First Report of Leaf Spot Caused by Alternaria alternata on Cyclocodon lancifolius in China.

Cyclocodon lancifolius Bunge in the family Campanulaceae, and commonly known as Hong Guo Ginseng, is found in the Indonesia, Philippines, Vietnam, Japan, and China. The leaves and roots of C. lancifolius are widely used as tonics by ethnic minorities in Guizhou and Hunan Provinces in China. In addition, the fruit is edible, and it is a new resource for both medicine and food. In June 2022, symptoms of leaf spot (Fig 1 A and B.) were observed on C. lancifolius plants in the medicinal plant greenhouse of Guizhou University of Traditional Chinese Medicine (106°61'E, 26°39'N), Guizhou Province. The incidence of leaf spot on C. lancifolius was approximately 40 to 70% of all leaves in canopy. Early symptoms on leaves were small circular or irregular brown spots. As the disease progressed the lesions gradually expanded, and multiple lesions coalesced to form large irregular brown spots. Eventually the seedlings died and leaves of mature plants wilted. In order to isolate the pathogen, ten leaf pieces (5×5 mm) were cut from the junction of the diseased and the healthy tissues, surface sterilized with 75% ethanol for 30 s, 0.1% mercuric chloride (HgCl2) solution for 60 s, rinsed in sterile water three times, finally dried and placed on potato dextrose agar (PDA) and cultured in the dark at 27°C for 4 days. Five purified fungal isolates were obtained by single spore isolation. The colonies were olivaceous to dark olive with white margins and abundant aerial mycelia. On potato carrot agar (PCA) medium, these fungi produced septate conidiophores. Conidia were obclavate or ellipsoid, brown, with one to four transverse septa and one to two longitudinal septa. Spores measured 7.64 to 14.20 × 3.32 to 6.38 µm (n=50). These morphological characteristics are consistent with Alternaria alternata (S. P. Wiltshire. 1933). To further confirm the identification, four genomic DNA regions including the ribosomal DNA internal transcribed spacer (ITS), translation elongation factor 1-a gene (TEF), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase II second largest subunit (RPB2), and Alternaria major allergen gene (Alt a1) were amplified and sequenced with primers ITS1/ITS4 (White et al. 1990), TEF1-728F/TEF1-986R (Carbone and Kohn 1999), gpd1/gpd2 (Berbee et al. 1999), RPB2-5F/RPB2-7cR (Liu et al. 1999), and Alt-for/Alt-rev (Hong et al. 2005), respectively. Sequences were deposited in GenBank with accession Nos. ITS: OQ128111, OQ690707, and OQ690708; TEF: OQ200380, OQ700996, and OQ700998; GAPDH: OQ200378, OQ700993, and OQ700995; RPB2:OQ200379, OQ701002, and OQ701004; Alt: OQ675614, OQ700999, and OQ701001. In a BLAST search, the sequences were 99-100% identical with corresponding sequences of A. alternata. A maximum likelihood phylogenetic tree was constructed with the combined sequence data sets of ITS, TEF, GAPDH, RPB2, and Alt a1 using MEGA 11. The isolate DHY0, DHY1, and DHY3 clustered with A. alternata (J. H. C. Woudenberg et al. 2015) (Fig. 2). To fulfill Koch's postulates, leaves on three healthy 3-month-old potted C. lancifolius seedlings were wounded with sterile needles and inoculated with 5 mm diameter mycelium, which was covered moist by sterile cotton for 24 h. Sterile water was used as the control. After inoculation, the plants were incubated at 27°C, 85% relative humidity, and a 12 h photoperiod. The experiment was repeated three times. Fifteen days after inoculation, all the leaves showed leaf spot symptoms that were similar to those observed in the greenhouse, while control leaves were asymptomatic (Fig. 1). A. alternata was successfully re-isolated from the symptomatic leaves and identified by morphology and the molecular methods described above. This pathogen has been reported to cause a leaf disease in a wide range of vegetables (Zhang et al. 2021), flowers (Zhang et al. 2022), and medicinal plants (Xing et al. 2020). To the best of our knowledge, this is the first report of A. alternata causing leaf spot on C. lancifolius in China. The accurate identification of this pathogen will provide a basis for the prevention and control of C. lancifolius leaf spot disease in the future.

Modulating interfacial structure and lipid digestion of natural Camellia oil body by roasting and boiling processes.

Oil body (OB) is the lipid-storage organelle in oilseed, and its stability is crucial for oilseed processing. Herein, effects of roasting and boiling on the structure, stability, and in vitro lipid digestion of Camellia OB were studied. The interfacial structure and physical stability of the extracted OB were investigated by electrophoresis, confocal-Raman spectroscopy, zeta-potential, and surface hydrophobicity, etc. Boiling caused protein loss on the OB surfaces, forming a stable phospholipid interface, which resulted in coalescence of the droplets (d > 100 µm) and negative ζ-potential (-3 âˆ¼ -8 mV) values at a pH of 2.0. However, roasting partially denatured the proteins in the seeds, which were adsorbed on the OB surfaces. The random coil structure of interfacial protein increased to ∼20 % after thermal treatment. Besides, heating decreased the surface hydrophobicity of OB and improved lipid digestion. After boiling 60 min, the extent of lipolysis increased from 41.7 % (raw) to 57.4 %.

