Inouye, Tadao

Mitsuhata, Koji

Simplified Chinese

Okayama University Digital Information Repository

CARNATION NECROTIC FLECK VIRUS

Okayama University Scientific Achievement Repository

CARNATION NECROTIC FLECK VIRUS Tadao INOUYE and Koji MITSUHATA Among the viruses of carnation， carnation latent virus and carnation vein-mottle virus have been known as elongated viruses. The authors isolated an elongated virus different from the known two viruses from carnations collected in Okayama and Shizuoka Prefectur，回 thatshowed grayish white or reddish purple necrotic flecks (Plate 1; A). The virus was newly named as carnation necrotic fleck virus (CNFV) on the basis of the characteristics of the symptoms on carnation. This paper mainly deals with the investigation on transmission， host range， symptoms， and electron microscopy of the virus. MATERIALS AND METHODS lsolation 01 the Virus An isolate of CNFV mainly used in this study was isolated by an aphid， 品のIZUStersicae， .from a diseased plant of carnation var. Coral collected in Kurashiki in 1969 showing reddish purple flecks on leaves. The isolate was maintained successively on carnation and Dianthus barbatus in glasshouse by aphid inoculation. lnoculation Sap inoculation was conducted by carborundum rubbing method. Most of the inoculation test was carried out using an aphid， Myzus tersicae. As the indicator plant for virus testing D. barbatus was used. Electron microscoty Virus particles were observed under a Hitachi HS-6 and a Hitachi HU-12 electron microscope. Specimens for observation were prepared by dip method or by direct negative stain method using 2 % PTA or 1-2 % uranly formate. Small piec田 ofinfected leaf tissues of carnation and D. barbatus were fixed with Dalton's chrome-osmium solution. After fixation tissues were dehydrated by a graded series of ethanol， and embedded with epoxy resin. Thin sections were cut by Porter-Blum MT-1 microtome using glass knives， and stained with uranyl acetate and lead citrate. RESULTS 1. Transmission Sap lnoculation The virus was transmitted by plant sap with some difficulties. After about 10 days， local grayish white necrotic lesions developed on the in∞ulated leaves of the seedlings of carnation and D. barbatω(PlateI: H， 1). Most of the inoculated seed1ings were not in-fected systemically， but some of the seedlings of D. barbatωprodu印 dfaint veinal chlorosis on the upper leaves. 196 T. Inouye & K. Mitsuhata Athid Transmission CNFV was transmitted by M. tersicae. Mode of transmi話 ionby this aphid was investigated using D. barbatus as the virus source and indicator plants. To know the time of acquisition feeding the aphids were fed on田町ceplants for the period varying from 5 min to 24 hrs， and then transferred to indicator plants and allowed to f田dfor 24 hrs. Table 1 shows the results. The aphids transmitted the virus by acquisition feedings longer than 30 min， but not by shorter than 10 min. E田ciencyof transmission was increased by feeding longer than 4 hrs. TABLE 1. Eff民 tof varying acquisition fl白dingtime on the transmission of carnation necrotic fleck virus by Myzus抑rsicaeDuration of a伺uisitionfeeding* Aon phiedas ltfhey d Experiment 5min 10 min 30 min 1 hr 4 hrs 24 hrs plants I 0/3 0/3 0/3 0/3 1/3 3/3 0/3 E 0/3 0/3 1/3 1/3 2/3 1/3 0/3 E 0/6 0/6 116 0/6 1/6 4/6 0/6 Total 0/12 0/12 2/12 1/12 4/12 8/12 0/12 Denominator indicates the num国rof test plants colotized. the numerator the number that出cameinfected. 