Maize stripe virus: Characteristics of a member of a new virus class

VIROLOGY

112,99-PO8

(1981)

Maize

Stripe

Virus: Characteristics a New Virus Class’

R. E. GINGERY: Agricultural Research, and Departments

L. R. NAULT,

AND

Science and Education Administration, of Entomology and Plant Pathology, and Development Center, Wooster, Accepted

January

of a Member

of

0. E. BRADFUTE U. S. Department Ohio Agricultural Ohio 44691

of Agriculture, Research

20, 1981

An unusual filamentous nucleoprotein about 3 nm in diameter was consistently assoIciated with maize stripe-diseased maize. Antiserum to purified nucleoprotein neutralized .the infectivity of extracts from maize stripe-diseased plants suggesting that the nucleoprotein was the maize stripe virus (MStpV). The rate-zonal sedimentation pattern of the nucleoprotein on sucrose gradients was polydisperse between 51 and ‘70 S. CsCl isopycnic centrifugation of combined nucleoprotein zones from sucrose gradients resulted in a single band of 1.28 g/ml. The nucleoprotein consisted of 5.2% RNA and a single capsid protein of molecular weight 32,700 daltons. Large quantities of a noncapsid protein of molecular weight 16,300 daltons were also found in MStpV-infected tissue. MStpV was transovarially transmitted by its vector, Peregrinus maidis. Other species susceptible to MStpV in addition to Zea mays were Rottboellia exaltata and Sorghum bicolor. The similarities between MStpV and rice stripe virus are discussed. We conclude that these viruses represent a new virus class.

examinations of diseased plants from Florida or test plants serially inoculated with MStpV from Florida failed to reveal such particles. Closer scrutiny of the East African and Venezuelan reports revealed that in neither case was the pathogenicity of the isometric particles established. Furthermore, the MStpV antiserum from East Africa (Kulkarni, 1973) was prepared against material from sucrose gradients identified only as a light-scattering band that was not shown to contain isometic particles. Therefore, considerable doubt exists that MStpV is an isometric particle. In this report we describe a fine-stranded nucleoprotein that is consistently found in MStpV-infected plants and present evidence relating it to MStpV.

INTRODUCTION

A viruslike disease of sweet corn (Zea mays L.), caused by a planthopper [Peregrinus maidis (Ashmead)]-transmitted agent, was discovered in Florida in 1974 (Tsai, 1975). The symptoms of this disease resembl.ed those caused by the maize stripe virus (MStpV) from East Africa (Kulkarni, 1973), and a serological relationship between the Florida pathogen and MStpV was subsequently shown (Gingery et al., 1979). MStpV has also been identified serologically from Venezuela (Gingery et al., 1979) and Peru (Nault et al, 1979). Kulkarni (1973) reported isometric viruslike particles associated with MStpV-diseased plants from East Africa and Trujillo et al. (1974) reported a similar finding for the Venezuelan disease. However, repeated

MATERIALS

AND

METHODS

Virus source and propagation. We used the MStpV isolate collected by Tsai (1975) from naturally infected sweet corn in Florida. For routine production of infected tissue, P. maidis were given a 4- to 7-day access period on infected plants, held on

1 Cooperative investigation of AR, SEA, USDA, and Ohio Agricultural Research and Development Center. Approved for publication as Journal Article No. 113-80 of the Ohio Agricultural Research and Development Center, Wooster. ’ To wh,om reprint requests should be addressed. 99

