THE MOVEMENT OF TOBACCO MOSAIC VIRUS IN THE TOMATO

Transcription

1 [ 89 ] THE MOVEMENT OF TOBACCO MOSAIC VIRUS IN THE TOMATO BY JOHN CALDWELL Department of Botany, University College, Exeter {Received 9 March 1954) In a series of papers Caldwell (1930, 1931, 1934) pointed out that there was clear evidence that the virus of tobacco aucuba mosaic (A.M.V.) did not normally travel in the xylem, and that artificial introduction of it into the xylem resulted in no infection of the plant unless contact was made with broken protoplasts by deliberate damage of the living cells. He adduced evidence to show that this virus travelled apparently as fast through stems from which the portions of phloem had been removed as in intact stems, so long as other living tissue was present; and that the movement did not appear to be associated with the mass movement of food materials. In general, he considered that the virus did not enter an unbroken protoplast but passed from cell to cell through the plasmodesmata, and that the movement was through living cells rather than through specific tissues such as the phloem. The rate of movement he established to be rather slow, viz. 2 mm. per hour which was of the same order as that suggested earlier by Boning (1928). The difficulties of obtaining more precise data are formidable and at present seem insurmountable. After inoculation into a leaf of a tomato plant, some time elapses before any evidence is obtained of infection in the plant itself. This period is made up of two components: {a) the period immediately following the entry of the virus into the protoplast of a broken hair or of a punctured cell when the virus is being multiplied in that cell and/or those immediately associated with it, and [b) the period which is required for the movement of the virus from the primary centres of multiplication through the rachis and petiole into the main stem itself. It has been impossible to ascertain the first period exactly, since plants seem to be highly variable in this respect and the environmental conditions probably play a considerable part. It would appear, however, to be of the order of hr. and these figures are supported by the findings of Kunkel (1939). Assuming, therefore, some hr. for the preliminary stages of multiplication in the area of inoculation and more or less regular rates of movement, one can calculate the rate of movement after the virus has left the inoculation area. It is noteworthy, and may be significant, that inoculation of the leaves of Nicotiana glutinosa by material containing tobacco mosaic virus (T.M.V.) or tobacco aucuba mosaic results in the formation of necrotic lesions round the points of entry of the virus (probably a broken hair) with no further infection of the plant. These local lesions appear on the third or fourth day after inoculation. It is suggested that the effect on the cells of the 'inoculation-area' of A'", glutinosa leaves is so lethal as to destroy the protoplasmic connexions between these cells and the other mesophyll tissues and so to stop virus movement out of the groups of infected cells. The fact that a period of some 48 hr. appears to be required before the concentration of virus is sufficient to cause collapse of these cells may support the idea that that is the period required for multiplication in the case of tomato leaf tissue also. Samuel (1934) suggested in his paper that the

