アブラムシによるタバコモザイク・ウイルスの不活性化

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(1)Title. アブラムシによるタバコモザイク・ウイルスの不活性化. Author(s). 由崎, 俊道. Citation. 北海道教育大学紀要. 第二部. B, 生物学,地学,農学編, 37(1): 19-26. Issue Date. 1986-10. URL. http://s-ir.sap.hokkyodai.ac.jp/dspace/handle/123456789/6434. Rights. Hokkaido University of Education.

(2) ^MtfeH^iie^ (Hl2gPB) ^37^ ^l^- RS?61^10^. Journal of Hokkaido University of Education (Section D B) Vol. 37, No. 1 October, 1986. Inactivation of Tobacco Mosaic Virus by Aphids. Toshimichi YOSHIZAKI Biological Laboratory, Sapporo College, Hokkaido University of Education, Sapporo 064. 7 7'7 ^ y ^ ^ ^ ^ ^' ^ -^-f^ ^ • tj7 ^f ;L^ ®^'^'?<b. ES ^ ft M. wm^±^w^Kw^m Abstract Infection of Nicotiana glutinosa L. by tobacco mosaic virus (TMV) was inhibited by the addition of the homogenate of aphids (Myzus persicae Sulz.) to the inoculum. The inhibition was reduced,. however, by heating at 80 or 100 C for 10 min., although not by heating to 40 C or by dialysis against water. The inhibition was negated by the addition of bentonite suspension to the mixture of TMV and homogenate. With the addition of bentonite, it was found that the infectivity of TMV recovered almost completely from the effect of the mixture with homogenate. On the other hand, TMV-RNA. was easily inactivated in a short time by the addition of the homogenate of aphids which was diluted to 2 X 10 or 10~3. There was no inactivation of TMV-RNA which had been fed to aphids through a. membrane for 2 days. After the aphids had acquired TMV, the total amount of infectivity that remained, both in the aphids and that released into honeydew, decreased with increasing feeding periods on sucrose solution. Since no apparent decrease occurred in the infectivity in honeydew, we. concluded that after the aphids had acquired TMV a certain quantity was inactivated in the insect bodies before the release of TMV into the honeydew.. Introduction. It has been reported that the addition of bentonite to an inoculum allows the infectivity of tobacco. mosaic virus (TMV) to recover from the inhibiting effect of the homogenates of aphids and butterflies which have been fed on a TMV suspension (Yoshizaki, 1980 ; 1981a ; 1981b). Yoshizaki (1981a) has also suggested that TMV in dead aphilds is gradually inactivated by incubation. The present. (19).

