A Phylogenetic and Taxonomic Revision of Discula theae-sinensis, the Causal Agents of Anthracnose on Camellia sinensis

Tea (Camellia sinensis (L.) Kuntze) is one of the most important economic plants in China, and has many benefits for human health. Anthracnose is one of the most serious diseases of tea in China, and control of the fungus is important since most Chinese cultivars are susceptible to it. The agent of tea anthracnose was initially described as Gloeosporium theae-sinensis I. Miyake in Japan, which was later transferred to Discula, but this taxonomic position remains problematic. To shed light on these taxonomic and phylogenetic issues, the tea anthracnose pathogens were re-studied. Combining the morphological characteristics and a multigene phylogenetic analysis of nrITS, nrLSU, rpb2, and tef1 sequence data, a new genus Sinodiscula was proposed to accommodate the causal fungi of tea anthracnose, including a new species Sinodiscula camellicola and a new combination Sinodiscula theae-sinensis. Furthermore, the pathogenicity of the pathogens was determined according to Koch’s postulates. This study thoroughly resolves the long-standing taxonomic and phylogenetic problems of the tea anthracnose pathogens.


Introduction
Tea (Camellia sinensis (L.) Kuntze), as one of the most popular non-alcoholic beverages, contains various chemical ingredients that are beneficial to human health, which can effectively reduce the risk of human diseases [1][2][3][4][5][6], and is loved by consumers worldwide.At present, China is home to 18 major tea-producing provinces, with a total tea plantation area of approximately 3.3303 hectares [6,7].In 2022, China's total output of tea reached 3.1810 million tons, with a national total output value of CNY 318.068 billion, making it the leading producer and exporter of tea worldwide [7].Camellia sinensis has emerged as one of the most crucial economic crops in southern China, particularly in mountainous regions [6,8].
Anthracnose is a highly detrimental disease affecting tea plants in both Japan and China [8][9][10], and has also been reported in Sri Lanka [11].In Japan, tea anthracnose is regarded as one of the three major diseases affecting tea plantations, together with the tea exobasidium blight and the tea white scab [11].Anthracnose causes extensive necrosis and abscission of tea leaves, leading to adverse effects on tea plant growth, as well as decreased yield and quality of tea [8,12].

Fungal Collection and Isolation
Disease leaves of tea anthracnose were collected from the tea leaves collected in Anhui, Hubei, and Zhejiang Provinces, China.Each sample was marked and placed in kraft bags, brought back to the lab, and preserved at room temperature before being processed.The tissues at the junction of disease and health were cut into 5 mm 2 fragments and disinfected in 75% ethanol for 30 s followed by 10% sodium hypochlorite for 5 min and washed in sterile water three times.The fragments were then placed on potato dextrose agar (PDA) and incubated at room temperature for a month.The cultures are deposited in the China Forestry Culture Collection Center (CFCC) and the Mycology Laboratory of Capital Normal University (CNUML), Beijing, China.

Morphological Studies
Cultures were transferred to PDA culture medium at room temperature under a dark light environment and growth rates were observed daily for a month, including colony color, texture, and the conidiomata; the cultures' color was described according to the color charts of Rayner [21].Spores were produced at room temperature, naturally.Cultures were examined periodically for sporulation.Conidia were taken from pycnidium and placed in sterilized water.The diseased tissues with large black small spots were picked out from the tea leaves, then 3-5 mm 2 small fragments were cut out and placed under a stereo microscope to be cut as thin as possible.The cut fragments were placed in sterile water, and the darker part was picked for microscopic examination of the cross-section of the conidioma.Aniline blue (cotton blue) was used to stain colorless structures (conidiomata, conidiogenous cells, and conidia).The shape and size of microscopic structures were observed and noted using a light microscope (Olympus DP71, Tokyo, Japan).

