Aphid Transmission of Turnip Mosaic Virus and Cucumber

日植病報51: 562-568 (1985) Ann. Phytopath. Soc. Japan 51: 562-568 (1985)

Mosaic Virus 2. Transmission from Virus Mixtures

Ichiro FUJISAWA

Abstract

An investigation was undertaken to obtain further information on aphid transmission of

turnip mosaic virus (TuMV) and cucumber mosaic virus (CMV) from doubly infected turnip plants (Brassica campestris cv Yorii) to healthy ones. Both winged and wingless forms

of Myzus persicae (SULZER) transmitted TuMV alone at a higher rate, but failed to transmit or seldom transmitted CMV alone. Both forms of Aphis gossypii GLOVER, showed three

types transmission, TuMV alone, CMV alone and both viruses simultaneously, but they transmitted CMV alone more effectively. In the transmission test with successive transfers at 10-minute intervals, most of the M. persicae individuals transmitted TuMV alone to only

the first plant of the series. And A. gossypii also commonly transmitted TuMV alone or CMV alone to the first plant only. When M. persicae and A. gossypii had fed first on infected leaves and then fed on anti-TuMV ƒÁ-globulin through a stretched parafilm membrane,

the transmission of TuMV was prevented completely or decreased remarkably. And A.

gossypii treated with anti-CMV ƒÁ-globulin after feeding on the infected leaves, transmitted TuMV regularly, but seldom transmitted CMV.

(Received June 14, 1985)

Key Words: aphid transmission, non-persistent virus, CMV, TuMV.

Introduction

Turnip mosaic virus (TuMV) and cucumber mosaic virus (CMV) are easily transmitted in a stylet-borne, non-persistent manner by several aphid vectors. We have already described the transmission efficiency and retention period of TuMV or CMV in four aphid species, Myzus persicae (SULZER), Aphis gossypii GLOVER,Brevicoryne brassicae (LINNE) and Lipaphis erysimi (KALTENBACH)7).The results obtained indicated that TuMV was transmitted at a higher rate both by M. persicae and A. gossypii, and CMV was transmitted at a high rate by A. gossypii, but seldom by M. persicae. This report provides further information on aphid transmission of TuMV and CMV, using the plants doubly infected with these two viruses as the sources.

Materials and Methods

Virus source. TuMV and CMV used in this study were obtained from diseased Japanese radish (Raphanus sativus cv. Taibyosobutori) in Hokkaido, Japan6). Turnip plants (Brassica campestris cv. Yorii) doubly inoculated with TuMV and CMV developed clear

Vegetable and Ornamental Crops Research Station, Ano, Mie, Japan農林水産省野菜試験場 Ann. Phytopath. Soc. Japan 51 (5). December, 1985 563

mosaic symptoms 7 days after inoculation. Those systemically infected leaves were used

as virus source. There are some reports that relative CMV concentration in Japanese

radish which was doubly infected with CMV and TuMV was higher than that of the

single infection17,21). In this experiment, the back-inoculation to plants of Vigna sesquipedalis cv. Kurodanesanjaku and the microplates method of enzyme-linked immunosor- bent assay were used for determining the relative concentration of CMV and TuMV,

respectively. Both relative CMV and TuMV concentration in the source plants doubly

infected with TuMV and CMV, however, were the same or a litte high as compared

with the each case of single infection.

Transmission. Vigorous, young adult, wingless or winged aphids belonging to M. persicae and A. gossypii were reared on Japanese radish and cucumber, respectively7). Single aphids starved for 2hr were allowed to probe on the source leaves for 10min

and then transferred to young turnip seedlings. Test plants with individual aphids were

covered with cellulose nitrate cages in a greenhouse at about 20-25C, and sprayed with

an insecticide 2hr after inoculation. Inoculated plants were kept under observation for

15 days, and then checked the presence of the viruses. The presence of CMV was check-

ed by mechanical inoculations to primary leaves of V. sesquipedalis and the presence

of TuMV was checked by SDS-immunodiffusion test with antiserum against TuMV.

