Characterization of Nuclear and Mitochondrial Genomes of Two Tobacco Endophytic Fungi Leptosphaerulina chartarum and Curvularia trifolii and Their Contributions to Phylogenetic Implications in the Pleosporales.

The symbiont endophytic fungi in tobacco are highly diverse and difficult to classify. Here, we sequenced the genomes of Curvularia trifolii and Leptosphaerulina chartarum isolated from tobacco plants. Finally, 41.68 Mb and 37.95 Mb nuclear genomes were sequenced for C. trifolii and L. chartarum with the scaffold N50, accounting for 638.94 Kb and 284.12 Kb, respectively. Meanwhile, we obtained 68,926 bp and 59,100 bp for their mitochondrial genomes. To more accurately classify C. trifolii and L. chartarum, we extracted seven nuclear genes and 12 mitochondrial genes from these two genomes and their closely related species. The genes were then used for calculation of evolutionary rates and for phylogenetic analysis. Results showed that it was difficult to achieve consistent results using a single gene due to their different evolutionary rates, while the phylogenetic trees obtained by combining datasets showed stable topologies. It is, therefore, more accurate to construct phylogenetic relationships for endophytic fungi based on multi-gene datasets. This study provides new insights into the distribution and characteristics of endophytic fungi in tobacco.

Formation of α- and ß-Cembratriene-Diols in Tobacco (Nicotiana tabacum L.) Is Regulated by Jasmonate-Signaling Components via Manipulating Multiple Cembranoid Synthetic Genes.

Cembranoids are a group of natural diterpenoid compounds with pharmaceutical potentials, and the cembratriene-diols produced by Nicotiana (tobacco) species display activities in anti-nicotine addiction and neuron protection. Although the enzymes catalyzing cembratriene-diols' formation in tobacco have been investigated, the regulatory mechanism underlying this physiological process remains unknown. This study has investigated the roles of phytohormone jasmonic acid (JA) in regulating cembratriene-diol formation in N. tabacum cv. TN90 and found that JA and COI1, the receptor protein of the bioactive derivative of JA (i.e., JA-Ile), display critical roles in regulating cembratriene-diols' formation and the expression of cembranoid synthetic genes CBTS, P450 and NtLTP1. Further studies showed that over-expressing either the gene encoding bHLH transcription factor MYC2a or that encoding MYB transcription factor MYB305 could upregulate the cembranoid synthetic genes and enhance the cembranoid production in plants with dysfunction of COI1. Further studies suggest that COI1 and its downstream regulators MYC2a and MYB305 also modulate the trichome secretion, which is correlated with cembranoid formation. Taken together, this study has demonstrated a critical role of JA-signaling components in governing the cembratriene-diol formation and the transcription of cembratriene-diol synthetic genes in tobacco. Findings in this study are of great importance to reveal the molecular regulatory mechanism underlying cembranoid synthesis.

Angularly Prenylated Indole Alkaloids with Antimicrobial and Insecticidal Activities from an Endophytic Fungus Fusarium sambucinum TE-6L.

Bioactivity-guided isolation of the endophytic fungus Fusarium sambucinum TE-6L residing in Nicotiana tabacum L. led to the discovery of two new angularly prenylated indole alkaloids (PIAs) with pyrano[2,3-g]indole moieties, amoenamide C (1) and sclerotiamide B (2), and four known biosynthetic congeners (3-6). Their structures were determined by comprehensive spectroscopic techniques, electronic circular dichroism (ECD), and X-ray diffraction. Compound 1 containing the bicyclo[2.2.2]diazaoctane core and indoxyl unit is rarely reported. All the compounds were evaluated for their antimicrobial and insecticidal activities. Notably, compounds 1-3 showed potent inhibitory effects against three human- and one plant-pathogenic bacterium, and seven plant-pathogenic fungi. Compounds 2-4 also exhibited remarkable larvicidal activity against first instar larvae of the cotton bollworm Helicoverpa armigera with mortality rates of 70.2%, 83.2%, and 70.5%, respectively. Further toxicity tests on zebrafish embryos were performed to evaluate the potential toxicity of PIAs. Of significance was that compound 3 in particular exhibited the highest activities but the lowest effects on the hatching of embryos among all the compounds. This study provides a basis for understanding developmental toxicity of PIAs exposure to zebrafish embryos, and also indicates the potential environmental risks of other natural compounds exposure in the aquatic ecosystem.

[Analysis on recurrence factors associate with giant cell tumor of tendon sheath in upper extremity].

OBJECTIVE: To study the relation of the sex, age, location and chemotherapy with recurrence of the tumor. METHODS: From January 2000 to August 2010, 47 patients with giant cell tumor of tendon sheath in upper extremity were retrospectively analyzed. Statistical analysis of sex, age at presentation, lesion location, chemical inactivation, surgical complications, tumor recurrence and pathological findings were explored. There were 28 females and 19 males, ranging in age from 17 to 78 years, with an average of 38.15 years. All the patients underwent surgical excision. Fourteen patients received intraoperative chemically inactive treatment. All the patients had routine follow-up to observe the wound healing, pathological findings,tumor recurrence, and received necessary imaging examinations. RESULTS: All the patients were followed up, and the duration ranged from 22 to 129 months, with a mean time of 53.89 months. Four patients who received intraoperative alcohol inactivation appeared wound complications such as wound swelling, discharge of necrotic tissue, delayed wound healing. Fifteen patients had active growth of tumor tissue, 1 patient had low-grade malignant giant cell tumor of tendon sheath. The recurrence rate was significantly higher in the group which preoperative X-ray was found to have bone destruction (P = 0.003); patients receiving chemically inactivation had lower risk of recurrence after surgery than patients not receiving chemically inactivation (P = 0.042). CONCLUSION: The recurrence rate of giant cell tumor of tendon sheath in upper limb was closely related to tumor growth site, bone destruction and chemical inactivation. Local excision of giant cell tumor of tendon sheath was the effective treatment. How to identify the patients at high risk of recurrence, how to reduce the recurrence rate and the functional restoration after wide resection are the priorities and difficulties of future researches.