Antifungal activity of caffeine against B. dothidea and C. gloeosporioides
Caffeine exhibited strong antifungal activity against both B. dothidea and C. gloeosporioides. B. dothidea was a fast-growing fungus, and it covered 9-cm control culture plates within 5 days incubation, its mycelial growth was measured up to 3 mg/mL of caffeine concentration, above this dosage, fungal growth was completely inhibited. The MIC and EC80 of caffeine against B. dothidea were 3 and 1.4 mg/mL, respectively. The mycelial growth rate of C. gloeosporioides was slower than B. dothidea, taking 7 days to cover control PDA plates, its mycelial growth was observed at the caffeine concentration of up to 7 mg/mL, above this dosage, the growth was arrested. The MIC and EC80 of C. gloeosporioides were 7 and 3.2 mg/mL, respectively (Fig. 1A and 1B). On the basis of their MICs, the fungi were classified as caffeine sensitive (B. dothidea) and tolerant (C. gloeosporioides). Subsequent investigations used the following caffeine concentrations: 1 and 3 mg/mL for B. dothidea, 3 and 7 mg/mL for C. gloeosporioides.
Effect of caffeine on cell structural changes
SEM results revealed that B. dothidea and C. gloeosporioides hyphae were thicker and serrated with roughened walls in caffeine treated samples, whereas healthy hyphae, with smooth surfaces and uniform growth were noticed in the control (Fig. 2A and 3A). An ultrathin section of the treated B. dothidea and C. gloeosporioides revealed that the cell wall and cell membrane were damaged, and some cell organelles were abnormal and degraded, such as the mitochondria, nuclei, and endoplasmic reticulum (Fig. 2B and 3B).
Effect of caffeine on bioactivity
Bioactivity − namely alkaline phosphatase (AKP), superoxide dismutase (SOD), and methanedicarboxylic aldehyde (MDA) − was investigated in both fungal isolates, and changes in bioactivity were observed between the caffeine-treated and control samples. The obtained results indicated that except MDA content was significantly higher in high-dose caffeine treated C. gloeosporioides, there were no significant differences in AKP, SOD and MDA activity between the high-dose and low-dose caffeine treated samples (Figure S1).
RNA-seq summary and caffeine response
RNA-seq profiling was performed to investigate the gene regulation of B. dothidea and C. gloeosporioides in control and caffeine-treated environments. The distribution of clean reads between the control and caffeine-treated RNA sequences exhibited relatively little difference (Table S2). In B. dothidea and C. gloeosporioides, most DEGs were observed in higher caffeine treated fungal culture (3 and 7 mg/mL caffeine, respectively), followed by lower caffeine treated fungal culture (1 and 3 mg/mL caffeine, respectively). The numbers of up-regulated and down-regulated genes are presented in Fig. 4. The similar results and trends were noticed in the gene ontology (GO) of B. dothidea and C. gloeosporioides (Table 1).
Table 1
Functional annotation of genes with significant transcript changes
GO terms | No. of genes (B. dothidea) | No. of genes (C. gloeosporioides) |
1 vs 3 mg/mL | 0 vs 1 mg/mL | 0 vs 3 mg/mL | 3 vs 7 mg/mL | 0 vs 3 mg/mL | 0 vs 7 mg/mL |
Biological regulation | 2 | - | 7 | 1 | 10 | 32 |
Cellular component organization or biogenesis | 1 | 2 | 8 | 1 | 5 | 25 |
Cellular process | 25 | 22 | 63 | 22 | 70 | 198 |
Detoxification | 1 | - | 1 | - | - | 2 |
Developmental process | 1 | - | - | - | - | 2 |
Growth | - | - | - | - | - | 1 |
Localization | 13 | 7 | 22 | 21 | 22 | 92 |
Metabolic process | 43 | 33 | 104 | 51 | 105 | 304 |
Multicellular organismal process | 1 | 1 | 1 | 1 | 1 | 1 |
Negative regulation of biological process | 1 | - | 1 | - | - | 1 |
Positive regulation of biological process | 1 | - | 3 | - | 2 | 5 |
Regulation of biological process | 2 | - | 6 | 1 | 6 | 22 |
Reproduction | - | - | 2 | 1 | - | 3 |
Reproductive process | - | - | 2 | 1 | - | 3 |
Response to stimulus | 6 | 3 | 11 | 1 | 5 | 3 |
Signaling | - | - | 2 | - | 1 | 3 |
Single-organism process | 21 | 19 | 61 | 33 | 59 | 188 |
Cell | 16 | 8 | 43 | 8 | 1 | 122 |
Cell part | 16 | 8 | 43 | 8 | 1 | 122 |
Extracellular region | - | 1 | 1 | 1 | 1 | 2 |
Macromolecular complex | 1 | 2 | 15 | 1 | 1 | 21 |
Membrane | 13 | 8 | 31 | 22 | 28 | 108 |
Membrane part | 12 | 6 | 24 | 20 | 26 | 93 |
Membrane-enclosed lumen | 1 | - | 4 | - | 2 | 5 |
Organelle | 7 | 2 | 19 | 1 | 18 | 71 |
Organelle part | 4 | - | 11 | 1 | 6 | 28 |
Supramolecular fiber | - | - | - | - | - | 2 |
Virion | - | - | - | - | 1 | 1 |
Virion part | - | - | - | - | 1 | 1 |
Antioxidant activity | 1 | - | 2 | 2 | 3 | 7 |
Binding | 24 | 26 | 72 | 33 | 67 | 219 |
Catalytic activity | 50 | 50 | 117 | 72 | 123 | 344 |
Electron carrier activity | - | - | - | - | - | 1 |
Molecular function regulator | - | - | - | - | 2 | 4 |
Nucleic acid binding transcription factor activity | 1 | - | 3 | 1 | 5 | 15 |
Nutrient reservoir activity | 1 | 1 | 3 | - | 1 | 1 |
Signal transducer activity | - | - | - | - | - | 1 |
