Abstract

   Blister blight, as one of the most threatening and damaging disease
   worldwide, mainly infects young organs and tissues seriously affecting
   tea growth and quality. In this study, the spread of pathogen on tea
   leaves were examined by toluidine blue staining, scanning electron
   microscope and transmission electron microscope analysis. The
   composition and abundance of fungal community on leaf tissues were
   firstly analyzed. Sensory evaluation and metabolites analysis indicated
   that diseased tea leaves had strong sweet taste and soluble sugars
   contributed significantly to the taste, while metabolites showing
   bitter and astringent taste (caffeine, catechins) were significantly
   decreased. According to the biological functions of differential
   metabolites, sugars including 7 monosaccharides (d-xylose, d-arabinose,
   d-mannose, d-glucuronic acid, glucose, d-galactose and d-fructose), 2
   disaccharide (sucrose and maltose) and 1 trisaccharide (raffinose) were
   the main differential sugars increased in content (>2 fold change),
   which was of great significance to sweet taste of diseased tea.

Introduction

   Tea, as a beverage, is popular for its healthy benefits and pleasant
   flavor. Tea plant is a perennial economic crop which is susceptible to
   various kinds of destructive foliar diseases due to the warm and humid
   growth environment in tea plantations that provides a suitable
   microclimate for the breeding of pathogens ([29]Baby, 2002). Among
   these diseases, blister blight caused by Exobasidium vexans Massee is
   considered as the most serious disease in the world that mainly damages
   tender leaves, buds and young fruits, resulting in yield loss and
   quality decrease ([30]Punyasiri et al., 2005, [31]Sen et al., 2020). At
   the tea plant experimental plots of Sri Lanka, the disease first occurs
   through the formation of appressoria and penetration of the cuticle,
   after penetration visible translucent spots are formed, and with the
   development of spots, characteristic circular blisters appear on the
   surface of young tissues, finally resulting in the infected tea leaves
   distorted and infected stems broken off at the point of infection
   ([32]Punyasiri et al., 2005). Blister blight disease occurring in
   almost all the tea plantation regions of Asia, has become the major
   problem on tea plants that causes serious economic losses ([33]Sinniah
   et al., 2016, [34]Baby, 2002, [35]Guo et al., 2005). It is reported
   that blister blight disease is more serious in alpine tea gardens in
   southwest and south of China. In epidemic years, the incidence of
   blister blight in tea growing regions located in southwest of China can
   reach 40–50 %, and even reach 90 % in severe cases ([36]Chen and Sun,
   2013, [37]Sen et al., 2020). It was estimated to cause about 33 % crop
   loss in Sri Lanka ([38]De Silva et al., 1992), 25 % in Indonesia and as
   high as 50 % in South India, respectively, in tea fields in the absence
   of control measures ([39]Radhakrishnan and Baby, 2004). Besides yield
   loss, blister blight disease negatively affects the growth of tea
   trees, resulting in the quality decrease of processed tea which is even
   exceeded the economic threshold level 35 % ([40]Ponmurugan et al.,
   2016, [41]Radhakrishnan and Baby, 2004).

