Improved techniques for measurement of nanolitre volumes of phloem exudate from aphid stylectomy.

BACKGROUND

When conducting aphid stylectomy, measuring accurate rates of phloem exudation is difficult because the volumes collected are in the nanolitre (nl) range. In a new method, exudate volume was calculated from optical measurement of droplet diameter as it forms on the tip of a severed aphid stylet. Evaporation was shown to decrease the accuracy of the measurement but was countered with the addition of water-saturated mineral oil. Volume measurements by optical estimation of the volume of a sphere suspended in oil was affected by the curvature of the oil surface. In contrast, measuring the exudate volume from optical measurement of droplet-diameter as formed on the tip of a severed aphid stylet, removes any inaccuracies due to oil surface curvature. A modified technique is proposed for measuring exudate volumes without oil by estimating the flow rate from photo-sequences of the collection period; a correction for evaporation is applied later.

RESULTS

A change in oil volume of ±1.75% from an optimum volume of 285 μl had a statistically significant effect on droplet measurement, under or over-estimating droplet volume due to optical effects caused by the oil surface. Using microscope image capture and measurement software, a modified method for measuring phloem volume in air was developed, by reducing air exposure during measurement to approximately 5 s for each measurement. Phloem volumes were measured using both techniques with measurements in air being on average 19.9 nl less (SD 18.87, p<0.001) than those made in oil, and there was a strong linear relationship (R(2)=0.942) between the techniques. This linear relationship enabled the development of a correction equation with no significant difference at the 5% level between corrected volumes and actual volumes measured under oil.

CONCLUSIONS

This study showed that oil has a significant role in countering evaporation but oil volume must be carefully optimised for optical measurement of droplets to ensure measurement accuracy. A linear correction factor was generated to correct the volumes measured in air for loss due to evaporation and the method provides for a much simpler alternative to previous approaches for measuring exudation rates and volumes from a cut aphid stylet.