Optimization of chloroplast microsatellite PCR conditions and primer screening for endangered Rheum officinale, Rheum palmatum, and Rheum tanguticum.

Chloroplast microsatellite primers were developed in order to provide more population genetic information of endangered Rheum officinale, R. palmatum, and R. tanguticum for conservation. The dried roots and rhizomes of these plants are important in traditional Chinese medicine. The results showed that the optimum concentrations of Mg(2+), Taq DNA polymerase, dNTPs, template DNA, and primers in a 25-µL reaction system were 2.0 mM, 1.0 U, 0.10 mM, 20 ng, and 0.8 µM, respectively. Fourteen of 53 primer combinations were chosen for their high clarity and repetition in three species, and their annealing temperatures ranged from 56 to 58°C. These primers and the optimized polymerase chain reaction system may provide a tool for understanding the demography and genetic variation of these endangered plants.

Aspergillus fumigatus CY018, an endophytic fungus in Cynodon dactylon as a versatile producer of new and bioactive metabolites.

Aspergillus fumigatus CY018 was recognized as an endophytic fungus for the first time in the leaf of Cynodon dactylon. By bioassay-guided fractionation, the EtOAc extract of a solid-matrix steady culture of this fungus afforded two new metabolites, named asperfumoid (1) and asperfumin (2), together with six known bioactive compounds including monomethylsulochrin, fumigaclavine C, fumitremorgin C, physcion, helvolic acid and 5alpha,8alpha-epidioxy-ergosta-6,22-diene-3beta-ol as well as other four known compounds ergosta-4,22-diene-3beta-ol, ergosterol, cyclo(Ala-Leu) and cyclo(Ala-Ile). Through detailed spectroscopic analyses including HRESI-MS, homo- and hetero-nuclear correlation NMR experiments (HMQC, COSY, NOESY and HMBC), the structures of asperfumoid and asperfumin were established to be spiro-(3-hydroxyl-2,6-dimethoxyl-2,5-diene-4-cyclohexone-(1,3')-5'-methoxyl-7'-methyl-(1'H, 2'H, 4'H)-quinoline-2',4'-dione) and 5-hydroxyl-2-(6-hydroxyl-2-methoxyl-4-methylbenzoyl)-3,6-dimethoxyl-benzoic methyl ester, respectively. All of the 12 isolates were subjected to in vitro bioactive assays against three human pathogenic fungi Candida albicans, Tricophyton rubrum and Aspergillus niger. As a result, asperfumoid, fumigaclavine C, fumitremorgin C, physcion and helvolic acid were shown to inhibit C. albicans with MICs of 75.0, 31.5, 62.5, 125.0 and 31.5 microg/mL, respectively.