0042-6822/81/090099-10$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

100

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corn plants for l-2 weeks for virus incubation in vectors, and then transferred at the rate of 10 insects per two sweet corn test plants (Aristogold Evergreen Bantam) for a 3- to 4-day inoculation access period. Rearing of planthoppers has been described (Gingery et al., 1979). Purification of nucleoprotein. Nucleoprotein was purified as follows: 3 to 30 g of infected maize leaf tissue were ground in buffer using 10 ml buffer/g tissue. Buffers were 0.5 M potassium phosphate, 0.01 M EDTA, pH 7.0; 0.1 M potassium phosphate, 0.01 M EDTA, pH 7.0; 0.15 M NaCl, 0.01 M potassium phosphate, 0.01 M EDTA, pH 7.0 (PBS-EDTA); or 0.1 M NaCl, 0.05 M Tris, 0.01 M EDTA, pH 7.0. Buffers were supplemented with 0.5% 2-mercaptoethanol (ME) and 200 pg/ml bentonite just before grinding. After grinding, the extract was squeezed through cheesecloth and clarified by emulsification with onethird volume CHC13. The clarified extract was centrifuged for 3 hr at 10” at 35,000 or 40,000 rpm (Beckman3 Type 35 or Type 42.1 rotor, respectively). The resulting pellet was resuspended in extraction buffer and sedimented on lo-40% sucrose density gradients at 10” for 2-3 hr in the Beckman SW 50.1 or 3-4 hr in the Beckman SW 41 rotor at 40,000 rpm. The nucleoprotein zones were pooled, adjusted to a density of 1.28 g/ml with solid CsCl, and isopycnically banded by centrifuging for 48-72 hr at 10” at 35,000 rpm in the SW 50.1 or SW 41 rotor. Nucleoprotein from CsCl gradients was considered purified. All solutions and glassware were autoclaved or flamed to inactivate ribonuclease. Samples at Electron microscopy. various stages of purification were mounted on Formvar-coated grids and stained with phosphotungstic acid neutralized with KOH. Nucleic acid analysis. Purified nucleoprotein was hydrolyzed in 0.4 M NaOH for 24 hr at 37”. The hydrolyzate was neu3 Mention of a trademark or proprietory product does not constitute a guarantee or warranty of the product by the USDA, nor does it imply approval to the exclusion of other products that may also be suitable.

AND

BRADFUTE

tralized with 4 M HCl, spotted on ammonium sulfate-impregnated Whatman No. 3MM filter paper, and developed by descending chromatography (Lane, 1973) with 70% ethanol as the developing solvent. Nucleotides were located with ultraviolet light, eluted in 0.01 MHCl, and their concentrations determined using the extinction coefficients of Knight (1963). Purified nucleoprotein was tested for RNA by orcinol (Shatkin, 1969) and for DNA by diphenylamine (Burton, 1956). Phosphorus content was determined by the method of Nakamura (1952). Polyacrylamide gel electrophwesis. Protein was released from nucleoprotein by boiling for 1 min in 0.01 M sodium phosphate, 1% sodium dodecyl sulfate (SDS), 1% ME, pH 8.0. Reference proteins (bovine serum albumin, ovalbumin, and pancreatic ribonuclease) were treated similarly. Electrophoresis was carried out by the procedure of Weber and Osborn (1969) except that 7.5% polyacrylamide gels were used and gel tops were formed by trimming gels to 6 cm with a scalpel. Stained gels were scanned in the Instrumentations Specialties Company (ISCO) (Lincoln, Nebr.) Model 659 gel scanner. Buoyant density. Combined nucleoprotein zones from sucrose gradients were dialyzed against PBS-EDTA and adjusted to a density of 1.28 g/ml with solid CsCl. After centrifugation for 60-65 hr at 36,000 rpm in the Beckman SW 50.1 rotor at 20”, the gradients were fractionated in the ISCO Model 640 density gradient fractionator. Densities of 0.4-ml fractions were determined by weighing 50-~1 aliquots in a micropipet previously calibrated with water. Gradient density at the peak of nucleoprotein concentration was considered to be the nucleoprotein density. Preparation of antiserum. Purified nucleoprotein or noncapsid protein was suspended in physiologically buffered saline (0.15 M NaCl, 0.01 M potassium phosphate, pH 7.4) emulsified with an equal volume of Freund’s complete adjuvant, and injected into lymph nodes in the hind legs of rabbits. About 1 mg in 1 ml of emulsion was injected weekly for 4 weeks. Rabbits were bled weekly (before, during, and for

