Prestic. Sci. 1979, 10, 369-372

Microscopy and Image Analysis in the Study of Plant Pathogens and the Application of Pesticides. Philip Jones

Plant Pathology Department, Rothamsted Experimental Station, Harpenden, Hertforhhire

(Manuscript received 12 January 1979)

Problems associated with the preparation of diseased plant material for transmission electron microscopy (TEM) and the uses of TEM to meet the specific needs of plant virologists are discussed. Methods of collecting spray droplets for analysis by an image-analysing computer method are described.

1. Introduction During the last two decades electron microscopy has provided important information and become widely accepted as a routine tool in pesticide research. This paper looks at two aspects of trans- mission electron microscopy: (a) some of the especial problems that can arise during the prepara- tion of diseased plant material for examination in the transmission electron microscope (TEM) ; and (b) the uses of the TEM to meet specific requirements of plant virologists. Finally some of the methods of collecting spray droplets for automated image analysis are considered.

2. Problems associated with the preparation of diseased plant material for electron microscopy Standard preparatory techniques have been used successfully for studying many aspects of plant diseases, such as the penetration processes of fungal pathogens1 and the morphology of plant viruses.2 However, diseased plant material often presents difficulties in fixation and embedding. The use of standard fixation techniques often leads to poor preservation of membranes, which results in loss of material from the cytoplasm and the degradation of cell organelles. The reasons for this are largely unknown but the effects can be decreased by using combinations of different fixatives. Two examples are (a) the fixation of coconut tissue infected with a protozoan flagellate using a buffer mixture of pH 7.2 containing sodium dimethylarsinate (0.05~), sucrose (0.15~), calcium chloride (2 mM), glutaraldehyde (30 g litre-1) and paraformaldehyde (30 g litre-1);3 and (b) the fixation of peanut root nodules with a buffer mixture of pH 6.8 containing trisodium phos- phate (25 mM), glutaraldehyde (30 g litre-I), paraformaldehyde (20 g litre-l) and acrylaldehyde 10 g l i ~ e - l ) . ~

The incomplete polymerisation of epoxy embedding resins, which is sometimes encountered during embedding of diseased material, often results from the presence of inhibitors, such as quin- ones, in thti tissues. This can sometimes be overcome by adding ascorbic acid, an antioxidant, to the fixatives and buffers used, after the method of Skene.5 Alternatively, it may be possible to remove inhibitors of polymerisation by repeated washings with phosphate buffer prior to post- fixation in osmium tetroxide.

3. Particle counting and electron microscope serology in plant virology A reliable estimate of the concentration of virus particles in a preparation, as required, for instance in studies of the inhibitors of virus multiplication, can be obtained by using the technique described

Presented at a symposium Microscopy and irs use in pesricide research on 7 November 1978, organised by the Physicochemical and Biophysical Panel (Pesticides Group), Society of Chemical Industry.

0031-613X/79/08004369 $02.00 0 1979 Society of Chemical Industry

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370 P. Jones

by Backus and Wil l iam~,~ modified by Nixon and Fisher.7 Briefly, a polystyrene latex suspension of known particle concentration is made up, mixed with the virus preparation and sprayed on a grid. The ratio of the numbers of latex to virus particles is determined from the droplets and from this ratio the concentration of virus particles in the original preparation can be calculated.8 Figure 1 is an electron-micrograph of a typical droplet prepared as described above.

Figure 1. Spray droplet used to assay the concentration of virus particles by electron microscopy The droplet contains six tobacco mosaic virus particles (about 300 nm long) and eight latex spheres.

Immune electron microscopy is the name given to the technique of detecting specific antibody/ antigen binding by electron microscopy. Techniques have been developed by plant virologists to detect immune reactions in negative-stain preparations using the transmission electron microscope. The advantages in using such a method are speed, greater sensitivity than the microprecipitin technique, that results are not obscured by the presence of other morphologically similar viruses, and that the technique can be used on viruses which are in low concentrations in the host plant. The technique involves coating electron-microscope grids with anti-serum, incubating these activa- ted grids with the virus preparation, recoating with anti-serum and then staining with a suitable negative stain (tungstophosphoric acid, 20 g litre-l, adjusted to pH 7.0 with sodium hydroxide solution) before examination in the microscope. A typical result of the procedure is shown in Figure 2. A more detailed account of the technique is given by Milne and L u i ~ o n i . ~

4. Image analysis in spray droplet assessment

Image analysing computers such as the Quantimet 720D (Cambridge Scientific Instruments Ltd) are able to count and measure the area, length and perimeter of specimens presented to them. They can also be programmed to carry out determinations of size distributions and to measure parameters for pattern recognition on those specimens.lO The features to be measured can be

Electron microscopy of plant pathogens and spray droplets 371

Figure 2. A mixture of potato virus Y and henbane mosaic virus treated according to the technique in the text using potato virus Y anti-serum only; the potato virus Y particles are decorated with antibody.

selected by the operator according to their optical density. Specimens can be presented to the computer in the form of light-microscope slides, transmission or scanning electron-micrographs, photographs or drawings. This technique has made many measurements feasible that were pre- viously unacceptably laborious.