(3) 20 Toshimichi YOSHIZAKI. experiment was designed to examine the inactivation of TMV or its ribonucleic acid (TMV-RNA) in the homogenate of aphids or in the release by aphid stylets, and in aphid bodies or in honeydew. As a result, it was found, by using bentonite, that some quantities of TMV were inactivated in aphids or slightly in the honeydew, whereas neither TMV nor TMV-RNA was inactivated by its release from the stylets of aphids.. Materials and Methods The green peach aphid, Myzi(s persicae Sulz. (green clone), was used for the experiments. Turnips (Brassica rapa L.) and the buds from tulip bulds were used for propagating the aphids. All experiments were conducted with apterous aphids. The plants used in these experiments were grown in pots and cultivated in a green house. The ordinary strain of TMV was propagated in tobacco plants (N. tabacum L. cv. White Burley), and purified by differential centrifuga-. tion (Steere, 1959). TMV-RNA was prepared from a purified TMV by the bentonite-phenol method (Fraenkel-Conra't et al., 1961). The concentration of TMV and TMV-RNA was determined spectrophotometrically (Takahashi, 1951). Two kinds of plastic ring, 2.9 and 1.8 cm in diameter, and 2.0 and 1.0 cm in height respectively, were used as cages for feeding the aphids through a Parafilm membrane. The upper end of the ring was covered with the parafilm, which was then sucked slightly by an aspirator on the under side of the ring to make a shallow hollow in the membrane. The test materials were placed in the hollow and covered with another membrane. Volumes of 0.7 and 0.3 ml were used for the 2.9 and 1.8 cm rings respectively. After the aphids had been placed in the cage, the lower end of the ring was covered with a net. All the feeding was carried out under lights in a room regulated at 20 C. The infectivity assay of TMV and TMV-RNA was made by inoculation to the leaves of Nicotiana glutinosa L. The bentonite suspension was prepared by the .procedure of Fraenkel-Conrat et al. (1961), and suspended in a 0.1 M phosphate buffer, pHi7.0, containing 0.02 M sodium oxalate. The oxalate has been found to cause the increase of TMV and TMV-RNA infection in N: glntinosa (Yoshizaki, 1976). Extreme care was taken in these experiments to prevent contamination by extraneous TMV. All glassware, cages, and the inoculating forceps were sterized before use.. Results 1. Inhibition of TMV by homogenates of aphids Aphids were homogenized in a phosphate buffer (0.1 M, pH 7.0) of 30 fold volumes per aphid weight. The homogenate was centrifuged for 30 min at 3,000 rpm to produce the supernatant which was used for the experiment. The homogenates were heated at 40, 80, or 100 C for 10 min. to determine the stability of the inhibitory agents to heat treatment, and were dialyzed against a large quantity of distilled water for 2 days. TMV was added to each homogenate at a concentration of 0.2 ,«g/ml, and the homogenate was then adjusted to a final dilution of 50 fold. As the control, TMV was. (20).

(4) 21. Inactivation of Tobacco Mosaic Virus by Aphids. added to a phosphate buffer (0.1 M, pH 7.0) at the same concentration. The infectivity of each preparation was examined in the usual way by the half-leaf method using N. glutinosa (Holmes, 1929). The results are shown in Table 1. No significant change of inhibitory effect was found either in the homogenates heated to 40 C or in those dialyzed against water. The homogenates heated to 80 or 100 C had a reduced inhibitory effect-about 75% below non-treated ones. The results suggest that the homogenate of aphids contains two or more kinds of inhibitory agent that are stable or unstable at high temperatrre. Table 1. Inhibition >f TMV by the homogenate of aph ids Numbi •r of local lesions. Treatment. Treatment. Control. Inhibi lion. None. 5. 345. 98.5. Heat. 40 C. 16. 291. 94.5. treatment. 80. 79. 342. 76.9. 100. 78. 307. 74.6. 22. 3.3.1. 93.7. Dialysis. %. The homogenate of aphids was diluted 30 fold with phosphate buffer (0.1 M, pH 7.0), and treated with heating or dialysis. The homogenate was mixed with TMV and inoculated to 12 half leaves of N. gtutinosa by the half-leaf metliod. TMV containing no homogenate was used as the control.. 2. Inactivation of TMV and TMV-RNA by the homogenate of aphids The mixture of TMV (1 mg/ml) and the homogenate of aphids was incubated at room temperature for 30 min. or 24 hours to test for inactivation. After incubation, the mccture was diluted to 10. with a phosphate buffer (0.1 M, pH 7.0) or the bentonite suspension. TMV (1 mg/ml) containing no homogenate and incubated for the same periods was used as the control. The resultant mixtures were inoculated into N. glutinosa by the half-leaf method. The results are shown in Table 2. The. infectivity of the TMV and homogenate mixture almost recovered after dilution with the bentonite suspenpion. No additional decrease was apparent in the infectivity of TMV when the mixture was incubated for 24 hours, as compared with those mixtures incubated for 30 min. ; all recovered to almost the same degree when TMV was incubated with the homogenate for either 30 min. or 24 Table 2. Inactivation of TMV by the hbmogenate of aphids Number of local lesions. Incubation period of the mixture of. Bentonite. TMV and homogenate 30 min.. 24 hrs.. Mixture of TMV and homogenate. Control. Inhibition (%). +. 26. 325. 92.0. 222. 252. 11.9. +. 25. 218. 88.5. 226. 258. 12.4. The homogenate of aphids was diluted 30 fold with phosphate buffer (0 .1 M, pH7, 0), and added to TMV (l.Omg/ml). TMV (l.Omg/ml) containing no homogenate was used as the cotrol. After incubation, they were diluted to 10~A with phosphate buffer (O.lM.pH 7.0) or bentonite suspension (5mg/ml), and inoculated to N. glutinosa by the half-leaf method.. (21).

