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Aphid Transmission, Serological Detection, and Identification of Cucumber Mosaic Virus

Hope Brooks, Plant Pathology: Microbes and Plants

INTRODUCTION Both cucumber mosaic virus (CMV) and brome mosaic virus (BMV), of the genera Cucumovirus and Bromovirus respectively, are causal agents of mosaic symptoms in infected plants. The host range of CMV includes 1200 species from over 100 families, commonly affecting cucurbits along with other vegetables, ornamentals, and woody or semi-woody plants. Vectors of CMV include over 80 aphid species which spread CMV via nonpersistent transmission, and CMV can survive in many weed and agronomic crop families. CMV consists of three 29 nm diameter, icosahedral particles, which each contain one single-stranded messenger-type RNA surrounded by a capsid. RNA1, the first RNA, is approximately 3,350 nucleotides long and produces the protein 1a, necessary for viral genome replication and protein synthesis. RNA2, the second RNA, is approximately 3,050 nucleotides long and produces proteins 2a and 2b. The 2a protein is used for CMV replication, and the 2b protein, a product of a subgenomic RNA strand, works to inhibit the host plants gene silencing mechanism in response to infection, facilitating the infection of new tissues. RNA3, the third RNA, is approximately 2,200 nucleotides long and produces proteins 3a and coat protein. The 3a protein is used for movement between cells and long-distance movement between plants, serving as a movement protein. The coat protein, a product of a subgenomic RNA strand (RNA4), controls the transmission by CMVs aphid vectors, indirectly regulates cell-to-cell movement, and directly regulates movement long-distance movement between plants (Murphy & Zitter, 2009). CMV contains 82% protein and 18% nucleic acid, with 24% G, 23% A, 23% C, and 30% U (Brunt et. al., 1996). Replication of CMV occurs in the cytoplasm, where viral RNA acts as a template for (-) RNA, from which (+) RNA are produced, and, ultimately, proteins are translated (Murphy & Zitter, 2009). The host range of BMV includes wheat, oats, maize, barley, rye, and species from the families Bromus, Lolium, Phleum, Agropyron, Agrostis, and Poa. Vectors of BMV include nematodes of the genus Xiphinema. Additional sources of additional inoculum and overwintering include Chenopodium spp. (International Maize and Wheat Improvement Center). BMV consists of one, 26 nm diameter, icosahedral particle, which contains three single-stranded RNAs surrounded by a capsid. RNA1, the first RNA, is 3234 nucleotides long and produces the protein 1a. Like in CMV, protein 1a is necessary for viral genome replication and synthesis. RNA2, the second RNA, is 2865 nucleotides long and produces the protein 2a, also necessary for viral genome replication and synthesis. RNA3, the third RNA, is 2117 nucleotides long and produces the

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protein 3a, a movement protein, and coat protein. The coat protein, a product of a subgenomic RNA strand (RNA4), controls the transmission by BMVs vectors, indirectly regulates cell-to-cell movement, and directly regulates movement long-distance movement between plants (Lane, L.). BMV contains 78% protein and 22% nucleic acid, with 28% G, 27% A, 21% C, and 24% U (Brunt et. al., 1996). Replication of BMV occurs on the endoplasmic reticulums surface, where viral RNA acts as a template for (-) RNA, from which (+) RNA are produced, and proteins 1a and 2a are (Ahlquist & Restrepo-Hartwig, 1999). CMVs is transmitted by over 80 prevalent aphid species, including Aphis gossypii (A. gossypii) and Rhopalosiphum maidis (R. maidis), by nonpersistent feeding through the stylet. For the snap bean, important aphid vectors include Aphids glycines, Therioaphis trifolii, and Acyrthosiphon pisum. The virus can be acquired within 60 seconds of feeding, but the virus transmission declines over a period of several hours (Murphy & Zitter, 2009 and Boley & Ferreira, 1992). As studied in this experiment, A. gossypii and R. maidis, are both vectors CMV. A. gossypii is supposed to have a host range of approximately 700 plants, with primary hosts being cucurbit vegetables, citrus, cotton, hibiscus, asparagus, pepper, eggplant, and okra, along with many weed species, like Chenopodium spp. also can be transmitted by seed, cucumber beetles, parasitic plants, and mechanical inoculation (Capinera, 2009). R. maidis is known to feed on sorghum, corn, small grains, and other grasses, like barley, causing reduced yields (The Pennsylvania State University, 2012). CMV is known to infect over 1200 species in over 100 families of monocots and dicots, and overwinters in many perennial weeds, flowers, and crop plants Studies were undertaken to compare aphid feeding mechanisms between two potential vectors of CMV, A. gossypii and R. maidis, and to determine whether beans, geraniums, barley, and/or lambsquarters are true hosts of the aforementioned organisms. Enzyme linked immunosorbent assays (ELISA) were carried out to determine whether beans from a local bean cooperative are truly infected with CMV, and to, ultimately, suggest how particular fields acquired CMV and provide control strategies for CMV. It was found that hosts of A. gossypii included bean, geranium, and lambsquarters; while, the sole host of R. maidis was barley. Farms A, B, and D were tested positive for CMV in their beans, while farms C, E, and F were likely affected by bean common mosaic virus, nitrogen deficiency, and bean bacterial blight. METHODS To identify feeding methods and host range of A. gossypii and R. maidis, A. gossypii and R. maidis (starved 2 hr) were obtained. One of each respective aphid was transmitted to bean, geranium, barley, and lambsquarter leaves and observed under a dissecting scope. The time

