First Report of Neopestalotiopsis piceana Causing Gray Blight in Camellia sinensis L. in China.

Tea plants (Camellia sinensis L.) are an important cash crop and are cultivated worldwide for their commercial value (Palanisamy et al. 2014). Tea gray blight is an important tea plant disease as it can cause a decline in tea quality and reduce yields by 20-30% (Sanjay et al. 2008). In August 2018, a disease survey was conducted on 400 ha of organic tea plantations in the Pu'er area of Yunnan Province (22.48° N, 100.58° E). The survey found that widespread disease was causing damage to 40% of the tea plantations and that the most seriously affected tea variety was Yunkang No. 10, which had an average disease incidence of 30-35%. The affected leaves grew small yellow-green spots on their tips or margins in the early stage that expanded into round or irregular brown spots with distinct concentric whorls and black conidial disks arranged in whorls when the humidity was high (Fig. 1A-B), which is consistent with tea gray blight disease (Zheng et al. 2021). Twenty-four diseased leaf samples were collected from four different tea plantations and transported to the Pu-Erh Tea Research Laboratory. Leaves with disease spots were cut into 4 mm ×4 mm square pieces, surface-sterilized with 75% alcohol for 1 min, disinfected with 1% sodium hypochlorite for 3 min, and washed thrice with sterile water. The tissue pieces were placed on potato dextrose agar (PDA) plates containing 100 µg ml-1 of chloramphenicol (Wang et al. 2021). After 3 d of culturing in the dark at 28 C, twenty pure cultures with similar morphology were obtained, and two representative isolates were selected and transferred into new PDA media. After 7 d, circular fungal colonies with dense aerial mycelium produced black, wet spore masses that grew on the PDA media (Fig. 1C-D). The conidia were spindle-shaped with four septa, measuring 25.0 (21.0-26.0) × 6.0 (4.5-7.0) µm (n=15). The conidia had three median cells, two of which were dark brown in color with unclear separations, with a single basal hyaline appendage 3.8 (3.5-4.5) µm (n=30) in length and 2-3 apical hyaline appendages 31 (27-35) µm in length (n=30) (Fig. 1E), similar to the conidial characteristics of Neopestalotiopsis piceana (Maharachchikumbura et al. 2014). Two isolates were selected for DNA extraction. The internal transcribed spacer (ITS) region, partial translation elongation factor 1-alpha (tef1-α) gene, and partial ß-tubulin (tub2) gene were amplified using the ITS1F-ITS4 primer set (White et al 1990), the EF-1α-F and EF-1α-R primer sets (Li et al. 2018), and the tub1 and tub2 primers, respectively (Chauhan et al. 2007). The ITS (OP535632 to OP535632), tef1-α (OP589285,OP589287), and tub2 (OP589286,OP589288) sequences were submitted to NCBI GenBank. Basic Local Alignment Search Tool analysis demonstrated that these sequences were 100% similar to those of N. piceana isolates available in GenBank. The sequences were compared using the Mafft software package, and sequences with the same ID were concatenated using scripts. A maximum likelihood phylogenetic tree was constructed using the MEGA (ver. 5.1) software package based on the concatenated sequences (ITS, tef1-α, and tub2). Phylogenetic analysis revealed that C-5 and B-3 showed 95% bootstrap support with N. piceana isolates in references (Fig. 2). According to the morphology and molecular characterization, C-5 and B-3 were identified as N. piceana. Pathogenicity tests on these two isolates were conducted using 36 healthy tea plants. The leaves were scratched slightly with sterile toothpick tips, after which pathogen cakes (6 mm diameter) were placed on the wounds with the mycelial side facing down and covered with sterile absorbent cotton to maintain a moist environment. Control leaves were wounded and covered with sterile PDA plugs (three replicates per treatment, three plants per replicate). Seven days later, the inoculated leaves exhibited similar symptoms observed under natural conditions, whereas the control leaves exhibited no symptoms. The same isolates as the introduced strains were isolated from the diseased tea leaves, completing Koch's postulates. To our knowledge, this is the first report of N. piceana causing gray blight on tea leaves in China. These results provide valuable information for the prevention and management of gray blight on tea leaves. References: Chauhan, J. B., et al. 2007. Indian J Biotechnol. 6: 404-406 Li, D. X., et al. 2018. J. Trop. Crops. 39:1827-1833. Maharachchikumbura, S. N., et al. 2014. Stud. Mycol. 79:121-186. Palanisamy, S., et al. 2014. Appl. Biochem. Biotechnol. 172:216-223. Sanjay, R., et al. 2008. Crop Protect. 27(3-5): 689-694. Wang, Q. M., et al. 2021. Front. Microbiol. 12:774438. White, T. J., et al. 1990. Academic, San Diego. 315-322 Zheng, S., et al. 2021. Plant Dis. 105:3723-3726.