勺 Afterthe a伺uisitionf，田dingon infected D. barbatus， aphids were transferred in groups in fives to individual test plants (D. barbatus) for 24 h四・Table 2 shows the results of experiments about the time of inocula-tion feeding. The aphids which had r配eivedsu伍cientfeeding for virus acquisition were transferred to indicator plants and allowed to feed for inoculation for the period varying from 5 min to 24 hrs. The virus was TABLE 2. Effect of varying in∞ulation f田dingtime on the transmission of 位 rnationnecrotic fleck virus by Myzusρersicae Duration of inoculation feeding* Aon phiedas ltfhey d Experiment 5min 10 min 30min 1 hr 4 hrs 24 hrs plants I 0/1 0/1 0/1 0/1 1/1 1/1 0/1 E 0/3 0/3 1/3 0/3 2/3 1/3 0/3 E 0/3 0/3 0/3 1/3 1/3 2/3 0/3 W 0/6 1/6 3/6 3/6 3/6 3/6 0/6 Total 0/13 1/13 4/13 4/13 7/13 7/13 0/13 Denominator indicates the number of test planta colonized， the numerator that加cameinfected. *) After being fed on source plants (D. barbatus) for a perlod sufficient for a司uisitionfor 24 hr・s.aphids were transferred in groups of fives to individual test plants (D. barbatus) and fed for given period宮・Carnation Necrotic Fleck Virus 197 transmitted by inocu1ation f田dings10nger than 10 min， but not by a short time feeding for 5 min. E自ciencyof transmission was increased by 10nger periods of feeding for 30 min or more. Resu1ts of dai1y serial transfer t田tsin Table 3 showed that M. tersicae retained their infectiviti回 for2 days after they 1eft from the source plants. TABLE 3. Retention of伺.mationn回roticf1eck Viru5 by Myzus tersicae in daily民rialtransfer Experlment Daily serlal tran免許)1 st 2nd 3 rd 4 thI 3/3 1/2 0/3 E 5/6 0/6 0/6 0/3 Total 8/9 1/8 。/9 0/3 Denominator indicates the number of test plants colonized， the numerator the number that加cameinfected. *) After being fed on田urceplants (D. barbatus) for a period suficient for virus acquisition for 24 hrs， aphids were transferred in groups of fiv田 toindividual test plants (D. barbatus) dai1y and fed for 24 hrs. 2. Host Range and Symttoms 50 far tested CNFV was infectious to 3 species in Callyothyllaceae， such as Dianthus caryothyllus， D. barbatus， and D. chinens;s. The fo1-lowing plants were not infected with the virus by sap and/or aphids: Nlcot;ana glut;nosa， N. tabacum， Daturσstramonium， Z;nn;a elegans， Arct;um latta， Beta vulgaris， Chenotod;um amaranticolor， Stinac;a olera-cea， Cucumis sat;vus， Pisum sativum， Phaseolus vulgaris， and Rhathanω sattvus. Symptoms on susceptib1e plants caused by aphid in∞ulation were briefly described be10w. on carnation， grayish white necrotic spots and flecks which were sometimes followed by reddish purp1e discoloration of leaves ap開ared2-3 weeks after the inocu1ation (Plate 1; B-D). Some of the seedlings severe1y affected were stunted and killed. However， most of the chroni-cally infected plants became masked. Symptoms on D. barbatus were characterized by chlorosis of veins and reddish discoloration of leav回 After2-3 weeks from the in∞ula-tion， chlorosis of main veins appeared on several fully expanded young leaves (Plate 1; E-G). Affected leav回 werefrequently showed yollow net symptoms (Plate 1; E， F). The 1回 ves，then， produ偲 dreddish dis-coloration followed by leaf blight from the tips (Plate 1; J). On several young leaves grown to the next， the symptoms were observed only at the leaf tips. The upper 1伺 V田 grownlater were almost symptomless. 198 T. Inouye & K. Mitsuhata Symptoms observed on D. chinensis are almost similar to th但 eon D. barbatus， but rather indistinct. 