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Annu. Rev. Phytopathol. 1998. 36:415–37
c 1998 by Annual Reviews. All rights reserved
Copyright °Annu. Rev. Phytopathol. 1998.36:415-437. Downloaded from arjournals.annualreviews.org
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CONTROL OF PAPAYA RINGSPOTVIRUS IN PAPAYA: A Case Study
Dennis Gonsalves
Department of Plant Pathology, Cornell University, New York State Agricultural
Experiment Station, Geneva, New York 14456; e-mail: dg12@cornell.edu
KEY WORDS:

pathogen-derived resistance, socioeconomic impact, technology transfer,transformation, cross protection

ABSTRACTThe papaya crop is severely affected by papaya ringspot virus (PSRV) worldwide. This review focuses on efforts to control the destructiveness of the disease caused by PSRV in Hawaii, starting from the use of cross protection toparasite-derived resistance with transgenic papaya expressing the PSRV coat protein gene. A chronology of the research effort is given and related to the development of technologies and the pressing need to control PSRV in Hawaii. The
development of commercial virus-resistant transgenic papaya provides a tangible
approach to control PSRV in Hawaii. Moreover, the development of transgenic
papaya by other laboratories and employment of a mechanism of effective technology transfer to different countries hold promise for control of PSRV worldwide.

INTRODUCTION
This review covers our efforts to develop control of papaya ringspot virus(PRSV) in papaya, with primary focus on Hawaii. The review traces the evolution of research to control PRSV, the use of transgenic papaya, and its recent
commercialization. It is not meant to be an in-depth review of either PRSVor alternate measures in use worldwide to control PRSV in papaya. The information, which is presented chronologically, points out the people involved,
interactions of technologies, timing of actions as related to disease occurrence,
potentials, and hurdles that must be overcome to use transgenic plants, all in
the context of controlling a widespread and severe disease.
415
0066-4286/98/0901-0415$08.00

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THE PAPAYA
Papaya (Carica papaya L.) is an important fruit crop grown widely in tropicaland subtropical lowland regions (31). It is largely consumed as a fresh dessert
fruit, and the green fruit is often used as salad. Papain is also recovered fromthe latex of green fruit. The tree is widely planted in home gardens because
it is relatively easy to grow from seed; the first mature fruits can be harvested
nine months after sowing seeds, and fruit is produced continuously year-round.
Although delicious and rich in vitamins A and C (31), the fruit is fragile,a characteristic limiting large-scale exportation to countries in temperate regions. Improvements in postharvest and shipping technologies should enhancecommercialization of this crop. In total production, papaya ranks above strawberries and below grapefruit (31). The FAO estimated that about 5.7 million
metric tons of fruit were harvested in 1995, almost double the 1980 harvest(18). Brazil, India, and Mexico are the largest producers of papaya. Hawaii
is the largest producer of papaya in the United States; about 66% of the total
fresh production is exported, primarily to the US mainland and to Japan (33).
Papaya is a herbaceous plant with a single stem bearing a crown of large
palmately shaped leaves. Flowers are produced at the axils of the leaf petiole. The plant is polygamous, with male, female, and hermaphrodite plants(31, 36). In the wild, dioecious plants predominate, whereas cultivated plants
are both dioecious and hermaphrodites. In breeding cultivars, the latter can beinbred, which results in stable characteristics from generation to generation (31).
The Hawaiian Solo types are gynodioecious cultivars (i.e. produce female andhemaphrodite plants) that are nearly isogenic as a result of years of selection andinbreeding. The dominant Solo cultivar grown in Hawaii is the yellow-fleshedKapoho, followed by the red-fleshed Sunrise, both of which produce small fruitof ca 454–1135 g. Sunrise is also widely grown in Brazil and Jamaica. Numerous other cultivars and selections have also been developed for particularcountries. In general, fruit production of hermaphrodite plants is adversely affected by such climatic factors as drought and wide fluctuations of temperature,
whereas female plants show more tolerance to these conditions (31).
Papaya trees are fast growing (they can be 12 feet tall in a year), and they
produce mature fruit within 9–12 months after seeds are planted. Commercially,
when trees are grown at a density of 1500 to 2500 per hectare, annual production
can range from 125,000 to 300,000 lbs per hectare. Fruit are harvested for 1 to
2 years, after which the trees are usually too tall for efficient harvesting.