The complete chloroplast genome of Laportea bulbifera (Sieb. et Zucc.) Wedd. and its phylogenetic analysis.

The circle complete chloroplast genome of Laportea bulbifera (Sieb. et Zucc.) Wedd. was sequenced for the first time. The genome length of L. bulbifera is 150,042 bp with 36.80% of GC content. The genome consists of a large single copy (LSC) region of 82,414 bp, a small single copy (SSC) region of 17,714 bp, and two inverted repeat (IRa and IRb) regions of 24,957 bp each. A total of 129 genes were annotated, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis was conducted by 29 species from the Rosales, the results presented a closed relationship between the species Laportea bulbifera and Poikilospermum lanceolatum.

Complete chloroplast genome of medicinal plant Sabia parviflora Wall. ex Roxb. (Sabiaceae).

Sabia parviflora Wall. ex Roxb., an evergreen climbing woody vine, is a Chinese herbal medicine commonly used by ethnic minorities in some areas of China. In this study, the chloroplast genome of S. parviflora was sequenced for the first time. Its genome is 162,054 bp in length with 38.6% of GC content. The genome consists of a large single copy (LSC) region of 90,001 bp, a small single copy (SSC) region of 18,887 bp, and two inverted repeat (IRa and IRb) regions of 26,583 bp each. A total of 130 genes were annotated, including 85 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Phylogenetic analysis was conducted by nine species from order Proteales, which demonstrated a close relationship between the family Sabiaceae and Nelumbonaceae.

Characterization of the Lycium barbarum fruit transcriptome and development of EST-SSR markers.

Lycium barbarum, commonly known as goji, is important in Chinese herbal medicine and its fruit is a very important agricultural and biological product. However, the molecular mechanism of formation of its fruit and associated medicinal and nutritional components is unexplored. Moreover, this species lacks SSR markers due to lack of genomic and transcriptomic information. In this study, a total of 139,333 unigenes with average length of 1049 bp and N50 of 1579 bp are obtained by trinity assembly from Illumina sequencing reads. A total of 92,498 (66.38%) unigenes showed similarities in at least one database including Nr (46.15%), Nt (56.56%), KO (15.56%), Swiss-prot (33.34%), Pfam (33.43%), GO (33.62%) and KOG/COG (17.55%). Genes in flavonoid and taurine biosynthesis pathways were found and validated by RT-qPCR. A total of 50,093 EST-SSRs were identified from 38,922 unigenes, and 22,537 EST-SSR primer pairs were designed. Four hundred pairs of SSR markers were randomly selected to validate assembly quality, of which 352 (88%) were successful in PCR amplification of genomic DNA from 11 Lycium accessions and 210 produced polymorphisms. The polymorphic loci showed that the genetic similarity of the 11 Lycium accessions ranged from 0.50 to 0.99 and the accessions could be divided into 4 groups. These results will facilitate investigations of the molecular mechanism of formation of L. barbarum fruit and associated medicinal and nutritional components, and will be of value to novel gene discovery and functional genomic studies. The EST-SSR markers will be useful for genetic diversity evaluation, genetic mapping and marker-assisted breeding.

Tyrosine-phosphorylated and nonphosphorylated sodium channel beta1 subunits are differentially localized in cardiac myocytes.

Voltage-gated sodium channel alpha and beta subunits expressed in mammalian heart are differentially localized to t-tubules and intercalated disks. Sodium channel beta subunits are multifunctional molecules that participate in channel modulation and cell adhesion. Reversible, receptor-mediated changes in beta1 tyrosine phosphorylation modulate its ability to recruit and associate with ankyrin. The purpose of the present study was to test our hypothesis that tyrosine-phosphorylated beta1 (pYbeta1) and nonphosphorylated beta1 subunits may be differentially localized in heart and thus interact with different cytoskeletal and signaling proteins. We developed an antibody that specifically recognizes pYbeta1 and investigated the differential subcellular localization of beta1 and pYbeta1 in mouse ventricular myocytes. We found that pYbeta1 colocalized with connexin-43, N-cadherin, and Nav1.5 at intercalated disks but was not detected at the t-tubules. Anti-pYbeta1 immunoprecipitates N-cadherin from heart membranes and from cells transfected with beta1 and N-cadherin in the absence of other sodium channel subunits. pYbeta1 does not associate with ankyrinB in heart membranes. N-cadherin and connexin-43 associate with Nav1.5 in heart membranes as assessed by co-immunoprecipitation assays. We propose that sodium channel complexes at intercalated disks of ventricular myocytes are composed of Nav1.5 and pYbeta1 and that these complexes are in close association with both N-cadherin and connexin-43. beta1 phosphorylation appears to regulate its localization to differential subcellular domains.