Serological Detection of Corn Stunt Spiroplasma and Maize

total of 100 samples. Leaves were Gordon, D. T., Nault, L. R., Gordon, N. H., and Heady, S. E. 1985. Serological detection of corn collected without regard to disease stunt spiroplasma and maize rayado fino virus in field-collected Dalbulus spp. from Mexico. Plant symptoms. Control leaf tissues were Disease 69:108-111. healthy and MRFV-infected maize maintained in the greenhouse at Wooster. Corn stunt spiroplasma (CSS) experimentally acquired from maize (Zea mays) was detected by enzyme-linked immunosorbent assay (ELISA) in extracts from individual as well as groups of five Preparation of extracts. Individual or Dalbulus maidis and in extracts from individual D. elimatus, D. gelbus, D. guevarai, and D. groups of five leafhoppers were ground in quinquenotatus. CSS was detected by ELISA in all exposed but none of the unexposed individuals glass tissue homogenizers (13 X 2.5 cm) of all species. Infectivity assays gave the following transmission rates: D. maidis 100%, D. elimatus with Teflon pestles in 0.2 or 0.3 ml of 83%, D. gelbus 0%, D. guevarai 25%, and D. quinquenotatus 50%. CSS infection of these species PBS-Tween (PBS 0. 15 M sodium was detected more reliably by ELISA than by infectivity assay. Purified CSS antigen was detected chloride + 0.02 M sodium phosphate + by ELISA at 25 but not at 10 ng/ml. CSS was detected by ELISA in about 10% of 436 individual 0.02% sodium azide, pH 7.4; Tween 20Dalbulus leafhoppers field-collected in Mexico in 1982, and maize rayado fino virus (MRFV) was 0.05% polyethylene sorbitan monolaur detected in 5.5% of 828 leafhoppers collected in 1981 and 1982. Incidence of MRFV in maize plants 0.05 p l o rbia n monate) in the field where the leafhoppers were collected in 1981 was 35-40%. As demonstrated by Wilk's and 0.1 ml of each homogenate was statistic (W), the absorbance (A 4 05 nm) values of the unexposed leafhoppers in ELISA were generally transferred to a single well of an ELISA normally distributed. On the basis of this distribution, when the mean of A405 nm values plus three microtiter plate (Dynatech Laboratories times the population standard deviation for the unexposed controls was used to calculate the lower Inc., Alexandria, VA). limit of acceptance for positive A 405 nm values, the type I error rate had an upper bound of 7% for a Extracts were prepared from leaves sample size (N) of 8 and of 1.3% for N = 30. (0.2 g) ground in PBS-Tween (0.8 ml) with a pestle in a mortar, and 0.1 ml of Enzyme-linked immunosorbent assay laboratory tests were taken from colonies each extract was added without further (ELISA) has been used to detect maize maintained on healthy maize in the treatment to each of two wells of a rayado fino virus (MRFV) in Dalbulus laboratory at Wooster, OH. CSSmicrotiter plate. maidis (DeLong & Wolcott) (4,10-12) exposed leafhoppers were given a 7-day ELISA procedures. Antisera to CSS and the corn stunt spiroplasma (CSS) in acquisition access period (AAP) and a strain 1747 was from R. E. Davis (USDA, both D. maidis (2) and Euscelidius 14-day inoculation access period (IAP) Beltsville, MD) and antisera to MRFV variegatus (Kirsch) (9). To date, no before assays. All field samples of was produced as described previously(8). reports of ELISA-detected CSS in fieldDalbulus leafhoppers were collected CSS and MRFV gamma globulins were collected vectors have been noted, but from maize plots at the Centro Interpurified and conjugated with alkaline Saavedra (12) reported detection of nacional de Mejoramiento de Maiz y phosphatase and ELISA was performed MRFV by ELISA in field-collected D. Trigo(CIMMYT)nearTexcoco, Mexico, as described previously (8). The coating maidis. In 1981 and 1982, we detected in early October. Leafhoppers were gamma globulin concentration was 1 CSS and MRFV by ELISA in extracts collected at night with an ultraviolet light pg/ml for antibodies to both CSS and from leafhoppers collected in a survey of (BioQuip 2800 Series, 15W blacklight MRFV, and the gamma globulin-enzyme leafhoppers on maize (Zea mays L.) and collecting light, BioQuip Products, Santa conjugate dilutions were 1:400 for on wild Zea and Tripsacum in Mexico Monica, CA) and a white sheet on which MRFV and 1:400 (leafhoppers) and (6). We also related the percentage of leafhoppers alighted after disturbance of 1:1,000 (purified CSS protein) for CSS. viruliferous leafhoppers to incidence of adjacent maize plants. Leafhoppers, Sodium hydroxide (3 N) was added to M RFV in maize in the field and report collected with aspirators, were placed on wells after a 2-hr incubation to terminate our findings in this paper. maize leaves in plastic refrigerator boxes reactions, and absorbances (A 4 o5 snm) were lined with moistened charcoal and plaster recorded. Measurements were made with MATERIALS AND METHODS of paris bottoms to prevent desiccation of either a Gilford Stasar II spectroSources of leafhopper and maize leafhoppers and host leaves during photometer (Gilford Instrument Laborasamples. All Da/bu/us leafhoppers for transport. Boxes were refrigerated until tories Inc., Oberlin, OH) equipped with a transported with ice in an insulated rapid sampling system or with a Bio-Tek Salaries and research support provided by state and container. Leafhoppers were identified to EIA Reader (Model EL307) (Bio-Tek federal funds (especially USDA competitive seisadsx l efopr rm IsrmnsIcBrigoV) Research Grant8lI-CRCR-0646)appropriated to the seisadsx l efopr rm IsrmnsIcBrigoV) Ohio Agricultural Research and Development laboratory tests and those from the 1981 Tests for ELISA sensitivity. To Center, Ohio State University, Wooster. Journal field collections were frozen until assayed determine the sensitivity of ELISA for article 177-83. by ELISA. For the 1982 field collection, detection of CSS antigen, a dilution series Accepted for publication 29 June 1984. only leafhoppers that survived transport of purified CSS protein (provided by R. __________________ to Wooster were assayed. E. Davis, USDA, Beltsville) was The publication costsof this articleweredefrayed in part In 1981, maize leaf samples collected prepared with PBS-Tween as the diluent by page charge payment. This article must therefore be hereby marked "advertisement' in accordance with 18 from the same field as the Da/bu/us to give 15 twofold or 2.5-fold dilutions U.S.C. § 1734 solely to indicate this tact. leafhoppers were taken from 33-34 plants ranging from 200 #eg/ ml to 5 ng/ ml. ©1985 The American Phytopathological Society in each of three rows about 6 m apart and These were tested in six replicates per 108 Plant Disease/Vol. 69 No. 2 dilution by ELISA. unexposed leafhopper population and mean A 40 5 nm was 0.993 (standard To compare detection of CSS by variations in the estimate of the deviation [SD] = 0.196 and N= 4) with a ELISA vs. infectivity assay, early-instar population variance were ignored, the minimum A 4 0 5 nm of 0.766 and a to mid-instar D. maidis, D. elimatus, D. type I error rate (ie, the probability of maximum of 1.240. For the unexposed gelbus DeLong, D. guevarai DeLong, classifying a pathogen-free sample as controls with five leafhoppers per well, and D. quinquenotatus DeLong & Nault positive for the pathogen) in this the mean A 40 5 nm = 0.072, SD = 0.0029, were assayed immediately after an AAP classification had an upper bound of and N = 4. For the single leafhopper and a 14-day incubation period by 11%. This rate was determined with the extracts per well, visual ratings averaged ELISA or tested for infectivity by caging Chebychev inequality. If an underlying 3.8, the mean A 4 05 nm= 0.538, SD = 0.373, on test maize plants (cultivar Aristogold normal distribution was assumed, the and N = 10. The corresponding Bantam Evergreen) (five per plant) for a estimated (95% probability) type I error unexposed controls had values of mean 7-day TAP. After the lAP, plants were rate had an upper bound of 7% for sample