3. Electγon Microsco卸Virus Partic1es. Partic1es of CNFV were long and very flexuous rods， 12-13 nm wide and 1.4-1.5μlong (Fig. 1 and Plate II; A). In leaf-dip preparations treated with uranyl formate， cross banding of particles with an average pitch of 3.4 nm was clear1y seen (Plate II; B). ????????』???'3 Fig. 1. Particle length of carnation necrotic f1eck virus. Length in nm U1trastructures of Infected Plant Ce11s. In thin sections of infected carnation and D. barbatus， virus particles were observed in phl田 mce11s (Plate II). Characteristic v田icularstructures were observed also in these ce11s (Plate II; C， D). Some of the phl偲 ma11s were almost fi1ed with large ma話回offlexuous particl田 (PlateII; A， B)， and/or particle aggre-gat田 andvesicular structur邸 (PlateII; C， D). Necrosis of some phloem alls were frequently observed. Aggregates of particles and/or vesiculer structures were seen in epidermal ce11s of inf配tedD. barbatus as well， which may well correspond to the X-bodies in epidermal strip in light microscopy (Plate I; C). In mesophyllぽl1sneither virus particles nor vesicular structures were so far found. 4. Distribution 01 CNFV in CarnattOn Diseased samples of 18 varieties of carnation were co11ected from farmer's fields at Kurashiki and Kasaoka， Okayama Prefecture， Doi， Kawazu， and Minami Izu. Shizuoka Prefecture， and from the stock nursery of Okayama Agricultural Ex開rimentStation. Electron micro-scopic observations on negatively stained dip preparations were conducted for detection of virus particles. In the samples of 150ut of 18 varieties isometric and/or rod-shaped particles were detected， which were presum-Camation Necrotic Fleck Virus 199 ably carnation mottle virus or carnation ringspot virus for isometric particl回 andcarnation latent virus or carnation vein mottle virus for rods. Particles of CNFV， long very flexuous and narrow rods， were det配tedin 13 out of 18 varieties， al of whose samples showing typical grayish white or reddish purple necrotic flecks were included. Double or triple infections with th田eparticles were frequently observed. DISCUSSION CNFV caus回 grayishwhite and/or reddish purple necrotic flecks or streaks in carna tion. J on偲(1945)reported that causal agent of carnation streak of which symptoms were reddish spots and streaks was trans-mitted by Myzωρersicae， but not by plant回 p. Carnation streak virus reported by Brierley and Smith (1957) was transmitted by grafting， but probably not by in詑cts. CNFV apparently di旺eredfrom Brierley and Smith' carnation streak virus in aphid transmission. Symptoms and aphid transmission of CNFV appeared somewhat re記 mbledto th侃 eof Jones' carnation str回 k. However， sap transmission of CNFV was not similar to Jon田， virus. The nature of the pathogen of carnation streak has not been defined (4)， and virus particl回 ofit also have not been reported. Therefore， the authors prefered CNFV rather than streak for the name of the virus in the present study. Several viruses of which particl回 werelong very flexuous rod a加ut10 nm wide and over 1μlong have b偲 nknown， such as国etyellows virus (1，3， 14， 17， 18)， citrus tristeza virus (9， 12， 13， 16)， Festuca n配 rosisvirus (15)， wheat yellow leaf virus (7)， and a virus from burd∞k (Bd-F) (5). Very flexuous rods with a helix pitch of 3.8 nm， 12 nm wide， and 600-620 nm long were also known to be particl田 ofa pple chlorotic 1伺 f-spot (10) and apple stem gr∞ving (11) viruses. Characteristics of the CNFV in particle morphology and aphid transmission probably in a田mi-persistent manner resembled to beet