Cycas Necrotic Stunt Virus, a New Member of Nepoviruses

日植病報 52: 302-311 (1986)

Ann. Phytopath. Soc. Japan 52: 302-311 (1986)

Cycas Necrotic Stunt Virus, a New Member of

Nepoviruses Found in Cycas revoluta

Host Range, Purification, Serology and

Some Other Properties

Manabu KUSUNOKI*, Kaoru HANADA**, Mitsuro IWAKI***, Moo Ung CHANG****,

Yoji DoI***** and Kiyoshi YORA******

A new disease of cycas (Cycas revoluta) characterized by dwarfing and twisting of young

leaves and chlorotic or necrotic spots on mature leaves was shown to be caused by a small

spherical virus of 28 nm in diam. By sap inoculation the virus was easily transmitted to

some Chenopodium spp. in which chlorotic local lesions and systemic mottlings were pro

duced. The host range of the virus was limited. Out of 39 species in 12 families tested,

only ten species in Aizoaceae, Amaranthaceae and Cycadaceae were susceptible to the virus.

The virus was transmitted through seeds from diseased plants of C. amaranticolor and C.

serotinum at the rate of 29 and 82 per cent, respectively. The virus was inactivated after

10 min at 60-65 C, and 18-24 days at room temparature. The dilution end point lay between

1•~10-3 and 5•~10-3. The virus particles were observed to show a linear arrangement in tu

bular structures in both cytoplasm and plasmodesmata, and to form crystalline aggregates

in cytoplasm. The virus was purified from systemically infected C. quinoa leaves, and the

purified virus was separated into three components after sucrose density gradient centri

fugation. The properties of the virus were very similar to those of nepoviruses so far des

cribed, but no serological reaction was found between the virus and nine known nepoviruses

tested. The virus was concluded to be a new member of nepoviruses, and was named the

cycas necrotic stunt virus. This disease designated as the cycas necrotic stunt disease was

newly described.

(Received December 2, 1985)

Key words: nepovirus, cycas necrotic stunt virus, Cycas revoluta, Gymnospermae plant.

Introduction

No virus disease of Gymnospermae plants has been reported with only an exception

of the detection of rod shaped virus-like particles in Picea excelsa and their graft and

* Forestry and Forest Products Research Institute, P. O. Box 16 Tsukuba-Norin-Kenkyu-Danchi,

Ibaraki 305, Japan林業試験場

** National Agriculture Research Center, Yatabe-machi, Tsukuba-gun, Ibaraki 305, Japan農

業研究センター

*** National Institute of Agro-Environmental Sciences, Yatabe-machi Tsukuba-gun, Ibaraki 305,

Japan農業環境技術研究所

**** Department of Biology, Yeungnam University, Gyonsan 632, Korea

***** Faculty of Agriculture, UniverSity of Tokyo. Bunkya-ku, Tokyo 113, Japan東京大学農学部

****** Tachikawa college of Tokyo, Akishima, Tokyo 196, Japan東京都立立川短期大学

Ann. Phytopath. Soc. Japan 52 (2). April, 1986 303

Fig. 1. Symptoms of cycas plant A; dwarfing and twisting of young leaves, B;

necrotic spots on mature leaves.

insect (Sacchiophantes abietis) transmission 2).

Diseased plants of Cycas revoluta, an ornamental plant in Gymnospermae, showing

dwarfing and twisting of young leaves (Fig. 1A) and chlorotic or necrotic spots on ma

ture leaves (Fig. 1B) were found in Chiba Prefecture in 197318). The diseased plants

declined in growth year after year, and severely affected plants were finally killed. A

virus was transmitted from the diseased plants to some herbaceous plants by sap in

oculation. Small spherical virus particles were observed in diseased plants of both

original host plants and inoculated plants by electron microscopy. In this paper the

virus and the disease were named as the cycas necrotic stunt virus (CNSV) and the

cycas necrotic stunt, respectively, and some properties of the virus are described.

Materials and Methods

Virus source. Naturally infected cycas plants were collected at Tomiura-cho,

Chiba Prefecture, in 1973. Some of them were transplanted to pots and grown in a gre

enhouse. The virus was also kept in Chenopodium amaranticolor or C. quinoa plants by

serial sap transmissions. Infected leaves of C. amaranticolor or C. quinoa stocked in a

ultra-deep freezer (-70 C) were also used as virus source for inoculation and purifica

tion.