PAPAYA RINGSPOT VIRUS
Papaya ringspot virus (PRSV) is by far the most widespread and damaging virusthat infects papaya. The name of the disease, papaya ringspot, is taken from theP1: PSA/DAT

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ringed spots on fruit of infected trees (25). Trees infected with PRSV develop
a range of symptoms: mosaic and chlorosis of leaf lamina, water-soaked oily
streaks on the petiole and upper part of the trunk, and distortion of young leaves
that resembles mite damage (20, 40). Infected plants lose vigor and becomestunted. When infected at the seedling stage or within two months after planting,trees do not normally produce mature fruit. Production of fruit by trees infected
at progressively later stages is severely reduced and of poor quality, owing to
the presence of ringspots and generally lower sugar concentrations.
PRSV is transmitted by numerous species of aphids in a nonpersistent manner to a limited host range of cucurbits and papaya. PSRV also produces local
lesions on Chenopodium quinoa and C. amaranticolor. A number of investigations have failed to demonstrate that PRSV is transmitted through seeds of
papaya or cucurbits (40), except for one study (5), which showed that 2 of 1355
papaya seedlings from PRSV-infected fruit showed PRSV symptoms. However, seed transmission is not a significant way for spreading PRSV (personalobservations).
PRSV is grouped into two types (40): Type P (PRSV-p) infects cucurbits
and papaya, whereas type W (PRSV-w) infects cucurbits but not papaya. The
latter type was previously referred to as WMV-1 (40). Although both types
are serologically closely related, observations suggest that papaya is the major primary and secondary source for the spread of PRSV-p in large plantations and small orchards alike. For example, serological and biological tests
on 454 cucurbit plants growing in villages of Northeast Thailand that harbored PRSV-infected papaya showed that while 31% of the cucurbits were
infected with PRSV, all were of the W type (V Prasartsee, unpublished results).
Similar observations were noted in Hawaii (S Ferreira, personal communication).
Much progress has been made in the molecular characterization of PRSV.
Strains of PRSV-p from Hawaii and Taiwan have been completely sequenced(55, 65). The genomic RNA consists of 10,326 nucleotides and has the typical array of genes found in potyviruses (44). The genome is monocistronic
and is expressed via a large polypeptide that is subsequently cleaved to functional proteins (64). There are two possible cleavage sites, 20 amino acids
apart, for the N terminus of the coat protein (56, 65). These two sites may befunctional; the upstream site for producing a functional Nib protein, and theother, to produce an aphid-transmissible coat protein. Nucleotide sequences
have been reported for the coat protein genes of 16 PRSV-p and PRSV-w types
from different geographic regions (2, 3, 41, 55–57, 65). It is impossible tosegregate PRSV-p and PRSV-w types by their coat protein sequences. Within
the p-types, however, the coat protein sequences can diverge by as much as
12%. The implication of these sequence differences are discussed in a later
section.

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The 1991 discovery of PRSV infection of papaya in Southeast Queensland(52), Australia, has provided insights on the possible origins of PRSV-p.
Bateson et al (3) proposed that PRSV-p from Australia could have originatedthrough a mutation of the PRSV-w strain, which has been in Southeast Queensland since 1978 (22). Sequence analysis of the coat protein gene of these PRSV-p
and PRSV-w isolates showed at least 98% nucleotide sequence identity. Moreover, the coat protein sequences of the PRSV-p and PRSV-w isolates from geographic regions close to Australia (such as Thailand, Vietnam, and Sri Lanka)
diverged from PRSV-p from Australia by as much as 12%. These observations
also suggest that the Australian PRSV-p type originated from the PRSV-w typepresent in Australia.

