Characterization of a cucumber mosaic virus isolate ...

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Characterization of a cucumber mosaic virus isolate causing leaf crinkle and severe mosaic of Amaranthus in India a

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a

S.K. Raj , Aminuddin , B.P. Singh & M. Pal a

Plant Virus Laboratory

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Lucknow, 226001, India

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Available online: 23 Dec 2009

To cite this article: S.K. Raj, Aminuddin, B.P. Singh & M. Pal (1997): Characterization of a cucumber mosaic virus isolate causing leaf crinkle and severe mosaic of Amaranthus in India, Canadian Journal of Plant Pathology, 19:1, 97-100 To link to this article: http://dx.doi.org/10.1080/07060669709500581

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Note CANADIAN JOURNAL OF PLANT PATHOLOGY 19:97-100, 1997

Characterization of a cucumber mosaic virus isolate causing leaf crinkle and severe mosaic of Amaranthus in India S.K. Raj, Aminuddin, B.P. Singh, and M. Pal Plant Virus Laboratory and (M.P.) Cytogenetics Laboratory, National Botanical Research Institute, Lucknow-226001, India. Corresponding author: B.P. Singh. E-mail: [email protected]

Downloaded by [National Botanical Research Inst] at 04:19 06 January 2012

Accepted for publication: 1996 09 30

A virus causing leaf crinkle, severe mosaic of Amaranthus tricolor and seed yield losses in A. hypochondriacus was characterized as an isolate of cucumber mosaic virus on the basis of aphid transmission by nonpersistent manner, presence of 28 nm isometric particles, 26 kd viral coat protein subunits in SDS-PAGE and close serological relationship with CMV-C strain. Nucleic acid extracted from purified particles was ssRNA and infectious. The electrophoretic pattern of RNA species of the virus isolate revealed multiple RNAs and was similar to the CMV RNA pattern. Raj, S.K., Aminuddin, B.P. Singh, and M. Pal. 1997. Characterization of a cucumber mosaic virus isolate causing leaf crinkle and severe mosaic of Amaranthus in India. Can. J. Plant Pathol. 19:97-100, Un virus, provoquant des symptömes de feuille frisée et de mosaïque grave chez VAmaranthus tricolor, de même que des baisses de production de graines chez l'A hypochondriacus, a été identifié comme étant un isolat du virus de la mosaïque du concombre (CMV) en s'appuyant sur la transmission non persistante par des pucerons, l'observation de particules isométriques de 28 nm, ['evaluation par SDS-PAGE d'une masse moleculaire de 28 kd pour les sous-unités de la capside virale et une proximité sérologique avec la souche CMV-C. L'acide nucléique extrait de particules purifiées était de l'ARN monocaténaire infectieux. L'électrophorèse de l'ARN viral a montré que Ie genome était divisé et similaire a celui du CMV. The genus Amaranthus (Amaranthaceae) comprises valuable species used for grains, as vegetables or as ornamentals. On account of their nutritional and ornamental qualities, the amaranths have recently attracted the attention of research scientists world over (Iturbide & Lorence 1986, Bhag Mai 1994). Presently the grain amaranths are cultivated on a large scale in the Himalayas and the Andes mountains, since they form the staple diet of the inhabitants in these regions (Anonymous 1984, Joshi & Rana 1991). Joshi (1991) reported that around 60% of the nonirrigated areas were under its cultivation in the high North-West Himalayan region. Severe mosaic, leaf crinkle, and extreme stunting of Amaranthus tricolor L. and A. hypochondriacus L. (grain species) were observed in experimental fields at the National Botanical Research Institute, Lucknow. Cucumber mosaic virus (CMV) was suspected to be the responsible agent of the disease. CMV is known to infect experimentally numerous Amaranthus s p p . i n c l u d i n g A. tricolor and A. hypochondriacus (Douine et al. 1979; Horvath 1991a, b, c). A. deflexus, A. lividus, and A. retroflexus are known as natural hosts of CMV (Horvath 1991a). There is a single report of a mosaic disease of grain amaranth (A. hypochondriacus) caused by CMV (Ohashi & Kamiunten 1994). However, there is no record of naturally occurring CMV in Amaranthus tricolor. The disease has become important because it is responsible for a reduction in seed yield (75%) of

