Section B: Viruses, Epidemiology, and Virus-Vector Interactions - 1999

Section B (2000)

Plenary Session Summary:

Author: Jane E. Polston

Affiliation & Location: University of Florida, Gulf Coast Res. and Educ. Ctr., Bradenton, FL

The Appearance of Tomato Yellow Leaf Curl Virus (Geminiviridae, Begomovirus) in Florida

Tomato yellow leaf curl virus (TYLCV-Is) is a whitefly-transmitted geminivirus first described from Israel. This virus has caused economically significant yield losses in tomato in the eastern Mediterranean for many years. Much time and expense have been devoted to developing strategies for its management. For a geminivirus, the host range is broad, and includes both crop, ornamental and weed species, however tomato is the crop which is most often afflicted. Symptoms appear in tomato several weeks after inoculation and include severe stunting, marked reduction in leaf size, upward curling and chlorosis of leaf margins, mottling of leaves, and high rates of flower abscission.

In the early 1990's symptoms characteristic of TYLCV-Is were observed in Cuba, the Dominican Republic, and Jamaica (1, 2, 3, 4, 5). These symptoms were shown to be caused by a virus with a genomic sequence nearly identical to that of TYLCV-Is. It is not known how this virus came to be in Cuba and Jamaica, but in the Dominican Republic it is believed that the virus was introduced on transplants which had been purchased in the eastern Mediterranean for fruit production in greenhouses in northwestern Dominican Republic.

In July 1997 symptoms characteristic of TYLCV-Is were observed on one tomato plant in a field in Collier Co. and several tomato plants in a retail garden center in Sarasota Co. FL (7). Amplification with three sets of primers, restriction analysis of amplified fragments, and hybridization with a clone of TYLCV-Is indicated that TYLCV-Is was present in symptomatic plants. The sequence of a 1300 bp amplified fragment was 99% identical to TYLCV-Is from the Dominican Republic and 98% identical to an isolate from Israel (Gene Bank Acc. No. X15656). It appears that the virus entered the U.S. in Dade Co. Florida in late 1996 or early 1997, infected tomato plants in production for retail sale in at least two Dade Co. greenhouses, and was rapidly distributed via retail garden centers around the state. Infected plants were purchased by homeowners and in some cases the virus appeared to move from home gardens to nearby commercial nurseries and production fields.

Regulatory procedures as well as field management practices were implemented within weeks of identification in Florida to minimize the movement and incidence of this virus. Initial incidences of TYLCV-Is were low in most of the state (except Dade Co.) during the 1997-98 production season. Incidences throughout the state were significantly higher in the 1998-99 production season. Yield losses were experienced by some growers, and most growers are experiencing increases in production costs due to new practices to manage TYLCV-Is. Almost all growers are using imidacloprid in both transplants and field plants, in addition to multiple inspections to rogue infected-looking plants from fields. Pesticide applications to minimize whitefly populations have increased. New regulations have been imposed on transplant producers of known TYLCV-Is host plants, including lisianthus (Eustoma grandiflorum), tobacco (Nicotiana tabacum) and tomato, to minimize the occurrence of TYLCV-Is in certified transplants.


Investigator's Name(s): Hamed Doostdar, Moshe Inbar, & Richard T. Mayer.

Affiliation & Location: U.S. Horticultural Research Laboratory, USDA, ARS, 2120 Camden Rd., Orlando, FL 32803-1419.

Research & Implementation Area: Section B: Viruses, Epidemiology, and Virus-Vector Interactions.

