Tea Plants In China Research Articles

A Novel Strain Burkholderia theae GS2Y Exhibits Strong Biocontrol Potential Against Fungal Diseases in Tea Plants (Camellia sinensis).

Tea plants (Camellia sinensis) are widely cultivated cash crops. However, fungal diseases lead to significant reductions in both the yield and quality of tea. Therefore, searching for economical, eco-friendly, and efficient biological control measures is crucial for protecting tea plants from pathogenic fungi. The confrontation assays were performed to identify the antagonistic bacteria against tea pathogenic fungi and evaluate the antifungal activity of these bacteria. Here, three tea pathogenic fungi were identified: Colletotrichum siamense HT-1, Diaporthe phaseolorum HT-3, and Fusarium fujikuroi HT-4. Notably, D. phaseolorum was the first to be reported in tea plants in China. Some tea pathogenic fungi showed a high relative abundance, suggesting a potential disease risk in tea plantations. Strain GS2Y, isolated from tea rhizosphere soil, exhibited strong antifungal activity against tea pathogenic fungi and represented a novel species within the genus Burkholderia, designated as Burkholderia theae. GS2Y could directly inhibit tea pathogenic fungi by disrupting the cellular structures and protect tea plants from fungal diseases caused by C. siamense HT-1 and D. phaseolorum HT-3. B. theae GS2Y might function as a potentially valuable resource for biocontrol agents, laying the foundation for the development of strategies to manage fungal diseases in tea plants.

Genomic variation of 363 diverse tea accessions unveils the genetic diversity, domestication, and structural variations associated with tea adaptation.

Domestication has shaped the population structure and agronomic traits of tea plants, yet the complexity of tea population structure and genetic variation that determines these traits remains unclear. We here investigated the resequencing data of 363 diverse tea accessions collected extensively from almost all tea distributions and found that the population structure of tea plants was divided into eight subgroups, which were basically consistent with their geographical distributions. The genetic diversity of tea plants in China decreased from southwest to east as latitude increased. Results also indicated that Camellia sinensis var. assamica (CSA) illustrated divergent selection signatures with Camellia sinensis var. sinensis (CSS). The domesticated genes of CSA were mainly involved in leaf development, flavonoid and alkaloid biosynthesis, while the domesticated genes in CSS mainly participated in amino acid metabolism, aroma compounds biosynthesis, and cold stress. Comparative population genomics further identified ~730 Mb novel sequences, generating 6,058 full-length protein-encoding genes, significantly expanding the gene pool of tea plants. We also discovered 217,376 large-scale structural variations and 56,583 presence and absence variations (PAVs) across diverse tea accessions, some of which were associated with tea quality and stress resistance. Functional experiments demonstrated that two PAV genes (CSS0049975 and CSS0006599) were likely to drive trait diversification in cold tolerance between CSA and CSS tea plants. The overall findings not only revealed the genetic diversity and domestication of tea plants, but also underscored the vital role of structural variations in the diversification of tea plant traits.

Biological characteristics of the bacteriophage LDT325 and its potential application against the plant pathogen Pseudomonas syringae.

Bud blight disease caused by Pseudomonas syringae is a major bacterial disease of tea plants in China. Concerns regarding the emergence of bacterial resistance to conventional copper controls have indicated the need to devise new methods of disease biocontrol. Phage-based biocontrol may be a sustainable approach to combat bacterial pathogens. In this study, a P. syringae phage was isolated from soil samples. Based on morphological characteristics, bacteriophage vB_PsS_LDT325 belongs to the Siphoviridae family; it has an icosahedral head with a diameter of 53 ± 1 nm and nonretractable tails measuring 110 ± 1 nm. The latent period and burst size of the phage were 10 min and 17 plaque-forming units (PFU)/cell, respectively. Furthermore, an analysis of the biological traits showed that the optimal multiplicity of infection (MOI) of the phage was 0.01. When the temperature exceeded 60°C, the phage titer began to decrease. The phage exhibited tolerance to a wide range of pH (3-11) and maintained relatively stable pH tolerance. It showed a high tolerance to chloroform, but was sensitive to ultraviolet (UV) light. The effects of phage LDT325 in treating P. syringae infections in vivo were evaluated using a tea plant. Plants were inoculated with 2 × 107 colony-forming units (CFU)/mL P. syringae using the needle-prick method and air-dried. Subsequently, plants were inoculated with 2 × 107 PFU/mL LDT325 phage. Compared with control plants, the bacterial count was reduced by 1 log10/0.5 g after 4 days in potted tea plants inoculated with the phage. These results underscore the phage as a potential antibacterial agent for controlling P. syringae.

First report of Fusarium cugenangense causing root rot of tea plants (Camellia sinensis) in China.

