Tuatara: Volume 5, Issue 2, August 1953
Plant Virus Research
Plant Virus Research
What is a virus? The true answer is that nobody really knows. And who can blame them when it is something so small that it is beyond the range of the ordinary microscope. One thing is certain, viruses are responsible for many of the diseases in plants and animals that have no other tangible or visible cause, such as parasites or bacteria. In fact, viruses have usurped the place of bacteria as the scapegoats of the biological world.
In 1892 a Russian botanist showed that sap from a diseased plant, after passage through a filter so fine that it removed all bacteria, could still infect healthy plants. This was the first scientific demonstration of a virus. Since then much finer filters have been designed that hold back some virus particles, particularly animal ones. All viruses are beyond the visibility of the ordinary microscope but some have been photographed by the electron microscope. The electron microscope reveals virus particles of varying shape from spheres to needle-like forms. Also when one considers that the electron microscope magnifies to something like 30 times that of the ordinary microscope, i.e. an object is magnified about 30,000 times its natural size, some idea of the extremely minute size of the virus particle can be realised. Or putting the matter another way, most of the smaller bacteria can be seen by the ordinary microscope and they are of the size order of 1/25,000 inch, whereas a large virus is only about 1/1,000,000 inch.
Research into plant viruses has been virtually restricted to flowering plants, and several hundreds of different virus diseases have been described. As vet no satisfactory system of naming the different viruses has been produced. Virus diseases differ from nutritional ones, for they are highly infectious and capable of indefinite reproduction from diseased to healthy hosts. Viruses also differ from bacteria which can be cultured in nutrient broths while a virus can multiply only in the living cells of some host. Certain viruses have been extracted and purified as crystals without loss of infectivity. The crystals have been identified as nucleo-proteins, and because of this many regard viruses as more closely allied to molecules than to organisms. However, although they approach a purely chemical form, viruses have all the characteristics of living matter in their ability to reproduce themselves and to change their form or ‘mutate’. The presence of many strains or varieties of a virus is evidence of mutation. Some strains may be so mild as to cause little noticeable effect, while other strains of the same virus prove lethal, the infected tissues dying.page 53
There have been a number of theories as to the origin of viruses. Firstly, there are those who believe them living organisms descended from similar living types. Animal virus investigators favour a series descending from a typical bacteria to the smallest virus, the extreme degree of reduction being due to adaptation to their particular type of parasitism. The smallest unit possible is considered to be a nucleo-protein which is capable of multiplication, the energy and material required for this being obtained from the host cell. Other workers have suggested that viruses existed in Archæozoic times as free-living non-cellular forms which later became parasitic. With the discovery of the nucleo-protein nature of virus particles, there has been in recent years a revival of the free gene theory. This suggests that viruses arose from genes that escaped from the nucleus and continued independent multiplication in the cytoplasm. It would perhaps be better to compare a virus with a chromosome fragment, both having several inheritable factors, rather than with a gene which is supposedly a single inheritable factor.
The second type of origin suggested for viruses is that they arose from some self-duplicating cytoplasmic constituent of the cell such as protein molecules. It is possible therefore that they arose from something unlike the actual virus. This raised a problem. A virus causing disease in one plant could possibly therefore be a natural constituent of another. A potential virus could arise in one plant and remain undetected until a means of spread to other plants was evolved. The problem of spontaneous generation of viruses could thus be explained.
Culture and Transmission
Plant viruses are unable to multiply outside the living cells of host plants. This necessitates special methods of culture and transmission. In experimental conditions viruses are ‘cultured’ in special host plants and are regularly transferred from diseased to healthy hosts. Certain precautions are essential to prevent contamination of one virus ‘culture’ by another. Two quite unrelated viruses are able to exist together in one host plant, each being widespread through the tissues and showing their own symptoms. Contamination by strains of a virus is possible, but the two strains do not become equally distributed. The more vigorous strain predominates, the weaker strain being restricted to small areas of the leaves about the point of infection.
Virus transmission is by three main methods, the first of these being Inoculation. This means the direct application of virus containing sap on to the leaves of the host plant. Before inoculation the host plant is dusted finely with carborundum powder. A pepper pot with the hole size reduced by several threads of florists' wire is ideal for the purpose. Too much carborundum causes excessive damage to the plant tissues — there should be just sufficient carborundum to puncture the epidermal cells. This aids the immediate entry of the virus particles. Sap is applied gently to the upper leaf surface, using a glass spatula, or one's forefinger. Excess page 54 sap, which would be toxic to the plant, is removed by washing the plant gently under the tap. The plant should be well labelled and then placed in a glasshouse cubicle reserved for infected material. Symptoms should develop some 4 to 21 days after inoculation. Among mechanically inoculated viruses are potato viruses X and Y and cucumber virus No. 1. Experimental virus ‘cultures’ may be mechanically contaminated with the very persistent tobacco mosaic virus by research workers who smoke. Fortunately there is a simple remedy, merely that of washing one's hands with soap and water before and after handling a virus infection, being careful, naturally enough, not to dry one's hands or touch anything other than sterile equipment and the virus concerned.