MAIZE

STRIPE

3 weeks after the injection schedule) by nicking the marginal ear vein. Serum was recovered, mixed with an equal volume of glycerol, and stored at -20”. Determination of thermal inactivation point, dilution endpoint, longevity in vitro, and neutralization of infectivity. Virus for testing was extracted from infected leaf tissue by grinding in PBS-EDTA containing 0.5% ME and 200 pg/ml bentonite and the extract was clarified with CHC& or by centrifugation for 30 min at 12,000 g at 4”. For thermal inactivation point determinations, CHCl,-clarified extracts were held in a water bath at various temperatures for 10 min, cooled in ice, centrifuged to remove any coagulated material, and then were injected into P. maidis. For longevity in vitro studies, CHCl,-clarified extracts were held at room temperature (23-26”) and 4” for various times before injection. To estimate the dilution endpoint, dilutions (in PBS-EDTA) of CHCl,-clarified extracts were injected. For neutralization of infectivity studies, aliquots of centrifuge-clarified extracts were combined with equal amounts of preimmune serum or MStpV antiserum, incubated for 30 min at 4”, and then injected into P. maidis. Planthoppers were lightly anesthetized with CO, and held by vacuum on a microscope stage before injection. Mechanically pulled glass needles which delivered approximately 0.025 ~1 were used to inject planthoppers abdominally, After injection, planthoppers were held on plants for lo-12 days, and then were transferred singly to test plants for a 3- to 4-day inoculation access period. Test plants were held in a greenhouse for 14 days before results, based on symptoms, were recorded. Transovarial transmission. Eggs from P. maidis, confirmed as vectors, were dissected from corn leaves and placed on healthy corn leaf pieces in petri dishes. Dishes were held at 18” in an environmental chamber for 3-6 days until all eggs hatched. Nymphs were then placed sequentially on two series of test plants for 7 days each. Test plants were held in a greenhlouse for 2 weeks for symptom development.

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Purification of noncapsid protein crystals. MStpV-infected tissue was ground in a phosphate-citrate buffer, pH 5.5 (1 g tissue/3 ml buffer). Phosphate-citrate buffers of various pH’s were prepared by combining 0.2 M K2HP04 and 0.1 M citric acid. If crystals were observed by phase-contrast light microscopy, the extract was centrifuged at 12,000 g for 5 min. Pelleted crystals were dissolved in phosphate-citrate, pH 7.0 (1 ml/g tissue). This suspension was centrifuged at 12,000 g for 20 min. The pH of the supernatant was then lowered by adding an equal volume of phosphate-citrate, pH 3.0, and immediately centrifuged (40,000 rpm for 30 min at 10” in the Beckman 42.1 rotor). The supernatant was held at 4” overnight during which time recrystallization occurred. Crystals were pelleted (12,000 g for 15 min), washed two times with phosphate-citrate, pH 5.5, and adjusted to a density of 1.27 g/ml with solid CsCl. After overnight centrifugation at 34,000 rpm at 20” in the Beckman SW 50.1 rotor, the isopycnically banded crystals were removed with a syringe. Host range. Viruliferous P. maidis, prepared by placing early-instar nymphs on MStpV-infected corn for 3 weeks were placed 10 per test plant species for a 3-day inoculation access period. Sweet corn was included in each test as a vector-inoculativity check. Symptoms were recorded after 14 days. Agar-gel double-diffusion assays were performed as described previously (Gingery et al., 1979). RESULTS

PurQication

of Nucleoprotein

The extraction of nucleoprotein from maize stripe-infected maize leaf tissue was accomplished with several buffers (see Materials and Methods). The best were 0.1 M potassium phosphate, 0.01 M EDTA, pH 7.0, and 0.15 M NaCl, 0.01 M potassium phosphate, 0.01 M EDTA, pH 7.0. Emulsification with CHC13 was the most reliable clarification method. Other methods tested were heating to 50”, adjustment to pH 5.0, centrifugation (1 hr at 12,000 g), and the addition of butanol to 8%. Nucleoprotein concentration from clar-

