First report of Coniella koreana causing leaf blight on Loropetalum chinense var. rubrum (Chinese Fringe Flower) in Sichuan, China.

Loropetalum chinense var. rubrum (Chinese Fringe Flower) is widely distributed in the middle and lower reaches of the Yangtze River, as well as northern India. It is a popular landscape plant for its red evergreen foliage and its showy red flowers in the spring. In July 2020, This leaf blight was discovered in Chengdu city (30°42＇41＂N, 103°51＇58＂E). In June 2021, the disease incidence rate at two places in Wenjiang District of Chengdu was 76% and 64%, respectively. The symptoms began to appear from May to June, worsened from July to August, and then disappeared gradually in November. Initially, brown-edged irregular necrotic patches appeared at the leaf margins. Progressively, the patches increased in number, expanded to leaf middle, and turned grayish-white. The scattered black fruiting bodies (conidia) were appeared at patches under humid conditions. Eventually, the leaves tended to dry up and fall off. Infected tissues from five samples and collected were cut into small pieces 2×2 mm, surface sterilized for 30 s in 3% sodium hypochlorite, 60 s in 75% ethanol, rinsed three times in sterile water, placed onto potato dextrose agar (PDA), and incubated at 25℃ in the dark. A total of eight isolates were collected, five isolates exhibited similar culture characteristics while two were Nigrospora sp. and one was a Fusarium sp.. The five similar isolates produced sparse, grayish-withe mycelia with a flat elevation and curled margin. Abundant globose and yellow pycnidia were formed on the PDA surface and arranged in irregular concentric zones. Conidia were 18.20 to 22.36 × 2.64 to 3.05 µm (average 20.36 × 2.82 µm, n=50) in size, fusiform, sickle-shaped, aseptate. DNA was extracted from the representative strain (HMcj B03), and the internal transcribed spacer (ITS) region, the large subunit of the nuclear ribosomal DNA (LSU), translation elongation factor 1-alpha (tef1-α), and the DNA-directed RNA polymerase II second largest subunit (rpb2), were amplified by polymerase chain reaction and sequenced with primers ITS1/ITS4 (White et al. 1990), LR0R/LR7 (Rehner and Samuels 1994; Vilaglys and Hester 1990), 728F/986R (Carbome and Kohn 1999), and 5F2/7cR (Alvarez et al. 2016), respectively. The sequences were deposited in GenBank, viz. OL468959, OL469170, OL489770, and OL855833, respectively. BLAST analysis showed >98.7% identity with several reference sequences of Coniella koreana strain CBS 143.97 and Coniella quercicola strain CBS 904.69, deposited in GenBank. A conidial suspension (1 × 107 conidia/mL) having 0.05% Tween 80 buffer was used for foliar inoculation of 6-year-old Loropetalum chinense var. rubrum plants for pathogenicity test. Ten leaves of each plant (10 pots in total) were inoculated with spore suspensions (20 µL onto the wounded sites). An equal number of control leaves were sprayed with 0.05% Tween 80 buffer to serve as a control. The experiment was repeated three times, and all plants were incubated in a growth chamber (a 12h light and 12h dark period, 25°C, RH > 80%). Twenty days later, all the inoculated leaves showed similar symptoms as the original diseased plants, however, the controls remained asymptomatic. The C. koreana was re-isolated from the infected leaves. To our knowledge, this is the first report of L. chinense var. rubrum caused by C. koreana in China. The discovery of this new disease will provide useful information for developing effective control strategies, and prove beneficial in reducing economic losses in floral product.

Neofusicoccum parvum Causing Stem and Branch Blight Disease of Zanthoxylum bungeanum in Sichuan, China.