A. hypochondriacus as well as the total loss of the ornamental qualities of A. tricolor plants (Raj et al., unpublished data). Disease incidence was about 10% and 12% in amaranthus experimental fields at NBRI, Lucknow, in 1992 and 1993, respectively. The present communication reports studies on symptomatology, host range, mode of virus transmission, serology, coat protein, and nucleic acid of a virus isolate obtained from plants of these fields. Leaf samples of naturally infected A. tricolor obtained from amaranthus experimental fields, were used for the initial sap inoculations. The inoculum was prepared by grinding the infected leaf tissues in potassium phosphate buffer pH 7.5 containing 1% sodium sulphite (1:2 w/v). Mechanical transmission tests on 17 plant species (Table 1) were done using carborundum as an abrasive. The virus was inoculated to and maintained in A. hypochondriacus and Nicotiana tabacum L. cv. Samsun NN plants for further studies. The aphids, Myzus persicae (Sulze) and Aphis gossypi (Glove), used for insect transmission were given a preacquisition starvation period of 2 h, an acquisition period of 2 min, and an inoculation period of 2 h on 10 A. hypochondriacus plants. To test seed transmission, 200 seeds of artificially infected A. hypochondriacus were collected and germinated. The seedlings were observed during two months for disease symptoms and presence of virus was checked by double diffusion tests (Noordam 1973) using a CMV-C antiserum. 97

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CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 19, 1997

Table 1. Reaction of some plant species to the amaranthus isolate Symptoms


Host species Capsicum annuum L, cv. Pusa Jwala Chenopodium album L. C. amaranticolor Coste & Reyn C. murale L. Cucumis sativus Mill. cv. Sutton Long Green Lycopersicon esculentum L. cv. Pusa Ruby Nicotiana glutinosa L, N. rustica L. N. labacum L. cv. Samsun NN N, tabacum L. cv. White Burley N. plumbaginifolia Physalis minima L. Pisum sativum L. cv. Aparna Solanum melongena L. cv. Pusa Purple Long S. nigrum L. Spinacea oleracea L. Vigna unguiculata cv. C-152 Viciafaba L. cv. Sutton

Inoculated leaves

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Systemic

NLL NLL

-

CLL

SM

-

-

NLL NLL NLL

M LC&SM LD&SM

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-

-

SM

NLL CLL CLL

-

-

CLL, chlorotic local lesions; NLL, necrotic local lesions; LC, leaf crinkling; LD, leaf deformaton; M, mosaic; SM, severe mosaic ; -, no symptoms,

The virus isolate was purified from systemically infected N. tabacum cv. Samsum NN leaves by the method of Lot et al. (1972). Virus particles were observed by transmission electron microscopy using 2% uranyl acetate (pH 4.2) as negative stain. Photographs (EM Film 4489; Eastman Kodak, Rochester, NY) were taken and diameter measurements were done on 123 particles using TMV particles as length standard. To free coat protein subunits, virus particles were disrupted by the addition of an equal volume of 0.5% SDS, 0.1% 2-mercaptoethanol, and 0.02% bromophenol blue to the purified virus preparations and by boiling the mixtures for 3 min in a water bath. Molecular weight (m.w.) of the coat protein was estimated after SDS-PAGE (Laemmli 1970) using trypsinogen (24 kd), carbonic anhydrase (29 kd), glyceraldehyde-3-phosphate dehydrogenase (36 kd), egg albumin (45 kd), and bovine serum albumin (66 kd) of Sigma Chemicals (St. Louis, MO) as standards. In addition Western immuno-blot assays were performed. After SDS-PAGE, the protein bands were electroblotted to a nitrocellulose membrane and protein detection was performed as described (Renart & Sandoval 1984). CMV-C antiserum (0.5 mg/mL) and anti-rabbit lgG alkaline phosphatase conjugate (Sigma Chemicals) were used at dilutions of 1:1000.