Root rot is an important disease of tea plants owing to its unobvious early symptoms and permanent damage (Huu et al. 2016). In 2019, 5% of tea plants displayed symptoms consistent with root rot in a tea plantation (28°09'N, 113°13'E) located in Changsha city, Hunan province of China. The symptoms of the diseased tea plants ranged from wilting leaves to entirely dead. The roots had black lesions and rot typical of this disease. Symptomatic roots were collected, washed with water and disinfected with 75% ethanol, then cut into pieces and sterilized with 0.1% mercuric chloride for 30 s, 75% ethanol for 1 min, and rinsed with sterile water five times. After drying on sterilized filter paper, root tissues were cultured on potato dextrose agar (PDA) medium at 25 oC for 7 days in the dark. Four isolates, CAGF1, CAGF2, CAGF3, and CAGF4 were purified by selecting single spores. All isolates were subjected to a pathogenicity test. A conidial suspension of each strain was collected at a concentration of 2×106 conidia/mL. For the pathogenicity test, two-year-old field grown tea plants were transplanted in plastic pots containing 240 g of the rice grain-bran mixture (inoculated with 4 mL of conidial suspension and cultured for 14 days) and 960 g of sterilized soil (Huu et al. 2016). The pots without inoculated mixture served as control group. All the pots were kept in illumination incubators at 25 oC and a 12L:12D photoperiod. The pathogenicity test for each strain was repeated three times with three repetitions. Only strain CAGF1 exhibited pathogenicity to tea plants. Symptoms appeared on the third day post inoculation (dpi) and gradually worsened by the 7 dpi. On the 14 dpi, most leaves had died and the roots were black and partially rotten, similar to field symptoms. The reisolated fungus from potted roots was identified as CAGF1 based on ITS region and colony morphology, while isolation was attempted, CAGF1 was not isolated from the control plants, which fulfilled Koch's postulates. On PDA, the colony center of CAGF1 was purple with white margin, while on carnation leaf agar (CLA) medium was white. On CLA medium, macroconidia have 0 to 3 septa, measured 19.1 μm to 41.2 μm × 4.2 μm to 5.4 μm (mean= 31.2 μm × 4.8 μm, n=30). The microconidia were measured as 6.7 μm to 12.8 μm × 2.4 μm to 4.9 μm (mean= 10.1 μm × 3.3 μm, n=30), with 0 to 1 septa. And the chlamydospores were measured as 6.0 to 9.7μm (mean= 7.7μm, n=30). Morphologically, strain CAGF1 was identified as Fusarium oxysporum (Leslie and Summerell 2006). Additionally, the genomic DNA of strain CAGF1 was extracted by cetyltrimethylammonium bromide (CTAB) method, the internal transcribed spacer (ITS), elongation factor 1 alpha (EF-1α) and second largest subunit of RNA polymerase II (RPB2) were amplified using the primers ITS1/ITS4 (White et al. 1990), EF-1/EF-2 (Geiser et al. 2004) and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. Sequences were deposited in GenBank (ITS, OK178562.1; EF-1α, OK598121.1; RPB2, OP381476.1). BLASTn searches revealed that strain CAGF1 was 100% (ON075522.1 for ITS and JX885464.1 for RPB2) and 99.6% (JQ965440.1 for EF-1α) identical to Fusarium oxysporum species complex (FOSC). Based on phylogenetic analysis, the strain CAGF1 was identified as Fusarium cugenangense, belonging to FOSC. To our knowledge, this is the first report of F. cugenangense causing root rot of tea plants in China. The findings are important for the management of this root rot and the improvement of economic benefits of tea cultivation.

First report of Fusarium proliferatum causing leaf spot on tea plants in Jiangxi Province, China.

Tea (Camellia sinensis (L.) O. Kuntze), a perennial evergreen shrub, is one of the most important cash crops in China. In September 2021, leaf spot symptoms were observed on approximately 30% of tea plants in a 2 ha commercial field of Lushan (29°33'0" N, 115°58'48" E), Jiangxi Province, China. The symptoms initially appeared as small, gray lesions, and later became larger (10-15 mm in diameter) circular to irregular spots with light brown centers and gray borders. To isolate the pathogen, small pieces (3×3 mm) cut from the margins of lesions were sterilized with 75% ethanol for 10 s, 0.1% HgCl2 for 20 s, and then rinsed three times with sterile water. The pieces were placed onto acidified potato dextrose agar (APDA) plates, and incubated in darkness at 28℃. Pure cultures were prepared by subculturing hyphal tips. A total of 16 fungal isolates were obtained, and the colonies of 15 isolates (isolation rate 93.8%) looked identical, resembling those of the genus Fusarium. The colonies were white to pink with purple woolly mycelium. After 10 to 15 days incubation, slightly curved macroconidia with three to four septa measuring 14.0 to 34.5 × 2.0 to 3.5 µm (n = 50), and oval, unicellar microconidia measuring 4.0 to 9.0 × 1.5 to 3.5 µm (n = 50) were observed. These morphological characteristics were similar to that described for Fusarium proliferatum (Leslie and Summerell 2006). Genomic DNA of representative isolates (LSZWY, LSZWY2, LSZWY3) was extracted with the Ezup Column Fungi Genomic DNA Purification Kit (Sangon Biotech Co., Ltd, Shanghai). The translation elongation factor 1 alpha gene (EF-1ɑ) was amplified using primers EF-1H / EF-2T (O'Donnell, et al. 2015). PCR product was sequenced and the sequence was 709 bp (Accession No. OL614004, ON357634, ON595710). BLAST search results showed that it had 99.9% identity with the EF-1ɑ gene sequence of F. proliferatum (MH341215, MT371378). To test pathogenicity, nine leaves from 5-year-old healthy tea plants (Ca. Luyun 3) were wounded using a sterilized needle and inoculated with a 20µl conidial suspension (2 × 107 conidia·mL-1) on one side of the plants and the other side with sterilized distilled water as a control. All leaves were incubated in a growth chamber at 28℃ and 80% relative humidity with a 12 h light/dark photoperiod. Seven days later, all inoculated treatments showed symptoms identical to those observed in the field, while the control remained asymptomatic. The experiment was repeated three times with similar results. Koch's postulates were fulfilled by successful re-isolation and morphological and molecular identification of F. proliferatum from the inoculated leaves. This pathogen can cause diseases of many crops, e.g. tobacco, Polygonatum cyrtonema and others (Li, et al 2017; Zhou, et al. 2021). However, this is the first report of F. proliferatum causing leaf spot on tea plants in China. This new disease poses a threat to the yield and quality of tea and methods need to be developed for its control and to prevent further spread.

