Section B: Viruses, Epidemiology, & Virus-Vector Interaction - 2000

Section B (1999)

Plenary Session Summary:

Investigator's Name(s): Henryk Czosnek, Shai Morin, Galina Rubinstein, Viviane Fridman, Muhamad Zeidan, and Murad Ghanim

Affiliation & Location: Department of Field Crops and Genetics, and the Otto Warburg Center for Biotechnology in Agriculture, Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot 76100, Israel

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

Dates Covered by the Report: 1997-1999.

Tomato yellow leaf curl geminivirus (TYLCV-Is), a sexually transmitted disease of the whitefly Bemisia tabaci (B. argentifolii)

Geminiviruses constitute the most important class of pathogens transmitted by B. tabaci Their single-stranded DNA (ssDNA) genome is encapsidated in ~ 20 x 30 nm geminate particles. Whitefly-transmitted geminiviruses (family Geminiviridae, genus Begomovirus) infect many economically important agricultural plants. Among them, tomato yellow leaf curl virus (TYLCV) has probably the most serious economic impact.

Begomoviruses and whiteflies have interacted for geological times. Multiple repeats of modern begomoviral-related sequences were found integrated in a single chromosomal locus of several Nicotiana species, suggesting that a unique geminiviral integration event occurred more than 100 MY ago. Fossil whitefly species, though not B. tabaci, have been found in 120-140 MY old amber from Lebanon. An assumed long-lasting virus-vector intimate relationship of this magnitude implies that the partners have developed co-evolutionary mechanisms that insure on one hand the survival and the efficient transmission of the virus, and on the other hand the safeguard of the insect host from possible deleterious effects of the virus. Novel data from our lab suggest that TYLCV-Is has retained, or acquired, some characteristics of an insect pathogen.

The whitefly B. tabaci acquires and transmits TYLCV-Is in a circulative manner. During the 8 h-long latent period, the ingested virus circulates in the insect along a path and with a velocity likely shared by all begomoviruses. Bacteria that have lived in symbiosis with B. tabaci for more than 100 MY are involved in the safe translocation of begomoviruses in B. tabaci. A GroEL chaperonin synthesized by B. tabaci endosymbiotic bacteria and released into the insect haemolymph ensures the safe transit of TYLCV-Is in the insect haemolymph. Disturbing this virion-GroEL interaction in the haemolymph lead to the destruction of virus particles and subsequently to a dramatic decrease in virus transmission (1).

Begomoviruses acquired during a short period of time by 1-2 day-old B. tabaci adults remain associated with the insect for several weeks. TYLCV-Is was present during the entire life of the insect (2). Although infectivity decreased with age, the insects were able to infect test plants for more than 4 weeks. Therefore, at least some of the acquired virions are able to remain as infective units, or to return, into in the circulative pathway several weeks after acquisition. It is likely that a large fraction of the ingested virus leaves the acquisition/ transmission pathway and invades insect tissues. This long-term retention of TYLCV-Is in its whitefly host was associated with a dramatic decrease in the life expectancy of the host and in its fecundity.

Invasion of tissues by TYLCV-Is and its effect on the insect fecundity was demonstrated for the reproductive system. The long-time retention of TYLCV-Is was accompanied by a ~ 40% decrease in the mean number of eggs laid by whiteflies. These negative effects were expressed several days after acquisition, as if the virus had first to invade the reproductive system and aborting part of the developing eggs laid (2). Indeed, eggs maturing in the ovaries of viruliferous whiteflies contained viral DNA detectable by PCR. Viral DNA was similarly detected in some of the adult insects that developed from these eggs (3). The way in which TYLCV penetrates the whitefly reproductive system is unknown. We presume but we do not have direct proof that TYLCV-Is invades tissues others than those of the reproductive system. Virus invasion may account for the reduction observed in the life span of viruliferous whiteflies compared to non-viruliferous insects. In both winter and summer, the viruliferous insects started to die earlier and at higher rates (up to day 30) than their non-viruliferous counterparts. As a result, the life expectancy of the viruliferous insects was significantly lower than that of the non-viruliferous controls. At the population level, the difference at the 50% mortality point was between 5 and 7 days (2).