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NAULT.

ified extracts by polyethylene glycol precipitation (optimum tested was 6% PEG, 4% KCl), resulted in yields lo-80% of those obtained by high-speed centrifugation. Concentration by precipitation with 25% ethanol or 50% saturated (NH&SO4 was unsuccessful. Nucleoprotein recovery from leaves was about twice as high as from roots. Little nucleoprotein was recovered from stems, leaf midveins, or symptomless regions of infected plants. The age of the tissue between about 1 and 6 weeks after inoculation had little influence on yield. Yields of nucleoprotein from frozen tissue were about 20% of yields from fresh tissue, which ranged from about 80 to 120 mg purified nucleoprotein/kg fresh tissue in the best preparations. Figure 1 shows typical ultraviolet absorption scans of centrifuged sucrose gradients layered with nucleoprotein preparations from healthy and MStpV-infected plants. The number of peaks varied from three to six, depending on the number of minor ones resolved. Isopycnic centrifugation of combined sucrose peaks in CsCl gradients resulted in a single peak of density 1.28 g/ml that accounted for all absorbance from the combined peaks. Characteristics of Maize Stripe Nucleoprotein The ultraviolet absorption spectrum of purified maize stripe nucleoprotein had a

SEDIMENTATION-

FIG. 1. Sedimentation of MStpV. Virus concentrated from a clarified extract was layered on a lo40% (w/w) linear sucrose gradient in PBS-EDTA. Sedimentation was in the Beckman SW 50.1 rotor at 45,000 rpm at 10” to 20,000 X lo7 rad’/sec (ca. 2.5 hr). The dashed line denotes the absorbance of a similar preparation from healthy tissue.

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maximum absorbance at 261-263 nm and minimum at 244-246 nm. There was no apparent tyrosine-tryptophan shoulder at 288 nm. The 260/280 ratio was 1.39 + 0.04. Fine-stranded material about 3 nm wide was found by electron microscopy of sucrose density gradient zones (Fig. 2), but it was not sufficiently dispersed to determine strand length or possible secondary or tertiary structure. Material viewed after high-speed centrifugation or CsCl isopycnic centrifugation was essentially similar. Positive reactions of purified nucleoprotein with orcinol and negative reactions with diphenylamine indicated that the nucleic acid was RNA. The nucleotide composition was estimated to be Ap, 26.3 -t l.l%, Up,37.8 f 1.3%, Gp 18.1 & 0.9%, and Cp 17.8 f 0.8%, based on three separate determinations. No species with Rf values different from authentic Ap, Up, Gp, or Cp were detected. The base ratios indicated that the RNA is probably single stranded. The nucleoprotein contained 0.51 rfr 0.3% phosphorus based on analysis of three separate preparations, each exhaustively dialyzed against water to eliminate buffer phosphorus. Based on the nucleotide ratios, the RNA contained 9.72% phosphorus by weight. From these data, the nucleoprotein contained 5.2% + 0.3% RNA. The percentage RNA was also estimated by determining the amount of protein (Lowry et al., 1951) and RNA (orcinol analysis) in purified nucleoprotein. The mean of two such analyses was 6.1% RNA. Sedimentation coefficients of the various nucleoprotein zones were estimated on linear-log sucrose gradients by the method of Brakke and Van Pelt (1970). Maize chlorotic dwarf virus (S = 183) (D. T. Gordon, personal communication) and an Ohio isolate of cucumber mosaic virus (S = 95) (D. T. Gordon, personal communication) were used as standards. Values ranged from 51 S for the slowest peak to 70 S for the fastest. The extinction coefficient of the nucleoprotein was estimated after exhaustive dialysis against distilled water. From the absorbance of the dialyzed preparation and the mass of nucleoprotein remaining