Dates Covered by the Report: January 1 - December 31, 1998

Effects of Tomato Mottle Virus Infection on Tomato Root Development

The effects of tomato mottle virus infection on the root development of tomato seedlings, Lycopersicon esculentum was investigated. Individual tomato seeds were planted in pots containing Metro Mix potting mixture containing no fertilizer. Pots were watered with 50 ml solutions of; A) fertilizer (9:45:15; N:P:K; 3.2 g/L); B) KeyPlex 350DP (5 ml/L); C) fertilizer + KeyPlex 350DP; at planting and then every two weeks for the duration of the experiment. Plants were separated into three sets (5 plants of each treatment per set), placed into enclosed cages and maintained in an environmental chamber at 26°C, 45% humidity and 12 h light/dark cycle. At the four leaf stage (three weeks after planting) 300 virus-free adult whiteflies, Bemisia argentifolii (WF) were introduced in to one of the cages (SET 1), the same number of viruliferus WF were added to the second cage (SET 2), and the third cage was maintained WF-free (SET 3). Plants were watered every other day with 50 ml of water during the course of the experiment. Three weeks post infestation visual symptoms of virus infection were detected in some of the plants, at which time the experiment was terminated. Plants were carefully removed from the pots, cleaned and the shoot and roots were weighed. Root and leaf samples were collected and the levels of chitinase, b-1,3-glucanase, peroxidase, lysozyme, and total phenolics were determined. No significant variation in the shoot weights (18 ± 1.3 g) was observed for the three SETs of treatment A plants. However, virus-infected plants in this treatment showed a 70% (P < 0.01) root loss compared to the other two SETs. Due to the lack of fertilizer the average weights of the shoots of treatment B plants were significantly less than those of treatment A (6.8 ± 0.3 g), but virus infected plants showed no reduction in root weights compared to plants from the other two SETs. Virus infected treatment B plants showed no visual signs of virus infection. Treatment C plants, exhibited only slight viral symptoms. The average weights of the shoots of treatment C plants (16.4 ± 1.1 g) were comparable to those of treatment A. But, the plants exposed to virus exhibited only a 38% reduction in root weight (P < 0.01) compared to the other two SETs. The levels of defensive proteins and total phenolics varied considerably in the roots and leaves depending on treatment and WF exposure. However, in all three SETs of both treatments B, and C, The level of root b-1,3-glucanase activity was significantly higher compared to plants in treatment A.

These results indicate that tomato mottle virus infection has an adverse effect on root development in tomato plants causing eventual severe decline and epinasty in infected plants. Addition of specific micro-nutrients (KeyPlex 350DP) to the soil appear to reduce this effect and allow the plants to tolerate infection.

Investigator's Name(s): Dale B. Gelman1, Michael B. Blackburn1, Jing S. Hu, & Jo-Ann Bentz2.

Affiliation & Location: 1Insect Biocontrol Laboratory, ARS-USDA, Beltsville, MD; 2Floral and Nursery Plants Research Unit, ARS-USDA, Beltsville, MD.

Research & Implementation Area: Section B: Viruses, Epidemiology, and Virus-Vector Interactions.

Dates Covered by the Report: 1998

Characterization of Fourth Instar Greenhouse Whiteflies, Trialeurodes vaporariorum, Developmental Markers and Ecdysteroid Fluctuations

A system of markers was devised to characterize the development of fourth instar T. vaporariorum. Measurement of body depth combined with size and color of the developing adult whitefly eye were used to divide the instar into ten stages. When reared on salvia, tomato or sunflower at LD 16:8 and 26.1oC, body thickness of 4th instars increased to reach a maximum of 0.3-0.02 mm in stage-5 larvae. Adult eye development was first observed at stage 6. The eye which previously had been a pinpoint became slightly diffuse. At stage 7, the eye was more diffuse and at stage 8, it became the bipartite solid red structure typically associated with the `pupal' stage. Ecdysteroid (molting hormone) levels of methanolic extracts of whitefly homogenates were determined by radioimmunoassay. The range of detection of the assay was 50-5000 picograms. Ecdysone, 20-hydroxyecdysone, 26-hydroxyecdysone, 20,26-dihydroxyecdysone and makisterone A had a high affinity for the antiecdysone antibody and thus, could be detected readily. In extracts prepared from whole body homogenates of between 15 and 55 whiteflies, ecdysteroid was undetectable. It was expected that ecdysteroid titers would have peaked in stage-5 insects, just prior to the onset of adult development which had been observed to occur at stage 6.

Histological studies suggested that apolysis (the separation of the larval cuticle from the epidermis) probably occurs after the whitefly body has grown to maximum thickness and just prior to the beginning of eye development, i.e., in stage-5 4th instars. Typically, in preparations of stage 6 or older 4th instars, the larval cuticle was either separated from the epidermis or had become detached during processing. Thus, apolysis, had probably occurred prior to this stage. In addition, in stage-6 4th instars, the wing buds were observed to have undergone considerable development, i.e., folding. By stage 7, spines were observed on the new cuticle, indicating that adult cuticle was well-formed by this stage. Results for Bemisia argentifolii 4th instars were similar.