2 9O JOHN CALDWELL fact that systemic infection occurs in A'^. glutinosa if the plants, after inoculation, are maintained at a temperature of 35 C. indicates that more virus is produced at that temperature. The opposite seems the more probable explanation, since 'masking' is usually a reaction to high temperature (cf. Kassanis, 1952). Workers with other viruses probably quite unrelated to tobacco mosaic virus have reported other and quite different rates of movement, notably Bennett (1927) for a virus of raspberries; Severin (1924) for the virus of curly-top of sugar beet; and Storey (1928) for the virus of streak disease of maize. A general summary of much of the available information is contained in a survey by Crafts (1951). It is not proposed in this paper to discuss the movement of viruses other than that of tobacco mosaic and the closely related tobacco aucuba mosaic. Samuel (1934) criticized the findings of Caldwell regarding the movement of T.M.V. which Samuel summarized, quite fairly, as being that: {a) the movement is in the living tissue, {b) it is a relatively slow movement, presumably from cell to cell, and (c) movement is both upward and downward from the point of insertion of the inoculated leaf. Samuel proceeded to show that the method of taking cuttings from the treated plant would be more reliable than that of inoculating leaves of A^. glutinosa with small quantities of expressed juice. He then described observations on the movement of T.M.V. in the tomato plant from which he concluded that: (a) the virus, on leaving the inoculated leaf, travels first to the roots ' with such speed that it can seldom be intercepted at intervening positions '; (i)' usually about a day later it travels with equal rapidity to the top of the plant'; (c) ' in pot plants, after the initial rapid infection of the developing leaves at the top of the plant the more mature leaves become successively invaded from the top downwards and from the bottom upwards until the plant is completely invaded by the virus...'; (^)' it is considered that these facts favour the theory of a slow cell to cell movement of the virus via the plasmodesmen, combined with a rapid distribution through the plant via the phloem and the value of the tobacco mosaic virus as an indicator of phloem movements is emphasized'. With the conclusions in the first part of section {d) the present writer, obviously, has no quarrel as they confirm his own previously expressed and still held views on the movement of T.M.V. Nor would he quarrel with the view that expressed juice inoculated into the leaves of A^. glutinosa may give inaccurate results if the concentration of virus in that juice is very low. It has been known for a long time that the presence of protein in a vims suspension may result in the absorption of the virus thus rendering it less active than it might otherwise be. A recent experiment by the present writer illustrated that point most strikingly. Mature leaves of A'^. glutinosa were rubbed respectively with: (i) 0-5 ml. of A.M.V. juice diluted i in 50 with the juice of healthy tomato plants expressed and filtered through muslin, or (ii) 0-5 ml. of A.M.V. juice diluted i in 50 with tap water. When a count of the local lesions was made it was found that 55 leaves on 11 plants had 33 local lesions in the material diluted with expressed tomato juice, an average of o-6 per leaf, while that diluted with water had resulted in the formation of 953 lesions on 51 leaves on II plants, an average of 18-7 per leaf. Kunkel (1939) published a paper on the movement of T.M.V. in tomato plants. He pointed out, as had been done before, that the difficulties of timing the movement of the virus out of the inoculated leaf made the exact assessment of the rate of movement in the stem very difficult. He tried using clonal material to ensure more constant results but apparently without success. He showed that the virus on reaching the stem could move

3 The movement of tobacco mosaic virus in the tomato 91 either up or down the plant or in both directions simultaneously. This confirms Caldwell's findings but not Samuel's. His data also show that ' in a considerable number of plants the virus that moved upward did not travel via the roots'. 'It is concluded', he writes, ' that virus particles either do not multiply or multiply very slowly as they move through stems.' 'Because of the speed with which tobacco mosaic virus moves in stems, it is presumed to travel in sieve-tubes, tissues through which food materials are known to move.' 'Virus particles that have remained for some time in a dormant condition in sections of a tomato or tobacco stem may move out into plants grown from such sections and there multiply and cause disease.' Kunkel (1939) postulates that hr. is the period required for movement of the virus out of his inoculated leaves, while noting that' all the tomato plants used... were of this clone but it cannot be said that they gave more uniform results than the seedlings (non-clonal) used previously'. In his presentation of the results he notes: 'Records for all inoculated plants used in the first 3 experiments and for 35 of 158 inoculated plants used in 12 other experiments are presented. In the plants for which no records are given, virus was either not found or was present in all parts tested.' In the 30 plants, details of which are recorded in Kunkel's ' table I (Expts. 1-3 inclusive)' 8 plants are recorded as negative in all parts which leaves 22 plants in which infection followed inoculation. In 10 of these 22 plants continuous distribution of the virus was noted, i.e. no infected areas were separated by those free of virus (plants 5, 6, 8 and 10 in Expt. I; plants, 7, 9 and 10 in Expt. 2; and plants, 5, 9 and 10 in Expt. 3). In some the virus had gone one way, in others both. In one instance it had not reached the apex of the plant though there was continuous distribution in the lower internodes. His 'table I (continued)' is more difficult to interpret. In 123 plants, according to the statement noted above, the virus had either not moved or had reached all parts of the plant. This makes for some difficulty because if all or most of the plants had virus distributed throughout their length, they should be added to those showing continuous distribution. The others, that is those in which infection had not taken place, would indicate, as do the 8 out of the 30 discussed above, the unprecise nature of the techniques of inoculation and the difficulties attached thereto. If we ignore these unknowns and subtract from the total of 35 recorded results the 3 plants in which all parts are given as negative except for the petiole of the infected leaf, we find that in 32 plants 20 show continuous distribution and that in 12 it was interrupted at some internode. If one adds the totals together one finds that plants in the whole of table I showed continuity of distribution and plants discontinuous distribution of the virus. As an unknown number up to 122 has to be added to the group of 30 and none to the group of 24, it is a little difficult to discover why one should assume from the data of Kunkel that there is a preponderance of evidence on the side of discontinuous distribution. In 'table H' very similar results are listed. In 33 plants out of 79 no movement took place at all. Of the 46 remaining 23 showed continuous and 23 showed discontinuous distribution. The data given in table 3 show a preponderance of instances of discontinuous movement, though Kunkel himself has called attention to the difficulties of inoculation into plants, both tomato and A'^. gluiinosa (see above), when small amounts of virus in the presence of large amounts of plant juice are used.

