The overall goal of the following experiment is to transmit plant viruses using white flies. This is achieved by establishing and maintaining a colony of white flies that supply newly emerged Adult white flies on a weekly basis, which are not contaminated with other potential vectors and pathogens. The colonies maintained by regularly adding white flies to young insect free plants from which new adults are produced.
After 17 to 20 days each week, the newly emerged adults can be transferred to test plants to either acquire or transmit virus transmission. Efficiencies of nearly 100%are obtained typically, and transmission rates will usually begin low and climb with systematic optimization. The most challenging aspect of using white flies to inoculate viruses is rearing the white flies in such a way that unwanted pathogens and insects are excluded.
Pathogens particularly root rotting fungi and leaf blights will reduce the quality of the virus host plants. Unwanted insects such as thrifts can suppress whitefly reproduction as well as compromise the quality of the virus host plant. Demonstrating the procedures will be Heather Capa Bianco, a technician from my laboratory.
Whitefly colonies should be maintained in a clean and controlled growth room. Control of relative humidity, temperature, photo period, and light intensity are essential for a colony that develops from egg to adult in 18 days, use the following settings. The temperature should be set between 26 and 30 degrees Celsius, and the relative humidity should ideally be between 30 and 50%If the relative humidity goes above 70%insect and plant fungal pathogens are likely to become a problem.
The ideal photo period is 14 hours. The light intensity should be fairly high, and VHO fluorescent bulbs can produce 800 to 1000 foot candles at the plant canopy. Fertilizer rates and watering must also be reduced to discourage fungal pathogens, as well as salt accumulation Whitely cages must allow for ventilation, ease of access, and sufficient space for a growing whitely population.
Good results have been obtained with organ organza plus aluminum screening, as well as whitely proof Screening the species of plant is an important consideration. Firstly, it should be quick growing, yet not overgrow the chamber. Secondly, the plant should be strong enough to support a high insect population without collapsing.
For these reasons, dwarf or bush cultivars make good plant choices. Thirdly, the plant must either be able to host or unable to host the virus being used depending on the type of colony being raised. Because the colony can collapse due to mites, THPs, wild white flies or other invasive insects, the chosen cultivars must be grown in a protected environment to exclude other insects.
The the first white flies of a new colony must be free of plant viruses, other insects, and insect pathogens. If white flies are collected from the field, rear them for at least eight weeks on plants that are not hosts to the virus during this time, check for absence of plant symptoms. Once clean white flies are obtained, introduce between 20 and 100 to a cage of plants by aspiration or gently shaking them from another source plant.
Every week a new cage needs to be established. For the first three weeks, use the whiteflies on non-ST plants to establish the new cage. Starting on the fourth week, use young adult whiteflies from the three week old cage to establish the next cage.
During the first week in a cage, the whiteflies will lay eggs on the underside of plant leaf surfaces. In the second week, immature white flies will become apparent and some of the adults will still be alive. In the third week, many new adult white flies will have emerged, and there should be a very noticeable increase in the adult whitefly population.
These whiteflies can be used for transmission experiments. In the fourth week, the one week old adults should be transferred to a new cage. The old cage should be removed and its plants discarded.Precise.
Gentle transfer of white flies is accomplished with an aspiration device and collection vials never aspirate. White flies that are feeding on plants when feeding the whitely styles are embedded in the plant and will break off if they're pulled away from the plant. Rendering the whitefly useless to collect white flies in one hand, hold a yellow plastic guard inside the colony cage with the other hand.
Gently tap the plants to encourage the adult white flies to fly. White flies will be attracted to the yellow card where they can be collected. Using the aspirator and very gentle suction add no more than 20 white flies to each collection vial.
Then tap the vial to disorient the flies and cap the vial with param. To ensure successful viral transmission, try 15 to 40 young adults from virus infected plants to healthy plants, depending on the virus and the host plant. Other protocols like resistant screening need larger starter populations.
In this case, shake the virus infected plants, which are infested with white flies over the plants that need to be inoculated. Once the white flies are placed on a virus infected plant, allow them to feed for 48 to 72 hours to acquire persistently transmitted viruses. More time does not generally increase the transmission rate for viruses transmitted in a non-persistent or savvy persistent manner.
A shorter acquisition access period of an hour to a day will suffice for experiments that require acquisition from specific leaves. Clip cages can be used, set up the cage so the leaf underside is accessible, and do not use more than 10 female White flies per 2.5 centimeter diameter cage, or the transmission rate will drop off due to crowding. For plant inoculation, select the appropriate cage considering the number of plants.
One plant, for instance, needs only a small cage just slightly larger than itself for multiple plants, choose between a smaller PVC tubing, framed cage, or a larger aluminum frame cage. The smaller the space around the plant canopy the better. Once the white flies are added to the new plant, the amount of time needed for inoculation depends on the virus and the host plant.
Just as for acquisition, the amount of time required for transmission is dependent upon the manner of transmission. Non-persistent transmission requires a much shorter access time than semi persistent or persistent transmission. Check that the whiteflies are probing or feeding during the inoculation access period.
Open the cage and investigate the underside of some leaves on each plant. For the longer inoculation. Access periods periodically gently disturb the plants to encourage the to redistribute on the plants.
This will increase the transmission rate by countering the natural tendencies of white flies to aggregate to end the inoculation period. Kill the white flies with two insecticides applied, one after the other. First apply a contact insecticide to quickly terminate adult white flies such as insecticidal soap.
Second, apply a systemic insecticide to terminate any white flies that develop in the following weeks. And any remaining adults emit, arid or pyrazine are effective choices. Using these procedures, it is possible to routinely obtain virus transmission rates of 100%Such high rates of transmission are essential when screening test plants for virus resistance and are desirable when evaluating the efficacy of chemical compounds for their ability to interfere with virus transmission.
After watching this video, you should have a good understanding of how to rear white flies and use them to transmit plant viruses. These procedures can be used for research purposes or for commercial development of virus resistant plants, or for the testing of compounds for their ability to interfere with virus transmission.
