Molecular Plant Pathology On-Line [] 1997/0116fuchs

Management of Virus Diseases by Classical and Engineered Protection

Marc Fuchs1, Stephen Ferreira2 and Dennis Gonsalves3

1 Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (Phone 315-787-2351; Fax 315-787-2389; e-mail, 2 Department of Plant Pathology, University of Hawaii, 3190 Maile Way, Honolulu, HA 96822 (Phone 808-956-2840; Fax 808-956-2832; e-mail, and 3 Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456 (Phone 315-787-2334; Fax 315-787-2389; e-mail

Corresponding Author:

Marc Fuchs, Department of Plant Pathology, Cornell University, New York State Agricultural Experiment Station, Geneva, NY 14456
telephone: 315-787-2351 fax: 315-787-2389 email:

Accepted: 9 December 1996

Table of Contents


2.1. Citrus Tristeza Closterovirus Cross Protection
2.1.1. Brazilian cross protection against stem pitting strains
2.1.2. Australian cross protection against stem pitting strains
2.1.3. Cross protection against decline inducing strains
2.1.4. Factors affecting effectiveness of CTV cross protection
2.2. Papaya Ringspot Potyvirus Cross Protection
2.2.1. Selecting a mild strain
2.2.2. Specificity of cross protection
2.2.3. Effect of mild strain
2.2.4. Implementing and commercializing cross protection
2.2.5. Factors affecting efficiency of cross protection
2.3. Zucchini Yellow Mosaic Potyvirus Cross Protection
2.3.1. Selecting a mild strain
2.3.2. Effectiveness of cross protection
2.3.3. Factors affecting effectiveness of cross protection
2.3.4. Commercialization of cross protection
2.4. Cucumber Mosaic Cucumovirus-Satellite RNA Cross Protection
2.3.1. China tests: Origination and deployment of CMV-satellite RNA isolates for cross protection
2.3.2. China tests: Effectiveness and extensive use of cross protection
2.3.3. China tests: Factors affecting efficiency of cross protection
2.3.4. Other field trials in the United States and Italy

3.1. Engineered Protection of Tomato
3.1.1. Control of tomato and tobacco mosaic tobamoviruses
3.1.2. Control of cucumber mosaic cucumovirus
3.2. Engineered Protection of Cucurbits
3.2.1. Transgenic cucumber and control of cucumber mosaic cucumovirus
3.2.2. Transgenic squash and control of zucchini yellow mosaic and watermelon mosaic 2 potyviruses
3.2.3. Transgenic cantaloupe and control of zucchini yellow mosaic and watermelon mosaic 2 potyviruses, and/or cucumber mosaic cucumovirus
3.3. Engineered Protection of Potato
3.3.1. Control of potato virus X potexvirus
3.3.2. Control of potato virus Y potyvirus
3.3.3. Control of PVX and PVY
3.3.4. Control of potato leafroll luteovirus
3.4. Engineered Protection of Papaya
3.5. Engineered Protection of Other Crops





The phenomenon of cross protection has been known for a long time and in theory it provides an efficient means of controlling virus diseases. McKinney (1929) was the first to show that plants already infected with a virus are normally protected against infection by a related strain of the virus. Thus, plants infected with a mild strain could be protected against infection by severe isolates or strains of that virus. However, cross protection referred to here as classical protection has been used to control virus diseases of a few crops (Fulton, 1986). There are a number of reasons for the limited use and application of classical protection: First, the fact that a "live" virus is used to deliberately infect plants makes researchers and growers reluctant to use this approach on a practical scale. Second, it is generally difficult to obtain mild strains that are of practical value. Third, there is some possibility that the mild strain might mutate and cause even greater problems than the control strain itself.

Engineered protection, which is a form of parasite-derived resistance (Sanford and Johnston, 1985), is being pursued in many laboratories as a strategy for controlling virus diseases. Numerous reports have shown that this approach is generally applicable to many plant viruses without the drawbacks of classical protection (Fitchen and Beachy 1993; Grumet 1994; Lomonossoff, 1995). In fact, it might be simply said that engineered protection is a means of inserting resistance genes into plants. The practical difference from conventional breeding for resistance is that the resistance is derived from the pathogen rather than the host itself.

Since recent comprehensive reviews have covered classical (Fulton, 1986) and engineered protection (Fitchen and Beachy 1993; Grumet 1994; Lomonossoff, 1995), this report will focus on the analysis of cases where these approaches have been used to manage viruses under field conditions. We cover reports on classical protection to control citrus tristeza closterovirus (CTV), papaya ringspot potyvirus (PRSV), zucchini yellow mosaic potyvirus (ZYMV), and cucumber mosaic cucumovirus (CMV). For engineered protection, we analysee reports on field trials of transgenic potato plants evaluated for resistance to potato virus Y potyvirus (PVY) and/or virus X potexvirus (PVX), transgenic papaya plants analysed for resistance to PRSV, transgenic tomato and cucumber plants investigated for resistance to CMV or tobamoviruses, and transgenic squash plants, including Freedom II, the first virus-resistant transgenic crop commercially released in the United States, and cantaloupes examined for resistance to mixed infections by ZYMV, watermelon mosaic virus 2 potyvirus (WMV 2), and/or CMV. Our overall objective is to glean some lessons from these reports which may facilitate better management of virus diseases.


Matthews (1991) and others have defined cross protection as the use of a viral strain to infect a plant which in turn prevents superinfection of that plant by closely related strains of the virus. However, this definition becomes less useful when cross protection is used as a practical approach to control or manage virus diseases because protected plants are usually superinfected. According to the strict definition, this would suggest that cross protection has not worked or has broken down once the challenge virus infects the protected plant regardless of whether it causes symptoms or damage. In this review, we use a definition of cross protection which reflects the practical effect that the protecting strain has on a plant subjected to challenge virus infections (Gonsalves and Garnsey, 1989): " the use of a mild strain of a virus to protect against the economic damage by severe strains of the same virus". With this definition cross protection can be effective even though a severe challenge strain of the virus has infected the protected plant.

2.1. Citrus Tristeza Closterovirus Cross Protection

Citrus spp. are among the most widely grown fruit crops in the world and citrus tristeza closterovirus (CTV) causes the most important virus disease of citrus. The magnitude of the citrus industry is apparent from the fact that the total estimated "on-tree" value is 10 billion dollars worldwide, and the fact that orange juice is one of the most widely consumed juices.

CTV is transmitted semi-persistently by several species of aphids, of which the brown citrus aphid, Toxoptera citricides, is the most effective (Lee and Rocha-Pena, 1992). CTV strains are broadly grouped according to how they affect certain plants or scion/rootstock combinations (Lee and Rocha-Pena, 1992), i.e., those causing mild symptoms, seedling yellows symptoms, decline on sour orange, stem pitting of grapefruit, and stem pitting of sweet orange. The mild forms do not normally cause noticeable effects on most commonly grown citrus cultivars whereas the seedling yellows strains cause severe chlorosis and dwarfing of inoculated sour orange under greenhouse conditions. Decline strains cause trees grafted on sour orange rootstock to decline and ultimately die. However, these strains do not cause damage on trees grown on tolerant rootstocks such as sweet orange or Rangpur lime. In contrast, stem pitting strains of grapefruit and sweet orange cause significant damage to grapefruit or sweet orange regardless of their rootstocks. Control of CTV by cross protection is largely aimed at the stem pitting strains of grapefruit and sweet orange, although work is progressing in Florida to identify mild strains that might give protection against the decline strains that affect trees grafted on sour orange rootstock.

2.1.1. Brazilian cross protection against stem pitting strains. The control of CTV strains causing stem pitting on sweet orange and grapefruit in Brazil is, by far, the largest and most successful use of cross protection to control a plant virus disease. This fascinating work was reviewed by Costa and Muller (1980). After the introduction of CTV to Brazil in the 1920's, the Brazilian citrus industry converted to growing trees on CTV-tolerant rootstocks. However, strains of CTV were still causing significant damage on lime, grapefruit, and Pera sweet orange grafted onto CTV-tolerant rootstocks. Cross protection efforts were aimed at these strains.