Because TYLCV-Is had several characteristics of an insect virus, we investigated the possibility that it could be transmitted horizontally, from insect to insect, without the mediation of an infected plant. TYLCV-Is was transmitted among whiteflies in a sex-dependant manner, in the absence of any other source of virus. TYLCV was transmitted from viruliferous males to females and from viruliferous females to males, but not among insects of the same sex. Transmission took place when insects were caged in groups or in couples, in a feeding chamber or on cotton plants, a TYLCV-non-host. The recipient insects were able to efficiently inoculate tomato test plants. Insect to insect virus transmission was instrumental in increasing the number of whiteflies capable of infecting tomato test plants in a whitefly population. TYLCV was present in the haemolymph of whiteflies caged with viruliferous insects of the other sex; therefore the virus follows, at least in part, the circulative pathway associated with acquisition from infected plants. Taken as a whole, these results implicate that a plant virus can be sexually transmitted among its insect vector.

Infection of plant hosts by begomoviruses is still not fully understood although the function of the geminiviral genes has been thoroughly investigated for the last decade. The way begomoviruses interact with their insect vectors is even less understood. The days when it was thought that whiteflies are mere go-between are gone. A begomovirus such as TYLCV-Is has many features of an insect virus. It is likely that this virus is not the only one with such extraordinary properties. Others may have retained, or gained, some pathogenic features during their co-evolution with B. tabaci which remain to be discovered.

References

1. Morin S, Ghanim M, Zeidan M, Czosnek H, Verbeek M and van den Heuvel JFJM (1999) A GroEL homologue from endosymbiotic bacteria of the whitefly Bemisia

tabaci is implicated in the circulative transmission of Tomato yellow leaf curl virus. Virology 30:75-84.

2. Rubinstein G and Czosnek H (1997) Long-term association of tomato yellow leaf curl virus (TYLCV) with its whitefly vector Bemisia tabaci: effect on the insect transmission capacity, longevity and fecundity. J. Gen. Vir. 78:2683-2689.

3. Ghanim M., Morin S, Zeidan M and Czosnek H (1998) Evidence for transovarial transmission of tomato yellow leaf curl virus by its vector the whitefly Bemisia tabaci. Virology 240:295-303.

4. Ghanim M, Morin S and Czosnek H (2000) Tomato Yellow Leaf Curl Geminivirus (TYLCV-Is) Is Transmitted among Whiteflies (Bemisia tabaci) in a Sex-Related Manner. J. Virology 74: 4738-4745, 2000.

Investigator’s Name(s): Robert L. Gilbertson

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

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

Dates Covered by the Report: December 1998 – December 1999

A New Bipartite Geminivirus (Begomovirus) Causing Cucurbit Leaf Curl and Crumpling Symptoms in the Imperial Valley of California

In the fall of 1998, volunteer watermelon plants, growing in a commercial honeydew melon field in the Imperial Valley of California, showed symptoms of leaf crumpling and yellowing that were suggestive of a geminivirus infection. Geminivirus infection in watermelon leaves showing these symptoms was established by squash blot hybridization analysis with a general probe for Western Hemisphere whitefly-transmitted geminiviruses (Family Geminiviridae, Genus Begomovirus) and by PCR analysis with degenerate PCR primers for the DNA-A and DNA-B components of bipartite begomoviruses. DNA-A (~1.2 kb) and DNA-B (~1.6 kb) fragments were amplified from DNA extracts prepared from symptomatic leaves and were cloned and sequenced. The DNA-A and DNA-B fragments had a nearly identical (99.5%) common region sequences, indicating they were from the same geminivirus. Database searches conducted with these sequences revealed no high degree of sequence identity (i.e., >90%) with other begomoviruses, including Squash leaf curl virus (SqLCV) from Southern California. Depending on the sequence used for comparison, the highest sequence identities were with Tomato severe leaf curl virus from Guatemala, SqLCV, Squash yellow mottle virus from Costa Rica, and Bean calico mosaic virus from Mexico. Evidence that this geminivirus was responsible (at least in part) for the disease symptoms in the watermelon volunteers came from experiments in which a DNA extract prepared from watermelon leaves with the crumpling and yellowing symptoms were inoculated into watermelon seedling by particle bombardment. Inoculated seedling developed leaf crumpling and distortion symptoms and geminivirus infection in these seedlings was demonstrated by PCR analysis. Watermelon seedlings bombarded with gold particles alone did not develop symptoms. These results establish that the watermelon volunteers were infected by a new bipartite begomovirus. Surveys of spring melons in the Imperial Valley indicated that leaf crumpling and/or yellowing symptoms were not common. However, the new begomovirus was detected from cantaloupe and watermelon leaves with crumpling and yellowing symptoms that were collected on July 2 and August 17, 1999. A survey of fall melons conducted September 23-24, 1999, revealed widespread symptoms of leaf curl and crumpling on new growth of muskmelon plants in all seven commercial fields examined (estimated incidence: 25-50%). No such symptoms were observed on honeydew melon plants. These plants were determined to be infected with the new begomovirus based on sequence analysis of PCR-amplified DNA-A fragments. These results suggest that a new cucurbit-infecting begomovirus has appeared in the Imperial Valley of California and that it has rapidly be spread through the area, presumably by whiteflies. Based on it’s most diagnostic symptoms and capacity to infect a range of cucurbits, the name Cucurbit leaf crumple virus (CuLCrV) is proposed. It will be important to carefully monitor for the incidence of CuLCrV to and to assess the capacity of CuLCrV to cause economic losses in desert melon production.

