Cornell Community Conference on Biological Control

Practical Control of Plant Viruses
through Pathogen-Derived Resistance

Dennis Gonsalves
Department of Plant Pathology
Cornell University NYSAES
Geneva, NY 14456

(prepared from the videotaped presentation*)

Although biological control doesn't usually include transgenic plants, today I will speak on something I consider to be biological control: parasite derived resistance used to develop viral resistant plants. I will deal with 3 systems (cucurbits, tomatoes, and papaya) in order to illustrate 3 related messages.

  1. Tomatoes: we have transferred resistance to tomato germplasm that already has resistance to at least 1 virus and other fungal pathogens. Genetic engineering was used to get resistance to cucumber mosaic virus (CMV) and tomato spotted wilt virus (TSWV), and then we crossed the transgenic plants and a combination of resistance between viruses was obtained.

  2. Cucurbits: we are working with Asgrow Seed Co. to get commercialized cucurbits. We are working on zucchini yellow mosaic virus (ZYMV), papaya ringspot virus (PRV), watermelon mosaic virus-2 (WMV-2), and cucumber mosaic virus, and we are pyramiding the genes of these 4 viruses to get multiple resistance without going through the traditional breeding.

  3. Papaya: here we have a good model system for research because papaya is an important crop and also because the problem (the recent infestation of PRV in Hawaiian papaya plantations) addresses the main point of using biological control in a real-world setting. This project allows us to
    1. try to address a serious problem and deal with all aspects of it,
    2. try to bring a product to commercialization, including the regulatory aspects,
    3. try to do the work that will give resistance to papaya, and
    4. try to save an endangered industry.

Tomato

Cucumber mosaic virus is, worldwide, one of the most important viruses affecting tomato. In mainland China, it is the most important virus. The other virus that is important in tomato is tomato spotted wilt virus.

In our research, we started with a plant, Geneva 80, that already had natural resistance to tobacco mosaic virus (TMV), verticillium, and phytophthera. Our strategy was to

Steps in the research process.

Our first effort was to develop plants resistant to CMV. After getting these resistant plants, we did field trials and found the plants were totally resistant to TMV and CMV.
CMV-infected nontransgenic tomato plants and healthy, transgenic plants, unaffected by the virus. The transgenic plant is heavy with fruit and robust; the nontransgenic plant is scraggly and has few fruits. A different group of plants was transformed with the nuclear protein gene of TSWV, and was found to be resistant to TSWV. We then crossed the 2 types of transgenic plants and derived a plant resistant to all 3 viruses.

We have utilized both transgenic and classical breeding plants to get multiple resistance. These genes are stable and inheritable, as far as we know, and can be used in germplasm to develop virus resistance plants or multiple virus resistant plants.

With cucurbits, we collaborated with Asgrow to get cucurbits resistant to 4 viruses important in cucurbits (ZYMV, PRV, WMV-2, CMV). In this case, to develop resistance, the genes from each virus are engineered in tandem in one cassette and then transformed into a plant to get a viral resistant plant. You get multiple resistance, but here the trait should be carried on in a single gene in the classical breeding. Electron microscope of PRV virus particles.

Cucurbits

In 1994, two plots were prepared and planted with squash plants.

Field Plot Design: Nontransgenic plants were put in border rows and inoculated with virus as a source for test plants inside the border rows. These inside plants were either transgenic plants or nontransgenic plants. Aphids spread the virus within the plots.
Both had border rows which were inoculated with ZYMV and WMV-2 which are transferred by aphids so that we can follow the progress of the infestation under natural field conditions. The control plot had standard plants within the inoculated plants, and the other had resistant transgenic plants. The plants were raised under heavy disease stress field conditions. The transgenic plants showed no virus symptoms; nearly all the controls were diseased within 3-4 weeks. Fruit collected from the diseased plants were often green, instead of the normal, healthy yellow, because of the virus. The harvest from the resistant plants was greater, more uniform, and the fruit were yellow.

Overall view of two field plots showing a healthy, green plot of ZYMV- and WMV-2-virus resistant transgenic squash and a sickly, yellow plot of infected, nontransgenic plants.
Close-up of two plants.
Yield comparisons of two plants.
Effect of virus on fruit distortion and coloration--comparison of infected and transgenic plants.

In 1995 field experiments, 4 plots were prepared in one area, all with border rows infected with virus. Two plots were planted with transgenic plants; two were not. Very good resistance was seen in the transgenic plots.

Table showing comparative fruit yield and marketability from transgenic plants with three, two, and one gene from resistant and from nontransgenic plants. The transgenic plants far outperform the nontransgenic plants. (Marketable yield was 97%, 90%, 77%, and 4%, respectively, for the four types).

The same gene can be transferred into melon, and the yield and health of commercial melon plants is similarly affected when this is done.

Papaya

Papaya is a very good example of what biotechnology can do on a worldwide basis. Papaya orchard severely affected with PRV. Papaya ringspot virus (PRV), a worldwide virus is now threatening the production of papaya, the 4th most important fruit crop in Hawaii. In 1992 PRV was discovered in to Puna, where, surprisingly, papaya trees grow on what is essentially lava rock. Ninety-five percent of the Hawaiian papaya crop is grown in Puna, and so a tremendous effort was made to eradicate PRV. Infected trees were cut and the virus was held at bay for a while , but by 1995, a third of the Puna crop, and thus of the Hawaiian crop, was infected. It seems probable that if nothing is done, by 2000 the Puna area will not be a major producer of papaya for Hawaii.

History of PRV in Hawaii.

Papaya field 9 months after planting showing healthy, green transgenic plants and yellow infected plants.
Comparison of green, transgenic plant and yellow, infected plant.
The papaya field trial about 11 months after planting. The nontransgenic plants are nearly dead while the transgenic plants are green and healthy.

There is tremendous amount of political pressure to find a way to control this virus. In this atmosphere, we undertook a field trial in 1992 to see if the transgenic plants would work against the PRV, to see if the plant could be deregulated, and to see if the plant would be of commercial quality. Two years later, there was no breakdown in the transgenic plants' ability to withstand the PRV virus and the fruit proved to be of commercial quality.

October 1995 Puna field trial design.

In October of 1995, we established a trial in Puna (above). Our challenge is to save an industry by working through the risk, the regulations, all the licenses needed, and other things that must be done to commercialize the crop. Can this be done in a timely way in order to save the papaya industry?

Ten years ago I introduced a crop-protected plant in Thailand in an effort to help the small farmers there. Although here at Cornell the talk is about expenses and the technology, the point is that biotechnology can help humans. I hope to be able to return to Thailand with something that will help the small farmers.

At Cornell we are very fortunate; biotechnology is going well at Cornell.

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