A4 05 m = 0.067, SD = 0.004, and N 10; sprayed with an insecticide, placed in an size (N) of 8 and of 1.3% for N= 30. no color was observed. All exposed insect-containment greenhouse, and To determine the lower limit for leafhoppers werejudged positive for CSS observed for symptoms. detection of CSS antigen, mean A 405 nm whether detected in extracts prepared Analysis of ELISA data. Reactions values for each dilution of purified from groups of five or from single were judged positive for the pathogen antigen were compared statistically with leafhoppers. Because only about one-half when A 405 nm values were greater than the means for the adjacent moreand lessof the extract was added to a well, CSS mean plus three times the population diluted preparations. Aone-tailedttestat was detected from about one-half standard deviation for the unexposed a level of a = 0.05 was used to determine of a leafhopper. There was no apparent leafhopper controls within the same significant differences between the paired interference from materials in leafhopper microtiter plate. A set of the latter means. extracts, because A405 nm values for controls was included within each extracts from groups of five unexposed microtiter plate, because previously, RESULTS leafhoppers were similar to those for when such controls were included in Detection of laboratory-acquired CSS. extracts from one unexposed leafhopper. several plates tested at the same time, the In the test of whether ELISA could detect In further tests of the sensitivity of CSS controls for different plates were CSS in D. maidis, wells that contained detection for individual D. maidis, D. significantly different (D. T. Gordon, extracts from five leafhoppers had visual elimatus, D. gelbus, D. guevarai, and D. unpublished). When no assumptions ratings that ranged between 5 and 6 on a quinquenotatus, all A 40 5 nm values were were made about the underlying scale of 0-7 (0 = no color and 7 = positive for CSS (Table 1). Results distribution of the absorbances for the maximum yellow coloration) and the confirmed the first test-that CSS could Table 1. Detection of corn stunt spiroplasma (CSS) in five Dalbulus spp. by enzyme-linked immunosorbent assay (ELISA) and infectivity assay D. maidis D. elimatus D. gelbus D. guevarai D. quinquenotalus Assay En NEb E NE E NE E NE E NE ELISA Mean (A 4 0 5 nm)c 0.36 -0.05 0.39 -0.05 0.51 0.06 0.39 0.06 0.23 0.06 SEd 0.020 0.003 0.063 0.002 0.037 0.004 0.048 0.003 0.037 0.005 No. positive' 19 0 25 0 20 0 12 0 10 0 No. total' 19 8 25 16 20 9 12 8 10 8 Infectivity assay 6/69 NTh 5/6 NT 0/6 NT 1/4 NT 4/8 NT 'E = leafhoppers given a 7-day acquisition access period on CSS-infected maize followed by a 14-day incubation period before assays. bNE = leafhoppers not exposed to infected plants. c Mean absorbance at 405 nm for extracts of leafhoppers within each column. d SE = standard error. 'Number of leafhoppers positive for CSS in ELISA. A partial summary of these results was published by Madden and Nault (5). 'Total number of leafhoppers assayed by ELISA. Numerator = number of plants showing symptoms of corn stunt after an inoculation access period (lAP) of 7 days with groups of five leafhoppers per plant. Denominator = total number of plants in test. All D. gelbus leafhoppers were dead by the end of the lAP and this probably explains the failure to transmit (5). h NT = not tested. Table 2. Numbers of individual Dalbulus spp. field-collected in Mexico that were positive and negative for corn stunt spiroplasma and the mean absorbance (A 405 nm) values for these leafhoppers and for the unexposed, laboratory-reared D. elimatus as determined in individual microtiter plates of enzyme-linked immunosorbent assay (ELISA) for 1982 collection ELISA for CSS in field-collected Mean A 405 nm for CSS detection by ELISA Dalbulus spp Field-collected Unexposed (no. of leafhoppers) leafhoppers laboratory-reared Plate no. Positive Negative Positive Negative D. elimatus 1 15 62 0.103a (0.006)b 0.063 (0.001)b 0.068 (0.002)b (12)c 2 9 69 0.162 (0.007) 0.100 (0.003) 0.081 (0.005) (12) 3 2 76 0.222 (0.038) 0.105d (0.003) 0.092 (0.007) (12) 4 4 74 0.192 (0.028) 0.087 (0.002) 0.079 (0.006) (12) 5 11 67 0.127 (0.014) 0.068 (0.001) 0.066 (0.001)(12) 6 3 44 0.161 (0.013) 0.071 (0.002) 0.087 (0.003) (12) 'Threshold value for positives of plate 1 was 0.074, based on three times the standard deviation plus the mean A 405 nmof the unexposed, laboratory-reared D. elimatus. bNumber in parentheses is the standard error. Number of individual leafhoppers assayed. 