Investigator’s Name(s): Moshe Lapidot, Rachel Ben-Joseph, Michael Friedmann¹, Meir Pilowsky¹, & Shlomo Cohen.

Affiliation & Location: Departments of Virology and ¹Plant Genetics, Agricultural Research Organization, The Volcani Center, Bet Dagan 50250, ISRAEL.

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

Dates Covered by the Report: 1999

The Effect of TYLCV-Resistant Tomato Plants on Virus Epidemiology

Tomato yellow leaf curl virus (TYLCV), transmitted by the tobacco whitefly (Bemisia tabaci Genn.), can be devastating to tomato (Lycopersicon esculentum L.) crops in tropical and subtropical regions, causing up to 100% crop loss. Control measures in infected regions are based on limitation of the vector population, which usually requires heavy pesticide use and physical barriers such as 50 mesh nets. Since control of the viral vector is difficult or nearly impossible, the development of resistant cultivars is the best option for control of TYLCV. However, all the resistant commercial cultivars tested at the Volcani Center, when infected with TYLCV, developed different levels of disease symptoms (1). Recently, we reported the development of a novel source of resistance to TYLCV, breeding line TY172, which is a symptomless carrier of TYLCV (2). Line TY172, whether infected in the greenhouse with virulifelous whiteflies, or when grown in the field under natural infection, showed no symptoms of the disease. Viral DNA was detected in infected TY172 plants, albeit at much lower levels than a susceptible infected control. In addition, grafting experiments utilizing infected susceptible scions grafted onto TY172 stocks, showed that even when exposed continuously to very high levels of virus, line TY172 did not develop disease symptoms, nor did it accumulate high levels of the virus.

In the present study, we tested the effect TYLCV-resistant tomato plants have on virus epidemiology. In order to understand the effect resistant tomato plants have upon TYLCV transmission by whiteflies (WFs), we have chosen three different TYLCV-resistant plants, cv. Fiona, cv. 8484, TY172, and as a control a TYLCV-susceptible tomato line, L27. Ten plants from each line or cv. were inoculated with TYLCV and served as source plants for TYLCV acquisition by WFs. Following a 48-hr acquisition access period, WFs were transferred to susceptible tomato plants, a single WF per plant, and allowed a 24-hr transmission access period. At the end of the transmission period, before removal of the insects from the plants, the plants were screened for WFs persistence, i.e., the presence of live and active WFs on the plants. Following inoculation the plants were sprayed and kept in an insect proof greenhouse for 4 weeks, while appearance of TYLCV symptoms were monitored. TYLCV level was determined in source plants and in WFs following acquisition. The effect different plant hosts have upon virus transmission efficiency by WFs was assayed as well. There were significant differences in TYLCV transmission efficiency; the highest rate of transmission, 59%, was by WF which acquired the virus from susceptible plants. The lowest level of transmission, 17%, was by WF which acquired the virus from TY172. Thus, although TY172 can serve as a source of TYLCV in the field, the acquisition rate of the virus from TY172 was low compared to other resistant plant lines tested. The correlation between TYLCV accumulation level in the source plant and acquisition, and subsequently transmission, efficiency by whiteflies will be discussed.

References

1. Lapidot et al. 1997. Plant Dis. 81: 1425-1428.

2. Friedmann et al. 1998. J. Amer. Soc. Hort. Sci. 123: 1004-1007.

Investigator’s Name(s): T.-X. Liu, K. Crosby, M. Miller, L. Gregg, and R. Hernandez.

Affiliation and Location: Texas Agricultural Experiment Station, Texas A&M University, 2415 E. Highway 83, Weslaco, TX 78596.