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STRIPE

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FIG. 2. Electron micrograph of negatively stained maize stripe nucleoprotein from sucrose density gradient zone showing fine strands, appproximately 3 nm wide (F). For comparison, note PVY-type flexuous rod (maize dwarf mosaic virus, strain B) (arrow) and phytoferritin molecules [light rings with dense centers approximately 11 nm in diameter (Hyde et al., 1963)]. The MDMV-B particle was introduced as a size and resolution standard; phytoferritin was frequently found in preparations at this level of purity. Magnification bar is about 100 nm long.

after lyophilization of a known amount of this preparation, the calculated extinction coefficient was 2.3 f 0.1 cm’/mg at 260 nm. This value is consistent with the estimated RNA content (56%). Protein released from purified nucleoprotein preparations migrated on SDSpolyacrylamide gels as a single protein species of molecular weight 32,700 & 500 daltons (Fig. 3A). Demonstration

of Pathogenicity

of Nu-

cleoprotein

Numerous attempts to mechanically transmit MStpV by air brush, pin pricking, or leaf rubbing with a variety of buffers were unsuccessful. The first transmission, other than from plant to plant by P.

maidis, were made after injection of P. maidis with crude extracts from MStpVinfected plants. In preliminary experiments, bentonite (200 pg/ml) in the extraction buffer raised the rate of transmission (transmitting insects/total insects injected) from 0.26 to 0.54; 2-mercaptoethan01 (0.5%) was essential for transmission. Both additives were used in all later injections. Results of experiments in which both infectivity and virus recovery were determined at various stages of purification are summarized in Table 1. Infectivity decreased markedly after high-speed centrifugation and was lost during subsequent purification steps. Transmission after high-speed centrifugation was greatly improved if the virus was centrifuged through a 40% sucrose cushion. The great-

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CA

RNase 05 E J 6 u‘ 2s

BRADFUTE

fraction of insects that transmitted MStpV at various temperatures were: 35/129 at 4”; 43/155 at 35”; 11/125 at 40”; l/157 at 45”; and O/99 at 50”. In longevity in vitro determinations, no infectivity remained after 24 hr at room temperature, but extracts kept at 4” retained infectivity up to 3 days. Dilution endpoints of initial extracts were about 10-z. Treatment of extracts with RNase (25 pg/ml, 37”, 30 min) eliminated infectivity; incubation at 37” without RNase did not.

hwpv

IO-

AND

I

Host Range

I

2

3

4

5

6

DEPTH,cm

FIG. 3. Migration of MStpV protein and noncapsid protein in polyacrylamide gel electrophoresis. BSA, Bovine serum albumin; OVA, ovalbumin; CA, carbonic anhydrase; RNase, ribonuclease; MStpV, protein released from MStpV nucleoprotein. Molecular weights used were BSA, 68,000; OVA, 43,000; CA, 29,000; and RNase, 13,700 (Weber and Osborn, 1969). Proteins were electrophoresed individually (not shown) and together in parallel gels. (A) MStpV and (B) noncapsid protein molecular weight determinations from two separate experiments. The small peak in (B) at 3.5 cm (molecular weight 27,000-27,500) was also present in gels containing only RNase and was assumed to be an RNase dimer.

est physical losses of virus (75-90%) also occurred after high-speed centrifugation steps. Attempts to improve recovery by varying the time or force of centrifugation had little effect. Further evidence for pathogenicity of the nucleoprotein was obtained by neutralization of infectivity. In five separate experiments, a total of 40/149 (26.8%) planthoppers injected with centrifugecleared extracts transmitted MStpV. Transmission after treatment of the extract with preimmune serum was 33/132 (25.0% ), but O/147 transmitted MStpV after treatment with antiserum to purified nucleoprotein. Properties of MStpV Determined by Injection of P. maidis The thermal inactivation point of MStpV was about 40”. From five experiments, the