4 Threshold for positives of plate 3 was 0.113, calculated as described in footnote a. Plant Disease/February 1985 109 be detected in extracts from individual parameters, and = the random error 0.092. Similarly, the average for the Dalbulus leafhoppers experimentally variable, former was 1.95 times that of the CSSexposed to a CSS source. Detection of CSS and MRFV in fieldnegative, field-collected leafhoppers. ELISA proved to be a more reliable collected leafhoppers. Mean A 405 nm Only about 10% of all the field-collected detector of CSS infection of these species values for CSS-positive, field-collected leafhoppers assayed were positive for than the infectivity assay (Table 1), D. maidis and D. elimatus (Table 2) were CSS (Table 3). Slightly more D. elimatus particularly with D. gelbus, which considerably lower (average of means = were positive for CSS than D. maidis, but probably succumbed to the pathogen 0.161) than those for the experimentally the difference was not significant. before transmission could occur (5). acquired CSS (average of means = 0.375). For 1981 collections, MRFV was Sensitivity of ELISA for detection of In all, six microtiter plates were used and detected in 31 of 464 (6.7%) fieldCSS antigen. Significant differences were there were appreciable differences in collected Dalbulus spp. by ELISA (Table obtained for antigen concentrations of A 405 nmvalues among plates, as judged by 4). For 1982, these figures were 16 of 364 adjacent pairs from 100,000 and 50,000 to the mean A 405 nm values for the unexposed (4.4%). The difference for the 2 yr was not 50 and 25 ng/ ml. Thus, the lowest leafhoppers of the six plates. Of special significant (Table 3). Absorbance means detectable CSS antigen concentration note, the mean A 405 nm for the CSSfor the MRFV-positive, field-collected was 25 ng/ml and the saturation point negative leafhoppers of plate 3 was Dalbulus spp. were 1.7-3.8 times those was 100,000 ng/ml for our conditions. slightly higher than that for the CSS for the unexposed, laboratory-reared D. Concentrations between 25 and 100,000 positives of plate 1 because of the maidis (negative controls) in 1981 and ng/ml were used to demonstrate the considerable difference in threshold 2.0-2.75 times those for negative controls relationship of A 4 0 5 nm to antigen values for the two plates. Mean A 405 nm in 1982. Means (A 405 nm) of the MRFVconcentration. The model A 405 nm = values for field-collected Dalbulus spp. positive, field-collected Dalbulus spp. a(C) t Ie (ie, log A405 nm was linearly related judged positive for CSS ranged from were about 2.9 times the means for to log C) was fit to the data yielding R2 = 0.101 to 0.222, with the average 2.04 times M RFV-negative, field-collected Da/bu/us 0.971, where C the concentration that for the unexposed leafhoppers, spp. in 1981 and about 2.1 for (ng/ml), a and /3 the regression which had mean values from 0.066 to comparable leafhoppers in 1982. In 1981, MRFV was detected in a significantly higher percentage of D. Table 3. Estimated percentage (^) of individual Dalbulus maidis and D. elimatus leafhoppers maidis than in D. e/imatus (Table 3). In field-collected in Mexico that were positive for corn stunt spiroplasma (CSS) and maize rayado 1982, D. elimatus was infected with a fino virus (MRFV) and number of each species tested by enzyme-linked immunosorbent assay significantly higher percentage of CSS (ELISA) for 1981 and 1982 collections than MRFV. Four of the 1982leafhoppers CSS MRFV (one female D. elimatus, one female D. 19111982 maidis, and two male D. maidis) were 1982 1981 N. positive for both MRFV and CSS. No. No. No. Detection of MRFV in field-collected Sc ted d te t P dmaize. Assays of leaf samples from the D. maidis 8.6(±5)d 163 10.4 (±4)d 250 7.4 (±5)d 149 Mexican maize field in 1981 gave 35 of D. elimatus 11.0 (±4) 273 2.3 (±2) 214 2.3 (±2) 215 100 positive for MRFV by ELISA. For Totals 10.1 (±3) 436 6.7 (±2) 464 4.4 (±2) 364 the positive samples, mean A 40 5 nm = 0.275 a Numbers of Dalbulus leafhoppers according to species and sex: 126 male D. maidis, 37 female D and SD = 0.163. For the negative, fieldmaidis, 207 male D. elimatus, and 66 female D. elimatus. collected samples, mean A 405 nm -0.072, bNumbers of Dalbulus leafhoppers according to species and sex: 223 male D. maidis, 27 female D. SD = 0.017, and N= 60. Five additional maidis, 167 male D. elimatus, and 47 female D. elimatus. samples were scored positive but I c Numbers of Dalbulus leafhoppers according to species and sex: 112 male D. maidis, 37 female D. sampl es were s maidis, 149 male D. elimatus, and 66 female D. elimatus. mean A405 am values that were only dNumber in parentheses when added to ^ yields the 95% confidence interval (CI) for the true slightly higher than the value for percentage positive for the pathogen as calculated by f±[tV'f(100-)/ (N1) + 1/2N], where statistical significance. For these five =estimated percentage positive for pathogen, N= no. tested, and t = t value from table for(N1) samples, mean A 40 5 nm = 0.116 and SD= degrees of freedom for 95% CI, as described by Cochran (1). 0.010. Table 4. Numbers of individual Dalbulus spp. field-collected in Mexico that were positive and negative for maize rayado fino virus (MRFV) and the mean absorbance (A 405 nm) values for these leafhoppers and for the unexposed, laboratory-reared D. maidis as determined in individual microtiter plates of enzyme-linked immunosorbent assay (ELISA) for 1981 and 1982 collections ELISA for MRFV in fieldMean A 405 nm for MRFV detection by ELISA collected Dalbulus spp. Field-collected Unexposed Plate (no. of leafhoppers) leafhoppers laboratory-reared Year no. Positive Negative Positive Negative D. maidis 1981 1 14 66 0.500 (0.130)a 0.172 (0.004)a 017(.0) 1) 2 2 78 0.786 (0.320) 0.190 (0.004) 0.206 (0.015) (10) 3 1 79 0.426c 0.165 (0.005) 0.248 (0.065) (10) 4 8 72 0.177 (0.017) 0.092 (0.002) 0.090 (0.005) (8) 5 4 76 0.139 (0.008) 0.068 (0.002) 0.065 (0.005) (8) 6 2 62 0.121 (0.001) 0.060 (0.001) 0.056 (0.004) (14) 1982 1 2 70d 0.374 (0.074) 0.132 (0.006) 0.149 (0.011) (18) 2 1 71 0.458c 0.261 (0.010) 0.224 (0.017) (18) 3 9 63 0.559 (0.05 1) 0.273 (0.008) 0.221 (0.017) (18) 4 2 70 0.401 (0.008) 0.190 (0.006) 0.146 (0.019) (18) 5 2 75 0.334 (0.019) 0.148 (0.007) 0.168 (0.014) (12) a Number in parentheses is the standard error. bNumber of individual leafhoppers assayed. SNo standard error with only one positive sample. dNumber includes one D. ge/bus leafhopper; the remainder were either D. e/imatus or D. maidis. 110 Plant Disease/Vol. 69 No. 2 Statistical evaluation of ELISA establishing CSS-positive values. The Statistical analyses of ELISA values absorbance data. To characterize the causes for these low values are unknown. for detection of pathogens in vectors have underlying distribution of the A 405 nm The delay between collection and assay or assumed a normal distribution of the values for the unexposed leafhopper the conditions for transport may have unexposed control A 40 5 nm values (4,10). controls in tests for CSS, values obtained reduced CSS titer. Serological differences Our demonstration of the reasonableness from an unexposed leafhopper sample of in the Mexican CSS may have decreased of the assumption of a normal distribution 30 individuals were tested by Wilk's (W) values, or simply a lower CSS titer for for most of our sets of negative control