DNA Extraction, PCR Amplification, and Sequencing
The diseased leaves with conidiomata were picked out from the dried tea leaves, then 2 mm 2 small fragments were cut out and crushed by shaking for 45 s at 30 Hz 2-4 times (Mixer Mill MM301, Retsch, Haan, Germany) in a 1.5 mL tube together with 3 mm diam.tungsten carbide balls, and total genomic DNA was extracted using M5 Plant Genomic DNA Kit (Mei5 Bioservices Co., Ltd., Beijing, China) following the manufacturer's instructions.Total genomic DNA was extracted from fresh mycelium that was gained by scraping the surface of 7-day-old colony on PDA, using M5 Plant Genomic DNA Kit following the manufacturer's instructions.This result is consistent with the positive control result, which proves the accuracy of the experimental data.PCR amplification and sequencing of the LSU nrDNA region using the primer pair LROR/LR5 [22,23], ITS nrDNA region using primer pair ITS1F/ITS4 [24,25], rpb2 region using the primer pair fRPB2-5f/ fRPB2-7cR [26], and tef1 region using primer pair EF1-728f/EF2 [27,28] were performed (Table 1).In order to accurately identify pathogenic fungi, the sequences of ITS, nrLSU, rpb2, and tef1 were amplified from the type strain of Discula theae-sinensis (MAFF 752003) for further research.
The temperature profile for both ITS nrDNA and LSU nrDNA was an initial denaturing step for 2 min at 94 • C, followed by 35 amplification cycles of denaturation at 94 • C for 60 s, annealing at 58 • C for 60 s and extension at 72 • C for 90 s, and a final extension step of 72 • C for 10 min [29].The temperature profile for the rpb2 was initial denaturation at 94 • C for 120 s, followed by 35 amplification cycles of denaturation at 95 • C for 45 s, annealing at 57 • C for 50 s, and extension at 72 • C for 90 s [24].The temperature profile for tef1 was initial denaturation at 94 • C for 120 s, followed by 35 amplification cycles of denaturation at 95 • C for 30 s, 58 • C for 50 s, 72 • C 60 s [30].PCR productions were estimated visually in agarose electrophoresis gel by comparing band intensity with a DNA ladder of 200 bp, purified and sequenced by Zhongkexilin Biotechnology Co., Ltd.(Beijing, China).

Phylogenetic Analyses
The new sequences were submitted to the GenBank database and additional sequences of ITS, nrLSU, rpb2, and tef1 included in this study were downloaded from GenBank (Table 2).Sequences of further taxa were included, and isolates were selected to represent each of the 31 known families in the Diaporthales based on the latest available literature.The ITS, nrLSU, rpb2, and tef1 datasets were aligned with MAFFT [31], and then manually corrected visually in Se-Al v.2.03a [32].Ambiguously aligned regions were not used in the analyses.A combined dataset of ITS, nrLSU, rpb2, and tef1 sequences was prepared and analyzed using the maximum parsimony method performed with PAUP * 4.0b10 [33].Maximum parsimony analysis was conducted using heuristic searches with 1000 replicates of random-addition sequence, tree bisection reconnection (TBR) branch swapping, and no maxtree limit.All characteristics were equally weighted and unordered.Gaps were treated as missing data to minimize homology assumptions.A bootstrap analysis was performed with 1000 replicates, each with 100 random taxon addition sequences.Maxtree was set to 1000, and TBR branch swapping was employed.For the Bayesian analysis, MrModeltest 2.3 with the Akaike information criterion (AIC) was used to choose the substitution model for each gene: GTR + I + G for ITS, nrLSU, and rpb2, HKY + I + G for tef1.The Bayesian analysis was performed with MrBayes 3.1.2[34,35].The analyses of four chains were conducted for 100,000,000 generations with the default settings and sampled every 100 generations, halting the analyses at an average standard deviation of split frequencies of 0.01.The first 25% of trees were removed as burn-in.Bayesian posterior probabilities (PP) were obtained from the 50% majority rule consensus of the remaining trees.Maximum likelihood (ML) analysis was performed with IQ-TREE 2.2.0 [36], the substitution model for each gene: TIM2e + I + G4 for ITS, TIM3e + I + I + R3 for nrLSU, TN + F + I + G4 for rpb2, and HKY + F + I + I + R4 for tef1, respectively.ML bootstrap replicates (1000) were computed in IQ-TREE using a rapid bootstrap analysis and search for the best-scoring ML tree.We only considered clades supported by bootstrap values (MLB) ≥70% for the ML analysis, supported by bootstrap values (MPB) ≥ 70% for the MP analysis, and supported by PP ≥ 95% for Bayesian inference.The final alignments and the retrieved topologies were deposited in TreeBASE (http://www.treebase.org,accessed on 13 January 2024), under accession ID: 31091.

