Litchi (Litchi chinensis) is an evergreen fruit tree grown in subtropical and tropical countries. China accounts for 71.5% of the total litchi cultivated area in the world. Anthracnose disease caused by Colletotrichum species is one of the most important diseases of litchi in China. In this study, the causal pathogens of litchi anthracnose in Hainan, China, were determined using phylogenetic and morphological analyses. The results identified eight Colletotrichum species from four species complexes, including a proposed new species. These were C. karsti from the C. boninense species complex; C. gigasporum and the proposed new species C. danzhouense from the C. gigasporum species complex; C. arecicola, C. fructicola species complex; C. arecicola, C. fructicola and C. siamense from the C. gloeosporioides species complex; and C. musicola and C. plurivorum from the C. orchidearum species complex. Pathogenicity tests showed that all eight species could infect litchi leaves using a wound inoculation method, although the pathogenicity was different in different species. To the best of our knowledge, the present study is the first report that identifies C. arecicola, C. danzhouense, C. gigasporum and C. musicola as etiological agents of litchi anthracnose.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H₂O₂, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

Anthocyanidin reductase (ANR) is a key enzyme regulating anthocyanin synthesis and accumulation in plants. Here, lychee ANR genes were globally identified, their sequence and phylogenetic characteristics were analyzed, and their spatiotemporal expression patterns were characterized. A total of 51 ANR family members were identified in the lychee genome. The length of the encoded amino acid residues ranged from 87 aa to 289 aa, the molecular weight ranged from 9.49 KD to 32.40 KD, and the isoelectric point (pI) ranged from 4.83 to 9.33. Most of the members were acidic proteins. Most members of the LcANR family were located in the cytoplasm. The 51 LcANR family members were unevenly distributed in 11 chromosomes, and their exons and motif conserved structures were significantly different from each other. Promoters in over 90% of LcANR members contained anaerobically induced response elements, and 88% contained photoresponsive elements. Most LcANR family members had low expression in nine lychee tissues and organs (root, young leaf, bud, female flower, male flower, pericarp, pulp, seed, and calli), and some members showed tissue-specific expression patterns. The expression of one gene, LITCHI029356.m1, decreased with the increase of anthocyanin accumulation in ‘Feizixiao’ and ‘Ziniangxi’ pericarp, which was negatively correlated with pericarp coloring. The identified LcANR gene was heterologously expressed in tobacco K326, and the function of the LcANR gene was verified. This study provides a basis for the further study of LcANR function, particularly the role in lychee pericarp coloration.

2,4-Dichlorophenoxyacetic acid (2,4-D) is a vital exogenous auxin for the induction and proliferation of litchi embryogenic callus. At present, its molecular regulation mechanism remains unclear. In this study, transcriptome sequencing samples were selected based on different cell growth phenotypes observed in ‘Feizixiao’ litchi embryogenic callus cultured in liquid medium with or without 2,4-D. By integrating transcriptome profiling with weighted gene co-expression network analysis (WGCNA), we identified key genes and signaling pathways dynamically responsive to 2,4-D concentration changes. We identified 558 commonly differentially expressed genes (DEGs), of which 117 were up-regulated and 387 were down-regulated; functional enrichment analysis revealed significant enrichment in the “plant hormone signal transduction” and “phenylpropanoid biosynthesis” pathways. In the former pathway, genes such as AUX28, GH3.17, GH3.6, and ARR5 were up-regulated; in the latter, by comparison, β-glucosidase 47 and Peroxidase 61 exhibited increased expression levels induced by 2,4-D. Furthermore, among these DEGs, 57 transcription factors belonged to 24 families. Notably, VRN1, FEZ, and DOF5.4 were significantly and rapidly induced by 2,4-D. WGCNA results demonstrated a significant positive correlation between the yellow module and 2,4-D treatment. Small heat shock protein (sHSP) genes constituted the core hub genes in the yellow module. Through Venn analysis of DEGs and key modules, 38 cross-genes were identified, of which non-specific lipid-transfer protein-like genes (nsLTP) were found to be specifically up-regulated without 2,4-D. The transcription factors and genes identified work in synergy to ensure the formation and sustained proliferation of embryogenic callus by precisely regulating the dynamic balance of auxin and cytokinin within cells and maintaining the stability of cell structure. Our findings provide a crucial theoretical foundation for understanding the molecular mechanism of 2,4-D in regulating litchi embryogenic callus proliferation.