EFFECT OF PRSV ON PAPAYA PRODUCTIONPRSV-p occurs worldwide wherever papaya is grown (40). The effect of PRSV-p
in Brazil, Taiwan, and Hawaii are discussed below to illustrate the significanceof the virus on the industry and the approaches taken to maintain papaya production.
Brazil is by far the largest producer of papaya (18), accounting for 45% of the
5.9 million metric tons produced worldwide in 1996. The commercial industry
started in the 1960s in the southeastern states of Sao Paulo and Rio de Janeiro,
where most of the country’s population resides. PRSV was detected in these
two states in 1969 (7). The destructive effects of PRSV forced the Brazilian
papaya industry to move from Sao Paulo and Rio de Janeiro to the northern,
more remote states, Espiritu Santo and Bahia. From 1973 to 1978, Sao Pauloand Rio de Janeiro accounted for 90% of the total papaya acreage, but for only
27% by 1984. By 1993, Sao Paulo and Rio de Janeiro grew only 309 hectaresof papaya compared to 4792 hectares in Espiritu Santo and 16,073 hectares in
Bahia. This migration of the industry is directly attributable to the effects of
PRSV-p. Unfortunately, PRSV has followed the industry, and it is more difficult
to establish new farms to temporarily escape the virus; rouging to manage thevirus is expensive and not always effective. Moreover, papaya is now grown inregions distant from the major markets of Sao Paulo and Rio de Janeiro. Thus
shipping costs to the major markets are much higher, and quality of the fruit
is lowered. PRSV-p has been declared the most important problem in papaya
cultivation in Brazil.
Unlike Brazil, Taiwan is a small island unable to escape PRSV by shifting
its industry away from infected areas. In Taiwan, the industry consists of many
farms with fewer than 20 hectares of papaya. PRSV was discovered in 1974(58) in southern Taiwan and spread throughout the entire island within several
years. The destructiveness of PRSV forced farmers to grow papaya as an annual

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crop. Seedlings are usually planted in October and November and harvestcommences in July or August until December, after which time the trees are
removed.
A number of farms in Taiwan have resorted to growing papaya under protective netting to eliminate aphids until the trees have produced a good canopyof fruit. The nets are then removed to allow more sunlight to the trees and thusincrease the sugar concentrations of fruit. Trees subsequently become infected,
but fruit production is assured for several months. Raising papaya under large
net houses is extremely costly but is economically viable because the returns on
papaya are very high. As in Brazil, PRSV in Taiwan is the major factor affecting
papaya production, aside from other natural disasters such as typhoons.

PRSV IN HAWAII
The effect of PRSV in Hawaii is similar to that in Brazil and Taiwan. The papaya
industry started in the 1940s on the island of Oahu, on about 500 acres (11).
The virus was discovered in 1945, and the term papaya ringspot virus coined
by Jensen in 1949 (25). By the 1950s, production on Oahu was affected andthe industry subsequently moved to Hawaii island into the area of Puna, which
had no commercial production. Acreage increased to 650 by 1960 and to 2250
in 1990. In contrast, the acreage on Oahu fell to less than 50 by 1990 (11).
Remarkably, Puna remained free of PRSV for over 30 years. One incidenceof PRSV was reported in Puna in 1965, but it was eradicated by rapid rouging
of trees (MOJ Isherwood, personal communication). Despite the presence of
PRSV in Hilo and Keaau, communities only 19 miles away, Puna remained freeof PRSV, thanks to an effective physical barrier of lava rocks and to the absenceof intervening plantings, which minimized host plants for PRSV. Diligence by
the Hawaii Department of Agriculture in surveying and rouging infected trees
in the Hilo and Keaau areas kept PRSV from spreading.