Sap inoculation and host range experiments. Carborundum dusted plants were

inoculated mechanically with crude sap prepared by grinding infected leaves of Cycas

304 日本植物病理学会報第52巻第2号昭和61年4月

revoluta, C. amaranticolor or C. quinoa in 0.1 M phosphate buffer, pH 7.0. For the host

range experiments, young seedlings of 39 species in 12 families were inoculated. To

detect latent infection, C. amaranticolor seedlings were used as a test plant.

Electron microscopy. Morphology of the causal virus was examined by the dip

negative staining method4). The preparations were obtained by grinding the small pieces

from infected cycas leaves in 2% phosphotungustic acid (PTA) solution pH 7 .0. In

addition, a small drop of the purified virus preparation was mixed with the same volume

of 2% PTA solution on a carbon stabilized collodion-coated grid, and examined under

an electron microscope (Hitachi HU-12).

For ultrathin sectionings, small pieces of infected cycas leaves were fixed with 1%

osmium tetraoxide in Dalton's solution, pH 7.2, at 0 C for 1 hr. After dehydration in

graded series of ethanol, these pieces were embedded in Epoxy resin. Ultrathin sec

tions were cut with glass knives on LKB ultramicrotome. Sections were stained with

uranyl acetate and lead citrate and examined under an electron microscope.

Stability in crude sap. Dilution end point (DEP), thermal inactivation point

(TIP) and longevity in vitro (LIV) of the virus in crude sap were determined by using

C. amaranticolor as an assay host. To prepare the crude sap, 5g of diseased C. amar

anticolor leaves was ground in 50ml of 0.1 M phosphate buffer, pH 7.0, and filtered

through several layers of cheesecloth. The crude sap was considered to be equivalent

to 10 fold dilution. In DEP tests the crude sap was diluted with 0.1 M phosphate buf

fer, pH 7.0. TIP was determined by heating 1ml of the crude sap in a test tube for

10min in a water-bath, and LIV was determined after incubating the crude sap at room

temperature (18-26 C).

Seed transmission. Seeds collected from diseased plants of C. amaranticolor, C.

serotinum and Tetragonia expansa were sown in sterilized soil. The developed progeny

seedlings were homogenized individually in a mortar containing 0.1 M phosphate buffer,

pH 7.0. Presence of the virus was examined by inoculating the homogenate to healthy

seedlings of C. amaranticolor.

Virus purification. Frozen diseased leaves of C. quinoa (100g) were homogenized

in 0.1 M phosphate buffer pH 7.0 (200ml) mixed with 10 mM sodium diethyldithiocar

bamate (Na-DIECA), 20 mM Na2SO3, 20 mM ethylendiaminetetraacetate (EDTA) and 100

ml of chloroform. The homogenate was filtered through several layers of cheesecloth,

and centrifuged at 8,800•~g for 10min. The supernatant was treated by three cycles of

differential centrifugation at 110,000•~g for 2hr and at 8,800•~g for 10min. The result

ant pellet (partially purified virus) was suspended in 0.05 M phosphate buffer, pH 7.0,

containing 5 mM EDTA, and placed on a sucrose density gradient column (10-40%) and

centrifuged at 55,000•~g for 3hr. The resultant oparescent bands were collected man

ually or by Isco fractionater.

Serology. Antiserum against CNSV was prepared in a rabbit by an intramuscular

injection of purified virus preparation emulsified in Freund's complete adjubant, and by

additional four weekly intravenous injections of purified virus. Blood was collected 10

days after the last injection. The serological relationships between CNSV and nine

nepoviruses were examined by the agar gel double diffusion method. The antisera

tested in this study were as follows: Antisera against cherry leaf roll virus (CLRV)


from golden elderberry, CLRV from cherry14) and crimson clover latent virus (CCLV)16)

were provided by Dr. A. T. Jones. Antisera against grapevine chrome mosaic virus

(GCMV)20), myrobalan latent virus (MLRV)5) and tomato black ring virus (TBRV)21) in

Italy were provided by Dr. G. P. Matelli. Antiserum against cacao necrosis virus

(CNV)15) was provided by Dr. R. H. Kenten. Antiserum against TBRV in Britain8) was

provided by Dr. B. D. Harrison. Antiserum against tobacco ringspot virus (TobRV)6)

was provided by Dr. Fukumoto. Antisera against TBRV in Japan12), tomato ringspot

virus (TomRV)11) and mulberry ringspot virus (MRV)10,25,26) were also used. In recip

rocal serological tests, partially purified preparations of TBRV, MRV, AMV and TobRV

were used.