   Tea has abundant non-volatile and volatile metabolites such as tea
   polyphenols, amino acids components, caffeine, soluble sugars, lipids,
   and aromatic compounds, which are proved to be closely associated with
   the flavor and quality of tea ([42]Cao et al., 2021). Some quality
   related metabolites such as caffeine, epicatechin (EC) and theanine
   (Thea) also have been reported to have potential effect against various
   kinds of viral or fungal diseases ([43]Sharma et al., 2021,
   [44]Punyasiri et al., 2005). The development of pathogen in various
   stages showed different effect on pathogenesis related protein,
   anti-oxidative enzymes and flavonoid pathway in tea, suggesting the
   possible role of some chemical compounds (i.e., reactive oxygen
   species, anthocyanins, lignins, catechins) and other synthesized
   compounds in acting as antifungal agents in different tea cultivars
   ([45]Nisha et al., 2018). A large amount of studies have validated that
   various kinds of biotic (ie, disease infection, insect attack) and
   abiotic (ie, mechanical damage, environment conditions) stresses can
   remarkably influence the quality and flavor of tea by significantly
   changing the composition and amount of metabolites, especially affect
   the aromatic compounds in tea plants ([46]Chen et al., 2017, [47]Cho et
   al., 2017, [48]Mei et al., 2017, [49]Zeng et al., 2019). Tea processed
   from blister blight infected fresh tea leaves is fragile and shows
   obvious bitter taste, and the level of chemical components related to
   tea quality has decreased significantly, especially tea polyphenols and
   catechins ([50]Guo et al., 2005, [51]Jayaswall et al., 2016).
   [52]Premkumar et al. (2008) reported that after the infection of
   blister blight disease, there were remarkable reduction in the amount
   of tea polyphenols, catechins, sugar, nitrogen, proteins, amino acids
   and other substances in infected tea leaves. [53]Mur et al. (2015)
   analyzed the chemical compounds including caffeine, flavan-3-ol,
   flavone and flavonol in blister blight infected tender tea leaves by
   using high performance liquid chromatography with online photodiode
   array detection and electrospray ionization-tandem mass spectrometry
   (HPLC-PDA-ESI/MS), and found that kaempferol and quercetin glucosides,
   kaempferol triglycosides and some catechin-class antioxidants were
   increased, while the level of caffeine and apigenin and myricetin
   glycosides were remarkably reduced as disease progressed. However,
   limited information are still available on the tea quality affected by
   blister blight disease.

   In this study, the development and spread of blister blight on tea
   leaves, and the composition and abundance of fungal community on leaf
   tissues were fully investigated. After the infection of the disease,
   major metabolite differences in healthy and diseased tea leaves were
   studied, centring on the metabolites that contributed to the tea taste,
   which is helpful to improve the understanding on the influence of
   blister blight on tea quality.

Materials and methods

Materials

   During 2018 to 2020, tea leaves displaying blister blight symptoms were
   observed in high mountain tea plantations above 1000 m altitude located
   in Quxian county, Dazhou city, Sichuan province, China (30°85′N,
   106°94′E). The typical blister blight symptoms were collected to show
   the symptom development. To analyze the characteristics of the blister
   blight disease, more than 100 fresh diseased tea shoots showing blister
   blight symptoms were harvested at April 2020, and used for microbial
   diversity analysis and microscopic analysis of pathogens including
   tissue section observation, transmission electron microscopy (TEM) and
   scanning electron microscopy (SEM) analysis. To investigate the effect
   of blister blight on tea quality and flavor, diseased tea shoots with
   one bud and two leaves (500 g) were sampled, while healthy tea leaves
   were used as a control. All the experiment had three replicates. Tea
   samples used for metabolites profile analysis were fixed by microwave
   (2–3 min), dried in a tea dryer machine (1 h, 80℃), finally milled and
   stored in a − 80℃ freezer.

Microscopic analysis

Toluidine blue staining (TBS)

   Tea leaf tissues (1 × 1 cm) were firstly fixed with FAA
   (Formaldehyde-acetic acid–ethanol) (Solarbio, Beijing, China), then
   dehydrated with ethanol, embedded in paraffin and routinely sliced. The
   slices were placed in xylene I (Sinaopharm Group Chemical Reagent Co.
   LTD) for 20 min, xylene II (Sinaopharm Group Chemical Reagent Co. LTD)
   for 20 min, anhydrous ethanol I (Sinaopharm Group Chemical Reagent Co.
   LTD) for 5 min, anhydrous ethanol II (Sinaopharm Group Chemical Reagent
   Co. LTD) for 5 min, 75 % alcohol for 5 min, and washed with double
   distilled water (ddH[2]O). The dehydrated slices were stained in
   toluidine blue solution (Wuhan Google Biotechnology Co. LTD) for 6 min,
   then washed with ddH[2]O, finally the qualified slices were dried in an
   oven. Transparent sealing: the dried slices were treated in xylene
   (Sinaopharm Group Chemical Reagent Co. LTD) for 5 min, then taken out
   to dry, finally sealed with neutral gum for microscopic examination.

