THE TRANSMISSION OF CAULIFLOWER MOSAIC BY APHIDS

By M. F. DAY* and D. G. VENABLES*

[Manuscript received October 20, 1960]

Summary Cauliflower mosaic virus (ClMV) has some characteristics of both non-persistent and persistent aphid-borne . It has been shown that ClMV is a true non­ persistent virus, and it is suggested that the unusual features of its, transmission characteristics are due to its relative stability and its distribution in the tissues of the . These considerations lead to more precise definitions of the terms "persistent" and "non-persistent".

1. INTRODUCTION Most of the phytopathogenic viruses transmitted by aphids may be classified as "non-persistent" or as "persistent" viruses. These terms, introduced by Watson and Roberts (1939), were subsequently somewhat modified in meaning (Watson 1946), and are now widely used. It is generally agreed that non-persistent viruses are transmitted by contaminated stylets, whereas persistent viruses are ingested, pass through the haemocoele, and are inoculated into a plant with the aphid saliva. The range of characteristics of these two groups of viruses is considerable, so that it has been thought that certain viruses do not readily fit into either category. Sylvester (1956, 1958) has suggested that an intermediate category be established to include these, which he called "semi-persistent" viruses. He considered beet yellows virus to be a typical member of the group, and thought that dandelion yellow mosaic virus and cauliflower mosaic virus should probably also be included in it. Cauliflower mosaic virus (ClMV) was first recognized by Tompkins (1937), or possibly earlier by European workers (see Klinkowski 1953). Early work on aphid transmission of ClMV seemed to indicate that it was a typical non-persistent virus. However, subsequent investigations (summarized by Broadbent 1956) have tended to show that it is atypical. Thus Hamlyn (1955) commented that "the ability to remain infective for at least 3 hours in the feeding vector distinguishes cauliflower mosaic virus sharply from non-persistent viruses studied previously". Chalfant and Chapman (1956) went further and stated that "transmission of virus 2 appears typically non-persistent by the green peach aphid, but shows some character­ istics other than those of non-persistent types when transmitted by the cabbage aphid." Van Hoof (1954) concluded that "the results ... would place cauliflower mosaic virus in the persistent viruses." Chalfant (1959), however, concluded that ClMV was non-persistent in both green peach aphid and cabbage aphid but that the virus was not carried on the stylets of the latter species after long feeds. * Division of Entomology, C.S.loR.O., Canberra. 188 M. F. DAY AND D. G. VENABLES

In view of the interest of CIMV in any discussion of mechanisms of transmission of viruses by aphids we have investigated its transmission, and the results are reported in this paper. It is concluded that cmv is a true non-persistent virus, and a revised description of this category is suggested.

II. MATERIALS AND METHODS The virus was that used by Day and Venables (1960). It was maintained by mechanical or insect inoculation in turnip (Brassica rapa L. cv. Flat Express) which served both as a source plant and as a virus indicator. No local lesion host is available. In the mechanical inoculations the leaves were lightly dusted with carborundum. Small squares of organdie wetted with the liquids to be inoculated were gently rubbed over the leaves which were then washed with water. A few experiments included wild mustard (Sisymbrium orientale L.) as an indicator, and this species was shown to be roughly equivalent to turnip in sensitivity. The aphids used were mature apterae of (Sulz.) and Hyadaphis (Brevicoryne) brassicae (L.). The former were maintained on a variety of hosts including chinese cabbage, turnip, stramonium L., and Physalis floridana Rydb. Colonies of H. brassicae were maintained on turnip. Both species were transferred from source to indicator by means of a fine camel-hair brush. Indicator seedlings were grown in boxes 14 by 14 by 4 in., and generally used in the cotyledon or one-leaf stage. Symptoms of viral infection first appeared 14 days after inoculation, and for about 7-14 days thereafter. Source plants invariably showed severe symptoms similar to those described by Broadbent (1956). The change in concentration of infective virus in infected plants with time was studied by bioassay by sampling at weekly intervals after mechanical inoculation. Infected leaves were harvested, treated as described in Table 1, and inoculated on to turnip seedlings. The results (Table 1) show that there is no marked change in virus content over a period of several weeks after symptoms have become marked. Indicator plants after infection were kept in glass-houses which were regularly sprayed with insecticides.