The following species were tested as hosts of the U. S. isolate of MStpV (the fractions are the number of plants with symptoms over the number of plants inoculated): Coix lacryma-jobi L., O/5; Oryxa sativa L., O/30; Paspalum notatum Fluegge, O/5; Rottboellia exaltata L., 7124; Sorghum bicolor (L.) Moench, l/27; S. halepense (L.) Pers., O/30; Tripsacum australe Cutler and Anderson, O/5; T. dactyloides L., O/9; and T. latifolium Hitchc., O/4. In these experiments, all maize plants used to check vector inoculativity became infected. In addition, four R. exaltata plants with maize stripe symptoms collected in January 1979 near Homestead (Dade Co.), Florida, were positive for MStpV by agar-gel double-diffusion assay. Transovarial

Transmission

Forty-six of seventy-nine (58.2% ) nymphs from eight viruliferous females transmitted MStpV, establishing transovarial passage of virus from adults to progeny. Noncapsid

Protein

We found large amounts of needleshaped crystals in infected tissue, but not in healthy tissue. The crystals were easily manipulated because they readily dissolved at about pH 6.0 and recrystallized below pH 6.0 (see Materials and Methods). Figure 4 shows purified crystals. Crystal preparations were markedly birefringent. The crystals were composed of a single protein of molecular weight 16,500 + 300 daltons (Fig 3B). This value is almost ex-

MAIZE

STRIPE TABLE

RECOVERY

Purification

AND TRANSMISSION

step”

Initial extract Clarified extract Resuspended pellet after high-speed centrifugation Resuspended pellet after high-speed centrifugation (cushion)f Supernatant after highspeed centrifugation Isolated virus zone from sucrose gradient Resuspended pellet after high-speed centrifugation (cushion)

1

OF MStpV

MStpV in sample as percentage of virus in starting material*

105

VIRUS

AT STEPS IN PURIFICATION Transmission Number of experiments

by injected

Peregrinus

maidis

Rate”

Ranged (%I

100 74.6

37 5

394/1057 29/92

14-67 18-53

ND”

5

l/154

o-4

14.0

3

12/84

-Q

1

O/26

6.7

3

O/67

0.9

ND

11-19

u Purification was performed as described under Materials and Methods. bThe amount of virus at each step was estimated by multiplying the volume of preparation times the dilution endpoint as determined serologically by a microprecipitin assay performed as described by Knoke et al. (1974). For each sample a twofold dilution series was tested against a 1:8 dilution of antiserium to purified nucleoprotein. Results given are the average of three experiments. ‘In each experiment approximately 40 P. maidis were injected. The fraction is the number of insects that transmitted MStpV to test maize plants over the number of injected insects that were placed on test plants. d The range of transmission among individual experiments. “Not done. fA 40% sucrose cushion was placed in the bottom of the centrifuge tube. o No virus detected

actly one-half that of the capsid protein (32,700 +- 500 daltons), but there was no serological reaction between nucleoprotein antiserum and crystal protein or between crystal protein antiserum and nucleoprotein. Noncapsid protein had a typical protein absorption spectrum with a maximum at 278-279 nm, a minimum at 254-255 nm, and a 260/280 ratio of 0.62 f 0.02. The density of the crystals determined ‘by isopycnic CsCl centrifugation was 1.27 g/ml. By weighing lyophilized preparations from known amounts of tissue, we estimated the crystal concentration to lbe as high as 2 mg/g tissue. Their function is not known. DISCUSSION

The maize stripe virus fine-stranded, filamentous

seems to be a particle about

3 nm in diameter and of uncertain length. The only other virus of similar morphology is the rice stripe virus (REX) (Koganezawa et al., 1975; Koganezawa, 1977). Besides morphology, other similarities between MStpV and RSV include: (1) transovarial transmission by delphacid planthoppers, (2) slow, heterodisperse sedimentation in sucrose density gradients, (3) single-stranded RNA, (4) single species of capsid protein, and (5) large quantities of a noncapsid protein in infected leaves. Coincidentally, before the discoveries of the fine-stranded nucleoproteins, both MStpV (Kulkarni, 1973) and RSV (Koganezawa, 1977; Kitani and Kiso, 1968) were reported to have isometric morphology. In preliminary experiments, RSV antiserum reacted with MStpV-infected extracts (Gingery, unpublished). In view of the similarities between MStpV