(5) 22. Toshimichi YOSHIZAKI. hours.. To examine the inactivation of TMV-RNA by the homogenate of aphids, homogenates diluted to 2 X10-2, 10-2, or 10~3 were added to TMV-RNA of a given concentration. After incubation at room temperature for 1, 3, or 5 minutes, the bentonite suspension was added to the mixture of TMV-RNA and homogenate to stop the reaction. Each preparation of TMV-RNA was inoculated into the leaves of N. glutinosa. The results are shown in Table 3. It was found that TMV-RNA was easily inactivated by the homogenate in a short time, although the degree of inactivation varied with the concentration of both TMV-RNA and homogenate. The infectivity of TMV-RNA did not recover by the addition of bentonite after incubation. The inactivation of TMV-RNA was irreversible and different from the case of TMV. Table 3. Inactivation of TMV-RNA by the homogenate of aphids Time of treatment with homogenate (min. ). 1. Dilution of homogenate. Number of local lesions/number of leaves inoculated Cone. of. TMV-RNA. 20. 40 pg/m\. 0/6. 2 X10-2. 0/4. 2/5. 10-2. 0/4. 8/7. 3. 2 X10-2. 0/5. 0/7. 10-2. 0/7. 0/6. 1/6 4/6 0/5. 10-3. 5. 1/9 118/6. 10-3. 3/5. 2 X10-2. 0/8. 0/6. 10-2. 0/8. 0/5. 0/5 4/6. 321/6. 218/6. 217/6. 10-3 Control. The homogenate of aphids \vas diluted with phosphate buffer (0.1 M, pH 7. 0) and added to TMVRNA. After a given time, the TMV-RNA preparations were diluted with bentonite suspension (5mg/ml) and inoculated to N. g/udnosa.. 3. The infectivity of TMV and TMV-RNA when presented by feeding to aphids Groups of 100 aphids were allowed to feed through a membrane on a 0.7 ml volume of TMV (1 mg/ml) or TMV-RNA (0.1 mg/ml) containing 10% sucrose for given periods. The preparations of TMV or TMV-RNA in the membrane were diluted to a concentration of 0.1 /ug/ml for TMV and 5 p.'. g/ml for TMV-RNA with the bentonite suspension. The infectivity of both TMV and TMV-RNA diluted preparations was compared to those of TMV and TMV-RNA which had had no access to the aphids. The results are shown in Fig. 1. No apparent decreases in infectivity were produced by. either TMV or TMV-RNA which were fed to aphids for 1 or 2 days, although the infectivity of TMV-RNA was completely inactivated after feeding to aphids for 3 days. TMV was not inactivated by the feeding for 3 days.. (22).