Mild strains were obtained by recovering CTV isolates from trees that grew well in orchards where severe infection was prevalent; the logic being that these trees were growing well because they were protected by mild strains. Indeed, six of 45 isolates induced only mild symptoms and protected sweet orange, grapefruit, or lime trees against damage from stem pitting strains after challenge inoculation by aphids. Furthermore, protected plants produced good fruit yields. Once the efficacy of the mild strains was established, protected trees were obtained rapidly by grafting scion buds from mild strain-infected trees to healthy CTV-tolerant rootstocks. By 1980, over 8 million trees of Pera sweet orange were cross protected. Cross protection to control CTV in Pera sweet orange in Brazil is still practiced (Lee and Rocha-Pena, 1992).

2.1.2. Australian cross protection against stem pitting strains. Stem pitting induced by CTV is a severe problem affecting grapefruit in New South Wales. Stem pitting strains reduced yield and, more importantly, fruit size which makes them less marketable. Cross protection experiments conducted at two locations over a 21-25 year period showed the beneficial effects of cross protection as well as the effects of the climate (Broadbent et al., 1991). As in Brazil, mild strains of CTV were selected from healthy appearing and non-stem-pitted grapefruit trees growing in severely affected orchards. The ability of two mild strains to protect inoculated grapefruit trees against a severe strain was compared at Somersby (humid area on the coast) and at Daredon (hot dry inland area), both in New South Wales.

The overall data showed clearly that cross protected plants yielded more and better quality fruits than severe strain-inoculated plants or plants that became aphid-infected by severe strains after planting in the field. The difference between cross protected and severe strain inoculated or initially uninoculated trees were much more obvious at Somersby than at Daredon. Mean yield of mild strain no. 3135-inoculated trees was 204 kg as compared to 63 kg for the severe strain in the 19th year after planting at Somersby. In comparison, the same mild strain-inoculated plants at Daredon had a mean fruit yield of 239 kg versus 145 kg for severe strain-inoculated plants at the same time period. Measurements in fruit size also showed significant differences. Cross protected trees had a much lower proportion of small fruit than the severe strain-inoculated trees. Measurement in breakdown of cross protection from infection by severe strains indicated that 10 of 117 (8.5%) plants showed deterioration of fruit quality at the cooler more humid Somersby site whereas no marked evidence of breakdown was noticed at the hotter and more dry Daredon site.

2.1.3. Cross protection against decline inducing strains. As mentioned earlier, some CTV strains cause decline of sweet orange trees grafted on the susceptible sour orange rootstock. In areas where these strains are prevalent, the industry generally adapts by abandoning the sour orange rootstock and switching to CTV-tolerant ones. However, there are still important citrus growing areas, such as Florida, where sour orange is still used because the dominant strains are the mild type, that do not cause quick decline.

Florida researchers have initiated experiments for selecting mild strains that might protect against CTV decline (Lee and Rocha-Pena, 1992). These efforts are timely since decline-type isolates are present in Florida and have caused severe losses in certain areas (Brlansky et al., 1986). Furthermore, should the expected invasion of the brown citrus aphid occur it will facilitate the spread of decline type isolates, and cause even more severe damage to citrus planted on sour orange (Lee and Rocha-Pena, 1992). Although trials are in the early stages, there is hope that selected strains may help to control CTV decline of trees grafted on sour orange rootstock. The development of a monoclonal antibody, MCA13, which reacts preferentially to decline-type isolates will help the researchers in their selection and evaluation of mild strains (Permar et al., 1990). Interestingly, efforts are being made to save mature trees on sour orange by deliberately infecting them with mild strains. If successful, this approach will be of significant economic benefit because about a third of the producing trees in Florida are still on sour orange rootstock.

2.1.4. Factors affecting effectiveness of CTV cross protection. A number of articles have been published on CTV as it relates to factors affecting cross protection (Gonsalves and Garnsey 1989; Lee and Rocha-Pena, 1992). As with any cross protection system, the availability of an effective mild strain is paramount. Intuitively, one might think that selection of mild strains of CTV would be difficult because this virus is poorly mechanically transmitted, and a local lesion host is not available, therefore greenhouse screenings are substantially reduced (Garnsey et al., 1977). Despite these limitations, many potential mild CTV strains have been found in nature. Thus, evaluating potential mild strains under proper field conditions seems to be more limiting than selecting mild strains. Although greenhouse evaluation is useful, field evaluation is mandatory because mild strains that appear promising under greenhouse conditions may not be effective in the field. Since CTV strains are defined by their reaction on various citrus host combinations, a mild strain must be evaluated for the type of strains targeted for control. In this respect, one would expect that it is easier to evaluate for cross protection against stem pitting strains since this involves evaluation of the growth of the tree, as compared with decline strains that cause a reaction at the scion/rootstock interface.

2.2. Papaya Ringspot Potyvirus Cross Protection

Papaya is a popular fruit crop grown in commercial farms in household backyards throughout the tropics and subtropics. Papaya ringspot potyvirus (PRSV) causes one of the most serious diseases limiting the economic viability and production of papayas and cucurbits around the world (Gonsalves, 1994). When it is established in papaya (Carica papaya L.), it has proven difficult to control in many areas including Hawaii, Taiwan, the Caribbean, Mexico, Brazil, Guam, the Philippines, and most recently, Australia. PRSV is transmitted nonpersistently by many species of aphids, and has a relatively narrow host range that is confined to members of the Caricaceae, Cucurbitaceae, and Chenopodiaceae. PRSV strains are divided into two groups: The PRSV strains that infect papaya are designated PRSV-p, and are differentiated from the PRSV-w strains (formerly known as watermelon mosaic virus 1) that are found only on cucurbits. Surveys in Thailand, Hawaii, Taiwan, Australia, and the Philippines suggest that PRSV-p strains do not occur naturally in cucurbits unless they are planted either within or near infected papaya orchards. This situation occurs despite the fact that PRSV-p is readily transmitted experimentally to cucurbits mechanically or by aphids. Interestingly, PRSV-w strains do not infect papaya. Most commercial cucurbits and nearly all papayas are susceptible to PRSV, although tolerance has been reported in a few papaya varieties and breeding lines (Conover et al., 1986), but these selections are not widely planted because of relatively poor fruit quality.

Management practices for PRSV in papaya include roguing when initial outbreaks are noted. This practice helps to slow down the spread of the virus (Namba and Higa, 1977) but the disease incidence eventually reaches unacceptable levels. Once this situation occurs, papaya production is moved to new areas if possible, only to repeat the roguing and movement cycle when PRSV comes into the new area. When these practices are not followed or cannot be used, production of papaya is generally not economical. These conditions make cross protection an attractive option to control PRSV in papaya (Yeh and Gonsalves, 1994).

2.2.1. Selecting a mild strain. Selection of mild PRSV strains has been difficult (Yeh and Gonsalves, 1994). Efforts to recover mild strains from "green islands" of infected leaves and from apparently healthy plants in the field have failed. The most successful attempt at producing a mild strain of PRSV was described by Yeh and Gonsalves (1984). They selected two mild strains, HA 5-1 and HA 6-1, following nitrous acid treatment of crude tissue extracts of squash plants infected by a severe PRSV-p strain from Hawaii. These strains gave good protection in papaya against the severe Hawaiian strain under greenhouse conditions and in small field plots in Hawaii. In addition, strain HA 5-1 was only poorly transmitted by aphids. Studies with strain HA 5-1 illustrated some of the unexpected problems and issues with respect to the practical use of cross protection to manage PRSV in the field as discussed below.

2.2.2. Specificity of cross protection. In Hawaii, field trials with several commercial papaya varieties demonstrated excellent protection (Ferreira et al., 1993; Mau et al., 1990). Trials in commercial farms indicated that a cross protected crop could be grown for 2-3 years and be economically viable despite high virus incidence in an area where economic papaya production was previously impossible. Breakdown of cross protection occurred in varying degrees and was dependent on the variety being protected and the incidence of native severe strains in the area. The highest level of breakdown from infection by severe strains was observed for 10% of mild strain-inoculated plants in 2.5 year.