III. EXPERIMENTAL RESULTS (a) Inoculation of OlMV into the Aphid Haemocoele ClMV, purified by the method described by Day and Venables (1960), was inoculated by the technique used by Day (1955) into 32 mature apterous M. persicae. No infections resulted when these aphids were allowed to feed on indicator seedlings for a period of 3 days. Twenty-four hours after inoculation 24 aphids remained alive and 8 were living at the end of the 3-day test period. A similar experiment was performed using H. brassicae. The results were the same, but H. brassicae tolerated the inoculation less well than M. persicae, only 16 remaining alive after 24 hr and 6 at the end of the 3-day feeding period. The poor survival may have been due to the toxicity of the purified ClMV when injected, because survival was much better in aphids inoculated by the same methods and at about the same time with plant extracts infected with potato leaf ron virus. TRANSMISSION OF CAULIFLOWER MOSAIC VIRUS BY APHIDS 189

Previous workers (Day 1955; Heinze 1955) have shown that blood from aphids infected with certain persistent viruses, when inoculated into non-infective aphids, caused them to become infective. Blood from 16 H. brassicae bred on CIMV-infected turnips was inoculated into 16 non-infective aphids. Each was then placed on separate indicator seedlings for 3 days. No infections resulted.

TABLE 1 EFFECT OF TIME AFTER INOCULATION ON YIELD OF ClMV FROM INFECTED LEAVES Tissue harvested regardless of symptoms, washed, frozen overnight, thawed, macerated in 2 vol. O' 5M pH 7· 8 potassium phosphate buffer, filtered, clarified (20 min at 12,000 g), filtered, and sedimented (90 min at 30,000 r.p.m. (No. 30 head, Spinco model L)). Pellet resuspended in O'IM pH 7·8 buffer at rate of 0·5 ml buffer/g tissue extracted. Dilutions were inoculated 24 hr after harvest. Numbers of infections recorded out of 16 indicators inoculated

Infections at Dilution of: T' ft ~!' n

1 1/10 1/100

21 7 3 1 28 16 6 35 14 8 4 42 15 15 2 50 13 14 2 57 14 9 3

(b) Time Required for Transmission of ClMV One of the characteristics of the non-persistent viruses is that the transmission time (the period from the beginning of the acquisition feeding period to the com­ pletion of the transmission feeding period) requires only a few minutes. Day and Irzykiewicz (1954, Table 1) showed, in fact, that M. persicae and H. brassicae could transmit ClMV when the transmission time was as short as 83 sec. In a more extensive test M. persicae transmitted ClMV when the acquisition feeding period and the transmission feeding period was 30 and 40 sec, respectively. However, only one transmission occurred out of 19 tests. It is therefore concluded that M. persicae is capable of functioning as a vector when the transmission time is short, but that it is relatively inefficient under these conditions.

(c) Acquisition and Persistence of Virus in Aphids (i) Influence of Acquisition Feeding Time on Transmission ofClMV by M. persicae and H. brassicae.-Two experiments were carried out to examine this aspect. In the first, individuals of both M. persicae and H. brassicae, previously allowed an acquisition feed in excess of 24 hr on infected turnips, were transferred to a series of five indicator plants allowing 5 min feeding time on each indicator. This was replicated 10 times for each species. 190 M. F. DAY AND D. G. VENABLES

The second experiment was of the same design except that a feeding time of 5 min was allowed before transfer to a series of indicator plants. The results of these experiments (Table 2) show: (1) no difference between short and long acquisition feeding periods with M. persicae, but suggest differences with H. brassicae; (2) no difference between the two species of vectors after short acquisition feeding periods; (3) the ability of both species to transmit without the intervention of an extrinsic incubation period; (4) the ability of both species to infect several plants in the series of transfers.

TABLE 2 ClMV INFECTIONS IN TURNIP SEEDLINGS BY SINGLE APHIDS MOVED TO FIVE SUCCESSIVE PLANTS AFTER 5 MIN TRANSMISSION FEEDING PERIODS

Infections per Plant Total Acquisition Aphid Species No. of Feeding Period Infections No.1 No.2 No.3 No.4 No.5 ------M. persicae > 24 hr 1 1 0 0 1 I 3 H. brassicae >24 hr 2 5 2 4 2 15 M. persicae 5 min 2 0 1 2 0 5 H. brassicae 5 min 3 2 0 0 0 5

(ii) Persistence of Virus in H. brassicae.-Forty-two individuals of H. brassicae taken from a colony raised on infected turnips were each transferred to a series of five indicator seedlings allowing 24 hr feeding of each aphid on each indicator, thus covering a 5-day feeding period for each aphid. At each transfer, note was taken of cast skins to show when each aphid moulted. From the results of this experiment (Table 3) it will be observed that H. brassicae can retain CIMV for 3 days. This period of retention is longer than for any previously described non-persistent virus and is of about the samy length of time for which a vector of beet yellows remains infective following a short acquisition feeding period. The data also show no instance of transmission of ClMV following a moult by the aphid vector. The conclusion that transmission of the virus did not occur following a moult was more conclusively demonstrated by the test reported by Day and Irzykiewicz (1954).

