The inhibitory action of mineral oil on the number of local lesions on Nicotiana glutinosa L. leaves inoculated with tobacco mosaic virus

VIROLOGY

65,

574-578

(1975)

SHORT The Inhibitory

Action

on Nicotiana

COMMUNICATIONS

of Mineral

Oil on the Number

ghtinosa

L. Leaves

Tobacco D. PETERS Laboratory

of Virology,

Agricultural

Mosaic AND

University, Accepted

Inoculated

of Local Lesions with

Virus

G. LEBBINK Binnenhaven

November

11, Wageningen,

The Netherlands

18, 1974

The effect of oil on the number of local lesions produced by tobacco mosaic virus (TMV) in Nicotiana glutinosa L. leaves was studied. Application of oil before or after inoculation of the leaves affected the number of local lesions produced to the same extent. An equivalent inhibition was found when nucleic acid of TMV was used, indicating that uncoating of the virus is not sensitive to or influenced by oil. Oil treatments at different times after inoculation showed that the lowest number of local lesions was produced by the leaves with oil at 15 min after inoculation. The results suggest that not every infecting particle is sensitive to oil at the same moment. Repeated treatments at intervals of 15 min over a period of 2 hr caused an almost complete inhibition.

Virus transmission by aphids in the style-borne manner is impeded on plants coated with various types of oils (1). This has been confirmed by Bradley (2) and others (3-6). As a result studies have been made on the possible prevention of the spread of viruses in the field and greenhouse by oil (3, 7-9). The mechanism by which oil counteracts the transmission of viruses by aphids is not understood (10). The effect of oil could not be explained by a change in the probing and feeding behavior of the vector (2, 11, 12). Bradley (2) suggested that oil may remove the virus from the stylets. Another suggestion made by Bradley was that oil may cause a stronger adherence of the virus onto the stylets. Finally, he suggested that oil, when introduced with the virus, could also affect the cell in such a way that the infection is inhibited. His experimental data supported the first hypothesis but did not exclude the other hypotheses. A possible effect of oil on the virus was 574 Copyright 0 1975 by Academic Press, Inc. All rights of reproduction in any form reserved.

studied by Loebenstein et al. (3). They showed that emulsification of virus suspensions of cucumber mosaic virus (CMV) and tobacco mosaic virus (TMV) with oil caused a reduction in the number of local lesions produced. The infectivity could be restored after breaking the oil-TMV emulsion by centrifugation. These observations suggest that oil may somehow interfere with the development of an infection. The present study was undertaken to examine the effect of oil, applied before and after inoculation of TMV on Nicotiana glutinosa L., on the number of local lesions produced. hoc&. Purified preparations of the Wageningen TMV isolate (W-Ul) were used in such concentrations (about 2 pg/ ml) that approximately 100 local lesions were obtained on each N. glutinosa leafhalf in the controls. RNA was extracted from the virus as described by Van Kammen (13). The inocula contained 50-100 pg of RNA/ml.

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Oil. We used Bayol 50 in our experiments. This oil is characterized by a specific gravity of 0.8 43 at 15”; a viscosity of 54 SSU at 37.8” and 33.5 at 98.9”; a refractive index of 1.4620 (20”); a cloud point of -33”; a color of +30 (Saybolt); a pour point at -39’C; its flash point is 120”; point analine point 96.1’; and distillation between 310’ (5%) and 357” (95%). This information and the oil samples were kindly supplied by Esso (The Hague). Testing the effect of oil on the number of local lesions. Leaves uniform in size and development were detached from N. glutinosa plants and cut along the midribs. Ten leaves were used for each treatment; in five the left leaf-half was used as the control and in five the right leaf-half. The leaf-halves were inoculated by rubbing the upper surface after first dusting them with Carborundum 500, and afterwards rinsed with tap water. The leaf-halves were immersed once, either at different times before, immediately after, or one or more times at intervals of 15 min after inoculation in an emulsion of 0.1% (v/v) Bayol 50 in water containing 0.1% (v/v) of the emulsifier Agral (I.C.I., The Netherlands) for 15 set and then rinsed with tap water. The emulsions were made by adding 1 ml of oil and 1 ml of emulsifier to 1 liter of water and were maintained by continuous stirring. The leaf-halves serving as controls were immersed in water containing 0.1% emulsifier and rinsed. Each leaf-half was placed with its corresponding half on a wet filter paper in petri dishes and kept at 20” under continuous illumination from fluorescent lamps. The lesions were counted 3 days after inoculation. Testing of the infectivity of TMV and TMV-RNA emulsified with oil. A TMV suspension (3 pg/ml) and a TMV-RNA suspension (150 gg/ml) were mixed in equal volumes with 0.2% oil emulsion, containing 0.2% emulsifier. These emulsions were inoculated onto leaf-halves as described above. The TMV and TMVRNA suspensions were mixed with an equal volume of 0.2% emulsifier for use as controls. The oil-virus emulsion was bro-