Tea green leafhopper-induced synomone attracts the egg parasitoids, mymarids to suppress the leafhopper.

The tea green leafhopper, Empoasca flavescens is the most important pest of tea plants in China. Mymarid attractants based on herbivore-induced plant volatiles (HIPVs) from leafhopper feeding and oviposition-induced plant volatiles (OIPVs) were formulated and tested as a novel pest control agent against the leafhopper in tea plantations. Results showed that two mymarid species, Stethynium empoascae and Schizophragma parvula, had a reducing effect on leafhopper populations. The HIPVs and OIPVs were identified and bioassayed to screen the key synomones showing strong attraction to the mymarids. They were formulated into different blends, of which Field Attractant 1, comprising linalool, methyl salicylate, (E)-2-hexenal, perillen and α-farnesene at ratio of 1:2:3:58:146 (20 mg/lure), showed the strongest attraction to the mymarids. In field trials with the attractant, the average parasitism rate (60.46 ± 23.71%) of tea leafhoppers by the two mymarids in the attractant-baited area was significantly higher than that (42.85 ± 19.24%) in the CK area. Also, the average leafhopper density (46 ± 30 per 80 tea shoots) in the attractant-baited area was significantly lower than that (110 ± 70 per 80 tea shoots) in the CK area. This study showed that a synthetic blend of key volatiles from HIPVs and OIPVs at an optimal ratio can be formulated into an attractant with the potential to attract and retain wild mymarid populations to suppress leafhopper populations in infested tea plantations, so as to reduce or avoid the spraying of insecticides. © 2023 Society of Chemical Industry.

Antennal transcriptomic analysis of carboxylesterases and glutathione S-transferases associated with odorant degradation in the tea gray geometrid, Ectropis grisescens (Lepidoptera, Geometridae).

Introduction: Carboxylesterases (CXEs) and glutathione S-transferases (GSTs) can terminate olfactory signals during chemosensation by rapid degradation of odorants in the vicinity of receptors. The tea grey geometrid, Ectropis grisescens (Lepidoptera, Geometridae), one of the most devastating insect herbivores of tea plants in China, relies heavily on plant volatiles to locate the host plants as well as the oviposition sites. However, CXEs and GSTs involved in signal termination and odorant clearance in E. grisescens remains unknown. Methods: In this study, identification and spatial expression profiles of CXEs and GSTs in this major tea pest were investigated by transcriptomics and qRT-PCR, respectively. Results: As a result, we identified 28 CXEs and 16 GSTs from female and male antennal transcriptomes. Phylogenetic analyses clustered these candidates into several clades, among which antennal CXEs, mitochondrial and cytosolic CXEs, and delta group GSTs contained genes commonly associated with odorants degradation. Spatial expression profiles showed that most CXEs (26) were expressed in antennae. In comparison, putative GSTs exhibited a diverse expression pattern across different tissues, with one GST expressed specifically in the male antennae. Disscussion: These combined results suggest that 12 CXEs (EgriCXE1, 2, 4, 6, 8, 18, 20-22, 24, 26, and 29) and 5 GSTs (EgriGST1 and EgriGST delta group) provide a major source of candidate genes for odorants degradation in E. grisescens.

First Report of Leaf Spot Caused by Phyllosticta capitalensis on Illiciumdifengpi in China.