Scanning electron microscopy (SEM)

   Tea leaf tissue blocks (3 mm^2) were harvested within 3 min and washed
   with phosphate buffer saline (PBS) (Servicebio, Wuhan, China) gently,
   then immediately immersed in electron microscopy fixative (Servicebio,
   Wuhan, China) at room temperature for 2 h, finally transferred into
   4 °C for preservation. After fixation, tissue blocks were rinsed with
   0.1 M phosphate buffer (PB, pH 7.4) for 3 times (15 min each),
   transferred into 1 % osmic acid (Ted Pella Inc.) for 1–2 h at room
   temperature, then washed in 0.1 M PB (pH 7.4) for 3 times (15 min
   each). Leaf tissue blocks were dehydrated with different ethanol
   concentrations (30 %, 50 %, 70 %, 80 %, 90 %, 95 %, 100 % and 100 %) in
   sequence for 15 min each, finally were placed in isoamyl acetate
   (Sinaopharm Group Chemical Reagent Co. LTD) for 15 min. After 0.1 M PB
   (pH 7.4) wash, the samples were dried with critical point dryer
   (Quorum, United Kingdom), then were attached to metallic stubs by using
   carbon stickers and sputter-coated with gold for 30 s, finally were
   observed with SEM (Hitachi, Japan).

Transmission electron microscope (TEM)

   Sampled fresh tea leaves were cut into 1 mm^3 tissue blocks within
   3 min, then quickly fixed in the electron microscopy fixative
   (Servicebio, Wuhan, China) at room temperature for 2 h, finally stored
   at 4 ℃. After fixation, leaf tissue blocks were rinsed with 0.1 M PB
   (pH 7.4) for 3 times (15 min each). Post-fixation: tissue blocks were
   fixed in 1 % osmic acid (Ted Pella Inc.) that prepared in 0.1 M PB (pH
   7.4) at room temperature for 7 h away from light, then washed three
   times with 0.1 M PB (pH 7.4), 15 min each time. Tissue dehydration:
   treated tissues were dehydrated in 30 %, 50 %, 70 %, 80 %, 95 %, 100 %
   alcohol in sequence, 1 h each time, and then in ethanol: acetone = 3:
   1, ethanol: acetone = 1: 1 and ethanol: acetone = 1: 3, for 0.5 h each,
   respectively, finally in pure acetone for 1 h. EMBed 812 (SPI, USA) was
   used for osmotic embedding, then the embedded plates were placed in 65℃
   oven to polymerize for 48 h. Treated blocks were cut into thin slices
   (60–80 nm) with ultra-microtome (Leica, Germany). The copper mesh
   slices were dyed in the dark with uranium acetate saturated alcohol
   solution (2 %) for 8 min, rinsed in ethanol (70 %) for 3 times and in
   ddH[2]O for 3 times, dyed in lead citrate solution (2.6 %, without
   CO[2]) for 8 min, washed with ddH[2]O for 3 times, finally put into a
   copper mesh box to dry overnight at room temperature. The prepared
   slices were observed with TEM (Hitachi, Japan).

Microscopic examination

   Diseased tea leaves exhibiting blister blight with abundant
   basidiospore were observed with a U-TV0.5XC-3 microscope (Olympus,
   Japan). The shapes and sizes of basidiospore were recorded by measuring
   at least 30 randomly selected basidiospore.

Microbial diversity analysis

   To analysis the composition of microbial communities in diseased lesion
   tissues showing blister blight in Quxian, Sichuan province, China,
   genomic DNA of lesion tissues was extracted by using HiPure Soil DNA
   Kits (Magen, Guangzhou, China) based on the manufacturer’s protocols.
   ITS gene region was used for the identification of fungal communities.
   Gene region of ITS2 was amplified with primer pair ITS3_KYO2
   (5′-GATGAAGAACGYAGYRAA-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The
   amplified products were purified and then quantified using Nanodrop
   procedures. Sequencing was conducted by Genedenovo Inc. (Guangzhou,
   China) by using Illumina Hiseq 2500 PE250 platform (Illumina, San
   Diego, CA, USA). The α-diversity indices such as Chao1, Simpson, and
   Shannon were quantified to show the fungal diversity in diseased tea
   leaves according to the Operational Taxonomic Units (OTUs) richness in
   QIIME (version 1.9.1) ([54]Caporaso et al., 2010). The species
   distribution river map of the fungal composition was plotted using R
   project ggplot2 package (version 2.2.1). The heatmap of species
   abundance was constructed based on pheatmap package (version 1.0.12) in
   R project. Functional group (guild) of the microbial communities were
   inferred by FUNGuild (version 1.0). The phylogenetic tree was
   constructed by Neighbor-Joining (NJ) method in MEGA6. The distances of
   evolutionary were computed by the method of Maximum Composite
   Likelihood.

