Identification of Autophagy in the Pine Wood Nematode Bursaphelenchus xylophilus and the Molecular Characterization and Functional Analysis of Two Novel Autophagy-Related Genes,BxATG1 and BxATG8

The pine wood nematode, Bursaphelenchus xylophilus, causes huge economic losses in pine forests, has a complex life cycle, and shows the remarkable ability to survive under unfavorable and changing environmental conditions. This ability may be related to autophagy, which is still poorly understood in B. xylophilus and no autophagy-related genes have been previously characterized. In this study, transmission electron microscopy was used to confirm that autophagy exists in B. xylophilus. The full-length cDNAs of BxATG1 and BxATG8 were first cloned from B. xylophilus, and BxATG1 and BxATG8 were characterized using bioinformatics methods. The expression pattern of the autophagy marker BxATG8 was investigated using in situ hybridization (ISH). BxATG8 was expressed in esophageal gland and hypodermal seam cells. We tested the effects of RNA interference (RNAi) on BxATG1 and BxATG8. The results revealed that BxATG1 and BxATG8 were likely associated with propagation of nematodes on fungal mats. This study confirmed the molecular characterization and functions of BxATG1 and BxATG8 in B. xylophilus and provided fundamental information between autophagy and B. xylophilus.     

Enhancement of Palmarumycins C12 and C13 Production in Liquid Culture of Endophytic Fungus Berkleasmium sp. Dzf12 after Treatments with Metal Ions

The influences of eight metal ions (i.e., Na⁺, Ca²⁺, Ag⁺, Co²⁺, Cu²⁺, Al³⁺, Zn²⁺, and Mn⁴⁺) on mycelia growth and palmarumycins C₁₂ and C₁₃ production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 were investigated. Three metal ions, Ca²⁺, Cu²⁺ and Al³⁺ were exhibited as the most effective to enhance mycelia growth and palmarumycin production. When calcium ion (Ca²⁺) was applied to the medium at 10.0 mmol/L on day 3, copper ion (Cu²⁺) to the medium at 1.0 mmol/L on day 3, aluminum ion (Al³⁺) to the medium at 2.0 mmol/L on day 6, the maximal yields of palmarumycins C₁₂ plus C₁₃ were obtained as 137.57 mg/L, 146.28 mg/L and 156.77 mg/L, which were 3.94-fold, 4.19-fold and 4.49-fold in comparison with that (34.91 mg/L) of the control, respectively. Al³⁺ favored palmarumycin C₁₂ production when its concentration was higher than 4 mmol/L. Ca²⁺ had an improving effect on mycelia growth of Berkleasmium sp. Dzf12. The combination effects of Ca²⁺, Cu²⁺ and Al³⁺ on palmarumycin C₁₃ production were further studied by employing a statistical method based on the central composite design (CCD) and response surface methodology (RSM). By solving the quadratic regression equation between palmarumycin C₁₃ and three metal ions, the optimal concentrations of Ca²⁺, Cu²⁺ and Al³⁺ in medium for palmarumycin C₁₃ production were determined as 7.58, 1.36 and 2.05 mmol/L, respectively. Under the optimum conditions, the predicted maximum palmarumycin C₁₃ yield reached 208.49 mg/L. By optimizing the combination of Ca²⁺, Cu²⁺ and Al³⁺ in medium, palmarumycin C₁₃ yield was increased to 203.85 mg/L, which was 6.00-fold in comparison with that (33.98 mg/L) in the original basal medium. The results indicate that appropriate metal ions (i.e., Ca²⁺, Cu²⁺ and Al³⁺) could enhance palmarumycin production. Application of the metal ions should be an effective strategy for palmarumycin production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12.