Recent greenhouse studies with severe strains from different geographical regions have shown that protection with mild PRSV strain HA 5-1 is largely specific to the Hawaiian isolates of PRSV (Tennant et al., 1994). Specificity of cross protection was evident in Taiwan where the first large scale field evaluations of the HA 5-1 strain were conducted (Wang et al., 1987). Protection was incomplete but could give economic benefit only if applied to whole blocks of plants that were isolated from severely infected papaya trees. For example, in one trial, the farmer obtained about twice the profit from protected compared to unprotected fields. By 1991, more than 1,700 hectares of cross protected orchards had been planted (Yeh and Gonsalves, 1994). However, cross protection with the mild PRSV strain HA 5-1 is currently not recommended in Taiwan because growing papaya under screen enclosures is apparently a more economical practice (Yeh, personal communication).

In Thailand, by contrast, the mild PRSV strain HA 5-1 did not provide a useful level of disease control (Yeh and Gonsalves, 1994). This was probably due to strain differences since comparative greenhouse tests showed that HA 5-1 did not protect against the severe Thai strain while it gave excellent protection to the severe Hawaiian strain. These greenhouse and field results clearly show that the specificity of the mild PRSV strain HA 5-1 will limit its usefulness to Hawaii and Florida (McMillan and Gonsalves, 1987).

2.2.3. Effect of mild strain. The mild PRSV strain HA 5-1 differentially affects Hawaiian solo papaya varieties indicating a mild strain x variety interaction (Ferreira et al., 1992; Ferreira et al., 1993). Fruits of varieties "Line 8", "Waimanalo", and "Kamiya" (an open-pollinated field selection from "Waimanalo") tested in Hawaii had a low to moderate number of ringspots depending on the time of year, whereas "Sunrise" had larger numbers of more severe ringspots on fruits that were also misshapen and distorted during part of the year. Because of its sensitivity to the mild strain, commercial cross protection of "Sunrise" was not recommended.

The effect of the mild strain on fruit quality and yield was also assessed. Sugar levels for all varieties were not significantly affected, and yield ranged from 275 to 606 kg of grade A fruit per hectares per month for the 1.5-2.0 years of harvest that most growers were able to obtain. Yield differences were attributed to inherent differences in yield potential of varieties, location of the field, and differences among individual growers in how irrigation and fertilization were managed. The initial field study in Hawaii indicated that production was reduced ca. 10-15% by the mild strain (Ferreira et al., 1993).

Symptoms caused by the mild strain, as judged by leaf chlorosis, varied substantially during the year. They were most severe during the winter months from December to March and moderated gradually to the summer lows in August and September. This was a consistent pattern from year to year.

Interestingly, while foliar symptoms peaked during winter months, the occurrence of ringspots on fruit was most prevalent about 4-5 months later in May or June. During the time foliar symptoms were most intense, fruit to be harvested in May and June were already set and in the initial stages of development. Similarly, fruit symptoms were least severe during the winter months because they were initiated during the summer when plants were least affected by the virus.

2.2.4. Implementing and commercializing cross protection. The situation in Hawaii and Taiwan has been instructive in large scale implementation of cross protection. Inoculating large numbers of plants is not a major problem because papaya seedlings are successfully and readily inoculated by spraying plants with crude extracts of mild strain-infected Cucumis metuliferus (Wang et al., 1987). A major concern is maintaining the purity of the mild PRSV strain and producing it under conditions which minimize contamination by severe strains. Consequently, the mild strain is produced at Cornell University and shipped to Hawaii as needed. This approach is possible because the mild strain is required on the island of Oahu, where only ca. 2% of the State's production occurs and little inoculum is needed. A more efficient production method would be necessary if cross protection is to be deployed on a large scale in the Puna area on the island of Hawaii, where 95% of Hawaii's papaya production occurs.

2.2.5. Factors affecting efficiency of cross protection. The specificity and unavailability of mild strains limits the widespread use of cross protection with PRSV. Unlike CTV, efforts to isolate naturally occurring mild strains have failed. Also, the available mild strain HA 5-1 causes significant symptoms on fruit of the popular 'Sunrise' solo variety. Nevertheless, cross protection has been economical on the island of Oahu because it ensures the production of papaya, despite the inherent decrease in yield, whereas orchards that are not protected would have very little productivity.

An interesting observation with implications on approaches that may affect the growth reducing effect of the mild strain HA 5-1 may be instructive. A solid block planting of 'Line 8' in which only ca. 50% of the seedlings were cross protected successfully was monitored for breakdown or superinfection. After 2.5 years, breakdown occurred, but affected less than 5% of the plants. Of those plants that were superinfected with severe virus strains, none of them was a healthy or unprotected plant. It appears that viruliferous aphids were attracted preferentially to the slightly more chlorotic leaves of cross protected plants which increased their chances of losing the severe viruses before feeding on the unprotected plants. By the time those aphids moved to unprotected plants, they were no longer infective for the severe strains. In effect, protection was maintained, even though less than 100% of the plant population had been protected. If these observations can be generalized, the debilitative effect of the mild PRSV strain might be reduced by inoculating only a portion of the orchard to be protected.

2.3. Zucchini Yellow Mosaic Potyvirus Cross Protection

Zucchini yellow mosaic potyvirus (ZYMV) causes severe losses to cucurbits worldwide (Lisa and Lecoq, 1984). As with PRSV, ZYMV is transmitted nonpersistently by several species of aphids. So far, ZYMV resistance genes from Cucurbita spp. have not been widely incorporated in commercial cultivars. The severity of the disease and the relatively short crop cycle makes cross protection an attractive control option.

2.3.1. Selecting a mild strain. The currently used mild strain was obtained by Lecoq et al., (1991) from a melon plant infected with a severe strain of ZYMV but having an axillary vine showing mild mosaic symptoms. The mild strain, designated ZYMV-WK, is transmitted poorly by aphids. Most importantly, ZYMV-WK, caused only mild leaf mottling and did not induce fruit malformation in cucurbits.

2.3.2. Effectiveness of cross protection. Unlike the PRSV mild strain, ZYMV-WK has effectively cross protected zucchini squash against isolates from different geographical regions such as France, Hawaii, Taiwan, and Great Britain (Cho et al., 1992; Lecoq et al., 1991; Walkey et al., 1992; 102 Wang et al., 1991). For all of these field trials, squash seedlings were inoculated with ZYMV-WK at the cotyledon stage and transplanted to the field at various times after inoculation. At all locations, cross protected plants produced 3 to 7 times more fruit, as judged by weight, than control plants. However, the most striking differences were observed in marketable fruits where cross protected plants yielded up to 40 times more fruit, by weight, than comparable unprotected plants.

Cross protection was also highly effective under conditions with high disease incidence, and the benefits were even more dramatic. In Taiwan, cross protected squash yielded two times more marketable fruit than unprotected plants in the initial planting, but 40 times more in adjacent plots established one month after the first (Wang et al., 1991). Although highly effective, Walkey et al., (1992) observed loss of protection at the latter stages of the trial in one location in Great Britain, but not in another. They attributed this gradual breakdown to inoculum levels in the area. Nevertheless, differences between protected and unprotected plots were much greater in the site with high disease incidence. In the French and Taiwanese trials, 15% of the protected plants also showed severe symptoms but these were attributed to infection by PRSV or WMV 2 (Lecoq et al., 1991; Wang et al., 1991). The usefulness of cross protection using ZYMV-WK was also demonstrated recently with cantaloupe plants tested in field enclosures in California (Perring et al., 1995). Cross protected plants conserved marketable fruit yield by nearly 75% upon subsequent infection with a severe ZYMV strain.

2.3.3. Factors affecting effectiveness of cross protection. It might be expected that cucurbits which have a short 8-12 week crop cycle would be more readily protected than longer term crops such as papaya (2-3 years) and citrus (greater than 10 years). However, published reports indicate several factors that affect the effectiveness of cross protection against ZYMV in cucurbits. Mild strain-inoculated plants were not fully protected until ca. 14 days after inoculation. Thus, in the trials reported so far, plants were inoculated in the greenhouse before transplanting to the field. Although this practice may be difficult for large scale implementation, it can be done, as shown by experience in Hawaii where cross protection of zucchini with ZYMV-WK has been commercialized (Cho et al., 1992). Inoculation of direct-seeded cucurbits is another approach. If precautions are taken to minimize the severe inoculum pressure, mild strain inoculated plants would be fully protected by the time severe strains are established in the field.