Pathogenicity Tests
The isolates CFCC 1914-2-2 and CFCC 107-4-1 were selected to fulfill Koch's postulates.Healthy one-year-old seedlings of Camellia sinensis with a height of approximately 0.2 m were obtained from Anhui Province.Each isolate was inoculated on three separate seedlings and three leaves were selected on each seedling for inoculation.Before the pathogenicity experiment, the surfaces of the leaves were sprayed with 75% alcohol 2-3 times, and then the above operation was repeated with sterile water to remove the residual alcohol; then, they were dried with absorbent paper, or we waited for the surfaces to dry [37].Sterilized needles (0.5 mm diam.) were used to wound five times in the middle parts of each disinfected leaf close to the margins on both sides.The 5 mm PDA medium plugs with mycelia from a 5-day-old culture were inoculated on the left side of the wounded leaves, and sterile PDA plugs without mycelia were inoculated in parallel on the right side of the wounded leaves as control, and repeated three times.A conidial suspension was also used for inoculation.When testing conidial suspensions (10 6 per mL in sterile distilled water), 10 µL of the suspension was deposited on one side of the tested leaves using a syringe, with 10 µL of sterile water acting as control on the opposite side.However, like the results obtained by Li et al. [18], the spore suspension could not bring about obvious disease spots.Each inoculated tea plant was placed in a light incubator at 28 • C and 75% relative humidity with a 12/12 h light/dark photoperiod, and the disease progression of the leaves was regularly observed.The experiment was repeated three times.To complete Koch's postulates, as previously mentioned, the fungi were reisolated from the margin tissue of the diseased lesions that developed from the inoculated tissue and were identified via molecular and phylogenetic analysis.
The multiple-gene phylogenetic analysis shows that sequence data obtained from specimens cited below for species of Sinodiscula form an independent clade with high support values (MLB = 100, MPB = 100, PP = 1.00).Morphologically, Sinodiscula can be distinguished from its closely related genera Greeneria, Melanconiella, Microascospora, Paraphomopsis, and Septomelanconiella.In contrast to the new genus, the asexual morph of Melanconiella usually consists of septate only at the base and hyaline to light brown conidiophores, annellidic or phialidic conidiogenous cells, dark brown melanconiumlike or hyaline discosporina-like conidia [46,47].Similarly, the genus Greeneria, which is typified by Greeneria uvicola (Berk.and M.A. Curtis) Punith., forms pale brown conidia, variously shaped ranging from fusiform, oval, to ellipsoidal, each with a truncate base and obtuse to bluntly pointed apex [48].The asexual morph of Paraphomopsis distinct from Sinodiscula by pycnidia with a slightly elongated, black neck, wider toward the apex at maturity [46].The asexual morph of Septomelanconiella distinct from Sinodiscula by mature conidia cylindrical to clavate, straight or slightly curved, brown, 1-euseptate, more often with six unequal lumina, guttulate, dark brown at the base [49].Although the asexual morph of Microascospora remains undetermined, Microascospora is distantly related to Sinodiscula in the phylogeny presented (Figure 1).Additionally, the sexual morph of Microascospora is distinct from other genera in the same family having immersed, solitary ascomata with narrow papilla with smaller hyaline, aseptate ascospores bearing long appendages [40,41,46].
Diagnosis: The new species is similar to Sinodiscula theae-sinensis, but differs by the scattered and dark-blown conidiomata with slight raising above the surface of the host tissue at maturity, the bigger conidiomata pycnidial, and the L/W ratio of conidia.
Etymology: Referring to the host plant, Camellia sinensis.On leaves of Camellia sinensis: Conidiomata scattered, round to elliptical or slightly irregular, 160-270 µm diam., dark brown, slight raising above the surface of the host tissue at maturity, opening by an ostiole to liberate the conidia.In the vertical section, conidiomata intraepidermal.Etymology: Referring to the host plant, Camellia sinensis.On leaves of Camellia sinensis: Conidiomata scattered, round to elliptical or slightly irregular, 160-270 µm diam., dark brown, slight raising above the surface of the host tissue at maturity, opening by an ostiole to liberate the conidia.In the vertical section, conidiomata intraepidermal.
Culture characteristics: Colony at first white, covered with medium after 15-20 d, becoming olivaceous after 25-30 days.The colony is flat, felty with a thick texture at the center and marginal area, aerial mycelium unconspicuous.Conidiomata sparse, irregularly distributed over agar surface, yellowish mucous conidia were produced on the colony.
The  On leaves of Camellia sinensis: Conidiomata scattered or coalesced, round to elliptical or slightly irregular, 130-260 µm diam., black, strongly raising above the surface of the host tissue at maturity, opening by an ostiole to liberate the conidia.In the vertical section, conidiomata intraepidermal.