The Chinese pepper Zanthoxylum bungeanum Maxim is a special economically important species and a traditional spice in China. It is widely used in medicine, food, timber, tourism, soil and water conservation. In April 2019, A stem and branch blight disease of Z. bungeanum was discovered in Muli, Puge and Yanyuan counties, in Liangshan Prefecture (27°15'20″-27°19'38″N, 101°44'58″-102°04'10″E), causing approximately 15% yield loss in the three counties. Among all fields in Muli County, approximately 41.38%, 10.79% and 2% of Chinese peppers exhibited mild, moderate and severe branch blight, respectively. The symptoms started to occur from March to April. First, red-brown spots on the base of the stem, branches or main trunks of young trees observed but were not obvious. In May, the spots became gray-brown to dark brown ovals and gradually expanded into long strips (Figure 1a, b). When the spots surrounded the branches, the branches above them withered and died, and the spots gradually expanded downward. Around June or July, scattered black dot-shaped fruiting bodies were observed on the lesion. The branches of infected trees were sampled systematically by cutting the branch at the junction of infected and healthy areas in 5×5 mm sections. Each sample was surface-sterilized with 3% NaClO and 75% alcohol for 60 s before being rinsed three times with sterilized distilled water. The sterile filter paper was used to dry the tissue, and the samples were cultured on potato dextrose agar (PDA) amended with streptomycin sulfate (50 µg/ml). Plates were incubated at 25°C in the dark. From the five isolates obtained, four exhibited the morphology described by Yu et al. (2015) for Neofusicoccum parvum. The colonies were white fluffy at first and grew fast (Figure 1c). After five days, the colony diameter reached 75.2-84.8 mm, produced yellow pigment and the mycelium in the middle of the colony began to turn gray (Figure 1d). and the entire colony turned dark gray 7-8 days post culturing as observed previously (Javier-Alva et al. 2009) and formed a black fruiting body at 20 days (Figure 1e). The width of the mycelium measured 2.3-4.8 µm, and with the diaphragm (Figure 1f). The spores were round or fusiform, colorless, transparent, smooth, thin-walled, and measured 6.3-10.6×3.1-5.2 µm (Figure 1g, h), similar to N. parvum (Yu et al. 2013). For molecular identification, DNA was extracted from the mycelia of four fungal isolates using a plant genomic DNA extraction kit (Solarbio, Beijing). Polymerase Chain Reaction (PCR) was performed with the primers ITS1/ITS4 (White et al. 1990), EF446F/EF1035R (Inderbitzin et al. 2005), BTF/BTR2 and HspF3/HspR (Inderbitzin et al. 2010) for the ribosomal internal transcribed spacer region (ITS), elongation factor-1alpha (EF1-alpha), beta-tubulin (TUB) and heat shock protein (HSP) genes, respectively. BLAST searches in the GenBank database indicated that the ITS, TUB, HSP and EF-1α sequences had 100%, 99.0%, 99.7% and 99.7% identity to N. parvum, respectively. Representative sequences were deposited in GenBank (ITS: MT355871; TUB: MT409397; EF-1α: MT409399; HSP: MT460413). A pathogenicity test was performed using N. parvum on ten 2-year-old potted Z. bungeanum plants at 22-28°C and 60% humidity indoors. The conidial suspension (1×107 conidia/ml) collected 25 days old PDA cultures with 0.05% tween buffer was used for inoculation by brushing the wounded area of branch scratched by epidermis with a piece of sandpaper. Ten plants in pots were inoculated with sterile water and served as controls. Thirty days post-inoculation, the plants showed the same symptoms as the original diseased plants, and the controls remained asymptomatic. N. parvum was re-isolated from the infected tissues and identified by morphological characteristics and DNA sequence analysis. The pathogenicity test was repeated three times with similar results, confirming Koch's postulates. This fungus is an important pathogen on a variety of woody hosts, and represents a serious problem in the vineyards worldwide (Mélanie, et al. 2017). To our knowledge, this is the first report of N. parvum causing stem and branch blight of Z. bungeanum trees in China.

Comparative study of the composition of cultivated, naturally grown Cordyceps sinensis, and stiff worms across different sampling years.

Natural Cordyceps sinensis, which is a valuable anti-tumor, immunomodulatory, and antiviral agent in Asia, has been overexploited in recent years. Therefore, it is important for cultivated C. sinensis to be recognized in the market. In this research, the main components of entirely cultivated, naturally grown C. sinensis, and stiff worms across different sampling years were detected and compared by HPLC-MS and UV spectrometry. The results indicated that the mean levels of adenosine and cordycepin were significantly higher, whereas the mean levels of mannitol and polysaccharides were remarkably lower in the cultivated type than in the natural type. No distinct difference in the average soluble protein content was observed. The composition of the stiff worms was similar to that of the natural herb, except that the total soluble protein content was higher, and that of mannitol was lower. In addition, the ultraviolet absorption spectroscopy of the three types showed high similarity at 260 nm. This research indicates that the main nutritional composition of cultivated and natural C. sinensis is identical and that cultivated type can be used as an effective substitute.

SMRT sequencing analysis reveals the full-length transcripts and alternative splicing patterns in Ananas comosus var. bracteatus.