Investigator's Name(s): Wayne Hunter, Anand Persad, Moshe Inbar, Hamed Doostdar, & Richard T. Mayer.

Affiliation & Location: U.S. Horticultural Research Laboratory, USDA, ARS, 2120 Camden Rd., Orlando, FL 32803-1419.

Research & Implementation Area: Section B: Viruses, Epidemiology, and Virus-Vector Interactions.

Dates Covered by the Report: January 1 - December 31, 1998

Geminivirus-Mediated Interspecific Competition Between Whiteflies and Other Insects

This study examined the possibility of an induced response caused by a Begomovirus infection, and its effect on the systemic acquired response due to whitefly feeding. The combined interaction of whitefly feeding and virus infection of the host plant may provide a biologically significant advantage to whiteflies over other insect herb-ivores trying to utilize the same resources. The effects of an induced plant response by the infection of the Begomovirus tomato mottle virus, ToMoV, in tomato, on the feeding and development of the corn earworm, Helicoverpa zea, the leafminer Liriomyza trifolii, and the whitefly, Bemisia tabaci (Gennadius), B-biotype, are reported. Corn Earworm: The relative growth rate of H. zea larvae, late second instar, was significantly reduced when fed on leaves from the treatment of whitefly-infested, virus-infected tomatoes. However, H. zea growth rate was not significantly different when fed on either treatment of leaves from non-infested, virus-free tomato, versus whitefly-infested, virus-free tomato. Virus infection of the host plant appeared to negatively affect herbivore competition, producing conditions unfavorable to H. zea. Observations suggested that H. zea larvae consumed more of the virus-infected leaf material than those fed leaves from virus-free plants. This may have been due to the reduced quality of the food source. Whitefly: Oviposition was recorded on all three treatments, on newly emergent leaves of plants on days 10 and 30 postinfestation. At the 10 d sampling, there were significantly more eggs oviposited on the control treatments than on plants which had both a whitefly-infestation and virus-infection. At 30 d there were significantly more eggs oviposited on the control plants, over both other treatments, but there was no significant difference in the number of eggs produced between either treatment which had a whitefly infestation on tomato, whether the plants were virus-free, or virus-infected. There was an overall reduction in eggs oviposited on older plants regardless of treatment. Leafminer: Acceptability to leaf miner, Liriomyza trifolii, was based on oviposition and feeding punctures/cm2 recorded on leaves of tomato, L. esculentum at 30 days post whitefly infestation. This was conducted in a random choice experiment in a growth chamber. There was a significant difference among all three treatments in the number of oviposition and feeding punctures with the greatest number recorded on the non-infested, virus-free tomatoes (465 punctures, q-value = 6.761, P = 0.000), next on the whitefly-infested, virus-free tomatoes (125 punctures, q-value = 11.940, P = 0.000), with the lowest number recorded on the whitefly-infested, virus-infected tomatoes (31 punctures, q-value = 3.395, P = 0.003) (%= 0.05). PR-Proteins: Of the PR-proteins measured, peroxidase, lysozymes, glucanase, and phenolic acid, only peroxidase showed a significant increase with time. Plant tissues were sampled on days 10, 20, and 30 post whitefly infestation. The relative mean concentration (delta A550/min/g tissue) for levels measured in the controls, non-infested, virus-free tomatoes, showed low constant levels for samples taken on day 10, and 20, with a small, but significant increase on day 30, which was approximately double the previous levels. Levels from the whitefly-infested, virus-free tomatoes, showed a significantly greater level on day 10, approximately three-times the levels of the control and almost twice that of the other treatment (whitefly/virus), but levels dropped when sampled on day 20 becoming comparable to the control plants. However, on day 30 peroxidase levels were again significantly highest among all treatments increasing approximately three-fold previous levels. The treatment of whitefly-infested, virus-infected tomatoes, showed significantly different levels on day 10 being between the levels of the other two treatments, the control being the lowest. On day 20, levels were significantly greater being approximately twice the level at day 10, and this level was maintained to sampling on day 30. The relative mean concentration of phenolic acid (microgram Tannic acid equivalent/ g dry tissue) present in L. esculentum, showed an age effect. There was a significant drop in phenolic acid levels across all treatments when sampled on day 20, levels remained at this reduced level through to the last day sampled. The relative mean concentration of lysozyme (delta A550/min/g tissue) in L. esculentum sampled on days 10, 20, and 30, showed no significant difference in lysozyme levels. The relative mean concentration of Beta-1-3-glucanase (micro-mol Glc/min/microgram tissue) present in L. esculentum sampled at 10, 20, and 30 days showed slight variations, but were not significantly different among treatments over time.