The occurrence of other cucurbit viruses is perhaps the most important factor to be considered when using cross protection against ZYMV. Since cucurbits are severely affected by other viruses including PRSV, WMV 2, and CMV, deployment of cross protection would be dependent on the importance of other viruses on the crop. Surveys to determine what viruses are prevalent would help to make such decision.

The benefit of protecting zucchini squash in the field in treatments integrating cross protection with the use of reflective mulches to inhibit aphid landing or feeding activity has been investigated in Hawaii (Cho et al., 1992). Although mulches are beneficial, the effectiveness of cross protection alone is adequate so that cross protection is mainly used without mulches (Cho, personal communication).

2.3.4. Commercialization of cross protection. ZYMV has severely limited the year-round production of zucchini squash in Hawaii, especially on the island of Maui. Following extensive field trials, Cho et al. (1992) successfully implemented the commercialization of cross protection on Maui. The program includes collaboration between researchers and extension personnel at the University of Hawaii and individual farmers. Briefly, the primary source of the mild ZYMV-WK strain is maintained at the University of Hawaii and given to farmers who then have the responsibility to increase the mild strain and inoculate their seedlings. Farmers are not encouraged to maintain the mild strain beyond several passages but instead are advised to get fresh inocula from the University. After inoculations, farmers follow their normal commercial practice of transplanting and growing the zucchini for market. The success of the program is evidenced by the fact that 90% of the farmers utilize cross protection in their production which has increased many fold in recent years.

2.4. Cucumber Mosaic Cucumovirus-Satellite RNA Cross Protection

CMV infects numerous plant species and is one of the most important viruses affecting vegetables worldwide (Palukaitis et al., 1992). Many isolates of CMV contain satellite RNAs, some of which can cause severe necrosis on tomato plants whereas most actually attenuate symptoms of the infection. CMV is very difficult to control because of its wide host range and its transmission by numerous species of aphids in a nonpersistent manner. Vegetable crops infected include cucurbits, tomato, and pepper plants.

Interestingly, cross protection to control CMV has been through the use of CMV strains carrying satellite RNAs that attenuate symptoms on vegetables. This approach has been used over several thousand acres in The People's Republic of China, and much less extensively in fields in Italy and the United States.

2.4.1. China tests: Origination and deployment of CMV-satellite RNA isolates for cross protection. The rationale in using CMV-satellite RNA isolates [referred to here as biological control agents (BCA), as designated by Tien and Wu (1991)], is based on the observation that CMV satellite RNAs affect symptoms of infected plants by causing either disease exacerbation, disease attenuation, or no affect on symptom expression. Tien and colleagues exploited the properties of the satellite RNA and constructed four BCA by mixing genomic RNAs from CMV isolates with satellite RNAs that did not exacerbate symptoms on plants (Tien and Wu, 1991; Tien et al., 1987). Screening of inoculated tobacco, pepper, and tomato plants resulted in selection of four BCA. Further inoculations of these isolates to numerous plant species showed that they were symptomless or caused only very mild symptoms.

Large scale application of BCA in China are done under conditions which minimize the build up of aberrant satellite RNAs that may cause severe symptoms. Stock BCA are kept as dried samples in several replicates and propagated on tobacco to be used as inocula for large scale tests. However, the inoculum is not passed again through tobacco in order to minimize the build up of undesired satellite RNAs. Infected tobacco leaves are subjected to polyethylene glycol precipitation to partially purify the BCA. Target plants are mechanically inoculated by rubbing leaves or by dipping tomato roots into diluted inoculum.

2.4.2. China tests: Effectiveness and extensive use of cross protection. Extensive field tests with pepper, tomato, and tobacco plants carried out over different regions in China showed the economical benefits of the BCA. Tests conducted from 1981 to 1985 on pepper plants showed that BCA reduced the disease severity by 21 to 82%, and increased fruit yield by 10 to 55% (Tien and Wu, 1991). Similar effects were obtained for tomato plants. With protected tobacco plants, the disease severity was also lowered and three times more leaves of superior quality were produced compared to control plants. The higher yields obtained led to have a dramatic increase in the adoption of cross protection for pepper, tomato, and tobacco plants. In one province alone, about 3,335 hectares of pepper plants were cross protected in 1990.

2.4.3. China tests: Factors affecting efficiency of cross protection. Quality control of the BCA is a key factor. The prevailing concern with the use of BCA has been related to the inherent danger of using satellite RNA since it is known that they can cause severe damage, and "new" satellite RNAs are easily obtained, especially when CMV is passed successively through hosts such as tobacco. The large scale use of BCA in China enhances this concern. However, severe damage resulting from the inoculation of BCA that are contaminated with disease-exacerbating satellite RNAs has not occurred in the numerous trials (Tien and Wu, 1991). Apparently, this has largely been prevented by periodic and detailed quality control of the BCA stock. Furthermore, satellite RNA that can cause necrosis in tomato have not been detected in areas where cross protection has been used, and the low concentration of the BCA in protected plants minimizes their aphid transmission and thus their spread into uninoculated plants.

2.4.4. Other field trials the United States and in Italy. BCA have been also used in limited field tests in Italy (Gallitelli et al., 1991) and the United States (Montasser et al., 1991) to control the CMV necrogenic satellite RNA in tomato plants. Unlike the trials in China, control was not aimed at avoiding CMV infection but at reducing damage caused by CMV necrogenic satellite RNAs. Both sets of trials showed excellent protection against the satellite RNA-induced necrosis. In fact, the United States trials showed that protecting satellite RNAs gave better protection than cross protection utilizing CMV strains without satellite RNAs. Likewise, in Italy where necrosis of tomato plants due to satellite RNA has reached high incidence levels in some areas, satellite RNA-protected tomato plants had a protective effect of greater than 95% and the fruit yield was double that of the control plants. Thus, these trials extend on the work in China by showing that the protecting satellite RNAs were effective against CMV isolates that carry necrogenic satellite RNAs.


Development of virus-resistant varieties using classical breeding has also been limited for several crops due to the lack of known resistance genes and/or genetic barriers or complexity of the target crops. Engineered protection offers a new approach to manage virus diseases.

In this review, engineered protection is referred to as resistance or protection conferred in plants by viral-derived nucleic acid sequences that are introduced into the plant genome via genetic engineering. Transgenic plants developed by this approach express viral sequences and are likely to be protected against infections by the virus from which the resistance gene is derived, and closely related viruses.

Hamilton (1980) first suggested integrating the viral genome into plants for protection against viruses. Powell-Abel et al. (1986) were the first to demonstrate that constitutive expression of the tobacco mosaic tobamovirus (TMV) viral coat protein (CP) gene in tobacco plants provided a substantial level of protection against infection by this virus. Since this pioneering work on TMV, viral CP genes have been used extensively to engineer protection against numerous plant viruses. Coat protein-mediated protection is an example of a broad strategy proposed by Sanford and Johnson (1985) to genetically engineer resistance to pathogens by using parasite-derived genes.

In general, viral sequences encoding structural and nonstructural proteins have been used; these include genes coding for the CP, replicase, movement protein, and protease. Viral coding sequences have been used as sense, antisense, full length, truncated or untranslatable constructs. In addition, several viral noncoding sequences have been used including satellite RNA, defective interfering RNA, terminal untranslated sequences, and ribozymes. Viral CP genes have been the ones most frequently used to engineer protection against plant viruses. The level of protection conferred by CP genes in transgenic plants varies from immunity to delay and attenuation of symptoms, and for some cases the protection is broad and effective against several strains of the virus from which the CP gene is derived. A number of reviews have discussed the different approaches developed for engineered protection (Beachy, 1993; Beachy et al., 1990; Grumet, 1994; Lomonossoff, 1995). Our goal is to present a comprehensive review on the benefits that engineered protection offers to agriculture in managing virus diseases, and epidemiological implications.