Results

Host range and symptoms

The most sensitive test plant against CNSV was C. amaranticolor. It showed chloro

tic local lesions 10-15 days (in April and May) after inoculation, and then systemic

symptoms of mottling and necrosis (Fig. 2A). Similar symptoms were produced on C.

quinoa, C. serotinum and T. expansa (Fig. 2B). Dwarfing symptoms were produced on Spinacia oleracea. Systemic symptomless infection was detected in Beta vulgaris and

Gomphrena globosa, but not in other plants, by back inoculations to C. amaranticolor as

shown in Table 1. Cycas seedlings from true seeds showed chlorotic mottling symptoms

on their leaves by sap inoculation.

Fig. 2. Symptoms on test plants A; systemic mottlings on C. amaranticolor, B; chlorotic

spots and rings on T. expansa


Table 1. Host range of cycas necrotic stunt virus

a) ChS; chlorotic spot, D; dwarfing, M; mottling, N; necrosis, NS; necrotic spot, +; latent infection

Electron microscopy

The electron micrographs of dip preparations from leaves and roots of infected

plants of cycas and herbaceous plants revealed the presence of isometric, spherical virus

particles of about 28 nm in diameter. In ultrathin sections, the virus particles were

observed in cytoplasm and plasmodesmata of infected plants. In cytoplasm, mature

virions were observed in crystalline aggregates of capsid coat protein (Fig. 3B). Tubular

structures which contained virus particles in a linear arrangement were characteristic

ally observed in cytoplasm and sometimes in plasmodesmata (Fig. 3A). Vesicular bodies

associated with virus infection were also observed in cytoplasm (Fig. 3C).

Stability in crude sap

Infectivity was lost at a dilution between 1•~10-3 and 5•~10-3 (DEP), and after stor

age for 18-24 days at room temperature (LIV). The thermal inactivation point lays be


Fig. 3. Electron micrographs of thin sections. A; CNSV particles in tubular structures in cytoplasm and in plasmodesmata, B; crystalline aggregates of CNSV in cytoplasm, C; vesicular body associated with CNSV infection (bar represents A; 200 nm, B and C; 500 nm, T; tubular structure, CW; cell wall, V; virus particles, Ve; vesicular body)


Fig. 4. Sucrose density gradient sedimentation profile of CNSV.

Fig. 5. Electron micrographs of A; top component and B; bottom component after sucrose density gradient centrifugation (A and B; bar represents 200 nm).

tween 60 and 65 C (TIP).

Seed transmission

Twenty nine per cent of progeny seedlings out of 100 seedlings from infected C:

amaranticolor plants and 82% from infected C. serotinum were infected. Plants infected


Fig. 6. Immunodiffusion tests of CNSV with antisera prepared to nepoviruses isolated in Japan.

Purified CNSV (C) and partially purified tomato black ring virus (TN). Antisera against CNSV (C), arabis mosaic virus (AN), tobacco ringspot virus (TR), mulberry ringspot virus (MR), tomato ringspot virus (TM) and tomato black ring virus (TN).

by seed transmission showed no obvious symptoms.

Purification

In sucrose density gradient centrifugation, the partially purified virus preparation

was separated into three components (Fig. 4). By electron microscopy, top component

contained protein shells devoid of nucleic acid, and was contaminated with host cell

substance (Fig. 5A). Separation of protein shells from the host cell substance was very

difficult. The amount of the middle component was much less than the bottom com

ponent when the virus was purified from infected leaves of C. amaranticolor. Virus par

ticles from middle component were partially stained with 2% PTA and those of bottom

component were completely stained (Fig. 5). The ratios of A260/280 of top, middle and

bottom components were 1.00, 1.41 and 1.67, respectively.