4 92 JOHN CALDWELL On the whole it would appear that Kunkel's findings do not support Samuel's conclusion that T.M.V. first moves rapidly to the roots and thence upwards to the other parts of the plant. Nor, in the submission of the present author, do they give unqualified support to Kunkel's own conclusion that he had confirmed Samuel's observation that in the earliest stages of entry into the stem, virus particles may be separated by considerable distances. The present writer has, for many years, carried out experiments on the movement of tobacco mosaic virus in tomato plants, continuing the work to which reference has already been made. Difficulties have been regularly encountered in that the stem may be cut into portions to allow an internode in each portion too early, i.e. before the virus has left the inoculated leaf; or too late, i.e. when all the parts show symptoms of infection in the rooted cuttings. Every effort has been made to prevent accidental infection by using a sterile scalpel and handling each portion in a separate sheet of paper and so forth. On no occasion has there been any evidence of virus being ' missing' from an intermediate section, though on various occasions there has been evidence of upward or downward movement in single plants as well as the more usual 2-directional movements. On some half a dozen occasions the cutting of the stem into portions was sufficiently well-timed as to result in the virus not having yet reached the top of the plant though it appeared in all the intermediate portions. At no time has there been any evidence to suggest that the virus, having reached portions of the stem farther away from the insertion of the inoculated leaf, was not present in an intervening portion. One of the conclusions of Samuel (1934, p. no) may be important in the consideration of the movement of virus, viz. no. 7. ' In pot plants, after the rapid initial infection of the developing leaves at the top of the plant, the more mature leaves become successively invaded from the top downwards and from the bottom upwards until the whole plant is completely invaded by the virus. Complete invasion occurs very quickly in small vigorously growing plants: it may take 3 weeks or more in medium-sized plants and as much as 2 months in large fruiting plants' [my italics]. In other words, even if one queries the rapid movement through the tissues to the apex and, still more, following Kunkel, that the movement to the upper part of the plant is via the roots, Samuel's data do favour the view of 'a slow cell to cell movement via the plasmodesmen'. The observed fact which has been noted by many workers, including the present writer, viz. that young actively growing plants show evidence of rapid infection, i.e. of rapid movement, is significant. Put another way, the movement of the virus is easier and faster in plants in which many cells of the stem are still in an active metabolic stage; in which, in other words, some ceil 5 may even be in the embryonic stage. During the past few years we have been concerned in this Department with a study of the effect on nuclear division (both meiotic and mitotic) of virus disease in plants. The topic suggested itself because of the quite remarkably few cases of known and agreed true seed-transmission of virus in plants (i.e. through an infected embryo) as opposed to infected testas (Caldwell, 1934). Holmes gives only eleven instances out of some 140 diseases listed by him (Bergey, 1948). The almost complete absence of seed in fruits of tomato plants infected with ' Aspermy' virus led to a study of the effect of this virus on spore-production (Blencowe & Caldwell, 1949). The results, which are discussed in detail (Caldwell, 1952), are briefly that the effect of the virus is to induce the breakdown