Pathogenicity Tests
For each species of Sinodiscula, a representative isolate was selected for the pathogenicity test (CNUCC 1914-2-2 from Sinodiscula theae-sinensis, CNUCC 107-4-1 from Sinodiscula camellicola).Two isolates of Sinodiscula were pathogenic, and the inoculated tea leaves showed lesions similar to the previous symptoms that were observed naturally; nevertheless, the controls remained healthy 7 days after inoculation.Infection occurred from the wound, gradually forming significantly dark lesions on the tea leaf surface (Figure 4).The mean spot size of infected leaves and the incidence of infection are shown in Table 3.The fungi were re-isolated from the lesions and cultured on PDA to verify Koch's postulates.

Discussion
In this study, fresh collections of diseased specimens, pure cultures, and multi-locus phylogenetic analysis were used to address the taxonomic and phylogenetic challenges related to the causal fungi of tea anthracnose in China, contributing toward a better understanding of the causal fungi of tea anthracnose in China, and providing clear pathogen information for the further evaluation of the disease control strategies.
As research has progressed, the tea anthracnose pathogen Gloeosporium theae-sinensis has undergone several taxonomic changes and has been successively transferred to different genera, namely, Colletotrichum and Discula [9,14].However, the morphology of Gloeosporium theae-sinensis is characterized by the small conidia, which are much smaller than those of any other species of Colletotrichum [13,50].And as shown in the study of Moriwaki and Sato [9], the conidiogenous cells of the strains of Gloeosporium theae-sinensis examined were ampoule-to-tenpin-shaped, like those of the type species of Apiognomonia veneta (Sacc.and Speg.)Höhn., the teleomorph of Discula nervisequa (Fuckel) M. Morelet (Gno-