yellows and several other viruses mentioned a加verather than the viruses of apple. Further， the charac・teristics ob田rvedin thin s配 tions，the aggregation of virus particles in phloem cells and phloem necrosis， were very similar to those reported in beet yellows， citrus tristeza， and wheat yellow leaf virus回. Vesicular structt江田， usually ass∞iated with filamentous particl田 observed in epidermal and phloem cells infected with CNFV were simi1ar to those repo吋edin加etyellows virus (3). Acknowledgement The authors express their sincere thanks to Mr. H. Morita， Shizuoka Agricultural Experiment Station， and Mr. F. Yama-muro， Okayama Agricultural Experiment Station for their c∞peration in collecting di田asedcarnation samples. 200 T. Inouye & K. Mitsuhata SUMMARY A virus was isolated from carnation showing symptoms of grayish white or reddish purple necrotic flecks， and was newly named as car-nation necrotic fleck virus (CNFV). The virus was transmitted by plant回pwith some difficulties， and easily by an aphid， Myzusρersicae. The aphids， which had r配eivedacquisition feedings longer than 30 min， transmitted the virus， but not after feeding shorter than 10 min. After sufficient time of feeding for virus acquisition on source plants， the aphids trans'l1itted the virus when fed on t田tplants for 10 min or more. Efficiency of transmission was increased by longer period than 4 hrs and 30 min for acquisition and inoculation feeding， respectively. In daily serial transfer tests， the aphids retained their infectivities for 2 days. CNFV was infectious to 3 species in Caryothyl!aceae， such as Dianthus carythyl!us， D. barbatus， and D. chinensis， but not to 12 species in the 6 families examined. In carnation the virus caused n配 roticflecks， grayish white or reddish purple in colour， which usually became masked in chronically infected plants. D. barbatus produced yellowing of veins followed by reddish discoloration of leaves. Chronically infected plants usually became masked. Particles of CNFV were long very flexuous rods with helical sym-metry. Length of the particles was 1.4-1.5μ， width was about 12-13 nm. and pitch of helix 3.4 nm. In thin sections of CNFV infected plants. nerosis of some phloem cells was observed. Ma話回 ofparticles and/or vesicular structures with which some of the cells were almost filed were usuaIly observed in epidermal and phloem cells of infected plants. Cel-lular inclusions (X-bodies) observed in epidermal strip of infected D. barbatus in light microscopy probably corr白 pondedto the aggregat回 ofparticles and/or vesicular structures. Electron microscopic observations for CNFV particles proved that the virus was rather commonly distributed in 13 out of 18 varieties of carnation collected in Okayama and Shizuoka Prefectures. LITERA TURE CITED 1. Brandes，]. and Bercks， R. 1965. Gross morpho1ogy and sero1ogy as a basis for the c1assification of e10ngated p1ant viruses. Advance Virus Res. 11: 1-12. 2. Brier1ey， P.and Smith， F. F. 1957. Carnation viruses in the United States. Phyto-path. 47: 714・721.3. Eぉau，K. 1袋却B. Viruses ln P1ant Hosts. Univ. Wisc. Press 225 p. 4. G明 tha1s， M. 1969. Inf1uence de 1a temperature sur la camation mottle virus et le streak de rOEillet. Ann. Phytopathol. 1: 573-582. 5. Inouye， T.， and Mitsuhata. K. 1971. Viru現sin burdock. Arctium laρe L. (Studies on the viruses of p1ants in Comρosifae in ]apan lI) Nogaku Kenkyu日:1-14.， Ber. Ohara Inst.