ATTEMPTS TO CONTROL PRSVBY CROSS PROTECTION
Although Puna was still free of PRSV-p in the late 1970s, I and personnel at
the University of Hawaii were convinced that PRSV-p would inevitably invade
Puna. Thus, in 1979, we began to investigate the possibility of using crossprotection as a control measure. Cross protection is the phenomenon whereby
plants that are systemically infected with a mild strain of a virus are protectedagainst the effects of infection by a more virulent related strain (60, 63). This
practice (17) has long been known and has been used to control citrus tristeza(8), tobacco mosaic (42), and zucchini yellow mosaic viruses (54). The key

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component in cross-protection programs is the availability of a mild strain that
effectively protects against the target virus.
Shyi-Dong Yeh, a graduate student at Cornell University, was sent by the
Taiwanese government to investigate PRSV-p because of its devastating effects
in Taiwan. His task was to obtain a mild strain of PRSV-p. The attempt toselect a mild strain by isolating virus from trees with mild symptoms in heavily
infected orchards of Hawaii failed, as did the selection of mild variants throughanalyses of numerous isolates from local lesions. However, two mild strains(63), designated PRSV HA 5-1 and PRSV HA 6-1, were selected following
nitrous acid treatment of leaf extracts of squash infected with PRSV-HA, asevere strain from Hawaii that had been recently characterized (21). Greenhouse experiments showed that both strains were mild on papaya and afforded
protection against PRSV HA.
The mild strains were first tried in large-scale field plots in Taiwan (59–61). In
preliminary results, plants preinoculated with the mild strains did not show complete protection against the severe strain in the field, but did show a delay in thesevere effects of the challenge virus. Although this degree of protection was insufficient for plants under severe disease pressure, economical returns could beobtained by isolating orchards as much as possible and by rouging out severely
affected trees until the flowering period. About 100–200 hectares of papaya were
protected by PRSV HA 5-1 yearly from 1985 to 1991 (60). Currently, the mild
strain is sparsely used, mainly because it does not provide consistent economic
returns to the farmers. The failure of PRSV HA 5-1 to completely protect against
PRSV in Taiwan is due to differences between the mild strain and the wild-typevirus, as shown by greenhouse experiments (51). Similarly, these mild strains(PRSV HA 5-1 and 6-1) did not give good field protection against PRSV strainsin Northeast Thailand (V Prasartsee & D Gonsalves, unpublished results).
The mild strains performed much better in Hawaii, presumably because thestrains had been derived from a Hawaiian PRSV isolate. A series of field experiments was conducted with the Hawaiian Solo cultivars line 8, Kamiya,
and Sunrise on the island of Oahu (11, 34, 39). These studies showed that mild
strain PRSV HA 5-1 gave good protection against the local strains, although
PRSV HA 5-1 produced noticeable symptoms on leaves and fruit, with thedegree of symptom severity markedly dependent on the cultivar. Cultivars line
8 and Kamiya were the least affected and could be grown economically, but
Sunrise was too severely affected in fruit appearance. Protection of line 8 has
been the most successful (34). However, cross protection has not been widelyadopted on Oahu, for several reasons: (a) the adverse affects of the mild strain
on Sunrise and to a lesser extent on Kamiya, (b) cross protection requires extra
cultural management and care, and (c) the reluctance of farmers to infect theirtrees with a virus.

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DEVELOPMENT OF TRANSGENIC PAPAYATO CONTROL PRSV IN HAWAII

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Parasite-Derived Resistance: A New Approachfor Controlling PRSV
The concept of parasite-derived resistance (PDR), conceived in the middle
1980s (43), offered a new approach for controlling PRSV. Parasite-derived
resistance is a phenomenon whereby transgenic plants containing genes orsequences of a parasite (in this case, the coat protein gene of a virus) areprotected against detrimental effects of the same or related pathogens. The
application of PDR for plant viruses was first demonstrated by Beachy’s group(1): Transgenic tobacco expressing the coat protein gene of tobacco mosaic
virus was protected against infection by tobacco mosaic virus. Subsequent
reports have shown that this approach is effective in controlling many plantviruses (30).