Discussion
In this study, fresh collections of diseased specimens, pure cultures, and multi-locus phylogenetic analysis were used to address the taxonomic and phylogenetic challenges related to the causal fungi of tea anthracnose in China, contributing toward a better understanding of the causal fungi of tea anthracnose in China, and providing clear pathogen information for the further evaluation of the disease control strategies.
As research has progressed, the tea anthracnose pathogen Gloeosporium theae-sinensis has undergone several taxonomic changes and has been successively transferred to different genera, namely, Colletotrichum and Discula [9,14].However, the morphology of Gloeosporium theae-sinensis is characterized by the small conidia, which are much smaller than those of any other species of Colletotrichum [13,50].And as shown in the study of Moriwaki and Sato [9], the conidiogenous cells of the strains of Gloeosporium theae-sinensis examined were ampoule-to-tenpin-shaped, like those of the type species of Apiognomonia veneta (Sacc.and Speg.)Höhn., the teleomorph of Discula nervisequa (Fuckel) M. Morelet (Gnomoniaceae, Diaporthales, Ascomycota), rather than a cylindrical shape as in Colletotrichum spp.[51,52].Phylogenetically, the strains isolated from the lesion of anthracnose of tea indeed fell in the same clade of Diaporthalean fungi with high supports, but did not form a clade with any species in this family [9].Therefore, Moriwaki and Sato suggested that this fungus should belong to the genus Discula [9].In this study, the morphological and molecular phylogenetic analyses indicate that the isolates of the causal fungus of the tea anthracnose belong to Melanconiellaceae, but cannot be classified within any existing genus of Melanconiellaceae.Therefore, a novel genus Sinodiscula is proposed in this study, typified by the new combination Sinodiscula theae-sinensis, and a new species Sinodiscula camellicola is also described.Indeed, these two species exhibit remarkable morphological similarities and lack significant differences in terms of pathogenicity, which presents challenges in their differentiation.However, there is a noticeable molecular distinction between Sinodiscula camellicola and Sinodiscula theae-sinensis, underscoring the importance of molecular markers in distinguishing between these two species.Further comparative research using genomic approaches in order to gain a more comprehensive understanding of these species should be conducted.
The use of the name "tea anthracnose" has long been controversial, because the disease of tea caused by Discula theae-sinensis, a synonym of Gloeosporium theae-sinensis, is commonly referred to as "tea anthracnose" [8][9][10][53][54][55].However, the disease of tea caused by Colletotrichum spp. is also referred to as "tea anthracnose" [56][57][58][59][60].It is noteworthy that the phytopathogenic fungi causing "tea anthracnose" do not belong to the same family or order.While the disease of tea caused by Colletotrichum camelliae Massee is known as "tea cloud leaf blight" in China, this name is not widely used [11,[61][62][63].Phylogenetically, the species of Colletotrichum spp. that cause "tea anthracnose" are distantly related to Discula theae-sinensis [64].However, the symptoms of the disease they cause on the leaves of tea are very similar.The disease caused by these phytopathogenic fungi primarily affects mature leaves and typically begins at the leaf edge or tip.Initially, they produce dark green or yellowish-brown watery spots that later expand along the leaf veins, forming irregular-shaped spots.These spots gradually turn brown or reddish-brown and eventually become greyish-white.The edges of the spots have a yellowish-brown line and are clearly distinguishable from the healthy part of the leaf.The front of the spot is densely covered with numerous small black conidiomata [11,12,58].The visual similarity of disease symptoms caused by these pathogens makes it challenging to differentiate them with the naked eye, which contributes to the confusion surrounding their identification.The study of Li et al. [18] revealed that Discula theae-sinensis is the predominant species in tea leaves, serving as the primary causative agent of tea plant anthracnose.However, there are numerous studies on "tea anthracnose" caused by Colletotrichum spp., so we suggest that the tea disease caused by Colletotrichum spp. is referred to as "tea anthracnose" and the tea disease caused by Discula theae-sinensis as "the tea leaf blight", in order to differentiate between the two diseases.

Figure 1 .
Figure 1.Phylogenetic tree derived from maximum likelihood analysis of the combined ITS, nrLSU, rpb2, and tef1 sequences of Diaporthales, using Ceratosphaeria aquatica (MFLU 18.323) and Pyricularia grisea (CG4) as the outgroups.Bootstrap support values for RAxML and maximum parsimony greater than 70% and Bayesian posterior probabilities (PP) greater than 0.95 are given below and above the nodes.New species and new combinations from this study are in bold.

Table 1 .
Primers used in this study, with sequences and sources.ITS, the internal transcribed spacer regions and intervening 5.8S nrDNA; nrLSU, the present study uses a combined taxonomic approach based on morphology and DNA sequence analyses of the partial 28S nrDNA; rpb2, DNA-directed RNA polymerase II second largest subunit; tef1, translation elongation factor 1-alpha. a

Table 2 .
GenBank accession numbers and culture collection/isolate information for the molecular analysis of Diaporthales.Information written in bold refers to sequences generated in the context of the present study.

Table 3 .
The mean spot size of infected leaves and the incidence of infection.