Two groups were used to evaluate the impact of dichlorvos on the indigenous bacterial community in the rape phyllosphere (experiment 1), and the other two groups were used to evaluate the availability of phyllosphere microorganisms for dichlorvos degradation on rape leaves (experiment 2). Before the plants were sprayed with RGFP966 solubility dmso dichlorvos, 50 g of rape leaf tissue was collected from each group and
used as the day 0 sample. In experiment 1, one group was sprayed with 1 : 1000 water-diluted emulsifiable concentrated dichlorvos (80% w/v), which is the recommended dose for rape. The other group was sprayed with the same dose of the auxiliary solvent that had been added into the dichlorvos, as the control. To sample the leaves, 50 g of fully expanded young leaves was collected from the control
and treated samples on days 1, 2, 4, 6 and 7 after treatment, placed on ice and brought back to MK-1775 manufacturer the laboratory. Three replicates of each sample were included. Samples for DNA extraction, subsequent PCR–denaturing gradient gel electrophoresis (PCR–DGGE) and the construction of a 16S rRNA gene clone library were stored frozen at −20 °C until use. In experiment 2, one group of samples was surface sterilized with 1% sodium hypochlorite for 1 min and three times with sterile water for at least 1 min; these were designated the sterilized samples. The other was used as the control (unsterilized samples). These two groups were then sprayed with dichlorvos, as described above. Immediately after spraying, the leaf samples were collected to determine
the initial dichlorvos concentration. A sample (50 g) of rape leaf material was collected from each control and treated sample on days 1, 2 and 7 after spraying. All control and sterilized leaves were sent to the Beijing Center for Physical and Chemical Analysis for analysis of the dichlorvos residues. Part (10 g) of each leaf sample from experiment 1 was transferred aseptically into an Erlenmeyer flask containing washing buffer (0.1 M sodium phosphate buffer, pH 7.5) and sonicated for L-gulonolactone oxidase 7 min in an ultrasonic cleaning bath (40 kHz) to dislodge the bacteria from the leaves. The leaf debris was removed by low-speed centrifugation, as described by Zhang et al. (2008). The wash solution was centrifuged at 10 000 g for 15 min at 4 °C. The supernatant was discarded and the pellet was used for analysis of the cellular dichlorvos-degrading ability, the isolation of dichlorvos-degrading bacteria and DNA extraction. The microorganisms eluted from the day 0 samples were used to clarify how much the phyllosphere microorganisms devoted to dichlorvos degradation. Dichlorvos was added to mineral salt medium (MSM; Shelton & Somich, 1988) to a final concentration of 400 mg L−1 as the sole carbon source, and incubated at 30 °C on a shaker at 200 r.p.m. in the dark for 48 h. The cultures were analysed in triplicate to ensure accuracy. Uninoculated medium containing dichlorvos (400 mg L−1) was used as the control.