Sensory evaluation

   Quantitative descriptive analysis was conducted by a sensory panel with
   seven well-trained panelists (4 males and 3 females, 40–55 years old)
   from the Department of Tea Science, Southwest University, China. All
   the panelists had been informed of the sensory evaluation, and agreed
   to take part in this research and use their information. The rights and
   privacy of all the panelists were protected during the research, and
   ethical approval for the involvement of human subjects in this study
   was granted by Southwest university research ethics committee. Total
   3.0 g tea sample was weighed, and brewed with 150 mL boiling water for
   5 min, then tea infusion was used for taste evaluation. Taste
   evaluation included the common characteristics such as bitterness,
   astringency, umami and sweetness ([55]Cao et al., 2021). Sensory
   evaluation was conducted by the national sensory evaluation method for
   tea GB/T 23776–2018 ([56]Gong et al., 2018). Values of taste attributes
   were scored by a 10-point scale and 5 intensity were evaluated in given
   tea samples: 0–2 represents extremely weak, 2–4 represents weak, 5–6
   represents moderate, 7–8 represents strong, and 9–10 represents
   extremely strong. The highest and lowest scores for each attribute were
   removed and the mean value was used for the evaluation.

Sugars analysis by GC–MS

   The content and composition of sugars were analyzed using gas
   chromatography-mass spectrometry (GC/MS) ([57]Chen et al., 2023). Tea
   sample (20 mg) was mixed with 500 μL methanol: isopropanol: water (3:
   3: 2, V/V/V) solution, vortexed for 3 min, ultrasound for 30 min, and
   then centrifuged at 12,210 g (3 min, 4℃). Agilent 8890 gas
   chromatograph coupled to a 5977B mass spectrometer with a DB-5MS column
   (J&W Scientific, USA) was used for GC/MS analysis. Helium was the
   carrier gas with a flow rate of 1 mL/min. Injections were made with a
   split ratio 5:1 and the total injection volume for each sample was
   1 μL. The oven temperature was set at 170℃ for 2 min, then increased to
   240℃ at 10 ℃/min, finally raised to 280 °C at 5 °C/min, raised to
   310 °C at 25 °C/min and kept for 4 min. Selective ion monitoring mode
   was used for sample analysis. The orthogonal projections to latent
   structure-discriminant analysis (OPLS-DA) were applied to distinguish
   the significantly differential metabolites between different tea
   samples. Significantly changed metabolites between healthy and diseased
   tea leaves were determined by absolute Log2FC (fold change), variable
   importance in project (VIP) and P value (Student’s t test). When the
   fold change ≥ 2 or ≤ 0.5, P value ≤ 0.5 and VIP ≥ 1 between two groups,
   the metabolites difference was considered significant. The metabolites
   annotated in Kyoto Encyclopedia of Genes and Genomes (KEGG) compound
   database ([58]https://www.kegg.jp/kegg/compound/), were then mapped
   into metabolic pathway based on database search
   ([59]https://www.kegg.jp/kegg/pathway.html). The P value represented
   the metabolites enrichment in a pathway, and P ≤ 0.05 was indicative of
   significant enrichment.

