[Objective] This study aimed to develop a simple and effective DNA extraction method for citrus Huanglongbing pathogen detection.[Method] From aspects of preparing procedure,prepare time and the quality of DNA,advantages and disadvantages of three sample preparation methods were compared,include two Direct-PCR extraction methods and one universal genomic DNA extraction kit method. In addition,PCR amplification effect on specific primers for 16S rDNA of " Candidatus Liberibacter asiaticus"( CLas) had also been evaluated.[Result] The results showed that RT-qPCR detected CLas by using DNA obtained from one of Direct-PCR extraction method as templates. Under improved Direct-PCR extraction method,16S rDNA of CLas could also be amplified by routine PCR.[Conclusion]A simple and effective DNA extraction method of citrus Huanglongbing pathogen have been established,which provides technical supports for preparation of large number of samples for detection of CLas.
The red imported fire ant( Solenopsis invicta) is a global major invasive pest, and has caused significant economic, social and environmental impacts since its invasion to mainland of China in 2004. To date, chemical control has been the most effective measure. However, the long-term use of chemicals would lead to an unexpected rebound. To understand the risks and explore the mechanisms of detoxification or induction to insecticides in S. invicta, the O-demethylase activity and expression of cytochrome P450 genes of workers and queens, and the effects of chlorpyrifos and fipronil exposure in workers were investigated. Biochemical assays showed the O-demethylase activity of cytochrome P450 was significantly higher in workers than in queens(1.66-fold), and was significantly induced in workers exposed to chlorpyrifos and fipronil, reaching a maximum(3.00- and 1.95-fold) at 48 h and then decreasing dramatically compared to controls(exposed to acetone counterpart). The relative expression levels of 12 cytochrome P450 expressed sequence tags(ESTs) in workers were significantly higher than in queens(from 2.3- to 36.4-fold). Multiple cytochrome P450 genes(except 9E4) were co-up-regulated(from 1.5- to 2.86-fold) in workers exposed to fipronil. These results indicated that the increased O-demethylase activity may result from the increased transcription levels of cytochrome P450 related to detoxification of insecticides in S. invicta. It appears that cytochrome P450 plays an important role in enhanced metabolic detoxification of insecticides. At the same time, it also provides the theoretical basis for resistance management and rational usage of in secticides to control S. invicta.