3.1. Engineered Protection of Tomato

Several major virus diseases affect tomato production throughout the world including tomato (ToMV) and tobacco (TMV) mosaic tobamoviruses, CMV, potato virus Y potyvirus (PVY), tomato spotted wilt tospovirus (TSWV), tomato yellow leaf curl geminivirus (TYLCV), and tobacco etch potyvirus (TEV).

3.1.1. Control of tomato and tobacco mosaic tobamoviruses. These viruses cause significant yield losses in tomato. Their impact is more significant for tomato production under greenhouse than under field conditions because these two viruses are highly infectious, extremely persistent, and easily transmitted by mechanical inoculations. High sanitation standards and the use of virus resistant-varieties have provided effective control of these two tobamoviruses. The host gene Tm-22 is widely used and has proven very effective against ToMV and TMV in commercial plantings worldwide (Watterson, 1993). However, breeding for resistance against tobamoviruses has been laborious for some cultivars due to undesirable traits tightly linked to the single partially dominant Tm-22 gene. Engineered protection has been used to develop resistance to ToMV and TMV in order to improve elite tomato varieties without altering their desirable characteristics.

In the first field trial ever of transgenic plants engineered for virus resistance, Nelson et al. (1988) evaluated two tomato lines expressing the CP gene of the TMV U1 strain. Transgenic plants displayed nearly complete resistance to mechanical infections by TMV and only 5% were symptomatic by at the end of the trial compared with 99% of the control plants. Quantitative ELISA analysis corroborated visual observations indicating that very low amounts of virions accumulated in transgenic tomato plants. Fruit yield was identical for inoculated transgenic and uninoculated control plants, demonstrating that viral transgenes do not alter the horticultural performance of elite tomato varieties.

Sanders et al. (1992) extended the field characterization of these transgenic tomato plants and demonstrated that they exhibit excellent resistance not only to the homologous U1 strain but also to the more severe PV230 strain. Only 1% and 2.5% of the transgenic plants inoculated with TMV-U1 and TMV-PV230, respectively, became infected within 8 wk of inoculation. No yield loss due to TMV infections was recorded for the transgenic plants versus 20-69% for the control plants. However, TMV-resistant transgenic plants had limited ability to protect against ToMV since 56-89% of them were infected with ToMV causing 11-25% yield loss. Tomato plants expressing the CP gene of ToMV were developed to introduce ToMV resistance. Transgenic line 4174 was not infected by ToMV in the field, while 93% of the control plants were infected. Interestingly, transgenic line 4174 also showed substantial resistance to TMV and only 7% of the plants were infected by TMV-U1.

Considering that TMV and ToMV are only transmitted mechanically, it would have been interesting to evaluate these transgenic tomato plants under greenhouse conditions where natural infections, either by inadvertently handling plants or manipulating contaminated tools, occur more readily than in the field. Some mechanical transmissions of ToMV or TMV occurred in field tests and were likely to account for the unexpected infections of uninoculated plants (Sanders et al., 1992). Unfortunately, no experimental comparison has been made of transgenic plants with classically bred resistant tomato varieties containing the Tm-22 resistant gene. Furthermore, combination the CP genes of both ToMV and TMV should be a valuable approach to develop elite tomato varieties with high levels of resistance to both tobamoviruses.

3.1.2. Control of cucumber mosaic cucumovirus. Due to high CMV incidence, tomato production has been completely abandoned in some traditional growing areas of Spain (Jorda et al., 1992). Resistance factors to CMV have been identified in several wild tomato species (Watterson, 1993), but resistant varieties have not been developed yet because of the polygenic nature of the resistance and plant infertility problems.

Tomato plants expressing the CP gene of CMV strain WL were developed recently using a parent tomato cultivar containing the Tm-22 gene for resistance to TMV (Xue et al., 1994). These transgenic plants showed excellent CMV resistance in greenhouse tests and were subsequently evaluated under field conditions in New York over two consecutive years (Fuchs et al., 1996). Transgenic plants displayed high resistance to mechanical inoculations since none of them became infected, as opposed to 80% of the mechanically inoculated control plants. Transgenic plants grew vigorously whether or not they were inoculated, and had a 17-fold overall increase in fruit yield relative to CMV-infected control plants. However, conclusions could not be drawn on resistance to infection by aphid inoculations because only 9-14% of uninoculated controls became infected despite the high inoculum incidence from CMV-Fny, a strain that is efficiently aphid-transmitted in cucurbit fields. Given the very low frequency of natural CMV transmission to tomato plants in New York, additional field tests need to be conducted in locations where natural spread of CMV by aphids occurs readily in tomato.

To evaluate the spectrum of resistance conferred by the CP gene of CMV strain WL, transgenic tomato plants were challenge inoculated in the greenhouse with CMV isolates from different geographic regions, including 10 strains from CMV subgroup I (California, Florida, Hawaii, France, Australia, New Zealand, Egypt, China, Japan, Taiwan) and 3 strains from subgroup II (New York, Mexico), some of which carried satellite RNA. Transgenic plants were completely resistant to all CMV field isolates tested so far (Provvidenti and Gonsalves, 1995). CMV virions could not be detected nor recovered from uninoculated leaves, although some virus was recovered from inoculated leaves of transgenic plants. Interestingly, a unique CMV variant which developed in the greenhouse overcame the resistance (Provvidenti and Gonsalves, 1995).

CMV-resistant transgenic tomato plants should have large economic impact given the fact that CMV causes severe damage worldwide and host-derived resistance genes are not readily available. The broad resistance offered by the CMV-WL CP gene construct was unexpected since the CP shares only about 80% homology at the amino acid level with CMV subgroup I strains.

3.2. Engineered Protection of Cucurbits

Cucurbit production is impaired throughout the world by several viruses, the most prevalent being CMV and three potyviruses: ZYMV, WMV 2, and PRSV-w. These four viruses are readily transmitted by several aphid species in a nonpersistent manner. Conventional breeding has been used successfully to develop resistant varieties in some cucurbits, especially CMV resistance in cucumbers (Cucumis sativus L.) (Munger, 1993; Superak et al., 1993).

3.2.1. Transgenic cucumber and control of cucumber mosaic cucumovirus. Conventional breeding of cucumber has been successful in developing the CMV-resistant 'Marketmore' series, widely used by growers in the United States (Munger, 1993). The development of transgenic cucumber plants allowed Gonsalves et al. (1992) to compare viral CP-derived resistance and host gene-derived resistance under field conditions over 3 years. The experimental design used included CMV-infected nontransformed plants set at defined positions throughout the field to ensure an uniform distribution of virus inoculum sources accessible to natural aphid vectors.

Transgenic cucumbers showed a very long delay in symptom development with only 4-30% symptomatic plants 9 wk after transplanting, compared with 84% of nontransformed controls and 13% 'Marketmore' resistant plants. ELISA 12 wk post-planting showed significantly fewer transgenic plants containing virus (28-41%) relative to both control (85%) and commercial 'Marketmore' resistant plants (62%). Transgenic line T48 produced a significantly higher yield (42% increase) than the other three transgenic lines analysed or the nontransformed counterparts. Unlike resistance to CMV in tomato plants, CMV resistance in commercial cucumber cultivars developed by conventional breeding is effective and readily available. Therefore, CMV transgenic cucumbers are not currently recommended as replacement for the current resistant cultivars. Nevertheless, the CMV trials were instructive in establishing field design parameters for evaluating cucurbits for resistance to natural infection by aphids. These general approaches have been used in our recent squash and cantaloupe trials.

3.2.2. Transgenic squash and control of zucchini yellow mosaic and watermelon mosaic 2 potyviruses. Transgenic squash (Cucurbita pepo L.) engineered for virus resistance have been developed by the Asgrow Seed Company. Several transgenic lines which express single or combinations of CP gene constructs of CMV, ZYMV, and/or WMV 2 were evaluated at Cornell University.

