In this study, metagenomic sequencing technology was employed to analyze the ITS1 region sequence of the ITS rDNA gene of endophytic fungi and 16S sequence of endophytic bacteria in tea leaves with varying degrees of infection by tea blister blight disease as well as healthy tea leaves. Subsequently, a comparative analysis was conducted on the endophytic microbial diversity and the community structure in tea leaves. The findings of this investigation reveal a shift in the dominant endophytic fungal genera from Ascomycota to Basidiomycota as the disease progressed. Furthermore, a negative correlation was observed between Exobasidium and Talaromyce, with Talaromyce exhibiting potential as an antagonist against the disease. Meanwhile, our findings reveal that Proteobacteria, Firmicutes, and Actinobacteria were the three most abundant bacteria phyla in tea leaves. As the disease progressed, there was an increase in the relative abundance of Actinobacteria, while Variovorax, Sphingomonas, and Pseudomonas were found to have higher abundance in later stages. The diversity analysis results indicated that the endophytic microbial diversity and the community structure in tea leaves in the diseased group were lower than those in the healthy control group. In general, blister blight disease altered the community structure of endophytic microorganisms in tea leaves, resulting in a few species with high abundance. The study lays a foundation for investigating the pathogenic mechanism of tea blister disease and establishing a theoretical basis for controlling diseases in tea trees.

Blister blight (BB) disease is caused by the obligate biotrophic fungal pathogen Exobasidium vexans Massee and seriously affects the yield and quality of Camellia sinensis. The use of chemical pesticides on tea leaves substantially increases the toxic risks of tea consumption. Botanic fungicide isobavachalcone (IBC) has the potential to control fungal diseases on many crops but has not been used on tea plants. In this study, the field control effects of IBC were evaluated by comparison and in combination with natural elicitor chitosan oligosaccharides (COSs) and the chemical pesticide pyraclostrobin (Py), and the preliminary action mode of IBC was also investigated. The bioassay results for IBC or its combination with COSs showed a remarkable control effect against BB (61.72% and 70.46%). IBC, like COSs, could improve the disease resistance of tea plants by enhancing the activity of tea-plant-related defense enzymes, including polyphenol oxidase (PPO), catalase (CAT), phenylalanine aminolase (PAL), peroxidase (POD), superoxide dismutase (SOD), β-1,3-glucanase (Glu), and chitinase enzymes. The fungal community structure and diversity of the diseased tea leaves were examined using Illumina MiSeq sequencing of the internal transcribed spacer (ITS) region of the ribosomal rDNA genes. It was obvious that IBC could significantly alter the species\' richness and the diversity of the fungal community in affected plant sites. This study broadens the application range of IBC and provides an important strategy for the control of BB disease.

BACKGROUND: Tea (Camellia sinensis (L). O. Kuntze) is known as the oldest, mild stimulating caffeine containing non-alcoholic beverage. One of the major threats in south Asian tea industry is the blister blight leaf disease (BB), caused by the fungus Exobasidium vexans Masse. SSR DNA marker EST SSR 073 is used as a molecular marker to tag blister blight disease resistance trait of tea. The amino acid sequences were derived from cDNA sequences related to EST SSR 073 of BB susceptible (TRI 2023) and BB resistant (TRI 2043) cultivars. An attempt has been made to understand the structural characteristics and variations of EST SSR 073 locus that may reveal the factors influencing the BB resistance of tea with multiple bioinformatics tools such as ORF finder, ExPasy ProtParam tools, modeler V 9.17, Rampage server, UCSF-Chimera, and HADDOCK docking server.
RESULTS: The primary, secondary, and tertiary structures of EST SSR 073 coding protein were analyzed using the amino acid sequences of both BB resistant TRI 2043 and BB susceptible TRI 2023 tea cultivars. The coding amino acid sequences of both the cultivars were homologous to photosystem I subunit protein (PsaD I) of Pisum sativum. The predicted 3D structures of proteins were validated and considered as an acceptable overall stereochemical quality. The BB resistant protein showed CT repeat extension and did not involve in topology of the PsaD I subunit. The C terminal truncation of BB resistance caused the formation of hydrogen bonds interacting with PsaD I and other subunits of photosystem I in the modeled three-dimensional protein structure.
CONCLUSIONS: Camellia sinensis EST 073 SSR motif coding protein was identified as the PsaD I subunit of photosystem I. The exact mechanism of PsaD I conferring the resistance for blister blight in tea needs to be further investigated.

Blister blight disease, caused by an obligate biotrophic fungal pathogen, Exobasidium vexans Massee is posing a serious threat for tea cultivation in Asia. As the use of chemical pesticides on tea leaves substantially increases the toxic risks of tea consumption, serious attempts are being made to control such pathogens by boosting the intrinsic natural defense responses against invading pathogens in tea plants. In this study, the nature and durability of resistance offered by chitosan and the possible mechanism of chitosan-induced defense induction in Camellia sinensis (L.) O. Kuntze plants against blister blight disease were investigated. Foliar application of 0.01% chitosan solution at 15 days interval not only reduced the blister blight incidence for two seasons, but also maintained the induced expressions of different defense related enzymes and total phenol content compared to the control. Defense responses induced by chitosan were found to be down regulated under nitric oxide (NO) deficient conditions in vivo, indicating that the observed chitosan-induced resistance is probably activated via NO signaling. Such role of NO in host defense response was further established by application of the NO donor, sodium nitroprusside (SNP), which produced similar defense responses accomplished through chitosan treatment. Taken together, our results suggest that increased production of NO in chitosan-treated tea plants may play a critical role in triggering the innate defense responses effective against plant pathogens, including that causing the blister blight disease.