(d) Distribution of ClMV in Plant Tissues The distribution of CIMV in tissues of infected turnip leaves was studied by the fluorescent antibody technique. Antiserum to virus purified by the method of Day and Venables (1960) was produced in rabbits. The inoculum consisted of 0·5 ml of the virus preparation diluted 1 in 4 in standard saline to which 2 ml of Freund's adjuvant was added. The mixture was homogenized for 5 min at maximum speed in a Virtis homogenizer. Two doses, each of 0·8 ml, were inoculated intramuscularly into the flanks of 3-month-old rabbits. Forty days later a booster injection con­ sisting of O· 5 ml of virus solution and 1·5 ml standard saline was inoculated by the TRANSMISSION OF CAULIFLOWER MOSAIC VIRUS BY APHIDS 191 same route. The rabbits were bled 5 days later and the globulin fraction was coupled with fluorescein isothiocyanate (Sylvana Chemical Co.). Pieces of leaves were embedded in 10% gelatine, and frozen in dry ice. Sections approximately 5/k thick were cut on a Cambridge rocking microtome by the method of Louis (1957). The sections were dried for 30 min on the slide and fixed in acetone for 5 min to remove most of the chlorophyll. A Zeiss fluorescence microscope was used with an Osram

TABLE 3 DISTRIBUTION OF INFECTIONS OF CIMV WIlEN SINGLE INDIVIDUALS OF H. BRASSICAE WERE MOVED DAILY TO HEALTHY TURNIP SEEDLINGS OVER A 5-DAY PERIOD X = infections; M = moulted skin found. An additional 24 aphids did not transmit; four of these moulted during the 5-day period

Aphid Day 1 Day 2 Day 3 Day 4 Day 5

1 X X X 2 X M 3 X M 4 X X M 5 X M 6 X 7 X M 8 X X 9 X M 10 X X 11 X X M 12 X M 13 X xM 14 X X M 15 X 16 X X 17 X X 18 X

Total 17/42 8/42 3/42 0/42 0/42 Number of aphids moulting 0 5 5 3 2

HBO 200 mercury vapour lamp. Photographs were taken on HP3 (Ilford) 35-mm film. Antigen was readily localized in the sections and appropriate controls showed that staining was specific for cmv. Uninfected leaves showed only the fluorescence of residual chlorophyll and of cell walls. Fluorescein-coupled vaccinia virus antibody produced in rabbits failed to stain sections of infected or control leaves. In an attempt to remove chlorophyll, leaves were soaked in acetone for 6 weeks. This treatment, however, removed all trace of specific staining as well as the characteristic fluorescence of chlorophyll. Cotyledons mechanically inoculated 6 days previously with clarified sap from infected leaves showed very sparse specific staining. In systemically infected leaves, many cells from most leaf tissues contained antigen (Plate 1, Fig. 1). Only the 192 M. F. DAY AND D. G. VENABLES xylem vessels regularly failed to stain. A few phloem cells were intensely stained and in these the entire was strongly positive. Within individual epidermal mesophyll cells the fluorescence was confined to the cytoplasm. Neither nuclei nor chloroplasts were ever stained. The distribution of the virus in discrete aggregates may explain the irregularity in infection in aphids fed for short periods, because an aphid may probe one of these aggregates and become infectious, or the probe may

TABLE 4 HEAT STABILITY OF CIMV 180 g CIMV-infected plants were macerated with 360 ml O'IM sodium phosphate (Sorensen's) buffer, pH 7· 6, then filtered through organdie. 10-ml aliquots were heated for 5 min at the temperatures indicated. Mter clarification in the Servall centrifuge, each supernatant was inoculated on to 16 indicator seedlings

Temperature (OC) Unheated 50 55 60 65 70 75 80

No. of infections 2 5 5 10 8 7 9 8

miss a virus aggregate completely. The greater efficiency of transmission by H. brassicae after longer feeds is explicable if this species is more efficient than M. persicae in locating the phloem, or if the stylets of H. brassicae penetrate cells more often than those of M. persicae, rather than penetrating through epidermal cell walls, which contained no stained antigen.