Brooks between stylet insertion and removal along with number of test probes was recorded over a three minute period for each aphid and leaf combination.

To determine which plants were infected with CMV from the bean samples, ELISA was used. CMV-specific antibodies were diluted to 1:200 in Carbonate Coating Buffer, yielding a pH of 9.6, and were loaded 100 l into wells in rows A, B, E, and F, and were incubated 24 hours at 4oC, adhering antibodies to well walls. Samples 1 through 6 (numbers corresponded to the numbers of each of the six farms) and a negative healthy plant control were loaded into labeled grinding bags with 3 mL ELISA Sample Buffer (ESB) and homogenized. Unattached coating antibodies were removed with a PBS rinse three times, taking care to eliminate any leftover liquid in wells. 250 l of sample, along with buffer, CMV positive, BMV positive, and a negative healthy plant controls, were loaded into coated wells, see Figure 1. Plate was incubated 24 hours at 4oC to bind antibody to the virus. After 24 hours, wells were rinsed with PBS and antibody conjugated with alkaline phosphatase enzyme diluted to 1:200 in ELISA sample buffer (pH 7.5) and were loaded 100 l into wells and allowed to incubate 24 hours at 4oC, adhering antivirus alkaline phosphatase with virus particles. After 24 hours, the alkaline phosphatase-antibody conjugate was rinsed out five times with PBS. A solution of one tablet of dinitrophenyl phosphate and 20 ml Substrate Buffer (pH 9.8) were mixed, and 0.25 ml of substrate solution was added to each well and allowed to incubate over fifteen to thirty minutes. Plates were scored visually by yellow color of CMV positive samples and quantified by a microtiter plate reader at 405 nm. For microtiter plate reader quantification the minimum positive threshold was set at 5 times the healthy plant sample value. Methods taken from PPATH 405: Serological Detection and Identification of Virus Using Enzyme Linked Immunosorbent Assay (ELISA) lab.

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Figure 1: ELISA Plate Diagram

1 Group: JS, ER B C D Group: HB, DR F G H RESULTS E A

5 A

6 Farm B Farm B

7 Farm C Farm C

8 Farm D Farm D

9 Farm E Farm E

10 Farm F Farm F

11 12

BUF CMV BMV NEG Farm BUF CMV BMV NEG Farm A

BUF CMV BMV NEG Farm A BUF CMV BMV NEG Farm A

Farm B Farm B

Farm C Farm C

Farm D Farm D

Farm E Farm E

Farm F Farm F

Figure 2: Aphid Feeding Results, Hope Brooks

A. gossypii Number of probes attempted Duration of longest feeding Antenna position (forward, vertical, backward) Host or nonhost?

