Several Pestalotiopsis-like species cause gray blight disease in tea plants, resulting in severe tea production losses. However, systematic and comprehensive research on the diversity, geographical distribution, and pathogenicity of pathogenic species associated with tea plants in China is limited. In this study, 168 Pestalotiopsis-like isolates were obtained from diseased tea plant leaves from 13 primary tea-producing provinces and cities in China. Based on a multilocus (internal transcribed spacer, translation elongation factor 1-α, and β-tubulin gene region) phylogenetic analysis coupled with an assessment of conidial characteristics, 20 Neopestalotiopsis unclassified isolates, seven Pestalotiopsis species, including two novel (Pestalotiopsis menhaiensis and Pestalotiopsis sichuanensis), four known (Pestalotiopsis camelliae, Pestalotiopsis chamaeropis, Pestalotiopsis kenyana, and Pestalotiopsis rhodomyrtus) and one indistinguishable species, and three Pseudopestalotiopsis species, including two known (Pseudopestalotiopsis camelliae-sinensis and Pseudopestalotiopsis chinensis) and one indistinguishable species, were identified. This study is the first to evaluate Pestalotiopsis chamaeropis on tea plants in China. The geographical distribution and pathogenicity tests showed Pseudopestalotiopsis camelliae-sinensis to be the dominant cause of gray blight of tea plants in China. In vitro antifungal assays demonstrated that theobromine not only derepressed mycelial growth of the 29 representative isolates but also increased their growth. Correlation analysis revealed a linear positive relationship between the mycelial growth rate and pathogenicity (P = 0.0148).

Vacuolar invertases (VINs) have been reported to regulate plant growth and development and respond to abiotic stresses such as drought and cold. With our best knowledge, the functions of VIN genes little have been reported in tea plant (Camellia sinensis L.). Therefore, it is necessary to develop research in this field. Here, we identified a VIN gene, CsINV5, which was induced by cold acclimation and sugar treatments in the tea plant. Histochemical assays results showed that the 1154 bp 5′-flanking sequence of CsINV5 drove β-glucuronidase (GUS) gene expression in roots, stems, leaves, flowers and siliques of transgenic Arabidopsis during different developmental stages. Moreover, promoter deletion analysis results revealed that an LTRE-related motif (CCGAAA) and a WBOXHVISO1 motif (TGACT) within the promoter region of CsINV5 were the core cis-elements in response to low temperature and sugar signaling, respectively. In addition, overexpression of CsINV5 in Arabidopsis promoted taproot and lateral root elongation through glucose-mediated effects on auxin signaling. Based on physiological and RNA-seq analysis, we found that overexpression of CsINV5 improved cold tolerance in transgenic Arabidopsis mainly by increasing the contents of glucose and fructose, the corresponding ratio of hexose to sucrose, and the transcription of osmotic-stress-related genes (P5CS1, P5CS2, AtLEA3, COR413-PM1 and COR15B) to adjust its osmotic potential. Comprehensive experimental results suggest that overexpression of CsINV5 may enhance the cold tolerance of plant through the modification of cellular sugar compounds contents and osmotic regulation related pathways.

Cold stress is a major environmental factor that adversely affects the growth and productivity of tea plants. Upon cold stress, tea plants accumulate multiple metabolites, including ascorbic acid. However, the role of ascorbic acid in the cold stress response of tea plants is not well understood. Here, we report that exogenous ascorbic acid treatment improves the cold tolerance of tea plants. We show that ascorbic acid treatment reduces lipid peroxidation and increases the Fv/Fm of tea plants under cold stress. Transcriptome analysis indicates that ascorbic acid treatment down-regulates the expression of ascorbic acid biosynthesis genes and ROS-scavenging-related genes, while modulating the expression of cell wall remodeling-related genes. Our findings suggest that ascorbic acid treatment negatively regulates the ROS-scavenging system to maintain ROS homeostasis in the cold stress response of tea plants and that ascorbic acid’s protective role in minimizing the harmful effects of cold stress on tea plants may occur through cell wall remodeling. Ascorbic acid can be used as a potential agent to increase the cold tolerance of tea plants with no pesticide residual concerns in tea.

Protease inhibitors promote herbivore resistance in diverse plant species. Although many inducible protease inhibitors have been identified, there are limited reports available on the biological relevance and molecular basis of constitutive protease inhibitors in herbivore resistance. Here, we identified a serine protease inhibitor, CsSERPIN1, from the tea plant (Camellia sinensis). Expression of CsSERPIN1 was not strongly affected by the assessed biotic and abiotic stresses. In vitro and in vivo experiments showed that CsSERPIN1 strongly inhibited the activities of digestive protease activities of trypsin and chymotrypsin. Transient or heterologous expression of CsSERPIN1 significantly reduced herbivory by two destructive herbivores, the tea geometrid and fall armyworm, in tea and Arabidopsis plants, respectively. The expression of CsSERPIN1 in Arabidopsis did not negatively influence the growth of the plants under the measured parameters. Our findings suggest that CsSERPIN1 can inactivate gut digestive proteases and suppress the growth and development of herbivores, making it a promising candidate for pest prevention in agriculture.