Consecutive transmission. A single aphid was subjected to a preliminary starving

period of 2hr, fed for 10min on the source of infection, and then transferred to five successive healthy turnip seedlings. The aphids were fed for each 10min on the first

to the fourth seedlings, and left overnight on the fifth. The attempt to check the pre-

sence of viruses was carried out in the same manner as mentioned above.

Treatment with antibodies. Each of the antisera against CMV and TuMV used

in this study was described previously6). Partially purified anti-TuMV or anti-CMV ƒÁ-

globulins were prepared by precipitation with ammonium sulfate and dialysis in pho- sphate-buffered saline (PBS) pH 7.4. Each was then desalted by exclusion chromato-

graphy on Sephadex G 25 and was further purified by passage through a column of DE 23 cellulose. Each of the ƒÁ-globulins was adjusted to approx. 1mg/ml in PBS and

sucrose was added to the final concentration of 20%. To analyse the effect of treat-

ments with these ƒÁ-globulins on virus transmission, two transmission experiments such

as pre-acquisition and post-acquisition treatments were performed. In the pre-acquisi-

tion treatments, two aphids that had fed first either on anti-TuMV or anti-CMV ƒÁ-

globulin through a stretched parafilm membrane12) for 5min and then probed on infected leaves for 5min, were placed each on one test plant for 2hr. After inoculation

access, test plants were sprayed with an insecticide and placed in a greenhouse. The

post-acquisition treatments were conducted in a sequence opposite to that of the acquisition feeding treatment, i.e., probing first on infected leaves and then treatment with

either anti-TuMV or anti-CMV ƒÁ-globulin. Thereafter, these two aphids were transfer-

red each to one test plant.

Results

Selective transmission 564 日本植物病理学会報第51巻第5号昭和60年12月

Transmission rates of TuMV and CMV by M. persicae or A. gossypii from doubly infected turnip leaves, were compared. There was an appreciable variation in the per- centages of virus transmission by the different species (Table 1). For wingless M. persicae, 9 of 24 aphids transmitted TuMV alone (37.5%) and 3 transmitted both TuMV and CMV simultaneously (12.5%). Both wingless and winged M. persicae failed to transmit or seldom transmitted CMV alone. A gossypii, however, showed three types of

Table 1. Comparison of transmission of TuMV or CMV from doubly infected turnip to healthy turnip seedlings by a single aphida)

a) Acquisition feeding was made by placing a single aphid on doubly inoculated leaves. Given a 10-minutes acquisition feeding period and inoculation feeding for 2 hours. Number of infected plants out of 24 test plants.

Table 2. Retention of TuMV or CMV by a single aphid (M. persicae) transferred at 10-minute intervals to five successive healthy turnip seedlings

a) Forty-seven wingless and forty-four winged aphids were used. However, those aphids that failed to infect plants are not included. Each fifteen aphids that transmitted the viruses are shown. b) The infected and non-infected tests plants are as follows. T: turnip mosaic virus, C: cucumber mosaic virus, M: complex of these two viruses and -: no infection. Ann. Phytopath. Soc. Japan 51 (5). December, 1985 565

transmission. Three of 24 wingless A. gossypii transmitted TuMV alone (12.5%), 9 transmitted CMV alone (37.5%), and 5 transmitted both viruses simultaneously (20.8 %). These experiments showed that M. persicae probing on doubly infected leaves transmitted TuMV selectively, but A. gossypii transmitted CMV more predominantly than TuMV, though the latter transmitted TuMV at a high rate. Consecutive transmission An attempt was made to determine precisely how long M. persicae and A. gossypii retained TuMV or CMV. Both M. persicae and A. gossypii showed several types of transmission when single aphid probing on infected leaves was transferred at 10-minute intervals to five successive healthy plants. With M. persicae, many aphids transmitted TuMV alone to only the first plant of the series, regardless of the aphid form (Table 2). However, transmission of TuMV alone to the successive three plants was achieved by wingless M. persicae but not by the winged form. On the other hand, A. gossypii commonly transmitted TuMV alone or CMV alone to the first plant only (Table 3). One of the winged form of A. gossypii transmitted CMV alone to the successive three plants. The simultaneous transmission of the two viruses was less common in M. persicae than in A. gossypii. The simultaneous transmission to only one plant of the series was more

