18 research outputs found
Transmission of CaMV from infected protoplasts.
No full text<p><b>A.</b> Influence of acquisition time on transmission of CaMV from infected protoplasts. After a fasting period of 60 min, aphids were allowed access to protoplasts for 15–45 min (<60 min), 60 min, or 75–180 min (>60 min) before transfer to healthy test plants. Acquisition times shorter than 60 min diminished somewhat, but not significantly transmission (<i>P</i> = 0.142; df = 2; Kruskal-Wallis test). Also acquisition times longer than 60 min were not associated with any significant difference in transmission rate compared to a 60-min acquisition. The graph presents data from 57 assays of 24 test plants each (9 lots for acquisition times shorter than 60 min, 17 lots for an acquisition time of 60 min, and 31 lots for acquisition times longer than 60 min). <b>B.</b> Effect of fasting on CaMV transmission from infected protoplasts. Aphids were starved for between 15 and 45 min (<60 min), for 60 min, or between 90 and 240 min (>60 min). They were then allowed a 60-min acquisition access period on protoplasts before transfer to test plants for inoculation. Under our conditions, no prominent effect of starvation on transmission was observed, although starving times shorter than 60 min seemed to reduce transmission. However, the effect was not significant (<i>P</i> = 0.083; df = 2; Kruskal-Wallis test). Data are from 59 tests using 24 plants each (9 assays for fasting of less then 60 min, 19 assays for a fasting time of 60 min, and 31 assays with fasting times longer then 60 min. Note that the data for 60 min fasting contains data from (A). <b>C.</b> Transmission of CaMV from protoplasts absolutely requires living cells. Intact (live) or ultrasonicated (dead) infected protoplasts were offered to aphids in acquisition assays. Only living protoplasts supported transmission. Data are from 11 assays of 24 test plants for each condition. The difference between the two acquisition conditions was highly significant (<i>P</i><0.001; Mann-Whitney test). <b>D.</b> Ultrasonication does not inactivate CaMV. Infected protoplasts were disrupted by ultrasonication and then transmitted by rub-inoculation. The graph shows that ultrasonicated protoplasts performed at least as well as intact protoplasts in mechanical transmission. Three lots of 24 plants were tested per condition. <b>E.</b> Oryzalin inhibits transmission of CaMV. Protoplasts were incubated for 60 min with DMSO (Ctl) or 10–50 µM oryzalin (Ory) before being offered to aphids for virus acquisition. After a 60-min access period, aphids were transferred to test plants for inoculation. The data shown are from 11 sets of 24 plants for each condition. One set was excluded from analysis because it was an outlier. The difference between the two conditions is significant (<i>P</i> = 0.014; Mann-Whitney test). <b>F.</b> Oryzalin does not impair in vitro acquisition of CaMV. Purified CaMV particles were mixed with helper component P2 and P3 and offered with or without 50 µM oryzalin to aphids for a 30-min acquisition access feeding period, before the insects were used for inoculation of test plants (10 aphids per plant). Three 24 plant lots were used per condition. All graphs present mean values ± standard deviation.</p
Experimental set-up of a typical VAPA (virus-acquisition phenotyping assay) transmission experiment.
No full text<p><b>A.</b> To construct the aphid harvesting device, a 1000 µl pipette tip with the utmost 2–3 mm cut off is attached to a silicon tube, with a Miracloth net squeezed in between. The tube is connected to a vacuum source (mechanical pump or human respiratory system) and aphids are sucked up into the pipette tip by negative pressure and retained by the net. <b>B.</b> The virus-acquisition phenotyping apparatus consists of a copper ring sealed with a Parafilm M membrane. Aphids placed in the ring are attracted to the membrane by a light source (not shown); protoplasts are then deposited onto the membrane and spread evenly with a cover glass. After a defined acquisition access period, aphids are transferred with an artist's paint-brush to test plants for inoculation.</p
Protoplast transmission of TuMV.
No full text<p><b>A.</b> Effect of different acquisition times on TuMV transmission. After 60 min of starving, aphids were allowed different protoplast acquisition periods before being placed on test plants for inoculation. No effect of different acquisition time was observed (<i>P</i> = 0.942; df = 2; Kruskal-Wallis test). The graph combines data from 24 test plants per assay with 6 assays for acquisition times from 15 to 60 min (<90 min), 6 assays for an acquisition time of 90 min, and 4 assays for acquisition times between 120 and 180 min (>90 min). <b>B.</b> Pre-acquisition starving has no effect on TuMV transmission. Aphids were starved for 60, 120, or 180 min before being allowed a 60-min acquisition period on TuMV-infected protoplasts and subsequent transfer to test plants for inoculation. The graph shows that there is no measurable effect of pre-acquisition starving on transmission efficiency (<i>P</i> = 0.193; df = 2; Kruskal-Wallis test). 6 assays of 24 test plants were tested for 60 min starvation, 13 assays for the 120 min time point, and 4 assays for 180 min starvation. <b>C.</b> TuMV acquisition from protoplasts absolutely requires living protoplasts. Aphids were allowed to feed for 60 min on infected intact (control) or dead (sheared) protoplasts before transfer to test plants for inoculation. The difference between transmission rates from living and sheared protoplasts is highly significant (<i>P</i><0.001; Mann-Whitney test). The graph shows data from 18 assays (9 for each condition) of 24 test plants each. <b>D.</b> Shearing does not inactivate TuMV. Infected protoplasts were homogenised by repeated passage through a syringe needle and then rub-inoculated to turnip test plants. The graph shows that virions from sheared protoplasts were as infectious as those from intact control protoplasts. Three 24 plant cultivation trays were inoculated per condition. All graphs present mean values ± standard deviation.</p