575

ken by centrifugation (10 min at 5,000 g). Infectiuity of oil-TMV and oil-(TMVRNA) emulsions. In five replicate experiments the oil-TMV emulsion produced 90% fewer local lesions than the TMV suspension. When the emulsion was broken by centrifugation, the inoculum was as, infectious as the original virus suspension. A similar result was obtained when the oil had separated from the emulsion by storage in the cold for one day. It is clear from these experiments that oil does not inactivate the virus in uitro. The results confirm those obtained by Loebenstein et al. (3). In a single experiment it was demonstrated that the infectivity of TMV-RNA was inhibited by oil to the same extent as that of TMV. This finding makes it unlikely that uncoating of the virus is a process sensitive to oil. Application of oil before inoculation. Reduction of virus transmission by aphids is found when the source plants or the plants to be tested are coated with oil before the inoculation access of aphids. We tested whether the infectivity of TMV could be reduced by coating N. glutinosa leaves with oil prior to inoculation. Leafhalves were dipped in the oil emulsion for 15 set at periods of 24 and 1 hr and 30, 15 and 5 min before inoculation. Oil applied in advance caused a reduction of approximately 87% in the number of local lesions produced irrespective of the interval between applying the oil and inoculating the leaves. Application of oil before and after inoculation. In two replicate experiments it was demonstrated that application of oil onto the leaves directly after inoculation was as effective in the reduction of the number of local lesions as dipping the leaves before inoculation. The reductions obtained were 68 and 76%, respectively. It is evident from this that oil also affects the infection after inoculation. This phenomenon, which had not previously been reported in the literature, was further studied in detail. Application of oil after inoculation. Inoculated leaf-halves were immersed in oil emulsion at different intervals after inocu-

576

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lation. Figure 1 illustrates the effect of oil on the number of loci1 lesions produced on N. glutinosa leaf-halves as obtained in 17 replicate experiments. Oil treatment immediately after inoculation with TMV resulted in a smaller decrease in the number of local lesions formed than a treatment 15 min after inoculation. This difference was significant at the 5% level of probability by Student’s t-distribution ( to.oa = 2.120 < 2.278 calculated). At 15 min the reduction in the number of local lesions was maximal and decreased thereafter. No reduction was detected 4 hr after inoculation. The curve obtained for TMV-RNA had a shape similar to that for TMV (Fig. 1) except that no reduction was found 2 hr after inoculation. Repeated applications of oil after inoculation. We tested whether the number of

local lesions could be reduced further by several applications of oil at short intervals (2 min) or at long intervals (15 min) after inoculation. As shown in Fig. 2, repeated dippings, each made at an interval of 15 min after inoculation, resulted in additional losses of local lesions over the number lost in the first dipping. These extra losses may have been the result of exposing the infecting virus to more oil during the Lesions as % of those on control leaf-halves

0 Interval

I I I 15 30 45 between

‘I 60 75

produced

-

TMV

o-----o

TMV-RNA

I I I/ 90 105 120

inoculation

‘240 and

mirl. immersion

FIG. 1. The production of local lesions as a function of the time between inoculation and immersion of the leaf-halves in an oil emulsion. The maximum number of lesions induced in the controls is set at 100%.

Lesions as % of those produced on control leaf- halves

90 80 70 60 50-1 40

‘\

30

\ ‘\\ b,

20 10

‘\n -ai

---*----*

IIIIllTl,

0

15 30 45 60 75 90 105120 Time

of

last

min

immersion

FIG. 2. The production of local lesions as a function of several immersions of the leaf-halves in an oil suspension. The first immersions were made directly after the inoculation and repeated for a set of leafhalves one or more times. The points indicate the reduction in local lesions as percent obtained at times at which a group of leaf-halves was immersed for the last time.

following dipping. When, however, a series of dippings was made in a limited period (e.g., at 30,32, 34 and 36 min after inoculation) no extra loss in the number of local lesions was detected over the number lost at 33 min after inoculation. The explanation of these results might be that each developing infection is not sensitive to oil at the same moment after the inoculation. Evidence for this hypothesis was obtained in an experiment in which the first treatment was made at a certain time after inoculation and was then repeated every 15 min until 120 min after inoculation. From Fig. 3 it is evident that the later the first treatment was given, the higher the number of local lesions that developed. Repeated dippings starting at or after 90 min had only a small influence on the reduction of local lesions, as could be expected from the results shown in Fig. 1. Results obtained with treatment of leaves before or after inoculation with TMV indicate that oil somehow reduces the number of local lesions that may develop after inoculation. A reduction was also observed (Fig. 1) when the leaves were

SHORT Lesions as % of those leaf-halves on control

produced

100 go-

--o

80-

,PX4 F-

70,/’

6050-

?’

