Quantification of the fungal pathogen Didymella segeticola in Camellia sinensis using a DNA-based qRT-PCR assay.

The fungal pathogen Didymella segeticola causes leaf spot and leaf blight on tea plant (Camellia sinensis), leading to production losses and affecting tea quality and flavor. Accurate detection and quantification of D. segeticola growth in tea plant leaves are crucial for diagnosing disease severity or evaluating host resistance. In this study, we monitored disease progression and D. segeticola development in tea plant leaves inoculated with a GFP-expressing strain. By contrast, a DNA-based qRT-PCR analysis was employed for a more convenient and maneuverable detection of D. segeticola growth in tea leaves. This method was based on the comparison of D. segeticola-specific DNA encoding a Cys2His2-zinc-finger protein (NCBI accession number: OR987684) in relation to tea plant Cs18S rDNA1. Unlike ITS and TUB2 sequences, this specific DNA was only amplified in D. segeticola isolates, not in other tea plant pathogens. This assay is also applicable for detecting D. segeticola during interactions with various tea cultivars. Among the five cultivars tested, 'Zhongcha102' (ZC102) and 'Fuding-dabaicha' (FDDB) were more susceptible to D. segeticola compared with 'Longjing43' (LJ43), 'Zhongcha108' (ZC108), and 'Zhongcha302' (ZC302). Different D. segeticola isolates also exhibited varying levels of aggressiveness towards LJ43. In conclusion, the DNA-based qRT-PCR analysis is highly sensitive, convenient, and effective method for quantifying D. segeticola growth in tea plant. This technique can be used to diagnose the severity of tea leaf spot and blight or to evaluate tea plant resistance to this pathogen.

The Mitochondrial Distribution and Morphology Family 33 Gene FgMDM33 Is Involved in Autophagy and Pathogenesis in Fusarium graminearum.

The mitochondrial distribution and morphology family 33 gene (MDM33) regulates mitochondrial homeostasis by mediating the mitochondrial fission process in yeast. The wheat head blight Fusarium graminearum contains an FgMdm33 protein that is orthologous to Saccharomyces cerevisiae Mdm33, albeit its function remains unknown. We have reported here the roles of FgMdm33 in regulating fungal morphogenesis, mitochondrial morphology, autophagy, apoptosis, and fungal pathogenicity. The ΔFgmdm33 mutants generated through a homologous recombination strategy in this study exhibited defects in terms of mycelial growth, conidia production, and virulence. Hyphal cells lacking FgMDM33 displayed elongated mitochondria and a dispensable respiratory-deficient growth phenotype, indicating the possible involvement of FgMDM33 in mitochondrial fission. The ΔFgmdm33 mutants displayed a remarkable reduction in the proteolysis of GFP-FgAtg8, whereas the formation of autophagic bodies in the hyphal cells of mutants was recorded under the induction of mitophagy. In addition, the transcriptional expression of the apoptosis-inducing factor 1 gene (FgAIF1) was significantly upregulated in the ΔFgmdm33 mutants. Cumulatively, these results indicate that FgMDM33 is involved in mitochondrial fission, non-selective macroautophagy, and apoptosis and that it regulates fungal growth, conidiation, and pathogenicity of the head blight pathogen.

A Loop-Mediated Isothermal Amplification Assay for the Rapid Detection of Didymella segeticola Causing Tea Leaf Spot.

Tea leaf spot caused by Didymella segeticola is an important disease that threatens the healthy growth of tea plants (Camellia sinensis) and results in reductions in the productivity and quality of tea leaves. Early diagnosis of the disease is particularly important for managing the infection. Loop-mediated isothermal amplification (LAMP) assay is an efficient diagnostic technique with the advantages of simplicity, specificity, and sensitivity. In this study, we developed a rapid, visual, and high-sensitivity LAMP assay for D. segeticola detection based on sequence-characterized amplified regions. Two pairs of amplification primers (external primers F3 and B3 and internal primers FIP and BIP) were designed based on a specific sequence in D. segeticola (NCBI accession number: OR987684). Compared to common pathogens of other genera in tea plants and other species in the Didymella genus (Didymella coffeae-arabicae, Didymella pomorum, and Didymella sinensis), the LAMP method is specific for detecting the species D. segeticola. The assay was able to detect D. segeticola at a minimal concentration of 1 fg/µL genomic DNA at an optimal reaction temperature of 65 °C for 60 min. When healthy leaves were inoculated with D. segeticola in the laboratory, the LAMP method successfully detected D. segeticola in diseased tea leaves at 72 h post inoculation. The LAMP assays were negative when the DNA samples were extracted from healthy leaves. Leaf tissues with necrotic lesions from 18 germplasms of tea plants tested positive for the pathogen by the LAMP assay. In summary, this study established a specific, sensitive, and simple LAMP method to detect D. segeticola, which provides reliable technical support for estimating disease prevalence and facilitates sustainable management of tea leaf spot.

