain 1–6) were further subjected to ITS sequencing and evolutionary analysis. Molecular identification results revealed that Strains 1–6 are Cytospora mali, Fusarium cf. solani, Alternaria alternata, C. mali, Diplodia seriata and F. proliferatum, respectively. All these fungi have been reported to be causal agents of apple diseases. By inoculating fungal plugs onto trunks of ‘Fuji’ apple trees, the pathogenicity of the six fungi were accessed. Only the inoculations of the two C. mali strains (Strain 1 and Strain 4) and the A. alternata strain (Strain 3) resulted in typical apple canker symptoms in trunks. It is worth noting that Strain 1 caused much more severe canker symptoms and higher pathogenicity incidence than the other two fungi. A. alternata has been identified as a pathogen causing diseases on apple fruits and leaves. By further assessing its pathogenicity on apple fruits and leaves, we verified that it can also cause typical fruit rot and leaf spot symptoms. To the best of our knowledge, this is the first report on apple canker disease caused by A. alternata in China. Our present study can provide a theoretical foundation for the prevention and control of apple canker disease.

Piriformospora/Serendipita indica has been frequently proved to play a crucial role in enhancing plant adaptation to environmental stresses. However, its influence on blueberry (Vaccinium corymbosum) drought tolerance has not yet been studied. Here, we reported that P. indica colonization can significantly enhance the drought tolerance of blueberry. Physio-biochemical parameter determination results showed that, compared to non-colonized controls (CK), P. indica-colonized (PI) plants exhibited higher leaf chlorophyll and carotenoids contents, photosynthetic capacity, biomass and root antioxidant enzyme activities (superoxide dismutase and catalase), while also exhibiting lower root malondialdehyde content under drought stress (DS). To explore the underlying mechanism, comparative root transcriptome analysis of well-watered (WW) and DS-treated CK and PI blueberry plants was conducted. In total, we identified 14,587 differentially expressed genes (DEGs) across CK-WW vs. CK-DS, PI-WW vs. PI-DS, CK-WW vs. PI-WW and CK-DS vs. PI-DS comparisons. Under DS, stress-, metabolism- and regulation-related DEGs were overwhelmingly upregulated in PI, while being downregulated in CK. Weighted gene co-expression network analysis categorized DEGs into four modules. Of them, the MEblack module was significantly correlated with the PI-DS group, with DEGs enriched in the cell wall macromolecule catabolic process, carbohydrate metabolic process, phenylpropanoid biosynthesis, and so on. Several defense-related genes, including four thaumatin family proteins, were identified as hub genes of this module. DEGs in the MEblue module were expressed at the highest level in CK-DS, followed by in PI-DS. Hub genes of the MEblue module included DEG-encoding lipid transfer protein, abscisic stress ripening protein, and so on. This study demonstrates that P. indica enhances blueberry drought tolerance by enhancing antioxidant ability and mediating the expression of genes related to stress, carbohydrate and secondary metabolism, and cell wall metabolism.

Dihydroflavonol 4-reductase (DFR) genes contribute greatly to anthocyanin biosynthesis in plants. Up to now, however, research on the DFR gene family and the key anthocyanin-related DFR members in blueberries (Vaccinium corymbosum) has been limited. In this study, we performed a genome-wide identification of the blueberry DFR gene family, identifying 36 VcDFR genes categorized into five subfamilies. Gene expression analysis showed that three Subfamily III members (VcDFR11/29/34) and four Subfamily V members (VcDFR4/7/30/33) are highly expressed in blueberry fruits, particularly at late ripening stages. Transient overexpression analysis in apple fruits verified the contributions of VcDFR11 and VcDFR30 to anthocyanin biosynthesis, with VcDFR11 showing better promoting effects. Blueberry fruit-based transient overexpression further confirmed the promoting effects of VcDFR11 on anthocyanin accumulation and the expression of anthocyanin-related structural genes (especially its downstream anthocyanindin synthase (ANS) and UDP-glucose: flavonoid 3-O-glycosyltransferase (UFGT) genes). The VcDFR11 promoter contains binding sites for both bHLH and MYB transcription factors (TFs). Consistently, yeast one-hybrid and dual-luciferase assays confirmed that anthocyanin-related VcMYB-1 and VcbHLHs can bind to and activate the VcDFR11 promoter. Furthermore, co-overexpressing VcMYB-1/VcbHLHs with VcDFR11 led to much higher anthocyanin accumulation than overexpressing VcDFR11 alone, indicating that these TFs positively regulate anthocyanin biosynthesis by upregulating VcDFR11. In summary, our study characterized the blueberry DFR gene family and demonstrated the role of VcDFR11 in anthocyanin biosynthesis.

C-terminally encoded peptides (CEPs), a class of post-translationally modified peptides, have been discovered to be potential mediators of abiotic stress responses in several plants. In this study, to explore the possible roles of CEPs in blueberry salt and osmotic stress responses, a genome-wide identification of CEP genes was first conducted. In total, we identified 12 VcCEPs that can be further categorized into three subfamilies in blueberry. Collinearity analysis identified 14 segmental duplicated gene pairs involving four Subfamily I members and five Subfamily III members among VcCEPs, indicating that the expansion of the blueberry CEP gene family was promoted by the segmental duplication events of Subfamily I and Subfamily III members. Promoter analysis revealed that all the VcCEPs’ promoters contain drought-inducible and defence and stress-related elements, suggesting that they might contribute to the stress responses of blueberry. Gene expression analysis showed that, of the 12 VcCEPs, VcCEP1, VcCEP4, VcCEP5 and VcCEP6 expressed in blueberry root and/or leaf, with varied expression levels under salt and drought treatments, while others showed no expression or very low expression. We further identified CEP mature peptide (CEPm) sequences in VcCEPs, and found that nine VcCEPs contain one mature peptide, while VcCEP7, VcCEP2 and VcCEP10 contain 3, 2, and 2 mature peptide sequences, respectively. The CEPms of Subfamily III members (VcCEP7~11) share more than 60% similarity with reported stress-related CEP mature peptides from other plant species, indicating that they might function in blueberry stress responses. Additionally, exogenous application of different synthesized CEP mature peptides alleviated blueberry leaf yellowing/browning under 150 mM NaCl/500 mM mannitol treatments, with CEP7a*2 and CEP7a*2 + 7c showing the best effects. These findings indicate that their functions are concentration-dependent to some extent. Collectively, our study characterized the CEP genes in blueberry and can provide a basis for the future applications of CEPs in enhancing blueberry stress tolerance.