Bean 2

Geranium 12

Barley 3

Lambsquarters 3

30 s

152 s

72 s

180 s

Forward Non-host

Backward Host

Backward Host

Backward Host

Brooks R. maidis Number of probes attempted Duration of longest feeding Antenna position (forward, vertical, backward) Host or nonhost? Bean 14 Geranium 6 Barley 2 Lambsquarters 0

56 s

180 s

180 s

0s

Backward Host

Backward Host

Backward Host

Vertical Non-host

Figure 3: Aphid Feeding Results, Class

A. gossypii Number of probes attempted Duration of longest feeding Antenna position (forward, vertical, backward) Host or nonhost?

Bean ~ 2.1

Geranium ~ 2.2

Barley ~ 1.6

Lambsquarters ~ 1.3

~ 97 s Backward/ Vertical Host

~ 75.9 s

~ 60.9 s

~ 77.8 s

Backward Host

Backward Non-host

Forward Host

R. maidis Number of probes attempted Duration of longest feeding Antenna position (forward, vertical, backward) Host or nonhost?

Bean ~ 2.1

Geranium ~ 2.6

Barley ~ 2.3

Lambsquarters ~1.8

~40.7 s

~ 55.8 s

~ 71.7 s

~ 60.2 s

Backward Non-host

Backward Non-host

Backward Host

Backward Non-host

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Figure 4: ELISA Results, Hope Brooks

1 Group: JS, ER B C D Group: HB, DR F E A

5 A

6 Farm B Farm B

7 Farm C Farm C

8 Farm D Farm D

9 Farm E Farm E

10 Farm F Farm F

11 12

BUF CMV BMV NEG Farm BUF CMV BMV NEG Farm A

BUF CMV BMV NEG -+ ---

Farm A + Farm A +

Farm B + Farm B +

Farm C -Farm C --

Farm D + Farm D +

Farm E -Farm E --

Farm F -Farm F --

BUF CMV BMV NEG -+ ---

G H
Figure 5: ELISA Results, Class

Samples Results (# +/ total) BUF 0/9 CMV 7/7 BMV 1/8.5 NEG .5/9 S1 6.5/9 S2 8.5/9 S3 0/9 S4 9/9 S5 0/9 S6 0/9

There is some discrepancy between individual aphid host range data and the class data. For further discussion of unexpected results see the discussion below. In general, the discrepancy in results can be attributed to an aging population of A. gossypii and R. maidis that had fed less than two hours prior. In general, all plants that reported positive in individual data, including the CMV control and samples A, B, and D, reported positive in class data. Cases where no data was recorded, indicated by a sample number less than nine, could be due to misloading of wells or the microtiter plate misreading results. Cases of false positives seen in class data, as in the BMV control and the negative control, could be due to cross-contamination of wells. DISCUSSION CMV is a nonpersistently transmitted virus with a wide host range, and both A. gossypii and R. maidis serve as CMV vectors. Feeding by either of the above is likely to result in the development

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of a newly-CMV infected plant. Test probing behavior is likely to be more influential in the spread of disease through a field because probing is a relatively quick process and aphids can probe many plants. Upon testing on bean, geranium, barley, and lambsquarters, it was found that A. gossypii had a much broader host range than R. maidis. Hosts of A. gossypii included bean, geranium, and lambsquarters; while, the hosts of R. maidis included only barley. When feeding both A. gossypii and R. maidis fed for longer periods on hosts than non-hosts. In practical terms, A. gossypii is more important to control in bean fields and in greenhouses where growers are concerned about CMV, as A. gossypiis host range is very large. Preferences for selected plants can be attributed to feeding mechanism evolution. Monophagous aphids are favored by clonal parthenogenesis of aphids that perform best on a given plant which grows in abundance. Polyphagous aphids, like A. gossypii and R. maidis, as per this model, are favored by plant populations which are not abundant enough to sustain specific aphids or which vary in abundance, traits easily overcome by a large host range (Dixon & Kindlmann, 1994). It follows that aphids with a small host range, like R. maids as per this model, are favored by clonal parthenogenesis of aphids that perform best on a narrow range of plants, more specifically, large monocultures of barley, sorghum, corn, and other grasses. There is some discrepancy between individual aphid host range data and the class data. The number of test probes attempted by A. gossypii on geraniums was much greater in individual data (13 probes) than in class data (~ 2.2 probes). Duration of feeding by A. gossypii also varied. In beans, the duration was much lower in individual data (30 seconds) than in class data (~ 97 seconds). In geranium, the duration was much higher in individual data (152 seconds) than in class data (~ 75.9 seconds). In lambsquarters, the duration was also much higher in individual data (180 seconds) than in class data (~77.8 seconds). Antenna position of A. gossypii was recorded as forward in bean and lambsquarters, while class data indicated antenna as backward/forward in bean and backward in lambsquarters. Bean and barley were determined to be A. gossypii hosts in individual data, while class data indicated them to be non-hosts. The number of test probes attempted by R. maidis on beans was much greater in individual data (14 probes) than in class data (~2.1 probes). In geranium, the number of probes was much greater in individual data (6 probes) than in class data (~ 2.6 probes). In lambsquarters, the number of probes was much lower in individual data (0 probes) than in class data (~ 1.8 probes). Duration of feeding by R. maidis on geraniums was much greater in individual data (180 seconds) than in class data (~ 55.8 seconds). In barley, duration was much greater in individual data (180 seconds) than in class data (~ 71.7 seconds). In lambsquarters, the duration of feeding was much lower in individual data (0 seconds) than in class data (~ 60.2 seconds). Antenna position of R. maidis was recorded as vertical in