Catechins, caffeine and amino acids analysis by HPLC

   Catechins and caffeine contents were analyzed by using high performance
   liquid chromatography (HPLC) (Agilent 1200, Agilent Technologies, Santa
   Clara, CA, USA) ([60]Chen et al., 2023). Prepared tea infusions were
   analyzed by an Agilent Eclipse XD8 C18 column (250 mm × 4.6 mm i.d.,
   5 μm; Thermo Electron Corporation, Waltham, MA, USA). The volume of
   injected sample was 5 μL with 0.9 mL/min flow rate. The wavelength of
   UV detection was 278 nm. The composition and content of amino acids
   were analyzed with HPLC system ([61]Chen et al., 2023). The HPLC
   conditions were as follows: mobile phase A was 4 mM sodium acetate
   (pH = 5.5); mobile phase B was 80 % acetonitrile solution; column
   temperature was 35 °C; flow rate was set at 0.9 mL/min. The wavelength
   of UV detection was 360 nm. The results were expressed as mean
   value ± standard deviation (SD). The t test of independent sample was
   used to calculate the statistically significant difference between
   different groups (P ≤ 0.05) by IBM SPSS Statistics 20.0 (SPSS Inc.,
   Chicago, USA).

Results and discussions

Disease symptoms on tea caused by blister blight disease

   As an important tea foliar disease, blister blight mainly infected
   young organs and tissues including tender leaves, buds, petioles and
   stems ([62]Fig. 1), directly affecting tea industry qualitatively and
   quantitatively. The damage to tender shoots and leaves was the most
   serious, while mature leaves and stems were not susceptible to blister
   blight disease. The symptoms caused by blister blight were firstly
   described in details by [63]Petch (1923). Blister blight usually has a
   short life cycle of 11–28 days due to the different climatic conditions
   in different locations ([64]Sen et al., 2020). In this study, the
   initial symptoms of blister blight disease usually infected tender
   leaves that caused light green, yellow-green, light yellow or slightly
   red tiny water-soaked translucent spots after 5–15 days of infection,
   then the spots gradually developed into well-defined circular and
   larger lesions with the diameter of 1–13 mm ([65]Fig. 1A).
   Simultaneously, the spots were sunken into slightly concaves, and on
   the downside, they bulged correspondingly, finally resulting in typical
   blister lesions, but there were also a few lesions that bulged towards
   the upper of the leaves ([66]Fig. 1A). The concave upper surface of the
   blister was smooth and shiny, and the convex surface was generally
   thickened with powdery coating, then become pure white velvety due to
   the intensive growth of basidiospores ([67]Fig. 1B). At the most
   serious stage, the adjacent blisters might fuse into large irregular
   lesions, and diseased tender shoots became distorted or irregularly
   rolled. At late stage, the mature basidiospores were released and
   spread by airflow, and the blisters turned dark brown or purple-red
   ulcer-like and dried up, or even form holes ([68]Fig. 1A), and might be
   infected by other saprophytic fungi. When young stems and petioles were
   infected, the diseased tissues showed obvious swelling, which turned to
   dark brown withered spots in the later stage ([69]Fig. 1C). The damage
   to stems was more serious, as the infected stems bent over and broke
   off at the affected spot, affecting the growth of tea plants.

Fig. 1.

   [70]Fig. 1
   [71]Open in a new tab

   Symptoms of blister blight disease on tea plants. (A) Tea leaves
   infected with blister blight disease at different stages. (B) Blisters
   on lower surface of tea leaf. (C) Blisters on stems.