Development of Transgenic Papaya
Our laboratory began utilizing the PDR concept in 1986 by cloning the coat protein gene of PRSV HA 5-1 in collaboration with Jerry Slightom, of The Upjohn
Company, who sequenced the gene (41) in an effort to develop virus-resistanttransgenic vegetables for the Upjohn’s subsidiary, Asgrow Seed Company. Our
objectives were to control the important viruses that infect vegetables, including cucumber mosaic, watermelon mosaic virus 2, zucchini yellow mosaic,
and papaya ringspot viruses. Asgrow Seed Company has subsequently developed commercial squash with resistance to zucchini yellow mosaic virus andwatermelon mosaic virus 2 (16, 53).
The expertise and reagents obtained from the initial vegetable work werethen applied to papaya. As noted above, our specific goal was to control PRSVin Hawaii in anticipation of its invading the Puna district. The cross-protection
work had been consistently supported through funds from the USDA Section
406 grant program, which focused on agricultural problems of the Pacific region.
Funding from this program had led to cooperative work with scientists fromthe University of Hawaii: Mamoru Ishii, Ryoji Namba, Mau, and Ferreira.
Fortunately, this program also funded the research to use PDR to control PRSV
in papaya in Hawaii. Thus, the collaboration of Richard Manshardt and Maureen
Fitch from the University of Hawaii, Slightom from The Upjohn Company, andmyself from Cornell University was begun. We were also very fortunate totap the expertise and services of John Sanford at Cornell University, who had
recently co-invented the gene gun.
Our target gene of PRSV was the coat protein gene of PRSV HA 5-1, themild mutant of PRSV HA that had been recently cloned and sequenced for theP1: PSA/DAT

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vegetable work. The coat protein gene of PRSV HA 5-1 had a 97.7% identity
to PRSV-w from Florida (41). Because of various technical difficulties and therequirement that the gene be expressed as a protein, the gene was engineered
as a chimeric protein containing 17 amino acids of cucumber mosaic virus
at the N terminus of the full-length coat protein gene of PRSV HA 5-1 (28).
Whether this would enhance or decrease the chances of obtaining resistant
plants was unclear. However, in tobacco this gene construct had expressedhigh levels of the coat protein as measured by ELISA (28). The transgenic
tobacco developed only mild symptoms following infection with tobacco etchvirus, a potyvirus unrelated to PRSV (44). The transgenic tobacco could not
be tested against PRSV because this virus does not infect tobacco. The cpexp
vector was subsequently developed; this vector utilized sequences within the
Nco 1 restriction site as the start codon for convenient engineering of coatprotein genes into plant expression vectors (45). This vector is widely used
today.
The task of transforming papaya was taken up in 1987 by Fitch, a graduate student under the supervision of Manshardt. The target cultivars were thered-fleshed Sunrise, the yellow-fleshed Sunset (a sib selection of Sunrise), andthe yellow-fleshed Kapoho, the dominant cultivar grown in Puna. Protocols
for transforming and subsequently recovering transgenic papaya plants had notbeen developed. One report indicated that papaya cells could be transformed,
but not regenerated (37). Numerous efforts by Fitch to develop a papaya regeneration system via organogenesis failed. However, a technique to developtransgenic walnuts by transforming embryogenic cultures had recently beenreported (35). The research moved rapidly once the decision was made to shift
to transforming embryogenic tissue. A technique to produce highly embryogenic tissue starting from immature zygotic embryos was developed (13). In
1988–1989, embryogenic tissue were bombarded with tungsten particles coatedwith DNA of the PRSV HA 5-1 coat protein gene using the gene gun in Sanford’slaboratory. Transgenic plants were obtained and were growing in the greenhouse 15 months later (14, 15).
What were the important factors that helped to achieve this timely breakthrough in transforming papaya? One was the unending enthusiasm that a
graduate student brings into the project. Another was the emphasis on obtaining a sufficient number of transgenic plants to test for resistance, rather thanfocusing on refining the details of the transformation technique. We wanted toquickly get to the stage of testing transgenic papaya to determine whether PDR
would work for controlling PRSV in papaya.
Clones of nine R0 transgenic lines, six Sunset and three Kapoho, were sent
to Cornell for inoculation tests with PRSV HA, which is the severe parent of themild strain PRSV HA 5-1. Inoculations were not done with the homologous