Structural molecule activity | 2 | 1 | 4 | - | - | 4 |
Transporter activity | 7 | 2 | 15 | 10 | 10 | 39 |
The KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment pathways differed between the caffeine treatments and control. The number of pathways gradually decreased as the caffeine concentration decreased and number of genes was higher in increased caffeine concentration (Figure S2). The prominent biological functions were cell cycle, oxidative phosphorylation, glycolysis/glycogenesis, fatty acid metabolism and degradation, fatty acid biosynthesis, sphingolipid metabolism, ribosome biogenesis in eukaryotes, DNA replication, protein processing in the endoplasmic reticulum, and aminoacyl-tRNA biosynthesis. All genes were either accelerated or repressed in caffeine-treated B. dothidea and C. gloeosporioides. Significantly enriched critical KEGG pathways of both fungi were presented in Table S3 and S4.
A Venn diagram illustrated the shared and unique genes among the different caffeine-treated samples (Fig. 4). There were 13 shared genes in the B. dothidea samples and 25 in the C. gloeosporioides samples. Heatmaps of the relative expression levels of the genes involved in the caffeine-treated samples were exhibited in Fig. 5. Among them, 10 genes were selected for qRT-PCR analysis, and the relative expression levels and their fragments per kilobase of transcript per million mapped reads (FPKM) values from the RNA transcriptome data were also combined to illustrate the expression tendency (Fig. 5). In addition, 5 genes (both accelerated and repressed) were also chosen for confirmation through qRT-PCR, including genes related to ribosomes, protein processing in the endoplasmic reticulum, fatty acid synthesis, sphingolipid metabolism, and melanin synthetic pathway which were detected during the transcriptome analysis. The relative transcript abundance patterns for the control and caffeine treatments were compared using transcriptome data. The gene expression analysis results were consistent with the RNA-seq data. However, slight quantitative differences were noted in gene expression level (Figure S3).
Important genes in primary metabolic pathways were suppressed by caffeine
We observed that the number of key genes significantly downregulated in various primary metabolic pathways. Among them, ribosome biogenesis in eukaryotes, mRNA surveillance, RNA degradation and transport, ribosomes, and aminoacyl-tRNA biosynthesis were all involved in amino acid and protein synthesis mechanisms in both fungal species. Most of the genes in these pathways, such as integral membrane protein (IMP-jgi293974), heat shock protein (HP-jgi285255), phospholipase D (PLD-jgi295869), G protein subunit alpha transducin family (GNAT-18734207, 18745356), integral membrane protein (IMP-18734425), putative stress response protein (csbB-18743821), carbohydrate kinase family (fggy-18735662), basic leucine zipper (bZIP-18742633) and translation initiation factor (IF-18737650) were all repressed. Sphingolipid is the important components for fungal cell membrane formation [21]. Arylsulfatase (ARS-jgi294066) and sulfatase (ST-jgi292050) are involved in galactosylceramide formation in sphingolipid metabolism. Disturbance of these genes indicated that caffeine probably affected B. dothidea cell membrane. Fatty acid biosynthesis pathway has been a common target for antimicrobial agents in recent scenario [22]. Fatty acid biosynthesis mechanism was highly disturbed in the caffeine-treated fungi, 67% genes were downregulated in C. gloeosporioides. Though, there were no significant changes in B. dothidea fatty acid biosynthesis.
In summary, RNA-seq results revealed that caffeine treatment severely affected various physiological and metabolic processes of B. dothidea and C. gloeosporioides. And C. gloeosporioides can produce various secondary metabolites supported to fungal pathogenicity mechanism. Among them, melanin is one of the most important secondary metabolites and plays a major role in the fungus pathogenicity by influencing appressorium development. Melanin biosynthesis pathway in Colletotrichum and other fungi starts from malonyl-CoA/Acetyl-CoA, intermediates are as follow: 1,3,6,8-tetrahydroxynaphthalene, scytalone, 1,3,8-trihydroxynaphthalene, vermelone and 1,8-dihydroxynaphthalene [23, 24]. The short-chain dehydrogenase reductases (SDR) family, specifically 1,3,6,8-tetrahydroxynaphthalene reductase (T4HNR-18739341), was repressed in the caffeine-treated C. gloeosporioides and the result suggested the disturbance of scytalone formation in melanin biosynthesis pathway (Fig. 6). The downregulation of T4HNR affected melanin synthesis and led to interruptions of growth development and C. gloeosporioides pathogenicity. Based on the caffeine tolerance and importance of anthracnose pathogen (C. gloeosporioides) in tea plant, we further extensively investigated the physical characterization of melanin pigment and its inevitable role in fungal growth development and pathogenicity.