TABLE 5 LONGEVITY OF CIMV IN O' 1M SORENSEN'S BUFFER, pH 7· 0, AT 50°0 Number of infections per 16 indicators inoculated

Time No. of Time No. of (min) Infections (min) Infections

0 8 15 14

5 8 20 13

10 13 30 12

The distribution of antigen within epidermal cells was studied in strips from the epidermis of infected turnip leaves. Strips from freshly cut leaves were soaked in solutions of the stain for 20 min, washed in several changes of water, and mounted in glycerol. No viral antigen was found in guard cells, but stained granules occurred in the majority of epidermal cells (Plate 1, Fig. 2). It seems likely that the inclusion bodies described by Rubio-Huertos (1950) are the granules which contain the viral antigen. Under the oil-immersion lens these granules were seen to have smooth contours and frequently consisted of several lobes. Three or more granules per cell TRANSMISSION OF CAULIFLOWER MOSAIC VillUS BY APHIDS 193 were frequently seen, but in many epidermal cells they were absent. In mesophyll cells adhering to the epidermal strips viral antigen occurred between but not in chloroplasts. The distribution of antigen in the turnip leaf described above was compared with that of another ClMV strain (originally isolated from wild mustard). In this, the amount of virus in the epidermal layers appeared to be similar to that of the common strain. However, stainable antigen was virtually absent from the mesophyll and there was less in the conducting tissue than was observed in leaves infected by the common strain. This second type of distribution may be usual in non-persistent VIruses. (e) Resistance of OlMV to Thermal Inactivation Most strains of CIMV have been reported by earlier workers to be relatively thermostable. Tables 4 and 5 illustrate that infectivity in the strain used is retained after treatment at 50°C for 30 min, or 80°C for 5 min. These results confirm that the common strain of CIMV is a relatively thermostable virus. This is of significance in the transmission of the virus. It may be anticipated that transmission of the relatively thermolabile strain described by Wei et al. (1958) would differ from that of the strain used in the present work.

IV. DISCUSSION Eleven characteristics of non-persistent and persistent viruses are tabulated in Table 6. In the final column is shown the category into which CIMV falls in respect to each criterion. It will be observed that CIMV is a typical non-persistent virus in most respects, but that there are five characteristics in which it has some of the properties of a persistent virus. Each of these is explicable if CIMV is: (1) more stable than most other non-persistent viruses; (2) distributed in the plant tissues in a manner which differs from the distribution of most non-persistent viruses. It has been shown that ClMV is a stable virus both to thermal and chemical inactivation. Concerning the second point, Mulligan (1957) has evidence for the view that CIMV and cabbage black ring spot virus are differently distributed in leaves. This conclusion is borne out by the transmission data of Hamlyn (1955), and is suggested by the experiments using fluorescent-labelled antibody. This technique has been used on plant tissues previously only by Schramm and Rottger (1959) to study tobacco mosaic virus, and data on the distribution of aphid-borne viruses in leaf tissues is unfortunately limited. On the basis of experiments involving virus inactivation by ultraviolet light Bawden, Hamlyn, and Watson (1954) concluded that cabbage black ring spot virus and henbane mosaic virus were distributed mainly in the epidermal cells. However, Hitchborn (1958) concluded that potato virus Y, potato virus C, tobacco mosaic virus, and henbane mosaic virus were present in approximately equal amounts in mesophyll and in epidermal cells, and concluded that the ultraviolet inactivation experiments were of doubtful validity. These conclusions leave some of the aphid­ transmission data unexplained, and further experiments using the fluorescent antibody method on a variety of plant viruses would be very desirable. In the 194 M. F. DAY AND D. G. VENABLES TABLE 6 CHARACTERISTICS OF APHID·BORNE VIRUSES AND BIOLOGICAL PROPERTIES OF ClMV N = ClMV has the property of a non-persistent virus; P = ClMVhas the property of a persistent virus; ± = CIMV has some of the properties of both groups

Character- N on-persistent Persistent Properties Reference istic No. Viruses Viruses ofClMV

Short transmission Longer transmission N Severin & cycle (seconds) cycle (hours) Tompkins (1948 ); Van Hoof(1954

2 No extrinsic incubation Often an extrinsic N All authors period incubation period

3 Pre-acquisition No such effect P Martini (1956) starvation increases vector efficiency

4 Short acquisition Long acquisition feeding ± Tbis paper feeding periods more periods more efficient efficient than longer than short ones ones