In 1993, the resistance of three transgenic lines expressing CP gene constructs of ZYMV and/or WMV 2 was evaluated in the field under severe incidence of ZYMV and WMV 2 (Fuchs and Gonsalves, 1995). The transgenic lines tested were: ZW-20 expressing the CP genes of both ZYMV and WMV 2, Z-33 expressing the single CP gene of ZYMV, and W-164 expressing the single CP gene of WMV 2. Transgenic line ZW-20 showed excellent resistance to mixed infections by ZYMV and WMV 2 in that none of the plants developed severe symptoms, i.e. foliar mosaic, chlorosis, malformation or plant stunting. Only a few ZW-20 developed very mild leaf symptoms in the form of localized chlorotic dots or blotches. In contrast, all plants of the transgenic lines Z-33 and W-164 expressing single CP genes developed severe symptoms, as did the control plants. ELISA data on ZW-20 plants confirmed visual observations. ZYMV and WMV 2 were detected only in chlorotic dots (56% of plants), but were not detectable in asymptomatic leaves even 10 wk after planting. Transgenic Z-33 were substantially infected with ZYMV (21%) after 10 wk and heavily infected by WMV 2 (98%) after 7 wk. Both transgenic W-164 and control plants were totally infected with ZYMV and WMV 2 after 5 wk.

Differences between squash lines in yield and fruit quality were even more dramatic. ZW-20 fruits were symptomless whereas all fruits from transgenic squash Z-33 and W-164, as well as from control plants were unmarketable because of severe discoloration and distortion. The high resistance of transgenic squash ZW-20 to infections by ZYMV and WMV 2 has been confirmed in several field tests at different locations (Arce-Ochoa et al., 1995; Clough and Hamm, 1995).

Transgenic line Z-33 containing only the ZYMV CP gene showed excellent resistance to ZYMV, but not to WMV 2, while transgenic line W-164 did not show high resistance to either WMV 2 or ZYMV. The low level of resistance of W-164 is likely due to the WMV 2 isolate which was able to severely infect transgenic plants also under greenhouse conditions. Interestingly, the combination of the CP genes of WMV 2 and ZYMV apparently provided synergistic resistance because the transgenic line ZW-20, which contains both genes, was resistant to severe dual infections by ZYMV and WMV 2.

Development of the transgenic squash hybrid ZW-20 is a significant breakthrough for squash improvement considering the economic importance of ZYMV and WMV 2, and the difficulties in developing resistant cultivars by conventional breeding. Transgenic squash ZW-20, subsequently renamed Freedom II, was recently approved as the first virus-resistant genetically engineered crop to be deregulated by USDA-APHIS (Medley, 1994). Seeds of Freedom II were marketed by the Asgrow Seed Company in the spring of 1995.

Transgenic squash lines Z-33 and W-164 with single resistance to either ZYMV or WMV 2 may be valuable for regions where only one of these potyviruses is prevalent. For example, ZYMV is prevalent in Maui, Hawaii but WMV 2 is not a major problem. The opposite situation occurs in central Florida where WMV 2 causes serious problems while ZYMV is less prevalent.

Nevertheless, it is important to highlight the value of transgenic lines containing multiple CP genes to control several aphid-borne potyviruses because mixed virus infections are common. Resistance of the transgenic squash and cucumber lines described above can still be broadened by incorporating CP genes of other cucurbit viruses. Transgenic squash lines expressing the CP genes of ZYMV, WMV 2, and CMV have been developed (Tricoli et al., 1995) and recent field tests demonstrated the potential of such transgenic squash in controlling mixed infections by these three viruses (Fuchs et al., unpublished data; Tricoli et al., 1995; Arce-Ochoa, et al., 1995; Clough and Hamm, 1995). Recently, transgenic squash line CZW-3 received exemption status from USDA-APHIS (Acord, 1996), thus clearing it for commercial release. This is the second transgenic squash line to be deregulated in the United States.

3.2.3. Transgenic cantaloupe and control of zucchini yellow mosaic and watermelon mosaic 2 potyviruses, and/or cucumber mosaic cucumovirus. Cantaloupes (Cucumis melo L.) are severely affected by CMV, ZYMV, WMV 2, and PRSV-w. Melon varieties with multiple resistance to these viruses would be valuable to growers. Recently, cantaloupes containing multiple viral CP gene constructs have been developed by the Asgrow Seed Company. One of these transgenic lines (CZW-30) was evaluated under field conditions at Cornell University (Fuchs et al., 1997).

Cantaloupe line CZW-30 expressing the CP genes of CMV, ZYMV, and WMV 2 was tested against infections by CMV, CMV and ZYMV, and CMV, ZYMV and WMV 2. Across all trials, transgenic plants showed excellent resistance against single or mixed infections. Transgenic homozygous plants developed localized mild symptoms late in the growing season whereas all control plants showed severe systemic symptoms and had high virus titers 5-6 weeks after transplanting. Interestingly, transgenic hemizygous plants exhibited a significant delay (2-3 weeks) in the onset of disease compared to control plants but showed systemic symptoms 9-10 weeks post-planting. Strikingly, 3-10% of the homozygous and 31-35% of the hemizygous plants had dual or triple infections as opposed to the control plants which had 66-99% mixed infections with two or three viruses. Transgenic plants grew vigorously whereas control plants were severely stunted with a 34-50% reduction in shoot length. Although hemizygous plants had a 17% reduction in shoot length compared to homozygous plants, they yielded 7 times more marketable fruits than control plants. This was the first report on the evaluation of a cantaloupe line of commercial quality with resistance to three of the four major viruses affecting melon production.

High levels of resistance to a wide range of CMV strains was expected in cantaloupe plants expressing the CMV-WL CP gene based on earlier studies on tobacco (Namba et al., 1992) and tomato (Provvidenti and Gonsalves, 1995; Xue et al., 1994) plants containing this CP gene. However, transgenic cantaloupe lines expressing the CMV-WL CP gene were not highly resistant to infection by CMV in the greenhouse (Gonsalves et al., 1994). One of these lines designated H7-21 was further evaluated under field conditions. It showed a 4-wk delay in infection relative to the control plants, but ELISA performed 9 weeks after planting revealed that 88% of the transgenic plants accumulated CMV similar to nontransformed controls (98%) (Fuchs et al., unpublished data).

3.3. Engineered Protection of Potato

Potato production for fresh market and seed tuber is often severely affected by several viruses including potato leafroll luteovirus (PLRV), potato virus Y potyvirus (PVY), potato virus X potexvirus (PVX), potato virus S carlavirus (PVS), potato virus A potyvirus (PVA), and potato virus M carlavirus (PVM) occurring alone or in combination. PVY and PLRV are aphid-transmitted in a nonpersistent and persistent manner; PVX is mechanically transmitted. When PVX and PVY occur together, they produce a synergistic increase in disease severity. The frequency in which mixed virus infections occur in potato plants stresses the need for developing multiple resistance, especially since viruses are readily disseminated in tubers. Conventional breeding methods to develop virus-resistant varieties have proven difficult because cultivated potato is an autotetraploid and highly heterozygous plant species. Although host resistance genes have been identified and used extensively for PVX, PVY, PVS, PVM, and PVA (Foxe, 1992), major commercial potato cultivars generally lack virus resistance. Incorporation of resistance to PLRV has been more difficult because of the polygenic nature of the resistance (Foxe, 1992).

3.3.1. Control of potato virus X potexvirus. PVX usually causes mild or barely detectable symptoms in most potato varieties. This virus has a worldwide distribution and is found in most potato growing areas. Besides being mechanically transmitted, it is also disseminated through infected seed tubers. PVX infections can result in loss of seed tuber certification.

The first field test of transgenic potato expressing the CP gene of PVX was reported by Hoekema et al. (1989) for the varieties Bintje and Escort. This field test was designed to evaluate whether horticultural and morphological characteristics were maintained in transgenic potato plants, but virus resistance was not evaluated.

Jongedijk et al. (1992) examined the frequency of PVX transmission from tubers of transgenic potato varieties Bintje and Escort that became infected with PVX in the field. A significant reduction in PVX incidence was observed among clonal progenies from tubers of mechanically infected transgenic plants in the field. Among the twelve transgenic lines tested, two (MGE-32 and MGE-44) performed best giving no infected tubers as did the classically bred PVX-resistant varieties Bildstar and Sant. The other transgenic lines showed a range in reaction from moderate resistance (6-43% infection of tubers) to full susceptibility. The results obtained with lines MGE-32 and MGE-44 indicate that potato lines expressing the CP gene of PVX offer potential to control PVX in commercial production of resistant seed tuber stocks.