Didymellaceae species associated with tea plant (Camelliasinensis) in China.

Tea plant is one of the most important commercial crops worldwide. The Didymellaceae fungi can cause leaf blight disease of tea plant. In this study, 240 isolates were isolated from tea plant leaves of 10 provinces in China. Combined with multi-locus (ITS, LSU, RPB2 and TUB2) phylogenetic analysis and morphological characteristics, these isolates were identified as 25 species of six genera in Didymellaceae, including 19 known species Didymellacoffeae-arabicae, D.pomorum, D.segeticola, D.sinensis, Epicoccumcatenisporum, E.dendrobii, E.draconis, E.italicum, E.latusicollum, E.mackenziei, E.oryzae, E.poaceicola, E.rosae, E.sorghinum, E.tobaicum, Neoascochytamortariensis, Paraboeremialitseae, Remotididymellaanemophila and Stagonosporopsiscaricae, of which 15 species were new record species and six novel species, named D.yunnanensis, E.anhuiense, E.jingdongense, E.puerense, N.yunnanensis and N.zhejiangensis. Amongst all isolates, D.segeticola was the most dominant species. Pathogenicity tests on tea plant leaves showed that E.anhuiense had the strongest virulence, while E.puerense had the weakest virulence. Besides, D.pomorum, D.yunnanensis, E.dendrobii, E.italicum, E.jingdongense, E.mackenziei, E.oryzae, E.rosae, E.tobaicum, N.mortariensis, N.yunnanensis, N.zhejiangensis and R.anemophila were non-pathogenic to the tea plant.

Sensitive detection of piperazinyl phenothiazine drugs by field-amplified sample stacking in capillary electrophoresis with dispersive liquid-liquid microextraction.

A rapid, simple and sensitive method for the detection of piperazinyl phenothiazine drugs using dispersive liquid-liquid microextraction (DLLME) combined with field-amplified sample stacking (FASS) in CE was developed. Sensitivity parameters that affect the extraction and FASS efficiency, such as the type and volume of disperser solvent, extraction time, addition of salt, and efficiency of FASS, were investigated and optimized. Note that the conductivity ratio between BGE and sample zone was measured to be 2300. Under optimal extraction and stacking conditions, the calibration curve, which ranged from 0.3 to 160 ng/mL, demonstrated good linearity with a correlation coefficient r≧ 0.9900. The LODs of prochlorperazine (Pcp), trifluoperazine (Tfp), perphenazine (Ppa), and fluphenazine (Fpa) at an S/N of 3 were 0.1, 0.1, 0.07, and 0.08 ng/mL, respectively. An approximately 1000-fold to 2500-fold improvement in sensitivity was achieved for the four tested analytes compared to conventional CZE without DLLME. The recoveries of all phenothiazines in urine and plasma ranged from 85.7 to 107.6% and 95.6 to 105.4%, respectively. The proposed method was demonstrated to be a rapid and convenient method for the determination of four piperazinyl phenothiazine drugs in human urine and plasma.

Combination of poly(diallyldimethylammonium chloride) and hydroxypropyl-γ-cyclodextrin for high-speed enantioseparation of phenothiazines by capillary electrophoresis.

High-speed capillary electrophoresis (CE) enables the simple, rapid, and inexpensive analysis of large sets of chiral samples in the pharmaceutical industry. Hence, we developed a novel method for separating enantiomers of d,L-phenothiazines simply and rapidly, based on using poly(diallyldimethylammonium chloride) (PDDAC) as an additive and hydroxypropyl-γ-cyclodextrin (Hp-γ-CD) as a chiral selector in capillary electrophoresis. Adding 0.9% PDDAC to the background electrolyte generated a stable, high, and reversed electroosmotic flow (EOF). Hp-γ-CD not only worked as a complexing agent to increase the chiral resolution between d,L-phenothiazines but also decreased the effective electrophoretic mobility of these drugs. Combining PDDAC and Hp-γ-CD as buffer additives enabled CE to achieve a high-speed enantioseparation of five pairs of d,L-phenothiazines. A decrease in capillary length and an increase in the intensity of the electric field further shortened the separation time. When the background electrolyte contained 0.9% PDDAC, 5mM Hp-γ-CD, and 75 mM formic acid (pH 3.0), enantioseparation of the d,L-phenothiazines was attained within 230 s by applying a capillary length of 32.5 cm and an electric field of 292 V cm(-1). The limit of detection (LOD) of the d,L-phenothiazines at a signal-to-noise ratio of 3 ranged from 2 to 8 µM. We demonstrated the feasibility of this method by detecting the five pairs of d,L-phenothiazines in urine samples.