Not all viruses can be mechanically transmitted. There are many that depend upon aphids for transmission from plant to plant, e.g. the green peach aphid, Myzus persicæ, is a vector for many viruses. A few viruses such as potato virus Y are transmitted by both means. Aphid Transmission involves firstly the raising of virus-free aphids. Host plants not normally attacked by the virus being studied are chosen, and kept in the glasshouse under muslin cages. In addition, frequent fumigation of the glasshouses with nicotine sulphate is a necessary practice to prevent the entry of unwanted virus-carrying aphids. Aphids live and multiply best at temperatures between 60° and 80° F. so heated glasshouses are essential for winter experimental work. An aphid moves around relatively little and once it has found a suitable feeding spot it inserts its piercing mouth parts into the plant tissues, remains there and sucks the sap. It is obvious therefore that great care must be taken when transferring an aphid from one plant to another not to damage the mouth parts. The aphid is first ‘tickled’ so as to make it withdraw its stylets from the plant tissues. Once the aphid is moving it is easily picked up by a fine, damp paintbrush and lifted over to another plant. When feeding, the aphid inserts its mouth-parts directly into the phlœm and is as efficient as an hypodermic. Virus particles are therefore injected into the plant conductive system with the minimum amount of cell damage.
The aphid is starved for one to three hours before feeding upon infected material. In some way this helps successful virus transmission. After a three to twenty-four hours' feed upon an infected plant the aphid is placed on to a healthy plant. Next day the glasshouse is fumigated to kill the aphids. Viruses which are both mechanically and aphid-transmitted are found to be transferred directly the aphid begins to feed, and in a short time the aphids lose their power to infect without further feeding on infected material. However, another group of viruses, transmitted only by aphids, do not transfer as soon as the aphid feeds, and twenty-four hours mav elapse before transmission takes place. This may be accounted for by the time taken by the virus to move from the gut into the blood and back into the salivary gland. These aphids remain infective all their lives. Because potato-leaf-roll virus is in this category it helps to account for the widespread page 55 occurrence of leaf-roll disease among field crops. It is not known why some viruses should transfer immediately and others not.
The only method whereby all viruses are transmitted is that of Grafting. If the main plant (stock) is healthy, then the grafted shoot (scion) must be taken from a diseased plant for symptoms to be developed by spread to the healthy stock. A cleft-graft is commonly used, the wedge-shaped end of the scion being inserted into a vertical cut made in the decapitated main stem of the stock. The two pieces are bound firmly together with elastic tape or ideally the wide elastic material of old golf balls. An interesting feature discovered in studying potato viruses was the production of small tubers on the stem of a virus-immune stock when grafted with an infected scion.
For lack of other available factors the majority of virus identification must be by the use of the various symptoms produced in host plants. For example, the colour break in tulip flowers is the symptom produced by a virus infection. This particular virus disease is known from the sixteenth century. Old Dutch flower paintings of this period show clearly the striping effect caused by the virus and it is also recorded in the botanical literature of the time. Symptoms are generally systemic, i.e. where the virus is distributed throughout the host plant tissues. However, they may be localised, i.e. small spots or rings of infection. Localisation depends upon a severe reaction in the plant cells to infection by the virus particles, the host cells dying before the virus can spread.
The ideal experimental host plant is one that will react to a particular virus with clear and consistent symptoms. There are a number of such plants used by virus workers, and they are called Indicator Plants. The most universally used plant is the tobacco (Nicotiana tobacum) which reacts with a variety of quite unrelated plant viruses. Symptoms range from rings and spots for tobacco etch virus, green mottles for potato virus X, yellow veining for potato virus Y and many others. Mixtures of viruses are common, and there are some accommodating indicator plants that will select one particular virus from a mixture. Such a plant is the thorn apple (Datura stramonium) which will select all strains of potato virus X from the very common field combination of potato viruses X and Y. For a quantitative study of viruses the demand is for a more specialised type of indicator plant, one reacting with local lesions or spots only. Two such plants much in use are Nicotiana glutinosa for tobacco mosaic virus and Gomphrena globosa for potato virus X. Infection never spreads from the original point of entry of the virus. The number of lesions on the inoculated leaves is in proportion to the concentration of virus in the infected sap.
Indicator plants have their drawbacks. There is the very natural inclination to consider a plant showing no symptoms as one with no virus infection. Occasionally this may be the case, but it should never be taken page 56 for granted. It may be that the strain of virus is an especially mild one, or environmental conditions may be such as to mask or hide symptoms which should be showing. Therefore all the symptomless plants and a number of healthy plants as well are inoculated with a severe strain of the suspected virus. On something the same lines as vaccination, the already-infected but symptomless plants continue to show no reaction, but all healthy ones succumb to infection.