Viral nucleic acid was extracted from purified particles by phenol-chloroform extraction and ethanol precipitation. The ethanol precipitate was suspended in 0.01M EDTA and stored at -20°C. The nucleic acid (20 ug) suspended in 50 uL water was mixed with DNAse (20 ug/mL), RNAse (20 ug/mL) and SI nuclease (50 jjg/mL) separately and incubated at 37°C for 30 min. Enzymes in these mixtures were thereafter removed by phenol-chloroform treatments and the nucleic acids reprecipitated by ethanol. Tests for infectivity of these nucleic acids, as well as untreated nucleic acid, were done in N. tabacum cv. Samsun NN and C. amaranticolor indicator plants. In order to know the number of species present in the viral genome, electrophoresis of the extracted nucleic acid was done in 1.2% TBE agarose gel under nondenaturing conditions (Sambrook et al. 1989). Crude sap samples from naturally infected A. tricolor were used in double diffusion tests to establish serological relationships of the virus isolate with some cucumoviruses, namely the CMV-C, D, S, L, T, and Pet (Srivastava et al. 1991) strains. The virus was transmitted easily by sap inoculations from A. tricolor to a number of plant species (Table 1) which developed local lesions or systemic symptoms. Experimentally infected A. tricolor and A. hypochondriacus plants developed symptoms of leaf deformation, leaf crinkle, leaf curling, stunting, and late flowering followed by reduced seed setting. The symptoms on A. hypochondriacus and A. tricolor were similar to those observed from field plants. However, some plant species (Chenopodium album L., Capsicum annuum L., Lycopersicon esculentum L., Physalis minima L., Pisum sativum L., Solanum melongena L., N. glutinosa L., and N. plumbaginifolia) neither developed symptoms after virus inoculation nor could virus be detected by back inoculations to C. amaranticolor. They were regarded as nonhosts of the amaranthus isolate. Aphid transmission tests revealed that the aphids M. persicae and A. gossypi successfully transmitted the virus in a nonpersistent manner. Symptoms in A. hypochondriacus (6/10) plants were identical to those obtained after sap inoculations. In the seed transmission tests, nearly 11% of the seedlings showed symptoms of mosaic disease, whereas serology indicated the presence of the virus in 16.5% (33/200) of the seedlings. The high rate of seed transmission of the amaranthus isolate in A. hypochondriacus may play an important role in the epidemics of CMV. Electron microscopy of purified virus preparations revealed the presence of polyhedral virus particles of about 28.0 (± 0.4) nm in diameter. This diameter is identical to the recognized 28 nm diameter of CMV particles (Francki et al. 1979).


RAJ ET AL.: CMV/AMARANTHUS

SDS-PAGE and Western immuno-blot analysis revealed the presence of one band corresponding to a 26 kd coat protein in naturally infected A. tricolor as well as in artificially inoculated A. hypochondriacus plants. Another band of 52 kd, which seems to be a dimer of the 26 kd protein, was also observed. The 26 kd m.w. is close to the accepted 24.5 kd m.w. of the CMV coat protein (Francki et al. 1979). The nucleic acids isolated from virus particles remained infectious when treated with DNAse. Treatment with RNAse and SI nuclease abolished the infectivity completely. These results indicated that single stranded RNA (ssRNA) present in the virions was responsible for infectivity. During electrophoresis of the extracted nucleic acids, RNA species separated as three distinct bands which were identified as RNA 1 and RNA 2 altogether, RNA 3 and RNA 4. RNA 1 and 2 were very close and did not separate as two distinct bands (Fig. 2). The pattern of four RNA species was similar to the one usually observed for CMV (Francki et al. 1979). There was one more band of about 650 bases which was near the bromophenol blue front. It could well be a satellite RNA as the one observed by Ohashi & Kamiunten (1994) in a CMV isolate from A. hypochondriacus. Double diffusion tests showed serological relationships between the amaranthus isolate and CMV-C and D (Fig. 1) but not between the amaranthus isolate and CMV-L, S, T, and Pet strains. The isolate infecting A. tricolor and A. hypochondriacus was more closely related to CMV-C than to CMV-D.

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Figure 2. RNAs of the amaranthus isolate after 1.2% agarose gel electrophoresis. M: ribosomal RNA 23 S and 16 S from E. coli and 1: RNAs extracted from the particles of the amaranthus isolate.