106

GINGERY,

FIG. 4. Phase-contrast infection. This material bar represents 10 pm.

micrograph was purified

NAULT,

of recrystallized through CsCl

and RSV, and their unusual morphology, it is highly likely that they are members of a new class of viruses. One difference between the two viruses is the isolation of a branched filamentous particle for RSV, but not for MStpV. This particle is thought to be a slender (3 nm) filament in a super coiled configuration (Koganezawa, 1977). Also, infectivity has been preserved throughout purification for RSV, but not for MStpV. It may be that a branched filamentous configuration is essential for infectivity and, therefore, the loss of infectivity during purification of MStpV reflects the loss of such a structure. In any event, MStpV is labile, as shown by the increase in infectivity after minimizing RNase activity, incorporating antioxidants, and concentrating through a sucrose cushion, and by the loss of infectivity after treatment of infective extracts with RNase. The foregoing discussion assumes that the fine-stranded, filamentous nucleoprotein is the MStpV, or at least some form of the virus, which to us is the most likely hypothesis. Although we have not obtained infectious, purified virus (probably

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noncapsid protein associated with MStpV as outlined under Materials and Methods. The

because of extreme particle lability) and established the infectivity of the nucleoprotein directly, we do have evidence indirectly linking the nucleoprotein with the infective agent: (1) the infectivity of otherwise infectious extracts is specifically neutralized by antiserum to purified nucleoprotein, (2) partially purified nucleoprotein is infective, and (3) there is a serological relationship between maize stripe nucleoprotein and RSV, whose infectivity has been established. The different nucleoprotein zones after rate-zonal centrifugation probably represent various stages of breakage, aggregation, or configuration of essentially similar material because the contents of all zones have the same density and the combined zones contain only one protein. Koganezawa (1977) reported that for RSV the slow-sedimenting zone contained circular and linear strands about 800 nm long, and the middle and bottom zones contained some circular particles about 800 nm in contour length, but mostly branched, filamentous particles. We can not yet offer a similar explanation for the polydisperse sedimentation of MStpV, because we have

MAIZE

STRIPE

seen no clear-cut morphological differences among the contents of the different zones. Although particle morphology and sedimentation are unusual for MStpV and RSV, there is nothing particularly novel about other properties of either virus or the diseases they cause including other chemical and physical properties of the viruses, their relationships with their vectors, and the symptomatologies and disease development resulting from infection. We have confirmed sorghum, S. bicolor, and itchgrass, R. exaltata, as MStpV hosts. It may be epiphytologically significant that the tropical weed, R. exaltata, is a host for both P. maidis (Nault, unmb&shed) and MStpV, because it may serve as a virus reservoir between maize crops. Other species susceptible to a maize stripelike disease in West Africa are Brachiaria dejlexa, Hyparrhenia dissoluta, andSetaria sp. (Fajemisin and Shoyinka, 1977). The maize stripe virus may be common and of world wide distribution. In addition to the ypreviously noted reports of MStpV from the United States, Venezuela, Peru, and Africa, MStpV or pathogens that produce similar symptoms and have P. maidis vectors have been reported from the Philippines (Exconde, 19’77), Mauritius (Ricaud and Felix, 1976), and Australia (Grylls, 1979). Despite this widespread distribution, the disease probably poses no immediate threat to maize production in temperate regions because P. maidis has a tropical distribution. (Anon., 1973). ACKNOWLEDGMENTS The authors wish to acknowledge the capable assistance of Mrs. Julie Bamberger, Miss Dru Grant, and Mr. William Styer. The RSV antiserum was generously supplied by Drs. S. Yamashita and H. Koganazawa. This work was supported in part by Grant PCM7922663 from the National Science Foundation. REFERENCES Anonymous. (1973). Pest: Peregrinus maicEis (Ashm.). In “Distribution Maps of Pests,” Map 317. Commonw. Inst. Entomol. Ser. A (Agric.). BRAKKE, M. K., and VAN PELT, N. (1970). Linear-log sucrose gradients for estimating sedimentation