5 Aphids generally Aphids capable of ± Van Hoof (1954); capable of infecting infecting many plants; Hamlyn (1955) one or few plants often remain infectious this paper for life

6 Virus not recoverable Virus usually recoverable N Tbis paper from haemolymph of from haemolymph of vectors vectors

7 Virus not transmissible Virus transmissible by N Day and by vector following vector following a Irzykiewicz a moult moult (1954)

8 Vector not infective Vector capable of N This paper after inoculation of becoming infective purified virus into when purified virus is haemocoele inoculated into haemocoele

9 Vector specificity not Vector specificity often N Day and Bennett not marked marked (1954)

10 Virus generally Not generally trans- N All authors transmitted by mitted by mechanical mechanical inoculation inoculation of expressed sap

11 Virus generally affects Virus generally affects ± Mulligan (1957); mainly epidermal conducting tissues but see also tissues Hitchborn (1958) TRANSMISSION OF CAULIFLOWER MOSAIC VIRUS BY APIDns 195 absence of further data, the suggestion that CIMV is distributed in leaf tissues differently from that of some other non-persistent aphid-borne viruses remains a likely hypothesis. It should be stressed that no antigen could be seen in cell walls. If the suggestion of Van Hoof (1958) is correct, namely that aphids acquire virus mainly from the cell walls of epidermal cells, then the fluorescent antibody technique may not provide useful information about the distribution of aphid-accessible virus. Furthermore, Van Hoof's suggestion of the source of the infective virus may render Hitchborn's (1958) conclusions concerning the relative amounts of virus in different tissues irrelevant to the problem of aphid transmission. Cauliflower mosaic virus can certainly be transmitted for longer periods following the acquisition feed than most non-persistent viruses. But this "persistence" is not a good criterion for placing a virus in either the persistent or the non-persistent category. For example, myxomatosis virus is apparently transmitted by a comparable mechanism to that by which the non-persistent aphid-borne viruses are transmitted, yet it persists for months on the mosquito mouthparts. In view of the thermal stability of CIMV it is not unexpected that aphid vectors can remain infective for periods longer than the vectors of more labile viruses. Cauliflower mosaic virus is thus a true non-persistent virus. The two char­ acteristics in which it is unusual are primarily the result of two properties, its stability and its distribution in the plant. Some of the differences observed between M. persicae and H. braBsicae in their ability to transmit CIMV may result from differences in their feeding behaviour. Such differences are illustrated by the data of Day and Irzykiewicz (1953). The above considerations assist in the selection of the best criteria for assigning any new aphid-borne virus to either the persistent or non-persistent category. Of the 11 characteristics listed in Table 6, it will be apparent that Nos. 6, 7, and 8 are those which distinguish unequivocally between the two types of transmission, whereas Nos. 3,4,5, and 11 are features dependent upon the stability and distribution of the virus rather than its mechanism of transmission. The view has been held that the best criteria for placing a virus in the non­ persistent category are the effect of pre-acquisition starvation on transmission efficiency and the greater efficiency in transmission following short feeding periods compared with long feeding periods. These criteria undoubtedly hold for many non­ persistent viruses, but they do not hold for ClMV. If the aphid can acquire a virus from deeper tissues such as the phloem, then feeding periods in excess of 10 min will be necessary to reach the tissue (Roberts 1940), and this time will negate the effect of a pre-acquisition starvation period. Weare now able to define more precisely the terms "persistent" and "non­ persistent" viruses. A persistent virus is one in which: (a) the transmission time is long; (b) the virus is recoverable from the haemolymph of a vector; (c) the virus is transmitted following the moult of a vector; and (d) the vector is capable of becoming infective when purified virus is inoculated into the haemocoele. A non­ persistent virus is one in which (a) the transmission time is short; (b) the virus is not recoverable from the haemolymph; (c) the vector is not capable of transmitting following a moult; and (d) the vector does not become infective when purified virus is inoculated into the haemocoele. 196 M. F. DAY AND D. G. VENABLES

Persistent viruses may behave in one of two ways. They may either multiply in the vector or they may pass through the vector unchanged. Those in the latter category often have some of the characteristics of non-persistent viruses. It would seem desirable to re-investigateother apparently anomalously transmitted viruses in the light of the above conclusions. It seems likely that the category of "semi­ persistent" viruses will be found to be superfluous. It should be mentioned that the determination of whether a virus is persistent or non-persistent may be of more than theoretical importance. Several investigators (see Broadbent 1957) have shown that it is sometimes possible to control persistent viruses by the application of systemic insecticides, but success has rarely attended attempts to reduce the occurrence of non-persistent viruses by these methods.