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mild PRSV HA 5-1 strain because it produced very mild or no symptoms on
papaya, and we assumed that PRSV HA was nearly homologous to PRSV HA
5-1. R0-micropropagated plants of the first line, designated 55-1, that was tested
showed excellent resistance to PRSV HA (15). Three other lines showed varying degrees of delay on the onset of symptoms, while the other lines developed
symptoms at the same time as control plants. Line 55-1 was female and thus
progenies could not be obtained directly from the R0 plants, as would be thecase for a hermaphrodite. A two-pronged approach was instituted to movethe research ahead aggressively and to determine whether line 55-1 would beresistant to PRSV under field conditions and have suitable horticultural characteristics. First, a decision was made to conduct a field trial using R0 plants
instead of waiting a year to obtain R1 plants. Second, R1 plants were obtainedby crossing line 55-1 with nontransgenic Sunset under greenhouse conditions
both at Cornell University and the University of Hawaii and then the plants werescreened in the greenhouse for resistance to PRSV isolates from around world.

Transgenic R1 Plants of Line 55-1 are Highly Resistant
to Hawaii Strains But Largely Susceptible to Strains
Outside Hawaii
Research headed by graduate student Paula Tennant at Cornell University wasdone to assess the resistance of R1 plants of line 55-1 against 3 PRSV isolates
from Hawaii and 13 isolates from different parts of the world (51). Furthermore, since the coat protein gene in line 55-1 originated from the mild strain
PRSV HA 5-1 used in earlier cross-protection studies, experiments were also
conducted to determine the cross-protection effectiveness of PRSV HA 5-1
against some of the strains that were being used to challenge plants of line
55-1. Analysis clearly showed that 50% of the progenies were transgenic, withthe rest nontransgenic; this confirmed that transgenic plants had one insert of thenptII gene and, presumably, the coat protein gene. These experiments illustrated
several important points: (a) Resistance of transgenic plants was not correlated
to level of protein expression, (b) R1 plants were highly resistant to Hawaiian
isolates, and (c) line 55-1 showed variable levels of resistance (largely susceptible) to non-Hawaiian isolates, (for example, plants inoculated with an isolate
from Thailand developed severe symptoms with no delay in symptom appearance, whereas isolates from Jamaica infected line 55-1, but symptoms were
delayed and attenuated; isolates from Florida and Mexico infected only a percentage of the plants and symptoms were milder), and (d ) the cross-protection
results on nontransgenic plants roughly paralleled those observed for transgenic plants. That is, protection was complete against the Hawaiian isolates
but not against non-Hawaiian isolates. Also, isolates that rapidly infected line
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protected with PRSV HA 5-1 showed almost no protection against the Thailand
isolate, which also rapidly overcame the resistance of line 55-1. Overall, theresults with transgenic papaya reinforced the information previously obtainedwith cross protection: R1 plants of line 55-1 would not be effective against
all isolates of PRSV. However, the results clearly showed that line 55-1 had
potential to control PRSV in Hawaii.

