Improving our understanding of and our ability to manage many of the insect-transmitted plant viruses requires the use of the vector. Insect transmission of plant viruses is a tritrophic interaction, and as such requires the manipulation of insects, virus, and plant. Vectors must be reared in large numbers and manipulated in such a way as to insure high rates of transmission to test plants. The basics of rearing and manipulating the whitefly, Bemisa tabaci (Hemiptera: Aleyrodidae), a vector of plant viruses belonging to several emerging plant virus genera, representing a large number of economically significant viruses is presented.
Whiteflies, Hemiptera: Aleyrodidae, Bemisia tabaci, a complex of morphologically indistinquishable species5, are vectors of many plant viruses. Several genera of these whitefly-transmitted plant viruses (Begomovirus, Carlavirus, Crinivirus, Ipomovirus, Torradovirus) include several hundred species of emerging and economically significant pathogens of important food and fiber crops (reviewed by9,10,16). These viruses do not replicate in their vector but nevertheless are moved readily from plant to plant by the adult whitefly by various means (reviewed by2,6,7,9,10,11,17). For most of these viruses whitefly feeding is required for acquisition and inoculation, while for others only probing is required. Many of these viruses are unable or cannot be easily transmitted by other means. Therefore maintenance of virus cultures, biological and molecular characterization (identification of host range and symptoms)3,13, ecology2,12, require that the viruses be transmitted to experimental hosts using the whitefly vector. In addition the development of new approaches to management, such as evaluation of new chemicals14 or compounds15, new cultural approaches1,4,19, or the selection and development of resistant cultivars7,8,18, requires the use of whiteflies for virus transmission. The use of whitefly transmission of plant viruses for the selection and development of resistant cultivars in breeding programs is particularly challenging7. Effective selection and screening for resistance employs large numbers of plants and there is a need for 100% of the plants to be inoculated in order to find the few genotypes which possess resistance genes. These studies use very large numbers of viruliferous whiteflies, often several times per year.
Whitefly maintenance described here can generate hundreds or thousands of adult whiteflies on plants each week, year round, without the contamination of other plant viruses. Plants free of both whiteflies and virus must be produced to introduce into the whitefly colony each week. Whitefly cultures must be kept free of whitefly pathogens, parasites, and parasitoids that can reduce whitefly populations and/or reduce the transmission efficiency of the virus. Colonies produced in the manner described can be quickly scaled to increase or decrease population numbers as needed, and can be adjusted to accommodate the feeding preferences of the whitefly based on the plant host of the virus.
There are two basic types of whitefly colonies that can be maintained: a nonviruliferous and a viruliferous whitefly colony. The nonviruliferous colony is composed of whiteflies reared on virus-free plants and allows the weekly availability of whiteflies which can be used to transmit viruses from different cultures. The viruliferous whitefly colony, composed of whiteflies reared on virus-infected plants, allows weekly availability of whiteflies which have acquired the virus thus omitting one step in the virus transmission process.
1. Whitefly Colony Maintenance
2. Whitefly Colony Establishment
3. Method for Inoculation of Test Plants
These methods for whitefly colony establishment and maintenance, as well as manipulation to transmit plant viruses have been used successfully in a number of studies4,11,12,14 as well as many others not cited. Using these methods with persistently transmitted viruses, we have routinely obtained the desired rates of transmission of 100% for selection for resistance, screening insecticides for their ability to interfere with transmission (and evaluation of resistance inducing compounds)13,14,17,18 (Figure 9). The procedures described here can and have been be adapted to many locations in both public and private research facilities.
Type of Transmission | Acquisition Access Period (hour) | Inoculation Access Period (hour) | Ref. |
Nonpersistent | 1 | 2-24 | 10 |
Semipersistent | 6-24 | 8-24 | 10, 16 |
Persistent | 48-72 | 48-72 | 3, 6, 8, 9 |
Table 1. Estimated times that can be used to produce high rates of transmission of viruses with different types of relationships with their vector Bemisia tabaci species complex. Estimated times are based on published minimum times required for 80-100% transmission and modified in some cases to account for latent periods and differences among whitefly populations, virus, and host plants.
Figure 1. An example of a viruliferous whitefly colony. This growth room contains cages of plants infected with a begomovirus and infested with viruliferous whiteflies.
Figure 2. Left: Immatures stages of the whitefly, Bemisia tabaci MEAM clade sensu De Barro6 approximately 1 week post-emergence and Right: an adult whitefly, an effective vector of many plant viruses.
Figure 3. Types of collection devices that can be used to collect whiteflies for plant virus transmission.