statistic for normal distribution (13). The field-infected compared with laboratoryvalues and the positive skew of the W statistic was greater than the 98th infected Dalbulus spp. may have been distributions for these established an percentile and we concluded that there responsible. Also, the pathogenicity of error rate that was probably less than that was not sufficient evidence to reject the CSS to D. elimatus (5) may have resulted for the normal distribution. As pointed null hypothesis that the distribution of in early death of severely infected out in Materials and Methods, when the the A 4 05 nm values was normal. individuals; these may have had higher threshold value is established at three The W statistic (13) was also used to CSS titers and would not have been times the population standard deviation determine whether there was sufficient assayed because of early death. However, above the negative control means, the evidence to reject the hypothesis that the CSS is not pathogenic to D. maidis and distribution of these negative control distributions of A 40 5 nm values for the 22 thus reduced longevity of infected values determines the error rate that can sets of unexposed leafhoppers listed in individuals would not have been a factor range at most from 1.3% (N = 30) to 7% Tables 1, 2, and 4 plus the values for eight in reduced values. (N= 8) assuming a normal distribution or sets of unexposed D. maidis tested for For MRFV-positive, field-collected at most 11% with no distributional MRFV by Gingery et al (4) were normal. leafhoppers, mean A 405 nm values were assumptions. For these samples, 22 of 30 samples did about two to four times those for ACKNOWLEDGMENTS not show sufficient evidence to reject the unexposed controls compared with We wish to thank A. Juan-Rubink, W. E. Styer, S. null hypothesis of an underlying normal 2.5-3.33 times for MRFV-positive, S. Mendiola, and B. W. Triplehorn for technical distribution. The remaining eight laboratory-exposed D. maidis relative to assistance; R. E. Davis, USDA, ARS, Beltsville, MD, for the gift of antiserum and purified protein of the samples, for which the W statistic unexposed controls (D. T. Gordon, corn stunt spiroplasma; and J. Mihm for permission justified rejection of the null hypothesis, unpublished). Data for the latter to sample leaves and leafhoppers from maize plots at were right (positive) skewed. This implies comparison were from a previous study the Centro Internacional de Mejoramientode Maiz y that the mean was greater than the 50th (4) wherein leafhoppers also were assayed Trigo (CIMMYT), El Batan, Mexico. percentile (median). Thus, in each of individually. Thus, unlike the results for LITERATURE CITED these eight samples, the mean plus three CSS, the field-collected, M RFV-positive 1. Cochran, W. G. 1977. Sampling Techniques. 3rd popestandard deviations would be Dalbulus spp. had values comparable to ed. John Wiley & Sons, New York. 428 pp. population2. Eden-Green, S. J. 1982. Detection of corn stunt at a percentile less than that for a those for the laboratory-exposed, spiroplasma in vivo by ELISA using antisera to symmetric distribution, such as the positive leafhoppers. extracts from infected corn plants (Zea mayss). normal distribution. This percentile In our study, we detected MRFV and Plant Pathol. 31:289-297. decrease implies that for these samples, CSS coinfecting four of 364 (1%) 3. Gamez, R. 1973. Transmission of rayado fino virus of maize (Zea mays) by Dalbulus maidis. the error rate in this classification was leafhoppers tested. Gamez (3) obtained a Ann. Appl. Biol. 73:285-292. probably larger than that assumed for the higher rate (8%) of dual infection of D. 4. Gingery, R. E., Gordon, D. T., and Nault, L. R. normal distribution. maidis by infectivity assay. 