(6) Inactivation of Tobacco Mosaic Virus by Aphids. 23. TMV. 100. •S 50. 01. 23. Days of feeding by aphids. Fig. 1. Infectivity of TMV and TMV-RNA which were fed to aphids TMV (l.Omg/ml) or TMV-RNA (O.lmg/ml) containing 10 % sucrose was fed to aphids through a membrane for given periods. After TMV or TMV-RNA was diluted with phosphate buffer (0.1 M, pH 7.0), the infectivities were compared with that of control TMV-RNA on N. glntinosa.. 4. The inactivation of TMV acquired by aphids An experiment was performed to test the inactivation of TMV, acquired by aphids. Fifty aphids which had been fed on TMV (10 mg/ml) through a membrane for 24 hours were placed in 1.8 cm diameter feeding cages. The cages were covered with a membrane,containing 15% sucrose in a 0.02. M phosphate buffer (pH 7.0). Half the cages were kept in a freezer (—15 C) as the control, while the other half were kept at 20 C for feeding. After feeding for given periods, the aphids and the portion of membrane and gauze which had come into contact with them were collected in a watchglass, but the sucrose solution as well as the memberane and gauze which had not come into contact were discarded. The collected aphids were homogenized with the collected gauze and membrane by a small glass rod, after the addition of the 0.3 ml bentonite suspension and a small amount of Carborundum. The inside of the cage was repeatedly wiped with the collected gauze, soaked in the homogenate, to obtain the honeydew. The control homogenates were likewise obtained from cages maintained in the freezer. The infectiviy of the homogenates obtained from 20 C feeding materials was compared to that of the respective control homogenates by inoculation into N. glutinosa using the half-leaf method. The experiments were repeated 20 to 30 times. The average values obtained are shown in Fig. 2. It was found that the infectiviy in the cages decreased over. time during the feeding periods. The results indicate an inactivation of TMV in the aphids and/or the honeydew.. The infectivky in the honeydew of the TMV-acquired aphids, incubated at 20 C, were examined. Each group of 50 TMV-acquired aphids was transferred to cages containing 15% sucrose. in a 0.02 M phosphate buffer (pH 7.0). After the aphids had been fed for 24 hours, both they and. (23).

(7) 24. Toshimichi YOSHIZAKI. 100. Honeydew. 80. :t. 60. 40 20. 0. 1234 Days after acquisition of TMV. Fig. 2. Infectivity of TMV accquired by aphids Aphid + honeydew : Aphids which had been fed on TMV (10 mg/ml) were placed in cages containing 15 % sucrose. Half of the cages were kept at 20 C for feeding., and the other half were kept in a freezer as the control. After. feeding, the aphids and the portions of membrane and gauze which had come into contact with the aphids were collected and homogenized. The inside of the. cage was repeatedly wiped with the collected gauze. The control homogenates were likewise obtained from cages maintained in the freezer. The infectivities of homogenates obtained from 20 C feedind materials were compared to those of the respective control homogenates on N. glutinosa.. Honeydew : Aphids which had been allowed to acquire TMV were transferred to cages containing 15 % sucrose. After 24 hours, both the aphids and the. sucrose solution were removed. Half of the cages were incubated at 20 C and the other half were kept in a freezer as the control. After given periods, the. inside of cage was repeatedly wiped with the collected gauze. The incectivities of the samples obtained from the incubated and control cages were compared on N. glutinosa.. the sucrose solution were removed. Half the cages were incubated at 20 C and the other half were stored in a freezer as the control. After given periods, the membrane and the gauze of the cages were collected in watchglasses. The inside of each cage was repeatedly wiped with the collected gauze to which added the 0.3 ml bentonite suspension and a small amount of Carborundum. The infectivity of the samples obtained from the incubated and control cages were compared by inoculation into N, glutinosa using the half-leaf method. The results are shown in Fig. 2. The average infectiviy was obtained by repeating the experiment 20 to 25 times. No apparent decrease was found in the infectivity of the honeydew samples from the cages incubated for 1 or 2 days, but a small decrease appeared in those incubated for 3 or 4 days. These results indicate that infectious TMV acquired by aphids is inactivated before its release with honeydew, rather than in the honeydew itself.. (24).

