Colletotrichum, the causative agent of anthracnose, is an important pathogen that invades the tea plant (Camellia sinensis). In this study, 38 isolates were obtained from the diseased leaves of tea plants collected in different areas of Zhejiang Province, China. A combination of multigene (ITS, ACT, GAPDH, TUB2, CAL, and GS) and morphology analyses showed that the 38 strains belonged to two different species, namely, C. camelliae (CC), and C. fructicola (CF). Pathogenicity tests revealed that CC was more invasive than CF. In vitro inoculation experiments demonstrated that CC formed acervuli at 72 hpi and developed appressoria on wound edges, but CF did not develop these structures. Under treatment with catechins and caffeine, the growth inhibition rates of CF were remarkably higher than those of CC, indicating that the nonpathogenic species CF was more vulnerable to catechins and caffeine. Growth condition testing indicated that CF grew at a wide temperature range of 15–35°C and that the optimum temperature for CC growth was 25°C. Growth of both CC and CF did not differ between acidic and weakly alkaline environments (pH 5–8), but the growth of CC was significantly reduced at pH values of 9 and 10. Furthermore, the PacC/RIM101 gene, which associated with pathogenicity, was identified from CC and CF genomes, and its expression was suppressed in the hyphae of both species under pH value of 5 and 10, and much lower expression level was detected in CC than that in CF at pH 6. These results indicated that temperature has more important effect than pH for the growth of two Colletotrichum species. In conclusion, the inhibition by secondary metabolite is an important reason why the pathogenicity by CC and CF are different to tea plant, although the environmental factors including pH and temperature effect the growth of two Colletotrichum species.
Anthracnose causes severe losses of tea production in China. Although genes and biological processes involved in anthracnose resistance have been reported in other plants, the molecular response to anthracnose in tea plant is unknown. We used the susceptible tea cultivar Longjing 43 and the resistant cultivar Zhongcha 108 as materials and compared transcriptome changes in the leaves of both cultivars following Colletotrichum fructicola inoculation. In all, 9015 and 8624 genes were differentially expressed between the resistant and susceptible cultivars and their controls (0 h), respectively. In both cultivars, the differentially expressed genes (DEGs) were enriched in 215 pathways, including responses to sugar metabolism, phytohormones, reactive oxygen species (ROS), biotic stimuli and signalling, transmembrane transporter activity, protease activity and signalling receptor activity, but DEG expression levels were higher in Zhongcha 108 than in Longjing 43. Moreover, functional enrichment analysis of the DEGs showed that hydrogen peroxide (H2O2) metabolism, cell death, secondary metabolism, and carbohydrate metabolism are involved in the defence of Zhongcha 108, and 88 key genes were identified. Protein–protein interaction (PPI) network demonstrated that putative mitogen-activated protein kinase (MAPK) cascades are activated by resistance (R) genes and mediate downstream defence responses. Histochemical analysis subsequently validated the strong hypersensitive response (HR) and H2O2 accumulation that occurred around the hyphal infection sites in Zhongcha 108. Overall, our results indicate that the HR and H2O2 are critical mechanisms in tea plant defence against anthracnose and may be activated by R genes via MAPK cascades.