Image analysers have been used in agricultural research to study soil-pore measurementsll and root-area measurements, and to assess, by means of aerial photographs, the spread of crop diseases. In pesticide research they have potential uses in the study of pest damage and the application of sprays. The efficiency of pesticides applied as sprays depends partly on the distribution and size of the droplets. Artificial collecting surfaces cannot mimic natural targets faithfully12 and can therefore give only a rough estimate of the droplets arriving in the vicinity of the target. The image analysing computer enables the analysis of droplet deposits by counting numbers, and measuring area and mean inter-droplet spacing quickly and efficiently. Four methods in current use for col- lecting droplets are described below.

4.1. Recording on paper strips Paper strips (Kromekote, Kodak Ltd) can be used to record water droplets as blue spots but difficulties of analysis arise because colour density can vary between droplets. For droplets below 200 pm diameter it is sometimes necessary to enhance photographically the contrast of the droplets.

4.2. Sensitive plastic slides Phoroslides (preparation method available from G. A. Lloyd, Ministry of Agriculture, Fisheries and Food, Plant Pathology Laboratory, Hatching Green, Harpenden) give excellent results with droplets between 75 and 250 pm diameter. They register impacted droplets as white spots against

372 P. Jones

a dark background. This method of collection has been used successfully in trials of two types of sprayer applying a binapacryl formulation (Morocide) to orchards (Harris, L. D., personal com- munication).

4.3. Trapping in a viscous liquid Fisher and Douganl3 have described a method of trapping water-based droplets in viscous poly- butene. Using Indopol H-1900 (K. and K. Greef Chemical Corporation), droplets can be caught on coated microscope slides and viewed by phase contrast. Impacted droplets can be measured quickly with the Quantimet, although some sedimentation of the droplets within the coating does occur if analysis is delayed for more than 3 days after exposure to the spray.

4.4. Natural surfaces The fluorescent-particle tracer method, introduced by Himel14 and extended by Uk,I2 used drop- lets deposited on natural surfaces, such as leaves and stems, which on drying left traces as clusters of fluorescent particles. Unfortunately, the level of fluorescence emitted by the particles is too low to be detected by the Quantimet. This can sometimes be overcome by preventing the droplets from drying out, because intact droplets fluoresce more brightly than dried deposits. The droplets may be prevented from drying out by careful choice of formulation. A spray formulation which has been used successfully is a 1 % Saturn Yellow solution in cyclohexanone+hexylene glycol (1+1 v/v). This method has proved useful for droplets between 15 and 50 p m diameter.

5. Conclusions

Transmission electron microscopy has become widely accepted in pesticide research but automated image-analysing methods are still subject to cautious experiment. As more stringent restrictions are placed upon the use of pesticide sprays in response to environmental and economic pressures, the need to relate spray deposit characteristics to biological requirements increases.l5. l6 The improvement of conventional methods for assessing spray efficiency by the use of natural targets will increase our knowledge about the optimum droplet size for a given pestlpesticide system. Image- analysing computers can speed up this characterisation process and hopefully remove some of the speculation that is current. As more and better methods of tracing droplets on natural surfaces are developed, so more precise information about the relationships between target and droplet characteristics and behaviour will become available for the guidance of agrochemical and applica- tion machinery manufacturers and users.

References 1. Edwards, H. H.; Allen, P. J. Phytopathology 1970, 60, 1504. 2. Maramorosch, K. An atlas of insect andplant viruses Academic Press, London, 1977. 3. Waters, H. Ann. Appl. Biol. 1978, 90, 293. 4. Chandler, M. R. J. Exp. Bot. 1978, 29, 749. 5. Skene, D. S . Micron 1978, 9, 242. 6. Bacus, R. C.; Williams, R. C. J. Am. Chem. Soc. 1949, 71, 4052. 7. Nixon, H. L.; Fisher, H. L. Br. J. Appl. Phys. 1958,9, 68. 8. Watson, D. H. Biochim. Biophys. Acta 1962, 61, 321. 9. Milne, R. G.; Luisoni, E. Ado. Virus Res. 1977, 21, 265.

10. Fisher, C. Microscope 1971, 19, 1. 11. Murphy, C. P.; Bullock, P.; Turner, R. H. J. Soil Sci. 1977, 28, 498. 12. Uk, S. Pestic. Sci. 1977, 8 , 501. 13. Fisher, R. W.; Dougan, G. P. Can. Entomol. 1970, 102, 31. 14. Himel, C. M. J. Econ. Entomol. 1969, 62, 912. 15. Lake, J. R. Pestic. Sci. 1977, 8, 515. 16. Matthews, G. A. Pestic. Sci. 1977, 8, 96.