Field Trials with R0 Plants Show Line 55-1 Is Effective
for Controlling PRSV in Hawaii
In 1991, Animal Plant Health Inspection Service (APHIS) issued a permit fora field trial at the University of Hawaii’s experimental farm at Waimanalo, on
Oahu island, under Manshardt’s leadership. The importance of a field evaluation at an early stage cannot be overemphasized because it allowed us to appraise
the resistance and horticultural characteristics of line 55-1, to bulk up seeds bycrossing line 55-1 with nontransgenic plant cultivars, and to demonstrate thelong-term resistance of a transgenic fruit crop to infection.
Plants were set in the field by the end of June 1992. The field trial was
designed to determine the resistance of R0 plants to mechanical and aphid
inoculations of PRSV. Nontransgenic plants in border rows of the plot were also
inoculated with a PRSV isolate from Oahu island to create a high virus inoculumpressure for the field plot. Data were taken on total soluble solid levels of fruit,
growth characteristics, and virus symptoms. The transgenic papaya showed
excellent resistance throughout the two-year trial (29). Nearly all (95%) of thenontransgenic plants and those of a transgenic line that lacked the coat protein
gene showed PRSV symptoms by 77 days after the start of the field trial, whereas
none of the line 55-1 plants showed symptoms. Virus was not recovered fromline 55-1 plants except for two plants, which showed virus symptoms on sideshoots but none on the leaves of the main canopy. Plants grew normally andfruit appearance and total soluble solids of about 13% were within the expected
range. Thus, by the mid-1993, the trial had provided convincing evidence thatline 55-1 would be useful for controlling PRSV in Hawaii, or at least on Oahu
island.

CRISIS IN HAWAII: PRSV INVADES PUNA IN 1992
PRSV Severely Affects Puna Area in Three Years
The inevitable entry of PRSV into the Puna district on Hawaii island was
discovered during the first week of May in a papaya field in Pahoa, 1–3 miles
from the major papaya growing areas in Puna (23). Apparently, infection had
been established in this area for several months, as judged by symptoms on
the fruits and the fact that many plants were infected in one location. Surveys

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of the immediate area revealed PRSV in abandoned orchards, as well as inyoung orchards that were not yet producing fruit. PRSV was poised to invade
the major papaya growing areas of Puna, which included Kapoho, Opihikau,
Kahuawai, and Kalapana.
The Hawaii Department of Agriculture (HDOA) immediately launched aneradication program. The area was surveyed and infected trees were rouged
out. A suggestion to destroy all papaya in that area, however, was not approvedby the growers. Nevertheless, a HDOA program to mark trees to be rouged bygrowers was started in 1992 (23). By September 1992, 4915 trees had been
rouged in Pahoa, and the number of trees being cut each week had decreased
to below 85, providing hope that the virus had been contained (23).
However, the hope of containment was short-lived. The incidence of PRSV
increased dramatically in Kapoho, which was closest to Pahoa, as the program ofvoluntary cutting of trees was not strictly followed and as farmers experiencing
high infection rates abandoned their fields, thus creating huge reservoirs of
inoculum for aphids to acquire and spread the virus (24). By late 1994, nearly
all papaya of Kapoho was infected by the virus. In October 1994, the HDOA
declared that PRSV was uncontrollable and stopped the practice of marking
trees for rouging. In less than three years, a third of the Puna papaya area was
infected. By 1997, Pohoiki and Kahuawai were completely infected. Kalapana
was the last place to become heavily infected. In this area, the furthest fromthe original infection site in Pahoa, the spread of PRSV was slowed by constantrouging of infected plants by farmers, who are encouraged by a small bounty
paid by a packing house. Nevertheless, virus infection has increased, and manyheavily infected orchards have ceased to eliminate trees. Sixty trees were cut
in May 1996, 2905 in July, 4312 in December, and 14,493 in June 1997. By
September 1997, rouging was also discontinued in Kalapana (M Isherwood,
personal communication). Five years after the onset of the virus in Pahoa, theentire Puna area was severely affected.