Serology

Antiserum prepared against CNSV had homologous titer of 1/128 in agar gel double

diffusion tests. The antiserum also reacted with preparation from healthy plants of C

quinoa. Therefore, the antiserum was used in serological tests after absorption treatment by adding an equal volume of preparation from healthy leaves of C. quinoa. CNSV

antiserum thus obtained reacted positively only with CNSV antigen, and did not react

with AMV, MRV, TobRV and TBRV antigens (Fig. 6). Purified CNSV did not react

with antisera against CLRV (from golden elderberry), CLRV (from cherry), CNV, GCMV,

MLRV (in Italy), TBRV (in Japan, Italy and Britain), TomRV, CCLV, MRV, or TobRV.

Discussion

Not a few nepoviruses have been isolated from fruit trees, vegetables, flowering


plants and other crops including both herbaceous and woody plants. Generally speaking, nepoviruses have wide host ranges, and are transmitted by sap inoculation, by nem

atode vectors, and through seed. Definitive nepoviruses are isometric, spherical parti

cles about 28 nm consisting of three components. They have two RNAs and one coat

protein9). Among nepoviruses, TomRV from narcissus11) and melon28), TBRV from narcissus12) and gerberal17), TobRV from gladiolus6), AMV from narcissus13) and Japanese

butterbur24), MRV from mulberry25,26) and grapevine fanleaf virus from grapevine23) have

been reported to occur in Japan.

Properties of CNSV, namely development of tubular structures containing virus par

ticles, and of vesiculated membraneous inclusion bodies in infected host cell, particle

morphology, multicomponent particles, seed transmission and symptoms on some differ

ential hosts are similar to those of nepoviruses3,9,22). As shown in our next paper7), the

properties of nucleic acid and coat protein of CNSV indicate that CNSV is a member of the nepovirus group. In our previous paper we thought that the causal virus of the

cycas necrotic stunt disease was a strain of TBRV1), but it was our mistake due to

misreading of serological reactions. In the present study CNSV was shown to be dif

ferent from any one of nepoviruses tested here, because no serological reactions were

observed between CNSV and the antisera against nine kinds of nepoviruses, and between

four kinds of nepoviruses and the antiserum against CNSV.

From these results it is concluded that CNSV is a new virus having many proper

ties in common with those of nepoviruses. As compared with nepoviruses, the host range of CNSV is rather limited, because it can infect only some species in Aizoaceae,

Amaranthaceae, Chenopodiaceae and Cycadaceae. Viruses similar to CNSV have been

isolated from gladiolus (Gladiolus sp.) (Fukumoto, F. et al. unpublished results), aucuba

(Aucuba japonica), daphne (Daphne odora)19) and allium (Allium fistlosum)27). This sug

gests that CNSV may be a widely distributed virus in Japan.Concerning Gymnospermae plants, the association of rod-shaped particles with a

spruce disease has been reported2), but no characterization has been done with the par

ticles. The present paper is thought to be the first report on the virus disease of

Gymnospermae plants.

The authors wish to thank Dr. Y. Nagai (Chiba Prefectural Agricultural Experiment Station) for supplying diseased cycas plants, and Dr. S. Toriyama, Dr. S. Yamashita (Laboratory of plant Pathology, University of Tokyo) and Dr. T. Kobayashi (Laboratory of Forest Pathology, Forestry and Forest Products Research Institute) for their helpful advices. We also wish to thank Dr. A. T. Jones and Dr. B. D. Harrison (Scottish Crop Research Institute, England), Dr. R. H. Kenten (Rothamsted Experimental Station, England), Dr. G. P. Martelli (Instituto di Pathologia Vegetale, Universita di Bari, Italy) and Dr. F. Fukumoto (National Agriculture Research Center, Japan) for their providing of the antisera.

Literature cited

1. Chang, M. U., Kusunoki, M., Doi, Y. and Yora, K. (1976). Ann. Phytopath. Soc. Japan 42: 64.2. Cech, M., Kralik, O. and Blattny, C. (1961). Phytopathology 51: 183-185.3. Cropley, R. and Tomlinson, J. A. (1971). CMI/AAB Descriptions of Plant Viruses No. 80.4. Doi, Y., Toriyama, S., Yora, K. and Asuyama, H. (1969). Ann. Phytopath. Soc. Japan 35: 180

-187.