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

Dates Covered by the Report: 1999

Cucurbit Yellow Stunting Disorder Virus on Melon In the Lower Rio Grande Valley of Texas: Blame to Bemisia?

Cantaloupe and honeydew melons exhibited unidentified virus-like symptoms in the Lower Rio Grande Valley (LRGV) of Texas in the fall 1999. Plant materials were sent to Dr. J. McCreight, USDA-ARS in Salinas, CA, Dr. J. Brown, University of Arizona, and Dr. B. Falk, University of California at Davis. Eventually, the disease was identified as Cucurbit Yellow Stunting Disorder Virus (CYSDV) transmitted by Bemisia. CYSDV is a whitefly-transmitted virus, a member of closteroviruses. The vector whitefly is Bemisia tabaci (Gennadius) in Europe (Spain), and B. argentifolii Bellows & Perring in the US. CYSDV can be retained by the whitefly for at least 7 days, and has an experimental host range restricted to members of the family Cucurbitaceae. Unusual yellowing symptoms were widespread in cantaloupe and honeydew melon fields in the LRGV in late September. Plants with symptoms ceased growing and began to decline. Fruit from affected plants were lower in sugars than fruit from symptomless plants. Although we did not have the exact data to compare the number of fruits and yields from both virus affected plants and unaffected plants, as shown in Table 1, numbers of fruits from affected plants were much fewer than those from unaffected plants, and 30-50% of fruits in the affected plants were not marketable. Leaf samples exhibiting symptoms were sent to several plant virologists for diagnosis. With the efforts from all the scientists involved, the disease was eventually identified as the cucurbit yellow stunting disorder virus (CYSDV), which is recorded in the literature as transmitted by the silverleaf whitefly, Bemisia argentifolii (= B. tabaci). Yes, B. argentifolii is still one of the most important pests on cucurbits in the LRGV, and whiteflies were found on the plants showing virus disease symptoms. At present, we do not know how important CYSDV will be to the melon growers in the LRGV; how severe CYSDV may be on the spring melons; and how we can manage the CYSDV. We will appreciate any comments and information about the CYSDV.

Investigator’s Name(s): Y. P. S. Rathi.

Affiliation & Location: (Head Plant Pathology Dept.) G. B. Pant University of Agriculture & Technology, Pantnagar - 263145, (U.P) INDIA.

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

Dates Covered by the Report: 1999

Epidemiology Of Mungbean Yellow Mosaic Virus , A Yellow Plague Of Kharif Pulses

Yellow mosaic disease popularly known as yellow plague of Kharif pulses is currently the most important and wide spread in India. The pathogen Mungbean yellow mosaic virus is vectored by the whiteflies, Bemisia tabaci Genn. in circulative manner. In the last decade, there has been considerable progress in characterization of the virus, its relationship with the vector and the management aspects. However, certain aspects of epidemiology particularly the role of the weed hosts is not well understood.

Several naturally infected weeds and cultivated plants exhibiting yellow mosaic symptoms were tested for host range studies using the vector, B. tabaci and light microscopy (Azur A staining) technique. All except Cajanus cajan Mill sp. showed negative results. Of the various shapes and colours, bright yellow coloured cylindrical traps attracted the maximum whitefly adults. The population of whitefly increased with the increase in temperature and decreased in relative humidity. Heavy showers, frequent raining, strong winds and high RH were detrimental to the adults. The maximum adults were trapped in May and June with the minimum in November. The disease incidence was positively correlated with the whitefly population. Early spring and late rainy seasons planted curdbean crop showed less incidence of the disease. Increased row spacing increased the whitefly population as well as the disease incidence.

Investigator’s Name(s): Philip A. Stansly.

Affiliation & Location: University of Florida, Southwest Florida Research and Education Center, Immokalee FL 34142.