   Blister blight is a low temperature and high humidity type disease, and
   the mycelium is latent in the living tissue of the diseased leaves for
   over-wintering and over-summering. When the climatic conditions were
   suitable, the basidiospores fell to the water droplets on the young
   leaves or new shoots of the tea trees with the wind, germinated and
   invaded the tea leaves in the water environment, and then the
   basidiospores reproduced in large numbers, so repeatedly infected, thus
   causing large yield and economic loss ([72]Sinniah et al., 2016,
   [73]Mur et al., 2015, [74]Sen et al., 2020). It has been reported that
   a white powder coating on blister can produce two million spores in
   24 h which represents an enormous consumption of metabolites and energy
   of tea ([75]Baby, 2002). In Quxian county, Sichuan province of China,
   blister blight disease mainly occurred in high mountain tea plantations
   above 1000 m altitude, infection began to appear when the temperature
   was above 13℃, and with the increase of temperature and humidity, the
   disease aggravated. Higher temperature coupled with less rainfall and
   low humidity during August in summer resulted in the disappearance of
   blister blight disease, and during December to January in winter,
   blister blight disease also disappeared when the temperature was lower
   than 12℃ ([76]Table S1). As shown in [77]Table S1, In the main tea
   growing areas worldwide, the occurrence period and damage degree of
   blister blight was closely associated with the different climatic
   conditions, therefore, the disease incidence were remarkably different
   in different tea plantations such as Sichuan, Guizhou, Anhui, Yunan,
   Zhejiang and Hunan province of China, and Sri Lanka, India, etc
   ([78]Wang et al., 2013, [79]Liu et al., 2021, [80]Liu, 2017, [81]Shi et
   al., 2016, [82]Sinniah et al., 2016, [83]Mur et al., 2015, [84]Ran et
   al., 2021). The relationships between rainfall, temperature and
   humidity to the intensity of blister blight show a strong linear
   regression pattern, which strongly supports that blister blight
   intensity decreases with reduced intensity of rainfall, rising
   temperatures and low humidity ([85]Mur et al., 2015). Although the crop
   loss changes with the nature of tea varieties and geographical
   locations, there are no cultivars that are completely resistant to
   blister blight disease in China, Sri Lanka, India or elsewhere.
   However, tolerant and susceptible tea varieties showed various degrees
   of physiological and biochemical changes during the infection of
   blister blight. Prevention and control of blister blight disease in
   early stage has not been highly effective due to the lack of suitable
   biological, chemical and cultural methods. So far, a few fungicides
   (ie. copper oxychloride, nickel chloride hexahydrate, ergosterol
   biosynthesis inhibitors) were found to be effective in blister blight
   disease control ([86]Baby, 2002, [87]Sen et al., 2020). However, for
   tea quality, the chemical elicitors may be an more eco-friendly
   approach for disease control, which can improve the innate immunity of
   tea plants by significantly increasing the level of defense molecule
   ([88]Sen et al., 2020).

The development and spread of disease on tea leaf

   The pathogen Exobasidium usually infected young tissues and harvestable
   tea shoots resulting in serious crop loss, and the normal physiological
   metabolism and growth of tea was negatively affected by the infection
   of Exobasidium ([89]Fig. 1). To reveal the development and spread of
   the pathogen E. vexans on tea leaves, TBS, SEM and TEM analysis were
   applied. The structure of tea leaves from top to bottom was upper
   epidermis, palisade tissue, sponge tissue and lower epidermis. As shown
   in [90]Fig. 2A, in healthy tea leaves, the cells in the upper epidermis
   were closely arranged without gap between cells; palisade tissue was
   composed of closely arranged cylindrical cells, which was perpendicular
   to the upper epidermal cells; below the palisade tissue was sponge
   tissue that was consisted of loosely arranged parenchyma cells with
   irregular cell morphology and large cell gap; the lower epidermis was
   made up of a layer of flat cell, which was densely covered with villi
   and stomata. Generally, the pathogen of blister blight disease invaded
   through the stomata of epidermal cells and was stained dark blue
   ([91]Fig. 2A). With the proliferation of pathogens, basidiospores grew
   in clusters by consuming nutrients in tea tissue cells ([92]Mur et al.,
   2015), broke through the epidermis, and a large number of hyphae
   expanded into the cells of sponge tissue, resulting in the necrosis of
   tea leaf cells and the destruction of leaf tissue structure ([93]Fig.
   2A).

Fig. 2.

   [94]Fig. 2
   [95]Open in a new tab

   The development and spread of blister blight disease occurring on tea
   leaves. (A) Tissue structure of tea leaves analyzed by toluidine blue
   staining. up-EP: up-epidermis; ST: spongy tissue; PT: palisade tissue;
   down-EP: down-epidermis. The arrows indicate the hyphae of pathogens.
   bar = 20 μm. (B and C) Scanning electron microscope and transmission
   electron microscope analysis. CK: healthy tea leaves；CB: infected tea
   leaves；GC: guard cell；St: stoma；MC: mesophyll cells；Ch: chloroplast; V:
   vacuole; CW: cell wall; Gr: grana lamella; SL: stroma lamella; M:
   mitochondria; Ba: basidiospore, basidia. The arrows indicate the
   basidiospore and hyphae of pathogens. (For interpretation of the
   references to colour in this figure legend, the reader is referred to