V. ACKNOWLEDGMENTS Grateful acknowledgment is made to Dr. S. Fazekas de St. Groth, Department of Microbiology, Australian National University, for preparing antiserum to cauli­ flower mosaic virus, and to Dr. C. Mims of the same Department for performing the experiments using fluorescent-labelled antibody.

VI. REFERENCES BAWDEN, F. C., HAMLYN, B. M. G., and WATSON, M. A. (1954).-Ann. Appl. BioI. 41: 229-39. BROADBENT, L. (1956).-"Investigations of Virus Diseases of Brassica Crops." (Cambridge Univ. Press.) BROADBENT, L. (1957).-Annu. Rev. Ent. 2: 339-54. CHALFANT, R. B. (1959).-Diss. Abstr. 20(1): 432-3. CHALFANT, R. B., and CHAPMAN, R. K. (1956).-Bull. Ent. Soc. Amer. 2: 19. DAY, M. F. (1955).-Aust. J. BioI. Sci. 8: 498-513. DAY, M. F., and BENNETTS, M. J. (1954).-A review of problems of specificity in arthropod vectors of plant and animal viruses. (Mimeo: Canberra.) DAY, M. F., and IRZYKIEWU'Z, H. (1953).-Aust. J. BioI. Sci. 6: 98-108. DAY, M. F., and IRZYKIEWICZ, H. (1954).-Aust. J. BioI. Sci. 7: 251-73. DAY, M. F., and VENABLES, D. G. (1960).-Virology 11: 502-5. HAMLYN, B. M. G. (1955).-Plant Pathol. 4: 13. HEINZE, K. (1955).-Phytopath. Z. 25: 103-8. HITCHBORN, J. H. (1958).-Ann. Appl. BioI. 46: 563-70. KLINKOWSKI, M. (1953).-Z.PjIKrankh. 60: 260-4. LOUIS, C. J. (l957).-Stain Tech. 32: 279-82. MARTINI, C. (1956).-Z.PflKrankh. 63: 577-83. MULLIGAN, T. (1957).-Rep. Rothamst. Exp. Sta. for 1956. ROBERTS, F. M. (1940).-Ann. Appl. BioI. 27: 348-58. RUBIO·HuERTOS, M. (1950).-Microbiol. Esp. 3: 207-31. SCHRAMM, G:, and ROTTGER, B. (1959).-Z. Naturjorsch. (b)14: 510-15. SEVERIN, H. H. P., and TOMPKINS, C. M. (1948).-Hilgardia 18: 389-404. SYLVESTER, E. S. (1956).-J. Econ. Ent·. 49: 789-800. SYLVESTER, E. S. (l958).-Proc. 10th Int. Congr. Ent. Vol. 3. pp. 195-200. 0

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TRANSMISSION OF CAULIFLOWER MOSAIC VIRUS BY APHIDS 197

TOMPKINS, C. M. (1937).-J. Agric. Res. 70: 379-404. VAN HOOF, H. A. (1954).-Tijdschr. PlZiekt. 60: 267-72. VAN HOOF, H. A. (1958).-An investigation of the biological transmission of a non· persistent virus. Doctoral Dissertation, Wageningen 1958. WATSON, M. A. (1946).-Proc. Roy. Soc. B133: 200-19. WATSON, M. A., and ROBERTS, F. M. (1939).-Proc. Roy. Soc. B 127: 543-76. WEI,C. T.,ETAL. (1958).-Acta Phytopath. Sinica4: 94-111. (See Rev. Appl. Mycol. 39: 135 (1960).)

EXPLANATION OF PLATE 1 Parts of turnip leaves infected with CIMV, stained by the fluorescent antibody technique

Fig. I.-Transverse section of leaf. The light spots indicate the presence of viral antigen. Note that this is abundant in the epidermis and in parts of the vascular bundles. Less antigen is seen in the mesophyll. Sections from uninfected leaves show little except the red autofluorescence of chlorophyll which is barely recorded photographically. 10 X ocular, 10 X objective. Fig. 2.-Part of an epidermal strip. Specific fluorescence indicating the presence of viral antigen in granules, generally 1-3 per cell. Antigen cannot be detected in nuclei, cell walls, or in guard cells. The chloroplasts of these show the characteristic fluorescence of chloro­ phyll. Epidermal strips from uninfectedplants show a few very small granules with the characteristic fluorescence of fluorescein.