40-

: :

30-

:

2010i 0

d I’ d I I I 1 3 I I I 15 30 45 60 75 90 105 120 Time

of first

577

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mln

Immersion

FIG. 3. The production of local lesions as a function of several immersions of leaf-halves in an’ oil suspension. The points indicate the reduction of local lesions as percent at the times at which the first immersion was made. The last immersion of each group was made 2 hr after inoculation.

treated some time after inoculation with oil. The course of the curve in Fig. 1 shows that the effect of oil on the number of local lesions increases during the first 15 min after inoculation and decreases afterwards. One may wonder whether the virus is already sensitive to oil at the moment of inoculation or immediately thereafter. The results have shown that the process of uncoating is not sensitive to oil. This indicates that a step occurring later in the infection process than uncoating will be impaired by oil. This makes it unlikely that the virus becomes sensitive to oil at the moment of or immediately after inoculation. As shown by Kiilps and Hein (IO), oil moves from the place where it has been applied, via the walls, further and deeper into the leaf. The higher number of local lesions found when oil has been applied directly after inoculation may then be explained by a loss in concentration of oil at or around the infecting virus before it becomes sensitive to oil. The number of local lesions produced increases sharply 30 min after inoculation, indicating that not each infecting virus particle is affected at the same moment (Fig. 1). This suggests that the infected sites are either sensitive for a limited

period but at different moments after inoculation or become sensitive at the same moment, each remaining so for different lengths of time. The results presented in Figs. 2 and 3 show that the first possibility is the most likely one. For, when the immersion starts directly after inoculation and is repeated at every multiple of 15 min, only a small percentage of the infecting virus escapes from the detrimental effects of oil (Fig. 2). And when the immersion starts at a multiple of 15 min after inoculation and is then repeated every 15 min, it is shown (Fig. 3) that the later the first immersion has been made, the more virus particles escape from the inhibitory action of oil. Repeated immersions have a greater effect on the reduction of the number of local lesions than a single immersion (Fig. 2). This cannot be explained by a lack of oil at the infected site, as there was no extra reduction in the number of local lesions when the leaves were repeatedly treated at short intervals (2 min). It is difficult to reconcile why the extra immersions are necessary to hit a larger number of virus particles in the process of infection. As oil does not inactivate the virus (3; this report) but interferes in the development of infection, oil has to be classified as an inhibitor (14). Oil is not the only inhibitor that acts on the infection with TMV as well as with TMV-RNA. This property has also been shown for the inhibitor occurring in carnation sap (15). ACKNOWLEDGMENTS We thank Professor Dr. Ir. J. P. H. van der Want for his interest in this work and helpful criticism in preparing the manuscript. Our thanks are also due to Dr. Ir. L. Bos, Dr. J. Dijkstra, Dr. B. D. Harrison, and Dr. M. G. Schultz for reading the manuscript and Miss M. Boerjan for technical assistance. REFERENCES 1. BRADLEY, R. H. E., WADE, C. V., and WOOD, R. A., Virology 18, 327-328 (1962). 2. BRADLEY, R. H. E., Can. J. Microbial. 9, 369-380 (1963). 3. LOEBENSTEIN, G., ALPER, M., and DEUTSCH, M., Phytopathology 54, 960-962 (1964). 4. HEIN, A., Phytopathol. Z. 52, 29-36 (1965). 5. VANDERVEKEN, J., and SEMAL, J., Phytopathology

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56, 1210-1211 (1967). 6. VANDERVEKEN J., and DUTRECQ, A., Ann. Phytopath. 2, 387-402 (1970). 7. BRADLEY, R. H. E., MOORE, C. A., and POND, D. D., Nature (London) 209, 1370-1371 (1966). 8. ALLEN, T. C., Plant Dis. Rep. 49, 557 (1965). 9. LOEBENSTEIN, G., ALPER, M. and LEVY, S., Phytopathology 60, 212-215 (1970). 10. KULPS, G., and HEIN, A., Phytopathol. Z. 73, 149-162 (1972).

11. HEIN, A., Phytopathol. Z. 75, 241-249 (1972). 12. PETERS, D., and LEBBINK, G., Entomol. Exp. Appl. 16, 185-190 (1973). 13. KAMMEN, A. VAN, Virology 31, 633-642 (1967). 14. LOEBENSTEIN,G., In “Principles and Techniques in Plant Virology” (C. J. Kado and H. 0. Agrawal, eds), pp. 32-61. Van Nostrand-Reinhold, New York, 1972. 15. RAGETLI, H. W. J., Tijdschr. Plontenziekten 63, 245-344 (1957).