5 The movement of tobacco mosaic virus in the tomato 93 of reduction division in both embryo-sac and pollen-cells at prophase in a substantial number and in some cases of all the spore-mother-cells. It is clear that an infected sporemother-cell cannot give rise to normal spores. Eurther, in A^. glutinosa Wilkinson (1952) has noted similar pachytene collapse in the meiocytes, accompanied by an apparent increase of nucleolar material, and has been able to correlate the degree of seed sterility with abnormality of pairing during the first meiotic division. Eurther work by Wilkinson (1953) has shown that marked nuclear aberrations are found with this virus in divisions in somatic tissue also. Nor is this confined only to 'Aspermy' virus in its various host plants. Evidence is accumulating that it is common to most plant viruses, including the classical T.M.V. in cells of the tomato and other hosts during mitosis. This work, which is discussed in detail elsewhere, has led us to postulate, as a working hypothesis, that the virus in plants is associated with, and perhaps causes a breakdown in, the enzyme system catalizing the ribose-desoxyribose nucleoprotein formation. It is not claimed, in the absence of precise information on an aspect of the problem still under investigation, that the presence of actively dividing cells is necessary for the multiplication of virus; but all the available evidence from a wide range of observations suggests that, at least, actively metabolizing cells must be present before active virus multiplication is possible. This has been shown by many workers (cf. Bawden, 1950; Cook, 1947). There is no precise information on the mechanism by which the virus does move in the plant nor is any definite theory here postulated. It is generally agreed now that T.M.V. and most other viruses do not travel in dead tissue and even if injected artificially into the xylem are unable to leave that tissue unless it is intentionally damaged. Samuel and Kunkel both suggest that the path of movement may be by the phloem, though this appears to be a suggestion based on the view of many plant physiologists, who hold that nitrogenous material does travel in that tissue. It is well to bear in mind that even the physiologists who hold this view are normally considering the movement of amino-acids and the like and as noted by Stiles (1950, p. 305):' The large protein molecule is relatively immobile with a low coefficient of diffusion, and consequently is converted into more readily diffusible substances for translocation.' Since the largest plant proteins have molecular weights of some 38 x 10^ it would hardly seem likely that the plant physiologists who question the movement of protein molecules themselves through the phloem would subscribe to the movement through it of molecules of a calculated molecular weight of the order of 17 x io^ SUMMARY Current views on the method of the movement within the plant of tobacco mosaic virus are noted and discussed. The suggestion of Samuel that movement \&, always slowly upward after a rapid movement to the roots of infected plants is considered in the light of Kunkel's later observations, and Kunkel's conclusions from his own experiments are discussed. REFERENCES BAWDEN, F. C. (1950). Plant Viruses and Virus Diseases. Waltham, Mass: Chronica Botanica. BENNETT, C. W. (1927). Virus diseases of raspberries. Mich. Agric. Exp. Sta. Tech. Bull. no. 80. BERGEY, D. H. (1948). Manual of Determinative Bacteriology. Baillifere Tindall & Cox. BLENCOWE, J. W. & CALDWELL, J. (1949). Aspermy a new virus disease of the tomato. Ann. Appl. Biol. 36, BONING, K. (1928). Beitrag zum Studium der Infectionsvorgange pflanzlicher Viruskrankheiten. Z. Parasitenk., 1,

6 94 JOHN CALDWELL CALJJWELL, J. (1930). 1. The physiology of virus disease in plants. The movement of virus in the tomato plant. Ann. Appl. Biol. 17, CALDWELL, J. (1931). IL Further studies of the movement of virus in the tomato plant. Ann. Appl. Biol. 18, CALDWELL, J. (1934). V. The movement of the virus agent in tobacco and tomato. Ann. Appl. Biol. 21, CALDWELL, J. (1952). Some effects of a plant virus on nuclear division. Ann. Appl. Biol. 39, COOK, M. T. (1947). Viruses and Virus Diseases of Plants. Burgess Publishing Co. CRAFTS, A. S. (1951). Movement of assimilates, viruses, growth regulators and chemical indicators in plants. Bot. Rev. 17, KASSANIS, B. (1952). Some effects of high temperature on the susceptibility of plants to infection with viruses. Ann. Appl. Biol. 39, KUNKEL, L. O. (1939). Movement of tobacco-mosaic virus in tomato plants. Phytopathology, 29, SAMUEL, G. (1934). The movement of tobacco-mosaic virus within the plant. Ann. Appl. Biol. 21, SEVERIN, H. H. P. (1924). Leaf curl transmission experiments. Phytopathology, 16, STILES, W. (1950). An Introduction to the Principles of Plant Physiology. Methuen. STOREY, H. H. (1928). Transmission studies of maize streak disease. Ann. Appl. Biol. 15, WILKINSON, J. (1952). Some effects induced in Nicotiana glutinosa by the 'aspermy' virus of tomato. Ann. Bot., Lond., N.S. 17, WILKINSON, J. (1953). Virus-induced nucleolar abnormalities in tomato. Nature, Lond., 171, 658.

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