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coefficients of plant viruses and nucleic acids. Anal. Biochem. 38,56-64. BURTON, K. (1956). A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. EXCONDE, 0. R. (1977). Viral diseases of maize and national programs of maize production in the Philippines. In “Proceedings Maize Virus Disease Colloquium Workshop” (D. T. Gordon, L. R. Nault, and L. E. Williams, eds.), pp. 83-88. Ohio Agric. Res. and Dev. Center, Wooster. FAJEMISIN, J. M., and SHOYINKA, S. A. (1977) Maize streak and other maize virus diseases in West Africa. In “Proceedings Maize Virus Disease Colloquium Workshop” (D. T. Gordon, L. R. Nault, and L. E. Williams, eds.), pp. 52-61. Ohio Agric. Res and Dev. Center, Wooster. GINGERY, R. E., NAULT, L. R., TSAI, J. H., and LASTRA, R. J. (1979). Occurrence of maize stripe virus in the United States and Venezuela. Plant Dis. Rep. 63, 341-343. GRYLLS, N. E. (1979). Leafhopper vectors and the plant disease agents they transmit in Australia. In “Leafhopper Vectors and Plant Disease Agents” (K. Maramorosch and K. Harris, eds.), pp. 179-214. Academic Press, New York. HYDE, B. B., HODGE, A. J., KAHN, A, and BIRNSTIEL, M. L. (1963). Studies on phytoferritin: I. Identification and localization. J. Ultrastruct. Res. 9, 248258. KITANI, K., and KISO, A. (1968). Studies on rice stripe disease: Part 1. Purification of rice stripe virus. Bull. Shikoku Agr. Exp. Sta. lS, lOl-116. KNIGHT, C. A. (1963). Protoplasmatolgia Band IV. In “Chemistry of Viruses,” p. 81. Springer, Vienna. KNOKE, J. K., LOUIE, R., ANDERSON, R. J., and GORDON, D. T. (1974). Distribution of maize dwarf mosaic and aphid vectors in Ohio. Phytoputhology 64, 639-645. KOGANEZAWA, H. (1977). Purification and properties of rice stripe virus. In “Symposium on Virus Diseases of Tropical Crops, Tropical Agriculture Research Series No. 10,” pp. 151-154. Tropical Agriculture Research Center, Ministry of Agriculture and Forestry, Kitanakazuma Yatabe-cho, Tsukuba-gun, Ibaraki-ken, 300-21, Japan. KOGANEZAWA, H., DOI, Y., and YORA, K. (1975). PUrification of rice stripe virus. Ann. PhytopathoL Sot. Jpn. 41, 148-154. KULKARNI, H. Y. (1973). Comparison and characterization of maize stripe and maize line viruses. Ann. Appl. Biol. 75, 205-216. LANE, B. G. (1963). The separation of adenosine, guanosine, cytidine, and uridine by one-dimensional filter paper chromatography. Biochim. Biophys. Acta 72,110-112. LOWRY, O., H., ROSENBROUGH, N. J., FARR, A. L., and

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RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193,265275. NAKAMURA, G. R. (1952). Microdetermination of phosphorus. Anal. Chem. 24,1372. NAULT, L. R., GORDON, D. T., GINGERY, R. E., BRADFUTE, 0. E., and CASTILLO-LOAYZA, J. (1979) Identification of maize viruses and mollicutes and their potential insect vectors in Peru. Phytopathology 69, 824-828. RICAUD, C., and FELIX, S. (1976). Identification et importance relative des viroses du mais a l’ile Maurice. Rev. Agr. Suer. Ile Maurice 55,163-169. SHATKIN, A. J. (1969). Calorimetric reactions for

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