BACKGROUND: Ananas comosus var. bracteatus is an herbaceous perennial monocot cultivated as an ornamental plant for its chimeric leaves. Because of its genomic complexity, and because no genomic information is available in the public GenBank database, the complete structure of the mRNA transcript is unclear and there are limited molecular mechanism studies for Ananas comosus var. bracteatus. METHODS: Three size fractionated full-length cDNA libraries (1-2 kb, 2-3 kb, and 3-6 kb) were constructed and subsequently sequenced in five single-molecule real-time (SMRT) cells (2 cells, 2 cells, and 1 cell, respectively). RESULTS: In total, 19,838 transcripts were identified for alternative splicing (AS) analysis. Among them, 19,185 (96.7%) transcripts were functionally annotated. A total of 9,921 genes were identified by mapping the non-redundant isoforms to the reference genome. A total of 10,649 AS events were identified, the majority of which were intron retention events. The alternatively spliced genes had functions in the basic metabolism processes of the plant such as carbon metabolism, amino acid biosynthesis, and glycolysis. Fourteen genes related to chlorophyll biosynthesis were identified as having AS events. The distribution of the splicing sites and the percentage of conventional and non-canonical AS sites of the genes categorized in pathways related to the albino leaf phenotype (ko00860, ko00195, ko00196, and ko00710) varied greatly. The present results showed that there were 8,316 genes carrying at least one poly (A) site, which generated 21,873 poly (A) sites. These findings indicated that the quality of the gene structure and functional information of the obtained genome was greatly improved, which may facilitate further genetic study of Ananas comosus var. bracteatus.

Comparative transcriptomic and proteomic analyses of the green and white parts of chimeric leaves in Ananas comosus var. bracteatus.

BACKGROUND: Ananas comosus var. bracteatus has high ornamental value due to its chimeric leaves. However, the chimeric trait is very unstable in red pineapple plants, and transcriptional variation between the two types of cells (white/green cells) and the molecular mechanism responsible for their albino phenotype remain poorly understood. METHODS: Comparative transcriptomic and proteomic analyses of the white parts (Whs) and green parts (Grs) of chimeric leaves were performed. RESULTS: In total, 1,685 differentially expressed genes (DEGs) (712 upregulated and 973 downregulated) and 1,813 differentially abundant proteins (DAPs) (1,018 with low abundance and 795 with high abundance) were identified. Based on Gene Ontology (Go) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses, the DEGs were mostly involved in carbon fixation in photosynthetic organisms, porphyrin and chlorophyll metabolism and oxidative phosphorylation, while proteomic analysis revealed that DAPs were mostly related to ribosomes, photosynthesis, photosynthesis antennas, and porphyrin and chlorophyll metabolism. Combined analysis showed increased mRNA levels but low abundance of nine proteins level in Whs /Grs related to photosynthetic pigment and photosynthesis. Transcriptional changes, posttranscriptional regulation and translational alterations of key enzymes involved in chlorophyll biosynthesis and photosynthesis may play important roles in the albino parts of chimeric leaves.

Genome-Wide Identification, Characterization, and Stress-Responsive Expression Profiling of Genes Encoding LEA (Late Embryogenesis Abundant) Proteins in Moso Bamboo (Phyllostachys edulis).

Late embryogenesis abundant (LEA) proteins have been identified in a wide range of organisms and are believed to play a role in the adaptation of plants to stress conditions. In this study, we performed genome-wide identification of LEA proteins and their coding genes in Moso bamboo (Phyllostachys edulis) of Poaceae. A total of 23 genes encoding LEA proteins (PeLEAs) were found in P. edulis that could be classified to six groups based on Pfam protein family and homologous analysis. Further in silico analyses of the structures, gene amount, and biochemical characteristics were conducted and compared with those of O. sativa (OsLEAs), B. distachyon (BdLEAs), Z. mays (ZmLEAs), S. bicolor (SbLEAs), Arabidopsis, and Populus trichocarpa. The less number of PeLEAs was found. Evolutionary analysis revealed orthologous relationship and colinearity between P. edulis, O. sativa, B. distachyon, Z. mays, and S. bicolor. Analyses of the non-synonymous (Ka) and synonymous (Ks)substitution rates and their ratios indicated that the duplication of PeLEAs may have occurred around 18.8 million years ago (MYA), and divergence time of LEA family among the P. edulis-O. sativa and P. edulis-B. distachyon, P. edulis-S. bicolor, and P. edulis-Z. mays was approximately 30 MYA, 36 MYA, 48 MYA, and 53 MYA, respectively. Almost all PeLEAs contain ABA- and (or) stress-responsive regulatory elements. Further RNA-seq analysis revealed approximately 78% of PeLEAs could be up-regulated by dehydration and cold stresses. The present study makes insights into the LEA family in P. edulis and provides inventory of stress-responsive genes for further functional validation and transgenic research aiming to plant genetic improvement of abiotic stress tolerance.