On the basis of aphid transmission in a nonpersistent manner, presence of isometric particles of 28 nm diameter coat protein subunits of 26 kd in SDSPAGE, multiple ssRNA species in nucleic acid preparations and serological relationships with CMVC and D strains in gel double diffusion tests, the amaranthus isolate was identified as a CMV isolate. The two species A. tricolor and A. hypochondriacus were already known to be susceptible to CMV when artificially inoculated. This time these species were found as new natural hosts of CMV in India. This is the first report of a natural infection of A. tricolor by CMV. The virus is vertically transmitted by infected seeds to the next generation or through aphids from one host to another. The amaranthus plants might act as a potential reservoir of the virus. Thanks are due the Director, National Botanical Research Institute, Lucknow, for providing facilities. We acknowledge the gifts of antisera from the late R.I.B. Francki, Australia (CMV-T); and from A.A. Zaidi, CSIR Complex, Palampur (CMV- C,D,S,L), who obtained them from H.A. Scott, USA.

Figure 1. Gel double-diffusion tests using antisera of different CMV strains (C,D,Pet,T,S,L) in peripheral wells and crude sap from infected A. tricolor in the central well (A).

Anonymous. 1984. Amaranth: modern prospects for an ancient crop. National Academy Press. Washington, D.C. 80 pp. Bhag Mai. 1994. Underutilized grain legumes and pseudocereals — their potential in Asia. RAPA (FAO) Bangkok, Thailand. 162 pp.


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Douine, L., J.B. Quiot, G. Marchoux, and P. Archange. 1979. Recensement des espèces végétales sensibles au virus de la mosaïque du concombre (CMV). Étude bibJiographique. Ann. Phytopathol. 11:439-473. Francki, R.I.B., D.W. Mossop, and T. Hatta. 1979. Cucumber mosaic virus. Descriptions of Plant Viruses, No. 213. Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England. 6 pp. Horvath, J. 1991a. Amaranthus bouchonii Thell. (Family: Amaranthaceae) a new adventive plant species in Hungary, and its reaction to some viruses. Acta Phytopathol. Entomol. Hung. 26:371-377. Horvath, J. 1991b. Amaranthus mangostanus L., A. mitchelli Benth. and A. quitensis H.B.K. (Family: Amaranthaceae) as new virus hosts. Acta Phytopathol. Entomol. Hung. 26:379-383. Horvath, J. 1991c. Amaranthus species (Family: Amaranthaceae) as hosts of plant viruses: a review. Acta Phytopathol. Entomol. Hung. 26:285-422. Iturbide, G.A., and F.G. Lorence. 1986. Cultivo del amaranto en Mexico. Coleccion cuadesnos universitarios serie agronomia. Universidad Autonoma Chapingo, Mexico. 12:18-245. Joshi, B.D. 1991. Exploitation for amaranth in North-West India. IBPGR, FAO, Rome, Italy. Plant Genetic Resources Newsletter 48:41-52.

Joshi, B.D., and R.S. Rana. 1991. Grain amaranths: the future food crop. National Bureau of Plant Genetic Resources (ICAR), New Delhi. NBPGR - Shimla Science Monograph 3, 152 pp. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685. Lot, H., J.B. Marrou, and C. Esvan. 1972. Contribution a l'étude du virus de la mosaïque du concombre (CMV). II. Methode de purification rapide du virus. Ann. Phytopathol. 4:25-38. Noordam, D. 1973. Identification of plant viruses. Methods & Experiments. Oxford & IBH Publishing Co., New Delhi. 207 pp. Ohashi, M., and H. Kamiunten. 1994. Mosaic disease of grain amaranth {Amaranthus hypochondriacus L.) caused by cucumber mosaic virus (CMV). Ann. Phytopathol. Soc. Japan. 60:119-121. Renart, J., and I.V. Sandoval. 1984. Western blots. Meth Enzymol. 104:455-460. Sambrook, J., E.F. Fritsche, and T. Maniatis. 1989. Molecular cloning - A Laboratory Manual (2nd Edition), Cold Spring Harbor Laboratory, New York. Srivastava, A., G. Chandra, R.K. Raizada, K.M. Srivastava, and B.P. Singh. 1991. Characterization of a virus causing necrosis in Petunia hybrida and establishment of its relationship using ELISA. Ind. J. Exp. Biol. 29:591-593.