Illicium difengpi B. N. Changet al., a shrub with aromatic odor in the Illicium genus, is extensively used as a medicinal plant in China. In June of 2020, a leaf spot on I. difengpi with incidence of about sixty percent was observed in a field located in Guilin (25°4'40"N; 110°18'21"E), Guangxi Province, China. Initialleafsymptoms were roundspotswith gray centers, surrounded by yellow halos. The spots gradually spread and merged. Six samples of symptomaticleaveswere collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 10 isolates were obtained from the affectedleaves.Two single-spore isolates (GX-1 and GX-2) were obtained and confirmed to be identical based on morphological characteristics. The representative isolate GX-2 was selected for further study on morphological and molecular characteristics. The colony of isolate GX-2 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white edge. Conidia were hyaline, unicellular, oval, narrow at the end with a single apical appendage, and 8.2 to 13.8 × 3.7 to 7.2 µm (n= 50). Spermatia were hyaline, bacilliform with swollen ends, 3.8 to 8.9 × 1.3 to 1.9 µm (n= 50). Morphological characteristics of isolate GX-2 were consistent with the description of Phyllostictacapitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), glyceraldehyde-3-phosphate dehydrogenase (GPDH) and actin (ACT) were amplified using primers ITS1/ITS4, EF-728F/EF-986R, Gpd1-LM/Gpd2-LM and ACT-512F/ACT-783R, respectively (Wikee et al. 2013). Sequences were deposited in GenBank with accession numbers OL505439 forITS, OL539429 forACT, OL539430 fortef1-α and OL539431 for GPDH. BLAST analysis in GenBank showed that these sequences were 99 to 100% similar to the correspondingITS(MT649668),ACT(MN958710), tef1-α(MN958711) andGPDH (KU716077) sequences ofP. capitalensis. Also, the phylogenetic tree based on genes of ITS, tef1-α, GPDH and ACTby the maximum likelihood method showed that isolate GX-2 clustered together withP. capitalensis.The pathogenicity tests were carried out on a healthy 3 year-old plant in the greenhouse with 80% relative humidity at 25 °C. Four sterilized leaves were wounded with a needle and inoculated with 20 μL spore suspension (1 × 106spores/ml). Another four sterilized leaves were inoculated with 20 μL sterile water as a control. All plants were incubated in a chamber with 98% relative humidity at 25 ± 1°C. After 12 days, disease symptoms similar to the field were observed on leaves, whereas control plants remained healthy.P. capitalensiswas successfully reisolated only from the inoculated leaves and identified based on morphological characters. P. capitalensiscausedleafspotson various host plants around the world (Wikee et al. 2013), including on tea plants in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on I. difengpi. Thus, this is the first report ofP. capitalensiscausingleafspoton I. difengpi. This study will provide an important reference for the control of the disease. The epidemiology ofthis disease should be investigated in further research.

Tea plant (Camellia sinensis) lipid metabolism pathway modulated by tea field microbe (Colletotrichum camelliae) to promote disease.

Tea is one of the most popular healthy and non-alcoholic beverages worldwide. Tea anthracnose is a disease in tea mature leaves and ultimately affects yield and quality. Colletotrichum camelliae is a dominant fungal pathogen in the tea field that infects tea plants in China. The pathogenic factors of fungus and the susceptible factors in the tea plant are not known. In this work, we performed molecular and genetic studies to observe a cerato-platanin protein CcCp1 from C. camelliae, which played a key role in fungal pathogenicity. △CcCp1 mutants lost fungal virulence and reduced the ability to produce conidia. Transcriptome and metabolome were then performed and analysed in tea-susceptible and tea-resistant cultivars, Longjing 43 and Zhongcha 108, upon C. camelliae wild-type CCA and △CcCp1 infection, respectively. The differentially expressed genes and the differentially accumulated metabolites in tea plants were clearly overrepresented such as linolenic acid and linoleic acid metabolism, glycerophospholipid metabolism, phenylalanine biosynthesis and metabolism, biosynthesis of flavonoid, flavone and flavonol etc. In particular, the accumulation of jasmonic acid was significantly increased in the susceptible cultivar Longjing 43 upon CCA infection, in the fungal CcCp1 protein dependent manner, suggesting the compound involved in regulating fungal infection. In addition, other metabolites in the glycerophospholipid and phenylalanine pathway were observed in the resistant cultivar Zhongcha 108 upon fungal treatment, suggesting their potential role in defense response. Taken together, this work indicated C. camelliae CcCp1 affected the tea plant lipid metabolism pathway to promote disease while the lost function of CcCp1 mutants altered the fungal virulence and plant response.

Complete genome sequence of a new badnavirus infecting a tea plant in China.

The complete genome of a novel virus, provisionally named "Camellia sinensis badnavirus 1" (CSBV1), was identified in tea plant (Camellia sinensis) leaves collected in Anhui Province, China. The genome of CSBV1 consists of 8,195 bp and possesses three open reading frames (ORFs), sharing 68.6 % nucleotide sequence identity with the genome of Camellia lemon glow virus (CLGV) from Camellia japonica. The genome organization of CSBV1 is highly similar to that of members of the genus Badnavirus (family Caulimoviridae). Phylogenetic analysis revealed that CSBV1, CLGV, and cacao swollen shoot virus form a separate clade within the genus Badnavirus, suggesting that CSBV1 is the first badnavirus infecting C. sinensis.

The Potential of Parapanteles hyposidrae and Protapanteles immunis (Hymenoptera: Braconidae) as Biocontrol Agents for the Tea Grey Geometrid Ectropis grisescens (Lepidoptera).