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lambsquarters, while class data indicated antenna as backward. Bean, lambsquarters, and geranium were determined to be R. maidis hosts in individual data, while class data indicated them to be nonhosts. As noted before, the discrepancy in results can be attributed to an aging population of A. gossypii and R. maidis that had fed less than two hours prior to testing. Given that an outbreak of R. maidis occurs in a cornfield between farms A and E, and an outbreak of A. gossypii in a tomato field between farms B and C occurs, the incidence of CMV will likely increase the incidence of CMV on all farms, as class data indicates that A. gossypii feeds and test probes beans. A. gossypii under normal environmental conditions has a flight distance of 6.1 km at the end of the bean growing season, a distance which far exceeds the distances between all five farms, indicating a strong probability of higher CMV incidence on all the farms (Heimpel et. al., 2008). In the case of grower E starting geraniums from cuttings in his greenhouse near an infected bean field and with R. maidis in a corn field between farms A and E, the grower need not be concerned about CMV in his geraniums because R. maidis has not been shown to feed on geraniums and, thus, does not appear to vector CMV to them. Populations of R. maidis are usually controlled by environmental factors, parasitic wasps, fungal disease, lady bettles, syrphid fly larvae, and lacewing larvae and adults (The Pennsylvania State University, 2012). Though, control of weeds, notably Chenopodium spp., in and around fields of cucumbers, beans, and other CMV hosts, is an important control method as numerous weeds serve as reservoirs for CMV and contribute to virus spread to crops at the beginning of the season and some perennials, biennials, and winter annuals harboer CMV in their roots, providing overwintering sources (Murphy & Zitter, 2009). In a related case study, a grower with a field of barley next to a CMV positive field of beans infested with unidentified aphids, it is unlikely that the CMV will transfer to his field as A. gossypii was not found to feed on barley, like R. maidis. Because the use of pesticides for an aphid treatment is expensive, the most cost-effective method of CMV prevention in this case is to create a weed-free zone in the farms fields. It was found that farms A, B, and D had high numbers of samples infected with CMV, while farms C, E, and F did not have any infected samples. Similar dates when the symptoms were noticed and common reports of symptoms along roads, as in farms A and D, and on the margins of the field, as in farm B, indicate that the distribution of CMV may be due to nonpersistent feeding by one of the many aphid vectors of CMV originating in one of the fields between farms A, B, and D, and exacerbated by the stress of a storm bringing windblown rain. Based on CMV symptomology, it is likely that beans from farm C may be afflicted with bean common mosaic virus (BCMV) because the described symptoms of yellow spots on foliage, twisting leaves, and very thin segments at the top