Colletotrichum infects diverse hosts, including tea plants, and can lead to crop failure. Numerous studies have reported that biological processes are involved in the resistance of tea plants to Colletotrichum spp. However, the molecular and biochemical responses in the host during this interaction are unclear. Cuttings of the tea cultivar Longjing 43 (LJ43) were inoculated with a conidial suspension of Colletotrichum camelliae, and water-sprayed cuttings were used as controls. In total, 10,592 differentially expressed genes (DEGs) were identified from the transcriptomic data of the tea plants and were significantly enriched in callose deposition and the biosynthesis of various phytohormones. Subsequently, 3,555 mass spectra peaks were obtained by LC-MS detection in the negative ion mode, and 27, 18 and 81 differentially expressed metabolites (DEMs) were identified in the tea leaves at 12 hpi, 24 hpi and 72 hpi, respectively. The metabolomic analysis also revealed that the levels of the precursors and intermediate products of jasmonic acid (JA) and indole-3-acetate (IAA) biosynthesis were significantly increased during the interaction, especially when the symptoms became apparent. In conclusion, we suggest that callose deposition and various phytohormone signaling systems play important roles in the tea plant-C. camelliae interaction. Tea plant (Camellia sinensis (L.) O. Kuntze) is an important economic crop in China. Its fresh shoots and leaves contain abundant inclusions and are used as raw materials of tea, which is popular among many people. Tea leaves are easily attacked by pathogens, leading to tree wilting and crop failure. There are many pathogens that attack tea plants, such as Colletotrichum spp., Pestalotiopsis spp., and Discula theae-sinensis 1-3 , of which Colletotrichum is the most important pathogenic genus. Colletotrichum species cause disease in an extremely wide range of hosts and live as endophytes in plants 4. To date, 17 species of Colletotrichum have been reported to infect tea plants in China 5. Previous studies have suggested that Colletotrichum camelliae is a host-specific taxon occurring on Camellia 6. C. camelliae is regarded as the dominant species in Ca. sinensis and has high virulence 2,5. Most Colletotrichum species are hemibiotrophs that initially develop biotrophic hyphae inside a living host, which later transition to necrotrophic secondary mycelia 7,8. The progression of Colletotrichum growth in host plants includes germ tube growth, appressoria development and sporulation. When a spore lands on the surface of a host, it rapidly germinates and adapts to its environment. Then, the initiation of appressorium development is activated when the fungus perceives surface hardness 9. After the transition to the necrotrophic stage, secondary
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).
Colletotrichum camelliae is one of the most serious pathogens causing anthracnose in tea plants, but the interactive relationship between C. camelliae and tea plants has not been fully elucidated. This study investigated the gene expression changes in five different growth stages of C. camelliae based on transcriptome analysis to explain the lifestyle characteristics during the infection. On the basis of gene ontology (GO) enrichment analyses of differentially expressed genes (DEGs) in comparisons of germ tube (GT)/conidium (Con), appressoria (App)/Con, and cellophane infectious hyphae (CIH)/Con groups, the cellular process in the biological process category and intracellular, intracellular part, cell, and cell part in the cellular component category were significantly enriched. Hydrolase activity, catalytic activity, and molecular_function in the molecular function category were particularly enriched in the infection leaves (IL)/Con group. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the DEGs were enriched in the genetic information processing pathway (ribosome) at the GT stage and the metabolism pathway (metabolic pathways and biosynthesis of secondary metabolism) in the rest of the stages. Interestingly, the genes associated with melanin biosynthesis and carbohydrate-active enzymes (CAZys), which are vital for penetration and cell wall degradation, were significantly upregulated at the App, CIH and IL stages. Subcellular localization results further showed that the selected non-annotated secreted proteins based on transcriptome data were majorly located in the cytoplasm and nucleus, predicted as new candidate effectors. The results of this study may establish a foundation and provide innovative ideas for subsequent research on C. camelliae.
Previous studies of amyloid diseases reported that the aggregating proteins share a similar conserved peptide sequence which can form the cross-β-sheet-containing nanostructures like nanofilaments. The template-assisted self-assembly (TASA) of peptides on inorganic substrates with different hydrophilicity could be an alternative approach to shed light on the fibrillization mechanism of proteins/peptides in vivo. To figure out the effect of interfaces on amyloid aggregation, we herein employed in situ atomic force microscopy (AFM) to investigate the self-assembling of a Parkinson disease-related core peptide sequence (TGV-9) on a hydrophobic liquid–solid interface via real-time observation of the dynamic fibrillization process. The results show that TGV-9 forms one-dimensional nanostructures on the surface of highly ordered pyrolytic graphite (HOPG) with three preferred growth orientations, which are consistent with the atomic lattice of HOPG, indicating an epitaxial growth or TASA. Conversely, the nanostructures formed in bulk solution can be free-standing nanofilaments, and the fibrillization mechanism is different from that on HOPG. These results could not only deepen the understanding of the protein/peptide aggregation mechanism but also benefit for the early diagnosis and clinic treatment of related diseases.
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