Simple SummaryThe tea grey geometrid Ectropis grisescens is a significant insect pest of tea plants in China. Parapanteles hyposidrae and Protapanteles immunis are Ectropis grisescens larval parasitoids. Here, we studied the parasitism performance of these two parasitoid species on different host densities under different temperatures as well as the interference effect of parasitoid density. We found that both parasitoid species, Pa. hyposidrae and Pr. immunis, exhibited a type II functional response towards the tea grey geometrid E. grisescens at four tested temperatures. With increasing the density of E. grisescens larvae, the number of parasitized larvae increased until a maximum was reached. Pr. immunis performed better than Pa. hyposidrae under higher temperatures. The parasitism rate by a single female parasitoid decreased with increasing parasitoid density at different temperatures, resulting in a reduction of searching efficiency. The findings of this study showed that Pr. immunis could be a better effective biocontrol agent than Pa. hyposidrae against the tea grey geometrid.The tea grey geometrid Ectropis grisescens has long been a significant insect pest of tea plants in China. Two parasitoids, Parapanteles hyposidrae and Protapanteles immunis (Hymenoptera: Braconidae: Microgastrinae), are the most important parasitoids in the larval stage of E. grisescens. Yet, the potential of these two parasitoids for controlling the tea grey geometrid is not known. Here, we studied the parasitism performance of these two parasitoid species on different host densities under different temperatures as well as the interference effect of parasitoid density. The results showed that both parasitoid species, Pa. hyposidrae and Pr. immunis, exhibited a Type II functional response towards the tea grey geometrid E. grisescens at four tested temperatures. With increasing the density of E. grisescens larvae, the number of parasitized larvae increased until a maximum was reached. The highest number of hosts parasitized by Pa. hyposidrae or Pr. immunis reached 14.5 or 14.75 hosts d−1 at 22 °C, respectively. The estimated values of instantaneous searching efficiency (a) and handling time (h) for Pa. hyposidrae or Pr. immunis were 1.420 or 3.621 and 0.04 or 0.053 at 22 °C, respectively. Pr. immunis performed better than Pa. hyposidrae under higher temperatures. The parasitism rate by a single female parasitoid decreased with increasing parasitoid density at different temperatures, resulting in a reduction of searching efficiency. The findings of this study showed that Pr. immunis could be a better effective biocontrol agent than Pa. hyposidrae against the tea grey geometrid.

First Report of Epicoccum nigrum Causing Brown Leaf Spot in Tea in Guizhou Province, China.

Brown leaf spots were observed on tea [Camellia sinensis (L.) Kuntze] in Sinan County (27.74 °N, 108.35 °E) and Kaiyang County (27.96 °N, 107.34 °E), Guizhou Province, China, from 2018 to 2020. For the leaf spots with the typical symptoms, the disease incidence was estimated to range between 56% and 61%, respectively. The disease severity was estimated to range from 39 to 43 across 12 tea plantations, respectively. The disease initially occurred at the margins of leaf tips, and the lesions expanded gradually, being dark brown and irregularly shaped and became necrotic. To identify the causal organism, two leaves from each of 15 tea twigs, one or two per plantation, were detached from 8- or 10-year-old tea plants on each of 12 plantations. Samples taken from the lesion margins were sterilized with 75% ethanol followed by 0.5% NaOCl, placed on potato dextrose agar (PDA), and then incubated at 25oC in darkness for 5 days (Wang et al. 2020). For each sample, hyphal tips from the margin of a growing colony were successively transferred to fresh PDA, and pure cultures were obtained. Three representative strains were grown on PDA, malt extract agar (MEA), and oatmeal agar (OA) plates. The colonies had smooth margins and abundant mycelia on all three media, with the colony colors being from gray to light purple on PDA, white on MEA, and purplish-red on OA at 5 days post-inoculation. At 20 days post-inoculation on MEA, stromata began to gradually form, which were droplet-like, 100 to 2,000 μm in diameter, and semi-immersed on the medium's surface. Black sporodochia were produced on the surfaces of stromata. Conidiophores were aggregated in sporodochia, densely compacted, and dark brown. Conidia were globose or pyriform, dark, multicellular, and measured 22.95 ± 3.59 × 19.82 ± 3.13 μm (n = 50) in diameter. The morphological characteristics of the mycelia and reproductive structures of the strains were identical to those of Epicoccum nigrum. The internal transcribed spacer (ITS) region of rDNA, and the partial 28S large subunit rDNA (LSU), RNA polymerase II second largest subunit (RPB2), and beta-tubulin (TUB) genes of these strains were amplified using the primers V9G/ITS4 (De Hoog and Gerrits van den Ende 1998; White et al. 1990), LR0R/LR5 (Rehner and Samuels 1994), RPB2-5F2/fRPB2-7cR (Sung et al. 2007), and TUB2Fd/TUB4Rd (Woudenberg et al. 2009), respectively, and deposited in GenBank (accession no. MW646378, MW291537, MW602293, and MW602295 for ITS, LSU, RBP2, and TUB, respectively). A maximum parsimony phylogenetic analysis indicated that the representative strains clustered with E. nigrum CBS 173.73 (Chen et al. 2017). Pathogenicity tests were performed on 5-year-old potted tea and on 10-year-old C. sinensis cv. Fuding-dabaicha in the field. Mycelial plugs (6-mm diam.) and a conidial suspension (106 conidial/mL) were applied on punctured leaves using a sterile needle and non-punctured leaves. Inoculation with only a PDA plug or sterile water served as controls. Brown spots appeared on the wounded sites of tea leaves at 2 days post-inoculation. No symptoms were observed on the non-wounded leaves or wounded leaves inoculated with PDA plugs lacking mycelia. The re-isolated pathogen from diseased plants was identical to the purified strain ACCC39731 used for inoculation, with re-isolation frequency being 85.0%. To our knowledge, this is the first report of E. nigrum causing leaf spot on tea plants in China, and our findings will be useful for its management and further research.