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of plant closely resemble the BCMV symptoms, including yellow to green mosaic on leaves, varying degrees of leaf distortion, and stunted growth of plants infected at a young age (Frate et. al., 2007). Although yellowing of leaves is symptomatic of CMV, beans from farm E with yellowing areas on leaves, leaves generally appearing smaller, and with smaller plants as well distributed generally across a field are likely suffering nitrogen deficiency from the previous N-intensive crop, sweet corn (Johnson, 2009). The symptomatic brownish spots on leaves, surrounded by yellow areas, and pods [that] are streaked suggests that beans from farm F are suffering from bean bacterial blight, an infection with symptoms including brown spot lesions surrounded by yellow halos and irregular lesions on the pods (Hardman et. al., 2009). Although nine samples were successfully processed from each farm by ELISA, samples is not an adequate number of samples for a representative study. In order to facilitate more informative sampling, leaf samples should be taken from regularly spaced intervals throughout each field. The number of leaf samples should exceed nine. In the future, growers can purchase virus-free plants, carry out strict aphid control, manage CMV and aphid-harboring weeds, discard virus infected plants, disinfect tools used for vegetative propagation, and propagate plants by seed. As a rule, any plants with CMV samples should be set aside and sent in for diagnosis. WORKS CITED Ahlquist, P. & Restrepo-Hartwig, M. (1999). Brome Mosaic Virus RNA Replication Proteins 1a and 2a Colocalize and 1a Independently Localizes on the Yeas Endoplasmic Reticulum. Journal of Virology, vol. 73 no. 12 10303-10309. Retrieved from http://jvi.asm.org/content/73/12/10303.full Boley, R. A. & Ferreira, S. A. (October 1992). Cucumber Mosaic Virus. Retrieved from http://www.extento.hawaii.edu/kbase/crop/type/cucvir.htm Brunt, A.A., Crabtree, K., Dallwitz, M.J., Gibbs, A.J., Watson, L. and Zurcher, E.J. (eds.) (1996 onwards). Plant Viruses Online: Descriptions and Lists from the VIDE Database. Version: 20th August 1996.' Retrieved from http://biology.anu.edu.au/Groups/MES/vide/ Capinera, J. L. (June 2009). Common Melon Aphid. Retrieved from http://entnemdept.ufl.edu/creatures/veg/aphid/melon_aphid.htm#host Davis, R. M., Frate, C. A., Hall, A. E., Temple, S. R. (2007).Dry Beans Bean Yellow Mosaic. UC IPM Pest Management Guidelines: Dry Beans, UC ANR Pulbication 3446. Retrieved from http://www.ipm.ucdavis.edu/PMG/r52101511.html

Brooks Dixon, F. G. & Kindlmann, P. (1994). Evolution of Host Range in Aphids. European Journal of Entomology, vol. 91: 91-96. Retrieved from http://www.eje.cz/pdfarticles/500/eje_091_1_091_Kindlmann.pdf Hardman, L. L., Lamey, H. A., Meronuck, R. A. (2009). Edible Bean Disease and Disorder Identification. North Central Regional Extension Publications. Retrieved from http://www.extension.umn.edu/distribution/horticulture/dg6144.html Heimpel, G. E., Wang, L., Wu, K., Wyckhuys, K. A. C., Zhang, Y. (2008). Flight Performance of the Soybean Aphid, Aphis glycines (Hemiptera: Aphididae) Under Different Temperature and Humidity Regimes. Physiological Ecology, vol 37(2): 301-306. Retrieved from

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http://www.entomology.umn.edu/prod/groups/cfans/@pub/@cfans/@ento/documents/ asset/cfans_asset_347342.pdf International Maize and Wheat Improvement Center. Brome Mosaic Virus. Retrieved from http://wheatdoctor.cimmyt.org/en/pests-a-diseases/list/187?task=view Johnson, G. (2009). Nitrogen Deficiencies in Lima Beans. Retrieved from http://agdev.anr.udel.edu/weeklycropupdate/?p=1288 Lane, L. Brome Mosaic Virus. Retrieved from http://lclane.net/text/bmv.html Murphy, J. F. & Zitter, T. A. (2009). Cucumber Mosaic Virus. The Plant Health Instructor, DOI: 10.1094/PHI-I-2009-0518-01. Retrieved from http://www.apsnet.org/edcenter/intropp/lessons/viruses/Pages/Cucumbermosaic.aspx The Pennsylvania State University (2012). CornLeaf Aphid on Field Corn. Retrieved from http://ento.psu.edu/extension/factsheets/corn-leaf-aphid