， Okayama Univ. 15(3): A19-A20 (1972). Camation Necrotic Fleck Virus 201 6. Inouye. T. and Mitsuhata. K. 1971. A new flexuous rod virus i釦 latedfrom cama. tion showing reddish purple or grayish white necrotic fleck symtoms. Ann. Phytopath.おc.Japan 37: 197-198. (A凶t.in Japanese) 7. Inouye. T.. Mitsuhata. K.. Heta. H.. and Hiura. U. 1973. A new virus of wh回 t.barley and several other plants in Gramineae. wheat ye110w leaf virus. Nogaku Kenkyu 54 (4) (In pr倒 s). .a Jon回.1. K. 1945. Mosaic. streak. and ye110ws of camation. Phytopath. 35: 37-46. 9. Kitajima. E. W.. Silva. D. M.. Oliveira. A. R.. Muler. G. W.. and Costa. A. S. 1964. Thread-like particles associated with tristeza disease of citrus. Nature 201: 1011-1012. 10. Lister. R. M. 1970. Apple chlorotic 1回 fs戸 tvlrus. C.M.I./ A.B.B. Descriptions of Plant Virus国 No.30.11. List忘れR.M. 1970. Apple stem gr∞ving vlrus. C.M.I./ A.B.B. Descriptions of Plant Viruses No. 31. 12. Price. W. C. 1966. Flexuous rods in phl僧 m偲l1sof lime plants infected with citrus tristeza virus. Virology 29:おら294.13. Prlce. W. C. 1970. Citrus tristeza virus. C.M.I./ A.B.B. D田criptionsof Plant Viru獄~.No.33. 14. Russell. G. E. 1970. Beet yellows virus. C.M.I./A.B.B. Descriptions of Plant Viruses No.32. 15. Schmidt. H. B.. Richter. J. Hertzsch. W.. und Klinkowski. M. 1963. Untersuchungen uber eine virusbedingte Necrose an Futtergrasem. Phytopath. Z. 47:随一72.16. Shikata. E. and Sasaki. A. 1969. Long flexuous threads a鰍 JCiatedwith Hassaku dwarf di路 aseof citrus. Jour. Fac. Agrlc. Hokkaido Univ. 56: 219-224. 17. S.v1vester. E. S. 1956. Beet mosaic and beet ye110ws vlrus transmission by green 開achaphid. J. Am.おc.Sugar beet Technol. 9: 56-61. 18. Varn叫ん.Gibbs. A. J. Wα刈s， R. D.. and Finch. J. T. 1968. Some observations on the structure of filamentous particles of several plant vIIuses. J. gen. Virol. 2: 1ぴ7-114.EXPLANATION OF PLATE Plate 1. Symptoms caused by camation necrotic fleck virus (CNFV) in camation and Dianfhus barbafus. A) Naturally infected camation (var. Coral) with grayish white and/or reddish purple necrotic flecks and streaks. B-D) Necrotic fl田 ksand streaks in camation inf，田tedwith CNFV by aphids. E) Veinal chlorosis on the lower leaf surfa田 ofD. barbatus infected with CNFV by aphids. F. G) Veinal chlor，佃isand necrosis on the upper leaf surface of D. barbatus infected by aphids with CNFV. H.I) L∞al lesions ln an in∞ulated leaf of D. baraatus by plant回 pafter 25 days (H). and 40 days (I) from the in∞ulation. J) Veinal necrosis and reddish discoloration in D. barbatus infected with CNFV by aphids. Plate 1. N Particles of CNFV in negatively stain回 dip-preparationmounted in PT A. Bar represents 1μ. B) Particl関 ofCNFV in uranyl foロnate. Bar represents 50 nm. C) Inclusion ¥)(到diesin epidermal cells of D. barbatus infected with CNFV in light microscopy. X: Iclusion bod)' n: Nucleus 202 T. Inouye & K. Mitsuhata Plate II. Ultrastructure of phloem cells of D. barbatus infected with CNFV A) Banded aggregates of CNFV partic1es in a phloem cell. Bar represents 1μ. B) A phloem cell almost fil1ed with masses of CNFV particles. Bar represents 1μ. C) A phl凹 mcell fi1ed with masses of CNFV particles and vesicular structures. Bar repr鎚 ents1μ. D) Part of a phl田 mcell fi1ed with masses of CNFV particles and vesicular structures. Bar repr飴 ents0.5μ. Abbreviations F: Virus particle V: Vesicular structure W: Cell wall Carnation Necrotic Fleck Virus 203 Plate 1 204 で.Inouye & K. Mitsuhata Plate n Carnation Necrotic Fleck Virus 205 Plate m 

CARNATION NECROTIC FLECK VIRUS

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