Temperature-dependent oviposition model of Scopula subpunctaria (Lepidoptera: Geometridae)

• Temperature significantly influence oviposition S. subpunctaria. • Male adults lived shorter than female did. • The temperature-dependent oviposition model was constructed. Scopula subpunctaria , is one of important defoliating insect pests of tea plants in China. However, the effect of temperature on its longevity and fecundity had not been reported, and the temperature-dependent oviposition model is lacking. The reproductive characteristics of S. subpunctaria were investigated under eight constant temperatures (13, 16, 19, 22, 25, 28, 31, and 33℃), with a 14L: 10D photoperid and 65–85% RH. The results showed that temperature significantly influence oviposition period, longevity, and fecundity of S. subpunctaria . The mean longevity of female adults increased from 10.8 days (33℃) to 33.9 days (13℃). Male lived shorter than female did. The mean longevity of males was shortest at 33℃ (8.1 days) and longest at 23.8℃ (17.6 days). Total fecundity of S. subpunctaria females ranged from 15.4 eggs at 33℃ to 279.9 eggs at 22℃. The temperature-dependent oviposition model of S. subpunctaria was constructed based on four sub-models (developmental rate model, total fecundity model, oviposition rate model, and survival rate model). The results of this study would be helpful in forecasting and predicting population occurrence of S. subpunctaria in tea plantations.

Electrophysiological and Behavioral Responses of Dasychira baibarana (Lepidoptera: Lymantriidae) to Tea Plant Volatiles.

Tea black tussock moth, Dasychira baibarana (Matsumura) (Lepidoptera: Lymantriidae), is a devastating pest species of the tea plant in China. Here, we evaluated the responses of D. baibarana to tea plant volatiles using gas chromatography coupled electroantennographic detection (GC-EAD), eleclectroantennography (EAG), and a Y-tube olfactometer. In total, 11 of 18 analyzed compounds elicited GC-EAD responses from test insects. GC-EAD bio-active compounds were further investigated using EAG and behavioral responses. In the EAG analysis, male moths had significantly greater responses to four compounds [(Z)-3-hexenyl butyrate, (Z)-3-hexen-1-ol, ocimene and benzyl alcohol] than female moths. For females, maximum EAG amplitudes, were recorded in response to linalool, (Z)-3-hexenyl hexanoate and (Z)-jasmone. In EAG and behavioral bio-assays, the responses of both sexes were dose independent. In behavioral bio-assays male moths responding significantly to (Z)-3-hexen-1-ol, ocimene, (Z)-3-hexenyl butyrate, linalool, benzyl alcohol, and (Z)-jasmone at various concentrations. For females, significant behavioral responses were observed to (Z)-3-hexenyl hexanoate, followed by (Z)-jasmone, linalool, ocimene, and benzyl alcohol. However, neither sex was sensitive to 4 of the 11 tested compounds, phenyethyl alcohol, phenylacetonitrile, (E)-nerolidol, and indole. The present results showed that tea plant volatiles influenced the behavior of D. baibarana moths, which will greatly contribute in developing eco-friendly control strategies for D. baibarana, through the application of a blend of compounds that showed significant EAG and behavioral responses or a blend combined with female-produced sex pheromones.

Accumulation pattern of catechins and flavonol glycosides in different varieties and cultivars of tea plant in China

Flavonoids are important phenolic compounds in tea plant since they have a large number of physiological functions for plants themselves and human beings. Tea samples (n = 151) harvested from 3 varieties, 9 plantation areas and 107 cultivars were analyzed to describe the attributes of catechins and flavonol glycosides in China. A support vector machine (SVM) classifier was built to recognize the assamica variety with 94.2 % accuracy. Accumulation of (-)-epicatechin gallate (ECG) and flavonol di-glycosides was especially observed in assamica variety, while (-)-epigallocatechin gallate (EGCG) and flavonol tri-glycosides were obviously accumulated in sinensis variety. The ratios of ECG/EGCG and flavonol tri-glycosides/flavonol di-glycosides could be used as indicators for distinguishing the different varieties and cultivars. Accumulation pattern of catechins and flavonol glycosides were mainly dependent on the genetic background of the cultivars rather than plantation locations. The increased hydroxyl groups at the C-ring of catechins and the extended sugar chain at the B-ring of flavonol glycosides could be considered as important rules of metabolite change during tea plant evolution. The mechanism of accumulation of flavonoids in various varieties and cultivars was also discussed.

Herbivore-induced DMNT catalyzed by CYP82D47 plays an important role in the induction of JA-dependent herbivore resistance of neighboring tea plants.

Herbivore-induced plant volatiles play important ecological roles in defense against stresses. However, if and which volatile(s) are involved in the plant-plant communication in response to herbivorous insects in tea plants remains unknown. Here, plant-plant communication experiments confirm that volatiles emitted from insects-attacked tea plants can trigger plant resistance and reduce the risk of herbivore damage by inducing jasmonic acid (JA) accumulation in neighboring plants. The emission of six compounds was significantly induced by geometrid Ectropis obliqua, one of the most common pests of the tea plant in China. Among them, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) could induce the accumulation of JA and thus promotes the resistance of neighboring intact plants to herbivorous insects. CsCYP82D47 was identified for the first time as a P450 enzyme, which catalyzes the final step in the biosynthesis of DMNT from (E)-nerolidol. Down-regulation of CsCYP82D47 in tea plants resulted in a reduced accumulation of DMNT and significantly reduced the release of DMNT in response to the feeding of herbivorous insects. The first evidence for plant-plant communication in response to herbivores in tea plants will help to understand how plants respond to volatile cues in response to herbivores and provide new insight into the role(s) of DMNT in tea plants.