3.3.2. Control of potato virus Y potyvirus. In the first field test reported in Europe with transgenic plants engineered for resistance to an insect-transmitted virus, Malnoe et al. (1994) relied on natural spread of two PVY strains, N and O, to evaluate the resistance of potato variety Bintje expressing the CP gene of PVYN. The transgenic line Bt6 was immune to the homologous PVYN under high inoculum levels, while 98% of the control plants were infected 15 wk post-planting. ELISA performed with monoclonal antibodies revealed that 23% of the transgenic plants became infected with PVYO, the most common PVY strain in potato fields. In a second-year field trial, the excellent resistance of line Bt6 was confirmed since PVY was detected in only 7-10% of the transgenic plants while 86% of the control plants were infected. Analysis of PVYO transmission in tubers from plants infected by aphids showed that although all the progenies of infected nontransformed control tubers were infected as early as 6 wk, only 28-41% of the transgenic progenies were infected 12 wk after planting. Potato plants expressing the CP gene of PVYN exhibited complete resistance to PVYN and some degree of protection to PVYO under natural aphid transmission in the field. In addition, the PVYN CP gene conferred some resistance in progenies from tubers of plants infected with PVYO by aphids. No yield data were presented in this study probably because of a significant contamination by PVS.

3.3.3. Control of PVX and PVY. Since mixed infections occur frequently in potato plants, development of cultivars with multiple resistance is a valuable objective for potato improvement. Lawson et al. (1990) and Kaniewski et al. (1990) demonstrated the usefulness of multiple genes to control mixed virus infections. Kaniewski et al. (1990) evaluated the resistance of potato plants expressing the CP genes of PVX and PVY following mechanical inoculations with both viruses. Transgenic line 303 was the only one that remained symptomless and was highly resistant to both PVX and PVY. At the end of the trial period, a very low percentage of plants in this transgenic line were infected with PVX (6%) or PVY (2%), and none was infected with both viruses. Although some spread of PVY occurred in the field via aphid vectors, no natural infection was detected in any of the plants of transgenic line 303. Moreover, the yield of transgenic line 303 was high and unaffected by virus inoculations, indicating its potential to control mixed infections by PVX and PVY while maintaining high yield. Virus incidence in tubers from the experimental plants was not reported in this study.

3.3.4. Control of potato leafroll luteovirus. Since host resistance genes available to control PLRV are polygenic, breeding PLRV-resistant varieties is difficult. Engineered protection offers an alternative. Potato plants expressing the CP gene of PLRV have been developed (Barker et al., 1992; del Vas et al., 1993; Kawchuk et al., 1990; Kawchuk et al., 1991; van der Wilk et al., 1991; Brown et al., 1995; Presting et al., 1995). Several transgenic potato lines tested under greenhouse conditions showed high levels of resistance to aphid inoculations of PLRV. Although greenhouse data appear to be very promising, resistance to PLRV has to be assessed under field conditions where plants are subjected to multiple random probing by aphid vectors throughout the growing season.

The previous examples illustrate that engineered protection of potato offers great value for virus management not only in commercial production but also in seed tuber production to ensure the continued commercial propagation of healthy seed tubers.

3.4. Engineered Protection of Papaya

Although several transgenic fruit crops have been developed and evaluated in the greenhouse for virus resistance, extensive field tests have only been conducted with transgenic papaya plants so far.

In Hawaii, PRSV has limited papaya production on the island of Oahu in Hawaii since the 1960's, and by the 1980's, the virus had moved within 40 kilometers of the Puna area on the island of Hawaii where 95% of the state of Hawaii's papaya is produced. Thus, efforts were made to develop transgenic papaya plants expressing the CP gene of PRSV. In 1992, Fitch et al., (1992) reported that transgenic papaya line 55-1 expressing the CP gene of PRSV strain HA 5-1 was highly resistant in the greenhouse to mechanical inoculations with this PRSV strain from Hawaii. The same year, the resistance of R0 clones of transgenic line 55-1 was tested against natural aphid infections in the field on the island of Oahu. The field trial data showed that transgenic line 55-1 was highly resistant to PRSV. None of the transgenic trees developed symptoms throughout the trial period (20 months), and virions could not be detected by ELISA whether the plants were mechanically inoculated or not (Lius et al., 1996). Also, transgenic plants grew vigorously and produced fruits of marketable quality (Lius et al., 1996). In contrast, all control plants became infected within 2-4 months. Tennant et al. (1994) subsequently showed that R1 plants of line 55-1 were highly resistant to PRSV isolates from Hawaii but less effective against other isolates from geographic regions outside of Hawaii.

Ironically, when the field trial was initiated on the island of Oahu in 1992, PRSV was discovered in the previously virus-free Puna area on the island of Hawaii where about 1,000 hectares of papaya were being grown. PRSV reached epidemic proportions, and by 1995, a third of the Puna area was devastated by PRSV. A subsequent field trial with homozygous and hemizygous plants of transgenic line 55-1 was established in Puna in 1995. The transgenic plants have shown excellent resistance so far. The transgenic line 55-1 is a good germ plasm source that offers excellent prospects for breeding PRSV-resistant papaya plants in Hawaii, especially since it is a 'Sunset' cultivar which is a sib of 'Sunrise', the most widely grown papaya cultivar worldwide. The 1995 field trial should provide useful information on the management of PRSV in a commercial orchard. It will also set the basis to evaluate the potential of transgenic papaya plants in controlling the disease in large-scale plantings before transgenic PRSV-resistant plants are made available to growers.

Transgenic line 55-1, which was recently named 'SunUp', and another transgenic line designated 63-1 were deregulated by APHIS/USDA in September 1996 (Strating, 1996). These two transgenic papaya lines are expected to clear regulatory status from EPA and FDA in 1997. Deregulation and subsequent commercialization of these papaya plants will provide Hawaii with a hope to control PRSV which is devastating the local industry. This may be the first case where a biotechnology product will have such a significant and timely impact in controlling a virus disease that threatens the economic survival of an industry.

As mentioned above, the resistance of transgenic line 55-1 is not effective against PRSV isolates from elsewhere (Tennant et al., 1994). Other genes from PRSV isolates collected in the Caribbean, South America and Asia are currently being engineered and transferred into commercial papaya germ plasms to develop plants resistant to PRSV strains outside of Hawaii (Gonsalves, unpublished).

3.5. Engineered Protection of other Crops

Several other transgenic vegetable, ornamental, cereal, and fruit crops, have been genetically engineered for protection against viruses. Some of these transgenic plants exhibit resistance under greenhouse or growth chamber conditions, however, no field data are available yet. Some of these examples include potato plants resistant to PVX, PVY and PLRV (Tacke et al., 1996), and to the carlaviruses PVS and, to some extent, to PVM (MacKenzie et al., 1991); alfalfa plants resistant to alfalfa mosaic alfamovirus (Hill et al., 1991); rice plants resistant to rice stripe tenuivirus (Hayakawa et al., 1992); cantaloupe plants resistant to ZYMV (Fang and Grumet, 1993); corn plants resistant to maize dwarf mosaic potyvirus and maize chlorotic mottle machlomovirus (Murray et al., 1993); tomato plants resistant to TSWV (Kim et al., 1994; Ultzen et al., 1995) or TYLCV (Kunik et al., 1994); petunia plants resistant to CMV (Kim et al., 1995); and plum plants resistant to plum pox potyvirus (Scorza and Ravelonandro, 1996).