Physical characterization of purified melanin pigment
The black colour pigment was noticed in control fungal culture, while it was absent in caffeine-treated fungal culture (Fig. 7A). And the purified black colour melanin powder is shown in Fig. 7B. Figure 7C shows the SEM image of the purified melanin. The appearance suggested that the extracted melanin was round edged and amorphous crystal shapes. Then, the melanin pigment was characterized by UV-visible, FTIR and NMR spectrum. The highest peak absorption was observed at 221 nm (Fig. 7D), which was close to the maximum absorption wavelength of standard melanin. Melanin in Lachnum YM226 (LM) and Rhizoctonia solani has maximum absorption peaks at 223 and 217 nm, respectively [25, 26]. The spectrum analysis of extracted melanin compared with commercial melanin was shown in Fig. 7E. FTIR showed broad absorption band at 3424 cm− 1, which indicated the presence of hydroxyl or amine groups. The broadband ranged from 3420 to 3400 cm− 1, which denoted that the presence of phenolics, carboxylic and aromatic amino functions in pyrrolic and indolic systems [27]. Another absorption band was noticed at 2924 cm− 1 contributed by aliphatic C − H groups. Stretching vibration of aliphatic C − H groups bands at 2922 cm− 1 and 2852 cm− 1 were observed in both synthetic and R. solani melanin [26]. We observed band at 1636 cm− 1, which denoted that the presence of the aromatic ring C = C and C = O in the sample. Olaizola [28] considered that the bands close to 1600 cm− 1 were attributed to stretching vibrations of C = C bonds of aromatic structures and this band was considered importantly for the identification of melanin. The important features of extracted melanin were almost on par with the procured standard melanin. The 1H NMR spectrum of C. gloeosporioides melanin pigment was presented in Figure S4. The signals at 6.0-8.5 ppm and 0.8–1.5 ppm indicated the presence of aromatic and aliphatic groups, respectively. The signals at 0.8-1.0 ppm and 1.3 ppm can be assigned to methyl and methylene groups of alkyl fragments, respectively. The signals between 7.5 and 6.6 ppm indicated the presence of indole, which is acted as a major precursor of melanin synthesis [29, 30]. Katritzky et al. [31] reported that sequence of four broad signals at 7.60, 7.35, 7.00 and 6.60 ppm in human hair melanin belongs to protons of indole or pyrrole.
Influence of melanin on fungal growth
The experimental results revealed that the mycelial dry weight significantly increased in melanin with caffeine-treated environment. Further, we noticed the dense mycelial structures and number of germinated and non-germinated conidia in 3 mg/mL caffeine with synthetic melanin but it was absent in 7 mg/mL caffeine with synthetic melanin (Fig. 8 and S5). Whereas, there were no conidia development noticed in caffeine treated fungal culture (3 and 7 mg/mL).
Pathogenicity test, histopathological observation and fungal biomass quantification
We performed pathogenicity assay to find symptoms production by the fungus on three different varieties tea leaves. The enlarged necrotic lesions were noticed around 14–16 days interval in all the wounded leaves of susceptible tea cultivar (Longjing 43) but no significant changes were noticed between moderately resistant (Longjing Changye) and resistant (Zhongcha 108) tea cultivars lesion size (Fig. 9a). The histopathological observations indicated that high fungal biomass production, germinated conidia and appressorium formation in susceptible tea cultivar, the fungal mycelium moved freely on infected tea leaves and the number of appressorium was higher. Whereas, the moderately resistant and resistant tea clone leaves, the spreading of fungal biomass, germinated conidia and appressorium development were limited (Fig. 9b). Aside, we performed fungal gDNA quantification in infected tea leaves of three varieties. The obtained results revealed that the amount of the fungal gDNA in susceptible tea leaves was higher than moderately resistant tea leaves, but there was no reasonable amount of gDNA quantified in resistant cultivar tea leaves (Fig. 9c).
Caffeine quantification and gene expression analysis
Caffeine content in chosen tea varieties was analyzed and the concentration marginally increased in the fungal inoculated tea leaves of susceptible and moderately resistant tea varieties compared with control. Though, there was no significant change between the control and resistant variety (Figure S6). The caffeine quantification results are closely associated with relative gene expression of caffeine biosynthesis genes (TCS1, TIDH and SAMS). The gene expression of SAMS and TIDH was higher in infected leaves of resistant variety, while the expression of TCS1 was higher in susceptible and moderately resistant varieties. Simultaneously, phenylpropanoid biosynthesis, plant hormone biosynthesis, signaling pathways and plant-pathogen interaction and other stress-responsive genes expression were analyzed to understand resistant response against anthracnose pathogen in tea plants through the molecular mechanism and the data were shown in Figure S7.