Table 3. Retention of TuMV or CMV by a single aphid (A. gossypii) transferred at 10-minute intervals to five successive healthy turnip seedlings

a) Forty-three wingless and forty-six winged aphids were used. However, those aphids that failed to infect plants are not included. Each fifteen aphids that transmitted viruses are shown. b) See foot-note b) in Table 2. 566 日本植物病理学会報第51巻第5号昭和60年12月

Table 4. Effect of antibody treatments on the transmission of TuMV and CMV by wingless aphids of M. persicae and A. gossypii

a) Number of infected plants were indicated when twenty-four test plants were inoculated with each two aphids.

frequently observed. One of the wingless form of M. persicae, however, transmitted

both viruses simultaneously to the successive three plants. Some of wingless M. per-

sicae and A. gossyyii transmitted the two viruses to the second plant after the transmis-

sion of TuMV alone or CMV alone to the first plant. On the contrary, in each one

case of A. gossypii, the aphid transmitted TuMV alone or CMV alone to the second

plant after the simultaneous transmission of the two viruses to the first plant, respectively.

Treatment with antibodies

Two experiments were carried out to evaluate the effect of the treatment of the aphids with each of the ƒÁ-globulins on the transmission of TuMV and CMV by M. persicae and A. gossypii (Table 4). The pre-acquisition treatments did not affect signifi-

cantly the ability of transmission of TuMV or CMV by the two aphid species. However,

post-acquisition treatments resulted in a clear decrease in virus transmission. When M. persicae was fed on anti-TuMV ƒÁ-globulin in post-acquisition treatment, the trans-

mission of TuMV decreased conspicuously. By the same treatment, A. gossypii failed

to transmit TuMV completely but transmitted CMV at a high rate. Aphids, M. persicae and A. gossypii, treated with anti-CMV ƒÁ-globulin transmitted TuMV regularly, and the

transmission of CMV by A. gossypii was remarkably reduced. When pre-acquisition or

post-acquisition treatments in winged aphids were evaluated, the results were similar to those obtained in wingless aphids.

Discussion

There are many reports on the mechanism of transmission, the sites of virus retention in aphid3,9,16), and various other aspects of the transmission process8,13,14,18,20). Use of doubly infected plant as virus source14) and treatment of aphids with specific anti- serum15) have helped overcome some of the limitations in the study of transmission of Ann. Phytopath. Soc. Japan 51 (5). December, 1985 567 plant viruses by aphids. In this study, both winged and wingless form of M. persicae selectively transmitted TuMV alone at high rates, but rarely transmitted CMV from source plants doubly infected with TuMV and CMV. On the other hand, A. gossypii transmitted CMV alone or TuMV alone, and often transmitted both viruses simultaneously. These features of transmission abilities by M. persicae and A. gossypii were in agreement with those obtained in transmission tests from single infections7). Similar phenomena have been recorded in the transmission tests from cucumber plants doubly infected with watermelon mosaic virus (WMV) and CMV. Those results suggested that M. persicae transmitted WMV alone at a high rate10), but A. gossypii transmitted CMV predominantly22). Numerous factors may affect the efficiency with which a par- ticular virus is transmitted8). It is most likely that the transmission efficiency within aphid species may be related to their probing behaviour11), inactivation of viruses by products secreted by aphids19,20), the surface configuration of stylets where viruses are adsorbed, and/or electrostatic charge of stylets1,2). Although A. gossypii transmitted not only CMV but also TuMV at a high rate, transmission rates of CMV from double infections were as high as in the case of single infections. Furthermore, in the transmission test with successive transfers at 10-minute intervals, some of the aphids showing simultaneous transmission of TuMV and CMV, transmitted TuMV alone or CMV alone to the former or subsequent test plants. These results suggest that each of the two viruses seems to be adsorbed to its own binding sites on or within the stylets20). The aphids treated with specific antibodies after acquisition access probes on infected plants, failed to transmit, or seldom transmitted the virus. These results coincided with those obtained by treatments of the stylets of vir- uliferous aphids with formaldehyde5) or ultraviolet irradiation4), and led to the hypothe- sis that the non-persistent viruses could not be carried by simple contamination of the stylets.