Tea Plants With Gray Blight Have Altered Root Exudates That Recruit a Beneficial Rhizosphere Microbiome to Prime Immunity Against Aboveground Pathogen Infection.

Tea gray blight disease and its existing control measures have had a negative impact on the sustainable development of tea gardens. However, our knowledge of safe and effective biological control measures is limited. It is critical to explore beneficial microbial communities in the tea rhizosphere for the control of tea gray blight. In this study, we prepared conditioned soil by inoculating Pseudopestalotiopsis camelliae-sinensis on tea seedling leaves. Thereafter, we examined the growth performance and disease resistance of fresh tea seedlings grown in conditioned and control soils. Next, the rhizosphere microbial community and root exudates of tea seedlings infected by the pathogen were analyzed. In addition, we also evaluated the effects of the rhizosphere microbial community and root exudates induced by pathogens on the performance of tea seedlings. The results showed that tea seedlings grown in conditioned soil had lower disease index values and higher growth vigor. Soil microbiome analysis revealed that the fungal and bacterial communities of the rhizosphere were altered upon infection with Ps. camelliae-sinensis. Genus-level analysis showed that the abundance of the fungi Trichoderma, Penicillium, and Gliocladiopsis and the bacteria Pseudomonas, Streptomyces, Bacillus, and Burkholderia were significantly (p < 0.05) increased in the conditioned soil. Through isolation, culture, and inoculation tests, we found that most isolates from the induced microbial genera could inhibit the infection of tea gray blight pathogen and promote tea seedling growth. The results of root exudate analysis showed that infected tea seedlings exhibited significantly higher exudate levels of phenolic acids and flavonoids and lower exudate levels of amino acids and organic acids. Exogenously applied phenolic acids and flavonoids suppressed gray blight disease by regulating the rhizosphere microbial community. In summary, our findings suggest that tea plants with gray blight can recruit beneficial rhizosphere microorganisms by altering their root exudates, thereby improving the disease resistance of tea plants growing in the same soil.

Massilia puerhi sp. nov., isolated from soil of Pu-erh tea cellar.

A Gram-reaction-negative, yellow-pigmented, non-spore-forming rod, aerobic, motile bacterium, designated SJY3T, was isolated from soil samples collected from a Pu-erh tea cellar in Bolian Pu-erh tea estate Co. Ltd. in Pu'er city, Yunnan, south-west China. Phylogenetic analysis based on 16S rRNA gene sequences showed that the isolate belonged to the genus Massilia. The closest phylogenetic relative was Massilia arenae CICC 24458T (99.5 %), followed by M. timonae CCUG45783T (97.9 %), M. oculi CCUG43427AT (97.8 %), and M. aurea DSM 18055T (97.8 %). The major fatty acids were C16 : 0 and C16 : 1 ω7c and/or C16 : 1 ω6c. The major respiratory quinone was ubiquinone Q-8 and the major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Genome sequencing revealed a genome size of 5.97 M bp and a G+C content of 65.4 mol%. Pairwise determined whole genome average nucleotide identity (gANI) values and digital DNA-DNA hybridization (dDDH) values were all below the threshold. Although the 16S rRNA gene similarity of stain SJY3T and Massilia arenae CICC 24458T was more than 99 %, the gANI, dDDH values and genomic tree clearly indicated that they were not of the same species. In summary, strain SJY3T represents a new species, for which we propose the name Massilia puerhi sp. nov. with the type strain SJY3T (=CGMCC 1.17158T=KCTC 82193T).

One-Pot Fabrication of Hierarchically Bicontinuous Polystyrene Monoliths with Homogeneous Skeletons and Glycopolymer Surfaces.

The hierarchically bicontinuous polystyrene monoliths (HBPMs) with homogeneous skeletons and glycopolymer surfaces are fabricated for the first time based on the medium internal phase emulsion (MIPE) templating method via activator generated by electron transfer for atom transfer radical polymerization (AGET ATRP). The synergistic self-assembly of amphiphilic diblock glycopolymer (ADG) and Pluronic F127 (PF127) at the oil/water interface via hydrogen bonding interaction contributes to the formation of bicontinuous MIPE with deformed neighboring water droplets, resulting in the highly interconnected HBPM after polymerization. There is a bimodal pore size distribution in the HBPM, that is, through pores (150-5000 nm) and mesopores (10-150 nm). The HBPMs as prepared show excellent biocompatibility, homogeneous skeletons, strong mechanical strength, and high bed permeability, overcoming the practical limitations of the second generation of polystyrene (PS) monoliths. Glycoprotein concanavalin A (Con A) can be easily and quickly separated by the HBPM in hydrophilic interaction chromatography (HILIC) mode. These results suggest the HBPMs have great potentials in catalysis, separations, and biomedical applications.