5. Dunez, J., Delbos. R. and Dupont, G. (1976). CMI/AAB Descriptions of Plant Viruses No. 160.6. Fukumoto, F., Ito, Y. and Tochihara, H. (1976). Ann. Phytopath. Soc. Japan 42: 384.7. Hanada, K., Kusunoki, M. and Iwaki, M. (1985). Ann. Phytopath. Soc. Japan (in subscription)8. Harrison, B. D. (1958). J. Gen. Microbiol. 18: 450-460.9. Harrison, B. D. and Murant, A. F. (1977). CMI/AAB Descriptions of plant viruses No. 185.

10. Hibi, T., Yagita, H. and Iwaki, M. (1984). Ann. Phytopath. Soc. Japan 50: 445.11. Iwaki, M. and Komuro, Y. (1971). Ann. Phytopath. Soc. Japan 37: 108-116.

12. Iwaki, M. and Komuro, Y. (1973). Ann. Phytopath. Soc. Japan 39: 279-287.13. Iwaki, M. and Komuro, Y. (1974). Ann. Phytopath. Soc. Japan 40: 344-353.14. Jones, A. T. and Murant, A. F. (1971). Ann. appl. Biol. 69: 11-15.

15. Kenten, R. H. (1977). CMI/AAB Descriptions of Plant Viruses No. 173.16. Kenten, R. H., Cockbain, A. J. and Woods, R. D. (1980). Ann. appl. Biol. 96: 79-85.17. Kishi, K. (1973). Review of researches in Vegetables and Ornamental Crops Research Station

(shiken-kenkyu-kadai-ichiran): 222-243.18. Kusunoki, M., Nagai. Y., Yamashita, S., Doi, Y. and Yora, K. (1975). Ann. Phytopath. Soc.

Japan 41: 285-286.19. Kusunoki, M., Shirako, Y., Chang, M. U., Doi, Y. and Yora, K. (1979). Ann. Phytopath. Soc.

Japan 45: 571-572.20. Martelli, G. P. and Quacquarelli. A. (1972). CMI/AAB Descriptions of Plant Viruses No. 103.21. Murant, A F. (1970). CMI/AAB Descriptions of Plant Viruses No. 38.22. Stace-Smith, R. (1970). CMI/AAB Descriptions of Plant Viruses No. 17.23. Tanaka, H. (1977). Plant Protection Japan 31: 414-418.24. Tochihara, H. and Tamura, M. (1973). Ann. Phytopath. Soc. Japan 39: 217-218.25. Tsuchizaki, T. (1975). CMI/AAB Descriptions of Plant Viruses No. 142.26. Tsuchizaki, T., Hibino, H. and Saito, Y. (1971). Ann. Phytopath. Soc. Japan 37: 266-271.27. Yamashita, S. and Osaki, T. (1983). In Handbook of Plant Viruses in Japan (Shokubutsu-Virus

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和文摘要

楠木学・花田薫・岩木満朗・張茂雄・土居養二・與良清:ソテツえそ萎縮ウイルスーソテツに発生

したnepovirusグループの未記載ウイルス寄主範囲,純化,血清関係,その他の諸性質

新葉にねじれや黄化,成葉に黄色またはえ死斑を現わすソテツから径28nmの小球形ウイルスが分離され

た。このウイルスは汁液接種した12科39種の植物のうちソテツ, C. amaranticolor, C. quinoa, C. serotinum

ホウレンソウなどに全身感染したが,寄主範囲は狭かった。粗汁液中での不活化温度は60～65C(10分),希

釈限度1×10-3～5×10-3,保存限度は18～24日(室温)であった。また全身感染したC. amaranticolorやC.

serotinumでそれぞれ29, 82%の高率で種子伝染した。ウイルス粒子は感染細胞の細胞質や原形質連絡糸内で

さや状構造の中に配列したり,細胞質内で結晶状に配列して観察された。純化ウイルスはしょ糖密度勾配遠心

により3成分に分かれ, Amax=260nm, Amin=237nmの核タンパクの吸収値を示した。本ウィルスは純化

ウイルスを用いて作製した抗血清と特異的に反応し, 1/128の力価を示したが,わが国で既知のnepovirusグ

ループのいずれのウィルス抗血清とも反応しなかった。土壌伝染は認められなかったが,本ウイルスをnepo

virusグル-プの未記載ウイルスと考え,ソテツえそ萎縮ウイルス(cycas necrotic stunt virus: CNSV),

本病の病名をソテツえそ萎縮病(cycas necrotic stunt disease)と命名した。

Documents

Cycas Necrotic Stunt Virus, a New Member of Nepoviruses