Other transgenic vegetable, fruit, cereal and ornamental crops have been engineered for virus resistance but no resistance data are available yet. Some examples include: apricot plants expressing the CP gene of plum pox potyvirus (Machado et al., 1992), citrus plants expressing the CP gene of CTV (Gutierrez-E. et al., 1992; Moore et al., 1993); rapeseed (Herve et al., 1993) and cauliflower (Passelegue and Kerlan, 1996) plants expressing the CP gene of cauliflower mosaic caulimovirus; grape plants expressing the CP gene of the nepoviruses grapevine chrome mosaic (Le Gall et al., 1994), grapevine fanleaf (Krastanova et al., 1995; Mauro et al., 1995) and tomato ringspot (Scorza et al., 1996), chrysanthemum plants containing the nucleocapsid gene of TSWV (Urban et al., 1994; Yepes et al., 1995); chinese cabbage plants expressing the CP gene of TMV (Jun et al., 1995); wheat plants expressing the CP genes of barley yellow dwarf luteovirus or wheat streak mosaic potyvirus (Hansen et al., 1995); peanut plants expressing the nucleocapsid gene of TSWV (Brar et al., 1994), soybean and bean plants expressing the CP precursor gene of bean pod mottle comovirus (Di et al., 1996) and antisense constructs of the AC1-3 and BC1 genes of bean golden mosaic geminivirus (Aragao et al., 1996); lettuce plants expressing the CP gene of lettuce mosaic potyvirus (Zerbini et al., 1995) or the nucleocapsid gene of TSWV (Pang et al., 1996), and sweet pepper plants expressing the CP gene of CMV (Zhu et al., 1996).


Management strategies for plant virus diseases are generally dictated by control measures that can be applied to the specific virus-crop combination. Ideally, for crops cultivated in areas where viruses are not present, the management practices are directed to prevent the introduction of viruses into the crop. However, when viruses damaging to a crop are prevalent, the ideal management practice is to grow cultivars that are immune or resistant. If these options are not available, management strategies become more complex but, in general, they are aimed at limiting the spread of viruses within the crop, and/or delaying entry of viruses into the crop, thus reducing their incidence. In general, however, the inability to adequately control virus diseases by using crop management practices or by controlling insect vectors emphasizes the need for alternative methods of crop protection. Two non-conventional management strategies, classical cross protection and engineered protection, have been discussed in this review.

Classical cross protection has been used successfully in a few crops, and is not widely applied. Why is this so? Several reasons exist. First, relatively few mild strains that are suitable for practical application of cross protection have been identified. Second, there is a theoretical possibility that the mild virus strain or a benign satellite RNA may mutate to a severe form and cause significant damage, and lastly, the use of a 'live' virus to deliberately inoculate healthy plants is against the intuitive thinking of most plant pathologists and growers. Thus, cross protection is usually used only after a virus disease has reached severe levels and all conventional management practices have failed. Work with PRSV showed that it is unusually difficult to obtain naturally occurring mild strains, whereas potential mild CTV strains abound in nature. However, bringing the potential mild CTV strains to practical use is still a challenge, primarily because citrus is such a long term crop.

The cases discussed here of CTV, ZYMV, and CMV-satellite RNA cross protection illustrate the significant potential benefits that cross protection can have on a crop, whereas PRSV cross protection efforts show the somewhat unexpected and disappointing specificity of the mild HA 5-1 strain. In most cases where cross protection has been used under high incidence of severe strains, there has been breakdown of the protection towards the latter part of the crop cycle. In such cases, the practical success of cross protection has to be determined by assessing the economic benefits realized during the protected period of the crop life, which is weeks for zucchini squash and years for citrus.

Several factors influence the effectiveness and durability of cross protection including the incidence of severe strains, the duration of the inoculation of the mild strain as illustrated by the ZYMV experiments, the specificity of the mild strain and varietal interactions as illustrated by the PRSV experiments, and environmental conditions as shown by the Australian CTV experiments (likely because temperature has a direct effect on aphid population growth and activity, and on virus replication). From an epidemiological perspective, it is interesting to notice that the mild strains of PRSV and ZYMV are poorly transmissible by aphids.

Cross protected plants significantly limit the incidence of severe virus strains in that symptoms of superinfected plants are attenuated or completely inhibited, and profitable yield is obtained. However, distribution and titer of severe strains do not appear to be significantly reduced, if at all, in cross protected papaya plants (Tennant et al., 1994). Therefore, acquisition and transmission of severe strains by aphids from cross protected plants that become superinfected can still readily occur.

Cross protection is likely to remain out of favor given the rapid successes occurring with engineered protection for a wide range of different crops and virus diseases. However, cross protection is likely to remain dominant for controlling CTV in Brazil and should find continued use with ZYMV. The inherent advantage of cross protection is that once the mild strain is obtained, it can be put into practice quite easily, and it does not require extensive regulations as for engineered protection. The potential use of cross protection still will be determined by the severity of the disease and the effectiveness of other management practices, including engineered protection.

Engineered protection has been used in several crops to overcome some of the limitations of classical breeding, among them the difficulty of introducing resistance genes directly into existing elite cultivars or breeding lines without altering their desirable agronomic traits. Considerable and rapid progress has been made toward applied engineered protection to manage virus diseases. So far, transgenic vegetables (potato, tomato, cucurbits) have been the first crops extensively evaluated in the field. In the future, more reports on field trials are likely to be published and more field tests are likely to be performed on transgenic fruit crops and cereals.

Virus-resistant transgenic plants offer the possibility of excluding viruses or reducing their spread by limiting the availability of virus inoculum, by restricting the amount of virus-infected tissue, and also by reducing virus titers. Given the substantial reduction of virus incidence, transgenic plants are also likely to significantly lower the probability of transmission of vector-borne viruses by reducing the acquisition efficiency of the vectors. This aspect is of epidemiological importance.

Field trials are a prerequisite to critically evaluate the potential of transgenic crops to control virus. For crops that are commonly infected by several different viruses, evaluation of transgenic plants with engineered protection to a single virus may be more difficult since non-targeted contaminating viruses can easily interfere, especially in the case of aphid-borne viruses. So far, only small-scale field trials have been conducted with transgenic plants. However, to assess the environmental risks that have been raised on the use of transgenic plants, such as enhanced weediness and development of modified virions (de Zoeten 1991; Hull 1990; Tepfer 1993), large-scale field trials which simulate grower's conditions will be important (Stone, 1994).

For most crops, developing broad and durable resistance is the goal. Although limited reports are available on the spectrum of resistance of important transgenic crops, we discussed in this review several promising transgenic lines for different crops that show resistance to more than one virus, even when occurring as mixed infections. Transgenic crops which contain combinations of CP genes have performed well. For commercial purposes, transgenic hybrids will have to be developed. This approach is feasible, however, the resistance level of transgenic hemizygous lines needs to be evaluated to determine if it differs or not from that of homozygous lines.

Combining host-encoded and virus-derived resistance appears to be a strategy of choice to ensure durability and develop enhanced protection by taking advantage of any synergistic effect in resistance (Barker et al., 1994; Motoyoshi and Ugaki, 1993; Xue et al., 1994).

Considerable progress has been made in the past decade towards the development of applied engineered protection, since Powell-Abel et al. (1986) first demonstrated the use of CP gene to confer resistance to TMV. The next decade should be even more exciting given the recent commercialization of the first transgenic virus-resistant crop in the United States. Deregulation of the Freedom II transgenic squash resistant to ZYMV and WMV 2 is a very important breakthrough in agriculture, especially considering that several valuable transgenic crops engineered for virus resistance are in the development. Recently, transgenic squash line CZW-3 (Acord, 1996) and transgenic papaya lines 55-1 and 63-1 (Strating, 1996) were deregulated by USDA/APHIS. These products are expected to be commercialized in the near future.

More research efforts from the scientific community should be directed to critically evaluate, under field conditions, the risks and environmental concerns related to the use of transgenic plants engineered for virus resistance. This information is critical to determine if the risks outweigh the benefits that virus-resistant transgenic crops offer to agriculture, especially in crops for which other control measures are limited or non-existent. Active research is on-going towards cloning virus resistance genes from plants. This will add to a very exciting era that will widen our understanding of the mechanisms underlying both resistance for host-encoded and virus-derived resistance genes. Future efforts will likely focus on combining host and pathogen-derived genes to achieve a synergistic effect and ensure durability of the protection against virus diseases.


We are very grateful to Luz Marcela Yepes and Carol Gonsalves for help in reviewing and editing the manuscript. We thank L. V. Madden, J. M. Thresh, and B. Raccah for their initial interest in this review and their comments.


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