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

Dates Covered by the Report: 1999

Impact and Management of Tomato Yellow Leafcurl Virus in Southwest Florida

We evaluated yield reduction caused by TLYCV infection in tomato as well as the mitigating effects of chemical and cultural practices aimed at controlling the whitefly vector, Bemisia argentifolii. Tomatoes were transplanted into beds covered with colored or reflecting mulches and in a separate experiment treated with combinations of systemic or insect growth regulator insecticides. Plants showing virus symptoms were regularly marked and harvested once individually. Yield varied with day of first symptom expression in a linear relationship. Total fruit weight was reduced 88% and 71% for plants showing symptoms at 30 and 33 days after transplanting respectively, compared to uninfected controls. Whitefly numbers were reduced and the appearance of virus symptoms was delayed by chemical treatments, especially those including soil-applied imidacloprid, in contrast to foliar applied thiamethoxam. Insecticidal effects on yield were consistent with virus incidence. These results were confirmed two earlier trials showing that soil applied imidacloprid or thiamethoxam provided better whitefly control than these same materials or acetamidprid applied to foliage from 2 to 6 times using the same or more total active ingredient. Aluminum mulch provided early protection from whiteflies and reduced virus incidence, although yields on black and aluminum mulch were not significantly different, probably because benefits from decreased virus incidence on aluminum were counterbalanced by lower soil temperatures. Thus, TYLCV had a devastating effect on tomato yield, especially when symptoms appeared early in plant development. These effects were mitigated through cultural and chemical practices aimed at delaying infection by protecting plants from the whitefly vector.

Investigator’s Name(s): Gail C. Wisler & Arturo A. Cortez.

Affiliation & Location: USDA--ARS, Crop Research & Improvement Unit, Salinas, CA.

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

Dates Covered by the Report: 1998-1999

Differential Transmission Characteristics Among Four Whitefly Vectors of Tomato Chlorosis Crinivirus

Two bipartite, whitefly-transmitted viruses belonging to the genus Crinivirus in the family Closteroviridae have been characterized infecting tomato. These are Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV). TICV is transmitted only by the greenhouse whitefly (GHWF; Trialeurodes vaporariorum), whereas ToCV is transmitted by the GHWF, the sweet potato whitefly (SPWF; Bemisia tabaci biotype A), the silverleaf whitefly (SLWF; B. tabaci biotype B; B. argentifolii), and the banded wing whitefly (BWWF; T. abutilonea). Since ToCV is transmitted by four vectors rather than one, it is expected to be more widespread than TICV. TICV was first identified as a distinct crinivirus in California in 1993. Since then it has been found in Italy, Taiwan and North Carolina. ToCV has been identified in Florida, Louisiana, Colorado, Spain, South Africa, and Taiwan. Identifications have been made by transmission using specific vectors and/or by dot blot hybridization using DIG-labeled riboprobes.

ToCV has a wide host range which includes 24 plant species in 7 plant families, and include some important crops and ornamentals, i.e., tomatillo (Physalis ixocarpa), tobacco, (N. tabacum), spinach (Spinacea oleracea), aster, calendula, and petunia. Unlike TICV, ToCV does not infect lettuce. Neither TICV nor ToCV infects members of the Cucurbitaceae.

Studies have been initiated to determine the transmission properties among the four whitefly vectors of ToCV. The efficiency of the four vectors differs significantly. Tests for transmission efficiency involved allowing aviruliferous whiteflies to feed on diseased plants in groups of 1, 5, 10, 20, and 40 per leaf cage for a 24 hr acquisition period, and then transferring them to Physalis wrightii Gray, an excellent indicator for most criniviruses tested. Ten replications were used for each test group and each test was replicated five times. All four vectors were tested concurrently. The SLWF is the most efficient vector. One SLWF transmitted ToCV 12.5% of the time. Five SLWFs transmitted at an efficiency of 48%, 10 transmitted at 40%, 20 at 88%, and 40 at 98%. The SLWF is expected to be an efficient vector because of its high incidence at the locations where ToCV was first identified in Florida. Surprisingly, the BWWF was equally as efficient as the SLWF at 7.5%, 40%, 43%, 84%, and 100% for 1, 5, 10, 20, and 40 whiteflies. In contrast, the SPWF and the GHWF were inefficient vectors. The efficiencies for the SPWF ranged from 0 to 68%, and the GHWF ranged from 0 to 28% for 1, 5, 10, 20, and 40 whiteflies, respectively. Preliminary tests using only the SPWF showed a persistence of 1 day. All SPWFs lost the virus during the first 24 hr feeding period. Additional studies are underway to determine the persistence, acquisition threshold, and inoculation threshold of all four whitefly vectors.

A third bipartite, whitefly-transmitted crinivirus in tomato has been identified from the Canary Islands. This new virus is very similar to ToCV according to the size of the double stranded RNA pattern and the fact that it is also transmitted by all four whitefly vectors. However, it is distinct from ToCV in that it infects lettuce. It is possible that other tomato infecting criniviruses exist. The vector specificity and transmission properties are valuable methods for identifying new viruses in combination with molecular and serological assays.