Investigator's Name(s): L. P. Sharp, Y. M. Hou, E. R. Garrido-Ramirez, P. Guzman, & R. L. Gilbertson.

Affiliation & Location: Department of Plant Pathology, University of California-Davis, Davis, CA 95616.

Research & Implementation Area: Section B: Viruses, Epidemiology, and Virus-Vector Interactions.

Dates Covered by the Report: January 1 - December 31, 1998

Synergistic Interactions Among Components of Whitefly-Transmitted Geminiviruses

A replication-competent Begomovirus (whitefly-transmitted geminivirus) DNA-A component (referred to as chino-A) was cloned from tomatoes with symptoms of chino del tomate disease from Sinaloa, Mexico. The role of chino-A in chino del tomate disease was investigated by co-inoculating chino-A alone or in combination with the cloned DNA components of two tomato-infecting begomoviruses, pepper huasteco (PHV) and tomato leaf crumple (TLCrV), into Nicotiana benthamiana, tomato, and pepper plants. The chino-A component alone was not infectious. In combination with PHV, chino-A was infectious in all three hosts, and plants infected with this combination had more severe symptoms than plants infected with PHV alone. Furthermore, the symptoms in tomato and N. benthamiana plants infected with PHV+chino-A were similar to those associated with chino del tomate disease. In combination with TLCrV, chino-A was infectious only in N. benthamiana and no increase in symptom severity was observed. Together, these results demonstrate a novel synergistic interaction between chino-A and PHV, and provide evidence that chino del tomate disease may be caused by a complex of geminivirus components.

Investigator's Name(s): Cica Urbino1, Marie-Line Caruana2, Nicolas Sauvion,1 & Claudie Pavis1.

Affiliation & Location: 1INRA, Unit de Recherches en Productions Vegetales, F-97170 Petit-Bourg, Guadeloupe (F.W.I.), 2 CIRAD-FLHOR, same location.

Research & Implementation Area: Section B: Virus Diseases, and Virus-Vector Interactions.

Dates Covered by the Report: January 1, 1997 - November 30, 1998

Potato Yellow Mosaic Virus (PYMV) on Tomato in Guadeloupe: Characterization of the Virus and its Vector

Since 1989 in Guadeloupe, tomato fields have been infested by a geminivirus disease. At the same time, severe whitefly outbreaks were recorded.

Four viral isolates were collected and maintained in greenhouse by vector then grafting to tomato in order to conduct a biological (graft, mechanical and vector transmission, host range) and molecular (PCR, RFLP, hybridization) characterization of the virus. The viral isolates were transmitted by stem grafting to tomato, tobacco, datura, potato and sweetpepper. Mechanical and vector transmission were obtained on tomato. Cloning and sequencing of an isolate (Polston et al., 1998) led to the identification of a bipartite geminivirus already known as potato yellow mosaic virus in Venezuela, Trinidad, and Puerto Rico.

The vector was identified as Bemisia tabaci B biotype, using isoenzyme (esterases) electrophoresis, PCR and silverleaf symptom induction. Virus transmission studies showed that in the population raised in the laboratory, 25% of adults transmit the virus from tomato to tomato. Female are twice more efficient than males.

Field experiments revealed that yield losses caused by PYMV reach 40% on a susceptible variety when 4 week-old tomato plants were infected and could be higher if plants were younger. No yield loss occurred on 9 week-old inoculated plants. The use of pesticides to reduce the vector population did not allow to limit the spread of the disease in fields with high viral pressure conditions.

Specific tools as primers and probe are now developed for the detection of PYMV. They will be used to determine the virus reservoirs in natural conditions and to evaluate the level of resistance in tomato genotypes. Cloning and sequencing of viral isolates are in progress to estimate the variability of the virus in Guadeloupe.

Polston J. E., D. Dubois, G. Ano, F. Poliakoff & C. Urbino, 1998. Plant Disease, 82 (1): 126.

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