First Report of Castor Dark Leaf Spot Caused by Phyllosticta capitalensis in Zhanjiang, China

Castor (Ricinus communis L.) is an important oil crop. Leaf spots on castor with disease incidence of about 22.5% have been observed on about 2,000 plants in a field located in Zhanjiang (21.17N, 110.18E), China, since 2016. Initial leaf symptoms were round spots with gray centers, surrounded by yellow halos. Ultimately, the spots then gradually spread and merged. Six samples of symptomatic leaves were collected from six diseased plants, and they were surface disinfested before isolation. Potato dextrose agar (PDA) was used to culture pathogens. Successively, pure cultures were obtained by transferring hyphal tips to new PDA plates. A total of 15 isolates were obtained from the affected leaves. Two single-spore isolates (RiL-1 and RiL-2) were obtained and confirmed to be identical based on morphological characteristics. Therefore, the representative isolate RiL-1 was selected for further study on the morphological and molecular characteristics. The colony of isolate RiL-1 was about 4 cm in diameter on a PDA plate in 5 days, dark green with a granular surface, and irregular white (later turning black) edge. Conidia were single-celled, oval, narrow at the end with a single apical appendage, and 8.9 to 12.6 × 4.0 to 7.5 µm (n = 40). Morphological characteristics of isolate RiL-1 were consistent with the description of Phyllosticta capitalensis (Wikee et al. 2013). The internal transcribed spacer (ITS) region, translation elongation factor 1-α (tef1-α), and actin (ACT) were amplified using primers ITS1/ITS4, EF1-728F/EF1-986R (Druzhinina et al. 2005), and ACT-512F/ACT-783R (Wikee et al. 2013), respectively. Sequences were deposited in GenBank with accession numbers MN548089 for ITS, MN565576 for tef1-α, and MN565573 for ACT. BLASTn analysis demonstrated that these sequences were 99 to 100% similar to the corresponding ITS (MF170677), tef1-α (KU716086), and ACT (KY855659) sequences of P. capitalensis. Also, by the neighbor-joining method, a phylogenetic tree showed that isolate RiL-1 (MN548089) clustered together with P. capitalensis (MF800912) from China. From the combination of the morphological and molecular characteristics, isolate RiL-1 was determined to be P. capitalensis. A pathogenicity test was performed in a greenhouse with 80% relative humidity at 25 to 30°C. Sterilized cotton balls were immersed in spore suspension (1 × 10⁶ spores/ml) or sterile water for 1 min. Sixteen healthy plants were divided into two groups (wounded group and unwounded group). Spore-containing cotton balls covered the inoculated sites on four plants in the wounded group and four in the unwounded group, with the same procedure for the water-only cotton balls, and kept for 48 h before removing the cotton. After 15 days, disease symptoms were observed on both wounded and unwounded leaves, whereas control plants (both wounded and unwounded) remained healthy. Morphological characteristics and the ITS sequences of the fungal isolates reisolated from the diseased leaves were identical to those of RiL-1. P. capitalensis has caused leaf spot on various host plants around the world (Wikee et al. 2013), including on tea plant in China (Cheng et al. 2019) and oil palm in Malaysia (Nasehi et al. 2020), but it has not been reported on castor. Thus, this is the first report of P. capitalensis causing leaf spot on castor. This study will provide an important reference for the control of the disease. The epidemiology of P. capitalensis on castor should be investigated in further research.

First Report of Leaf Blight Caused by Arthrinium arundinis on Tea Plants in China