1982. Purification and properties of an isolate of Because 100% of laboratory-exposed maize rayado fino virus from the United States. Phytopathology 72:1313-1318. DISCUSSION D. maidis and 80-100% of D. elimatus 5. Madden, L. V., and Nault, L. R. 1983. Our lower limit of detection of purified were positive for CSS by ELISA and Differential pathogenicity of corn stunting CSS antigen was 25 ng/ml. Eden-Green infectivity assay, a high percentage of mollicutes to leafhopper vectors in Dalbulus and Baldulus species. Phytopathology 73:1608-1614. (2) reported a minimum detection level of field-collected individuals of the two 6. Nault, L. R., DeLong, D. M., Triplehorn, B. W., less than 105 CSS (about 1 ng of protein) species was expected to be positive in Styer, W. E., and Doebley, J. F. 1983. More on per milliliter by ELISA from in vitro ELISA. However, our data indicated that the association of Dalbulus (Homoptera: culture, and Raju and Nyland (9) were the CSS infection potential for these two Cicadellidae) with Mexican Tripsacum(Poaceae), , 1including the description of two new species of able to detect 0.01 .sg (l04-l05 cells per Dalbulus spp. was not realized in the leafhoppers. Ann. Entomol. Soc. Am. 76:305-309. milliliter) of CSS protein per milliliter of field. 7. Nault, L. R., Gingery, R. E., and Gordon, D. T. pure culture. Average MRFV infection rates for 1980. Leafhopper transmission and host range of maize rayado fino virus. Phytopathology The fact that ELISA would not laboratory-exposed D. maidis were 12% 70:709-712. discriminate between intact CSS cells and for single leafhoppers (N= 1,753) assayed 8. Nault, L. R., Gordon, D. T., Gingery, R. E., cell fragments or proteins suggests that it for infectivity (7), whereas leafhoppers Bradfute, 0. E., and Castillo Loayza, J. 1979. may overestimate CSS titer, but this lack assayed by ELISA in a previous study Identification of maize viruses and mollicutes and their potential insect vectors in Peru. of discrimination should not affect had a rate of 30% (4; D. T. Gordon, Phytopathology 69:824-828. detection of infected leafhoppers by unpublished). Average MRFV infection 9. Raju, B. C., and Nyland, G. 1981. Enzyme-linked ELISA. rates determined by ELISA for fieldimmunosorbent assay for the detection of corn We failed to confirm Eden-Green's (2) collected D. maidis of 8.9% for the 2 yr of stunt spiroplasma in plant and insect tissues. repot o inerfrene b D.maiis ur sudywer cosidraby lss hanthe Curr. Microbiol. 5: 101-104. repot o inerfrene b D.maiis ur sudywer cosidraby lss hanthe 10. Rivera, C. 1981. Multiplicaci6n del virus del extracts when the equivalent of four laboratory rates. Similarly, ELISA rayado fino del maiz en el insecto vector leafhoppers per sample extract was detected 11 and 25% MRFV-infected D. Da/bolos maidis (Homoptera: Cicadellidae). assayed for CSS by ELISA. However, maidis collected from earlyand lateM.Sc. thesis. Universidad de Costa Rica, San Jose. 53 pp. like Eden-Green, we were able to detect planted maize, respectively, in Costa 11. Rivera, C., Kozuka, Y., and Gamez, R. 1981. laboratory-acquired CSS in the equivalent Rica (12). Rayado fino virus: Detection in salivary glands of individual D. maidis leafhoppers For Costa Rica, incidence of MRFVand evidence of increase in virus titer in the without leafhopper extract interference, infected maize was 22.6 and 4 1.7%, lefhpervctr7ab8-8mids0Triab In our tests, the amount of leafhopper respectively, for the two plantings (12). 12. Saavedra, F. M. 1982. Epidemiologia del virus extract per assay was about two to three For the Mexican maize in our study, del rayado fino en plantaciones de maiz en times that used by Eden-Green. incidence was 35-40%. Thus, the data for Alajuela, Costa Rica. M.Sc. thesis. Universidad The relatively low A4 o5 nm values for MRFV incidence in maize and D. maidis 1.de Costa Rica, San Jose. 112 pp. 3.Shapiro, S. S.,and Wilk, M. B. 1965. An analysis field-collected compared with laboratoryfrom Mexico and Costa Rica were of variance test for normality (complete exposed leafhoppers posed a problem in similar. samples). Biometrika 52:591-611. Plant Disease/February 1955 111