Figure 4. Left: Yellow plastic card used to collect whitefly adults showing whiteflies ready for collection. Right: Collecting whiteflies from a yellow plastic card. Whiteflies can be seen on the card as small white/gray spots.
Figure 5. Whiteflies collected by aspiration (20 in a vial) from the colony ready for either acquisition or inoculation.
Figure 6. Upper Left: Single plant cages used for either acquisition or inoculation. Upper Right: Larger cages made of pvc pipe and organza. Lower: Aluminum/screen and organdy cages in a greenhouse containing plants intended for a whitefly colony.
Figure 7. Whiteflies being transferred to a single plant for inoculation (or acquisition).
Figure 8. Addition of a systemic insecticide drench to terminate the whitefly inoculation access period.
Figure 9. Field plants under evaluation for resistance to Tomato yellow leaf curl virus. All plants were inoculated as 5 week old seedlings and then transplanted to the field for evaluation. Plants in the foreground are a resistant germplasm line, plants in the background are susceptible germplasm line.
The methods described here were developed over a period of two decades and are based on the basic information provided by many studies of whitefly transmission, behavior, and biology. Since there are many publications, this manuscript refers primarily to reviews and to a few selected specific publications to illustrate the type of data used to develop this technique. It is very important in this procedure that the whiteflies are handled gently since breaking of legs and antennas can lead to abnormal behaviors and lower rates of transmission. Collection with vacuums and other mechanical suction devices has never produced good results. Another consideration for success is that the number of whiteflies used to obtain 100% transmission to test plants, must be determined for each combination of plant host and virus. This optimal ratio only needs to be established once. Best results have been obtained using young adult whiteflies (1-3 days past emergence) since they give the highest rates of transmission. Older adults can be used but larger numbers of whiteflies will be needed to compensate for their reduced rates of transmission. Females are known to transmit at higher rates than males, since they feed more often than males. However it is usually not worth the time to separate the sexes for transmission. This procedure can be modified in many ways to suit the resources and needs of the researcher.
When working with multiple viruses it is best to keep a single colony of whiteflies reared on nonhost plants of the virus. These whiteflies can be collected placed on virus-infected plants for acquisition and then after acquisition the whiteflies can be moved again to the test plants. Plants intended for the colony should be grown in greenhouses in cages to prevent the introduction of whiteflies that might infect them with another virus. Infestation of these plants with mites, thrips, and other plant pests can compromise the health of the colony and cause it to collapse. Finally watering of the colony plants has to be done with care. These plants are subject to extreme stress by the feeding of so many whiteflies and the lower than normal light conditions. Root rotting fungi which are introduced on the peat in the soilless mixes can become a problem if the plants are overwatered even once. Pretreatment of the plants with a fungicide drench can eliminate some of the problems.
While whitefly transmission of plant viruses can be time consuming and require valuable resources (such as growth rooms) it is essential for the transmission of some viruses for which we have no other means of transmission. It is also a valuable means of screening plants for resistance to viruses, as it uses the same type of transmission that plants in the field will be expected to resist. The use of more artificial means of transmission do not always yield the best results in evaluations of germplasm for virus-resistance.
The authors have nothing to disclose.
Name of the Item | Company | Catalogue No. | Comments (optional) |
Whitefly proof screening | Klayman Meteor Ltd. Petah Tikva 49130, Israel | — | BioNet 50 mesh screen Antivirus Insect Screen |
Whitefly proof screening | Hummert International (www.hummert.com) | 27-4050-1 – 27-4062-1 | BioNet 50 mesh screen Antivirus Insect Screen |
Whitefly Collection Devices | BioQuip | 1135A | Can also be made using eye droppers, flexible plastic tubing, plastic vacuum adaptors, and cheesecloth |
Collection vials | BioQuip | 8909 | Vials used depend on which aspirator is used. |
Yellow plastic cards | any company | — | Any bright yellow plastic card will work, can be purchased in many places. |
Whitefly Cages | — | — | These are not commercially available and must be constructed. 60 cm x 60 cm x 60 cm cages work well for us. Our preference is for those constructed of aluminum window screening, nylon organdy, vinyl plastic, with a structure created using aluminum window frames. Doors are attached using piano hinges and sealed with weather stripping; tops of the cages are vinyl plastic. Cages can also be made of 1) plexiglass glued together on the edges with ventilation provided by screened openings cut on the side pieces; 2) large bags of nylon organdy, organza, or 50 mesh whitefly screening with internal supports provided by PVC pipes and fittings cut to desired lengths and assembled inside the bags, 3) wood frames and nylon organdy, organza, or 50 mesh whitefly screening. |