The author would like to thank Dr. N. Iizuka, Hokkaido National Agricultural Experiment Station, for his useful suggestions.

Literature cited

1. Bradley, R.H.E. (1961). Virology 15: 379. 2. Bradley, R.H.E. (1962). Ibid. 17: 95-98. 3. Bradley, R.H.E. (1966). Ibid. 29: 396-401. 4. Bradley, R.H.E. and Ganong, R.Y. (1955). Can. J. Microbiol. 1: 775-782. 5. Bradley, R.H.E. and Ganong, R.Y. (1955). Ibid. 1: 783-793. 6. Fujisawa, I. and Iizuka, N. (1982). Ann. Phytopath. Soc. Japan 48: 121. (Abstr. in Japanese). 7. Fujisawa, I. and Iizuka, N. (1985). Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 141: 57-64. 8. Hariss, K.F. (1977). In Aphids as Virus Vectors. New York. Academic Press. pp. 166-208. 9. Lim, W.L., De Zoeten, G.A. and Hagedorn, D.J. (1977). Virology 79: 121-128. 10. Maejima, I. and Nishi, Y. (1974). Ann. Phytopath. Soc. Japan 40: 215 (Abstr. in Japanese). 11. Nakazawa, K. (1972). Bull. Hatano Tobacco Exp. Stn. 72: 1-127. 12. Pirone, T.P. (1964). Virology 23: 107-108. 13. Pirone, T.P. and Harris, K.F. (1977). Ann. Rev. Phytopathol. 15: 55-73. 14. Rochow, W.F. (1972). Ann. Rev. Phytopathol. 10: 101-124. 15. Rochow, W.F. and Muller, I. (1975). Virology 63: 282-286. 16. Taylor, C.E. and Robertson, W.M. (1974). Phytopathol. Z. 80: 257-266. 568 日本植物病理学会報第51巻第5号昭和60年12月

17. Tsuchizaki, T., Goto, T., Fujisawa, I. and Yoshida, K. (1981). Res. Bull. Hokkaido Natl. Agric. Exp. Stn. 131: 71-93. 18. Watson, M.A. (1971). Principles and Techniques In Plant Virology. New York. Van Nostrand pp. 131-167. 19. Watson, M.A. and Roberts, F.M. (1940). Ann. appl. Biol. 27: 227-233. 20. Watson, M.A. and Plumb, R.T. (1972). Ann. Rev. Ent. 17: 425-452. 21. Yamamoto, T. and Ishii, M. (1981). Bull. Shikoku Agric. Exp. Stn. 38: 1-16. 22. Yamamoto, T. and Ishii, M. (1983). Ann. Phytopath. Soc. Japan 49: 508-513.

和文摘要

藤澤一郎:カブモザイクウイルスとキュウリモザイクウイルスのアブラムシ伝搬,第2報,混合感染葉からの伝搬

カブモザイクウイルス(TuMV)および,キュウリモザイクウイルス(CMV)に混合感染したカブを接種源として,モモアカアブラムシとワタアブラムシの無翅虫および有翅虫による各ウイルスの健全カブへの伝搬様式を調べた。モモアカアブラムシはTuMVのみを高率に伝搬したが,CMV単独および両ウイルスの同時伝搬はまれであった。ワタアブラムシではCMVの単独伝搬が多く,またTuMV単独および同時伝搬もよく認められた。ウイルス獲得後10分毎に順次検定植物を変えて連続伝搬を調べたところ,モモアカアブラムシは1番目の植物のみにTuMVを単独伝搬するものが多く,ワタアブラムシでも1番目の植物のみにTuMV あるいはCMVを単独伝搬する虫が多かった。また,ウイルスを獲得させた後TuMVあるいはCMVの各抗体をパラフィルム膜法で吸汁させると,TuMV抗体吸汁アブラムシではTuMV伝搬が,CMV抗体吸汁アブラムシではCMV伝搬が著しく阻害された。