Tea (Camellia sinensis [L.] Kuntze) is one of the most important economic crops in China. During a survey in July 2018, symptoms of brown leaf patches (3 to 4 cm diameter) with irregular margins were observed on tea leaves of Longjing43 cultivar in Huoshan tea farm, Anhui province, China. The disease incidence reached 18 to 20% in the most severely affected tea gardens. Lesions first appeared as gray-brown dots, subsequently expanding to irregular dark brown, and then ended with large brown patches on the tea leaves. The infected tea leaf samples were collected from the tea plants, and the pathogenic fungus isolation was performed according to the method described by Thangaraj et al. (2018). Briefly, the tea leaf samples washed with tap water followed by distilled water. The margins of infected leaves were cut into small pieces (4 to 5 mm²) and surface sterilized with 0.1% HgCl₂ for 1 min followed by 70% ethanol for 30 s and rinsed three times in sterile distilled water. Then, these aseptic tissues were blotted dry on sterilized filter paper, transferred onto 2% water agar, and incubated in the dark at 28°C until fungal hyphae started to grow. Single hyphal tips were transferred onto potato dextrose agar (PDA) plates and incubated for 7 days at 28°C for morphological observations. A total of five isolates were obtained and identified as the same fungus on the basis of phenotypic characteristics. The colonial characters of the isolated fungus on PDA were dirty white and moderate aerial mycelium with dark colored patches. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were reduced to conidiogenous cells, size ranged from 4.2 to 11.0 × 2.7 to 5.7 μm (length and width, respectively) (average 7.4 × 3.8 μm; n = 30), pale brown, smooth, and ampulliform. Conidia were 4.5 to 12.2 × 3.4 to 9.4 μm (average 6.9 × 5.0 μm; n = 50), single celled, brown, smooth, anomalous shape (globose to ellipsoid shape), mostly globose in surface view and lenticular in side view with an equatorial slit. Based on morphological characteristics, the isolated plant pathogenic fungus, named ZZZ3, was identified as Arthrinium spp. (Corda) Dyko & B. Sutton (Crous and Groenewald 2013). To identify the isolate to the species level, genomic DNA was extracted (Fungal DNA extraction kit, BIOMIGA, China) from mycelial mats. The internal transcribed spacer region (ITS1/ITS4) (White et al. 1990) and D1/D2 region of 28S rDNA (LR0R/TW13) genes (Hamayun et al. 2009) were amplified. The obtained ITS (539 bp) and 28S rDNA (585 bp) (GenBank accession nos.: ITS, MN187298; 28S rDNA, MN187305) sequence BLAST analysis showed 100 and 99% similarity with Arthrinium arundinis (ITS, KF144883; 28S rDNA, KF144929), respectively. Based on these morphological and genetic characteristics, the fungus was identified as A. arundinis. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and the adaxial surface of matured leaves was injured (two needle punctures per leaf, using a sterile needle). The conidial suspension (10⁶/ml) was spread over the wounds, and sporeless sterile water was spread on wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 14 days following inoculation, lesions appeared on the wounded leaves, and the symptoms were similar to those described above, whereas the control and unwounded leaves remained healthy. The experiment was performed thrice, and the same fungus was reisolated from the infected leaves, showing similar morphological characteristics and molecular traits. Wang et al. (2018) identified eight new Arthrinium spp. from different host plants in China, of which A. xenocordella and A. camelliae-sinensis were isolated from tea plants. To our knowledge, this is the first report of A. arundinis causing tea leaf blight in China. The establishment of the disease in tea plants may require the development of effective control strategies to minimize its impact on the production of tea.

Primary screening and application of repellent plant volatiles to control tea leafhopper, Empoasca onukii Matsuda.

The tea leafhopper, Empoasca onukii Matsuda (Hemiptera: Cicadellidae), is a major pest of tea plants in China. Here, we evaluated the repellent properties of eight volatile chemicals alone and in various combinations as tools for the management of this pest in tea gardens. These chemicals were from the Alliaceae and other aromatic plants, and are known to repel various insect species. Among the eight volatile compounds, dimethyl disulfide (DMDS), 1,8-cineole and allyl methyl sulfide were significantly repellent towards E. onukii adults. DMDS and 1,8-cineole were mixed to formulate a binary repellent. Under field conditions, spraying and slow-release applications of the mixture significantly decreased the density of E. onukii adults. The repelling effect after spraying was very short, only ∼ 2 days, but the slow-release mixture had a longer term repelling effect on E. onukii adults. High emission of the slow-release mixture, which was achieved by increasing the number of slow-release bottles, had a stronger repellent effect than low emission. Moreover, when the amount emitted was sufficient, the slow-release mixture significantly decreased the number of leafhopper nymphs in a treated tea-plant line, and significantly decreased the number of leafhopper adults and nymphs in a tea-plant line adjacent to the treated area. This study demonstrates the repellent action of a mixture of DMDS and 1,8-cineole applied by a slow-release method against E. onukii in a tea plantation. This mixture has potential applications in integrated pest management schemes. © 2019 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Characterization and Pathogenicity of Pestalotiopsis-Like Species Associated With Gray Blight Disease on Camellia sinensis in Anhui Province, China.

Gray blight disease, caused by Pestalotiopsis-like fungi, is one of the deadliest threats to tea (Camellia sinensis) production. However, little information is known about the traits and characteristics of this pathogen. Here, a systematic survey was performed, and a total of 20 representative isolates were obtained from the leaves of tea plants affected by gray blight in two main tea plantations located in Anhui Province, China. Further analyses showed that two isolates were identified as Neopestalotiopsis ellipsospora, three isolates were regarded as Pseudopestalotiopsis chinensis, one isolate was considered as Pseudopestalotiopsis camelliae-sinensis, and the remaining isolates belonged to Pseudopestalotiopsis spp., on the basis of morphological characteristics and multigene phylogenetic analyses of the internal transcribed spacer, β-tubulin, and translation elongation factor 1-α. Pathogenicity tests indicated that there were significant differences in virulence among the Neopestalotiopsis and Pseudopestalotiopsis isolates when inoculated on the leaves of the tea plant (C. sinensis 'Shuchazao'). Furthermore, varied pathogenicity was also observed for the same isolate when inoculated on different varieties of tea plants. To our knowledge, this is the first record of Neopestalotiopsis ellipsospora and Pseudopestalotiopsis chinensis causing gray blight disease of tea plants in China.