Unfortunately identification of viruses by their symptoms takes time. From inoculation to full development of symptoms takes from seven to twenty-one days. In recent years a rather more speedy means of testing has been found. These are serological methods, using rabbits. Firstly sap from a diseased plant is clarified by standing in water at 55° C. for ten minutes. This precipitates most of the plant proteins which cause the rabbit undue pain. Five millilitres of clarified sap is next injected into the marginal ear vein of a large rabbit. White rabbits are best, as the ear veins are prominent and easy to see; also white rabbits tend to be larger in size. The blood of the rabbit reacts to produce antibodies which protect it from any harmful effects of the virus. Ten days after injection the rabbit is bled. The ear not used for injection is wiped with an irritant to bring the blood to the veins. A small cut is made, and the drops of blood collected in a tube. The blood is allowed to coagulate, and the colourless serum containing the antibodies separates from the red blood cells. This serum can be stored in a deep-freeze unit for two years without losing its effectiveness.
When plants are to be tested for virus infection, sap is extracted and clarified and added to a quantitv of serum, diluted with saline, and placed in a water bath at a temperature of 50° C. If the same virus is present in the sap as was injected into the rabbit, then the antibodies in the serum react with the virus particles and a fluffy white precipitate appears. This takes from two to ten minutes, so it is easy to see how time and space are saved by using the serum method.
There are two variations of the above technique, requiring less elaborate equipment, and one of them is just as speedy in its reaction. A drop of clarified sap and a drop of antiserum are placed on a slide and mixed together, then placed under a microscope. If the virus particles are present, a white precipitate appears at the edge of the liquid. One worker can do about 500 of these tests in a day, a great advance on the maximum of 100 for the inoculation technique. The second variation of the serological method is called the ring test, and requires a steady hand. Sap is added carefully to the antiserum so that the two liquids do not mix. If the sap is virus infected, a ring of precipitin appears where the two solutions are in contact. There are certain limitations to the use of antiserum. Suitable viruses are those which are more or less stable, i.e. able to survive the clarifying temperature of 55° C., and also transmissible at dilutions of 1 in 1,000 or more. There are some viruses that cannot be transmitted at dilutions greater than 1 in 20, and so far antisera for these have not been page 57 made. Fifteen plant viruses have been used successfully for the production of antiserum, and of these tobacco mosaic virus and potato virus have given the best results. The real advantage of serological testing lies in the time it saves. To determine the presence and identity of a particular virus is only a matter of minutes compared with several days or even weeks required for indicator plant reactions.
There are many other methods of testing for specific viruses. For example, infected potato tubers fluoresce under ultra-violet light, and stem sections from infected plants often stain with particular chemicals, e.g. phoroglucinol in hydrochloric acid, and show microscopic inclusions in the plant sap. However, there is still plenty of room for new and improved techniques of testing for virus infection and identification to further fundamental research into the nature of viruses.
Viruses have an importance in the nation's economy out of all proportion to their size. They affect the productivity of the back garden plot as well as many commercial crops. When one realises that all plant viruses are transmissible by grafting, the question immediately arises as to how this affects plant stocks that are normally propagated by this means. Every precaution must be taken to ensure that only grafts between healthy stock and scion are made. For example, it has been shown that ‘green-crinkle’, a virus disease of apples found in orchards in both North and South Islands, can be transmitted from infected trees to healthy scions and from diseased buds to healthy trees. Unfortunately the foliage and wood of infected trees appear normal, and it is not until the fruit appears that the virus disease is manifest. ‘Green-crinkle’ has caused appreciable loss in several varieties of apple, but principally Granny Smith.
Virus disease affecting tobacco, tomatoes and potatoes has been referred to above. It has been estimated that tobacco mosaic and potato viruses cost their respective commercial enterprises £50,000 and £20,000 per annum in New Zealand. These are conservative estimates for only two commercially grown crops. It takes very little imagination to realise that virus diseases of one kind or another are costing the country large sums of money. Naturally one wishes to know what can and is being done about control or treatment of virus diseases. Fundamental scientific research into the nature of viruses must come first. Then the information so gained can be put to practical use. Basic research is being carried out by various Government Scientific Departments. Reference to Bulletins of the Department of Scientific and Industrial Research and the Cawthron Institute, etc., will give readers more detailed accounts of the work being done. It is then up to all plant growers, including the home gardener, to put into practice the best means of treatment and control.page 58
Man is the most potent transmitter of virus diseases, so it does not seem out of place to give a few cautionary words of how best to avoid passing virus diseases to healthy plants. Certain obvious control methods come to mind when it is explained that infective material, e.g. in the form of plant sap, plant debris, etc., can be transferred by hands, clothing or implements during routine work in the garden. As explained above, soap and water is an effective cleansing agent. A few further control measures are as follows: Where possible (this is more applicable to the home gardener) burn the infected plants; isolate susceptible crops from likely sources of infection; use disease-free seed in case of seed-carried viruses (e.g. lettuce); use healthy stocks in plants propagated vegetatively (e.g. potato, iris); and control the insects responsible for transmission.
Viruses have a history of many hundreds of years, yet even so, how little we know about them. Just a little of the diseases they cause, and something of the way to handle and direct them in host tissues. There is still endless room for investigation.