In-situ grafting temperature-responsive hydrogel as a bifunctional solid-phase microextraction coating for tunable extraction of biomacromolecules.

A temperature-responsive solid-phase microextraction (SPME) coating was prepared via in-situ atom transfer radical polymerization (ATRP) method. By controlling the temperature of solution below and above the lower critical solution temperature (LCST) of the coating, it can switch between hydrophilic and hydrophobic, thus providing a convenient approach for the selective extraction of analytes with different polarities. The average extraction amount of temperature-responsive coating for polar analytes is about 1.5-fold to that of non-polar ones below LCST, and vice versa. Effective extraction of three biomacromolecules was also obtained by controlling the temperature below or above LCST. The adsorption capacity of the coating for the hydrophilic biomacromolecules at 15 °C is 1.5-2 folds that of 50 °C, whereas the adsorption capacity of the coating to BSA at 50 °C is about 3 folds that of 15 °C. This approach holds great promise for SPME because it provides a simple strategy to prepare bifunctional coatings for various applications.

Puerhibacterium puerhi gen. nov., sp. nov., a novel member of the family Promicromonosporaceae, isolated from Pu-erh tea pile-fermentation.

A Gram-staining positive aerobic bacterium, designated TLY-12T, was isolated from the Pu-erh tea pile-fermentation process in Pu'er city, Yunnan, China. Strain TLY-12T grew at 15-37 °C (optimum, 30 °C), pH 6.0-11.0 (optimum, pH 9.0) and 0-9.0% (w/v) NaCl (optimum, 3.0%). The major cellular fatty acids were anteiso-C15:0, C16:0 and iso-C16:0. The respiratory quinone were menaquinones MK-9 (H2) and MK-9 (H4). The polar lipids were phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylinositol (PI), phosphoglycolipid (PGL), glycolipid (GL) and an unidentified phospholipid (PL). The peptidoglycan contained glutamic acid, aspartic acid, alanine and lysine, with the last named being the diagnostic diamino acid. Whole-cell sugars of the isolate were ribose, galactose and glucose. Phylogenetic analyses of 16S rRNA gene showed that this strain belonged to the family Promicromonosporaceae, and was most closely related to Isoptericola cucumis DSM 101603 T, which gave sequence similarity of 97.9%. Genome sequencing revealed a genome size of 3.91 Mbp and a G + C content of 75.0%. Average nucleotide identity and digital DNA-DNA hybridization values were all below the species threshold of described Promicromonosporaceae species. Genome phylogenetic analysis showed that strain TLY-12T formed a separate evolutionary branch, and was parallel to other related genera of Promicromonosporaceae. Based on the phylogenetic, phenotypic, chemotaxonomic and genome pairwise data, strain TLY-12T is considered to represent a novel species in a new genus in the family Promicromonosporaceae, for which the name Puerhibacterium puerhi gen. nov, sp. nov. is proposed. The type strain is TLY-12T (= CGMCC 1.17157T = KCTC 49467T).

Optimisation of saponin extraction conditions with Camellia sinensis var. assamica seed and its application for a natural detergent.

BACKGROUND: Camellia sinensis var. assamica seed cake (a by-product of tea-seed oil) is an abundant resource with poor utilisation. C. sinensis var. assamica seed saponin (CSS) is one kind of non-ionic surfactant. In this study, the CSS extraction conditions were optimised by response surface methodology (RSM) and then the CSS detergent was developed. Additionally, the safety and decontamination ability of the developed detergent were evaluated. RESULTS: The optimised extraction conditions were including the extracting temperature of 40.04 °C, extraction time of 4.97 h, ethanol concentration of 64.11% and liquid-solid ratio of 14.57:1 mL g-1 . The formula of the CSS detergent was as follows: 20% crude CSS, 0.3% oxidised tea polyphenols (OTPs), 0.2% nisin, 0.3% sodium dehydroacetate, 0.7% sodium alginate and 0.5% sodium polyacrylate. The LD50 of the CSS detergent exceeds 14 g kg-1 in mice, indicating the detergent was non-toxic. Both of the emulsifying and the pesticide residues removal abilities of the CSS detergent were significantly stronger than the commercial detergent. CONCLUSION: A natural tea seed saponin detergent with good safety and decontamination ability was successfully developed. This can make better use of the tea seed cake, thereby creating added value in the tea seed oil industry. © 2017 Society of Chemical Industry.