(8) Inactivation of Tobacco Mosaic Virus by Aphids 25 Discussion. Black (1939) showed that extracts from two leaf-hoppers, five aphids, and mosquitoes, all. reduced the infectivity of TMV. None of the inhibitors from insects has been identified, however, although Black (1939) obtained evidence linking a protein or proteins with the inhibitory action of juice from the clover leaf-hopper. The ability to inhibit was not affected by dialysis, but it was destroyed by heating and strong acid or alkali. Smith (1941) reported that juice from caterpillars may contain more than one inhibitor, since boiling the juice decreases but does not abolish the inhibitory power. In the present experiment, the same results as those described above have been. obtained. The infectivity of TMV was strongly inhibited by the homogenate of aphids. The inhibition was reduced but not destroyed completely by heating at 80 or 100 C. The ability to inhibit was not affected by dialysis of the homogenate against water ; this is the same result as that reported. by Black (1939). It was found that TMV-RNA was easily inactivated after a short time by the homogenates of aphids diluted to 2 X 10 or 10- . An attempt was made to demonstrate the inactivating effect of saliva secreted into the TMV or TMV-RNA preparation. No apparent inactivation by aphid secretion could be proved by the present experimental procedure.. In previous papers (Yoshizaki, 1980 ; 1981a; 1981b), it has been stated that the addition of bentonite to an inoculum made homogenates of aphids and butterflies (Pieris rapae crucivora Boisduval. and Colias erate poliographus Motshulsky) which had been fed on the preparation of TMV allowed the infectivity of TMV to recover. Similarly, the infectivity of TMV in the haemolymph of larvae of Philosomia cynthia ricini Donovan, injected with TMV suspension, could easily be detected by the bentonite method (Yoshizaki, 1981b). The infectivity of TMV in the haemolymph was appreciably reduced with the increasing passage of days after injection with TMV. The infectivity that remained in the haemolymph at 3 and 5 days after the injection were reduced to only 7.6 and 2.6% of the control which was assayed immediately after the injection. It has already been reported that the. degree of infectivity of TMV in dead aphids decreases with increasing incubating periods (Yoshizaki, 1981a). Silmliar results for the inactivation of TMV in aphids were obtained in the present experiment. After the aphids had acquired TMV, the total amount of infectivity that remained, both in the aphids and that released into honeydew, decreased with increasing feeding periods on the sucrose solution. No apparent decrease in infectivity occurred in the honeydew. On the basis of these results, we conclude that, after the aphids have acquired TMV, a certain quantity is inactivated in the insect bodies before the release of TMV into the honeydew. In this connection, we require further experiments to confirm the aphids inactivation mechanism of TMV. It would also be. interesting to identify the inactivating agent(s) in insect bodies.. Literature cited Black, L. M., 1939. Inhibition of virus activity by insect juices. Phytopathology 29: 321-327. Fraenkel-Conrat, H., B. Singer, and T. Tsugita., 1961. Purification of viral RNA by means of bentonite. Virology. (25).

(9) 26 Toshimichi YOSHIZAKI 14: 54-58. Holmes, F. 0., 1929. Local lesions in tobacco mosaic. Bot. Gaz. 87: 39-55. Smith, K. M., 1941. Some notes on the relationship of plant viruses with vector insects. Parasitology 33: 110-116. Steere, R. L., 1959. The purfication of plant viruses. Adv. Virus Res. 6: 3-73. Takahashi, W. N., 1951. Ultraviolet absorption as a meansure of tobacco virus nucleoprotein. Phytopathology 41 : 142-145.. Yoshizaki, T., 1976. Effects of oxalate on the infectivity of tobacco mosaic virus and its ribonucleic acid. J. Hokkaido Univ. of Education IIB 26: 5-15. Yoshizaki, T., 1980. Recovery of infectious TMV from a single aphid by the use of bentonite. Ann. phytopath. Soc. Japan 46; 464-470.. Yoshizaki, T., 1981a Acquisition of tobacco mosaic virus by aphids (Myzns persicae Sulz.). Ibid. 47: 24-28. Yoshizaki, T., 1981b. Detection of tobacco mosic virus in insects by the use of bentonite. Seibutsu Kyozai 16 : 62-70.. (26).

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