LARGE-SCALE FIELD TRIAL IN DEVASTATED AREA
Establishment of Transgenic Field Trial in Kapoho
By 1994, personnel at the University of Hawaii (e.g. Ferreira and Mau) hadrecognized that PRSV was out of control in Puna and would significantly reduce
the state’s papaya production. To maintain production and eventually reclaim
the Puna area for papaya growth, a bold plan was proposed (10): Move new
papaya plantings to areas of Hawaii island where PRSV-p had not been detected,
completely eradicate papaya and cucurbits from the papaya area in Puna, andplace a one-year moratorium on planting papaya and cucurbits. Papaya acreage
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from the Puna area. The program was anticipated to be completed in 6–8 years.
Funding was provided by USDA to support the initial phases of the plan.
Several factors weighed against the program’s success. Data were neededon whether the Kapoho cultivar would grow well in the areas targeted for
expansion, as several observations had suggested that this cultivar does not
adapt well outside of Kapoho. More important, complete removal of every
papaya tree and cucurbit from the Puna area and maintenance of the area free of
infected plants for one year would be a monumental task, requiring widespreadcooperation. Nevertheless, this plan of action was a clear declaration of thecommitment to control PRSV, to save the papaya industry in Puna, and tomaintain papaya production in the state.
Given such a bold initiative, with the Kapoho area virtually abandoned and
PRSV out of control elsewhere in Puna, and the papaya industry severely threatened, the stage was set to establish a field trial in Kapoho to determine if thetransgenic papaya could be used to rescue the papaya industry. Arguments could
be marshaled for and against establishing such a field trial in the middle of a devastated commercial growing region of papaya: Line 55-1 had performed very
well in field trials on Oahu island and the line was resistant to PRSV-Panaewa,
a greenhouse isolate from Hawaii island; the industry needed drastic actions tosurvive; the plan for moving the industry might not succeed and PRSV might
not be eradicated from Puna; the risk of PRSV-resistant papaya becoming aweed was not relevant because papaya is not a weed in areas that do not have
PRSV-p; wild relatives of C. papaya are not grown in Hawaii; and the potential
benefits of transgenic papaya far outweighed the risks. Mitigating against thefield trial were the facts that pollen from the transgenic papaya might contaminate commercial plantings, resulting in the potential sale of commercial fruitwith a nonderegulated transgene; preventing pilferage in a trial installed on a
farmer’s field would be difficult, and thus there might be serious consequences
if stolen fruit ended up in commercial markets.
By late 1994, we submitted an application for a field trial to APHIS. Our
previous experience in applying for a field trial permit, combined with thehelpful cooperation of APHIS, facilitiated the review process. The field trial
was allowed with the stipulation that (a) the field must be sufficiently isolated
from commercial orchards to minimize the chance of transgenic pollen escaping
to nontransgenic material outside of the field test, (b) all abandoned trees in
the area must be monitored for the introgression of the transgene into fruits ofthese trees, and (c) all fruits had to be buried on site.

SunUp and UH Rainbow Cultivars
As noted above, early efforts to develop transgenic plants focused on transferring virus resistance into commercial papaya cultivars suitable for Hawaii.

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Line 55-1 is a transgenic Sunset, which is a commercial red-fleshed cultivar.
The dominant cultivar growing in Puna, however, is the yellow-fleshed Kapoho.
Unfortunately, we had not developed virus-resistant Kapoho and thus did nothave a yellow-fleshed cultivar as a substitute for this cultivar. Also, there was
the question of whether the transgenic plants could grow as well as Kapoho in
the Puna area. Manshardt crossed the homozygous transgenic line 55-1 with
Kapoho, anticipating the yield of a yellow-fleshed hybrid (because yellow isdominant over red) to substitute for Kapoho until such time that a transgenic
Kapoho could be developed. He had made these crosses with plants in the
1992–1994 field trial but had not yet evaluated them for their horticulturalcharacteristics. Thus the proposed field trial became a test not only of virus
resistance but also of fruit quality. The homozygous line 55-1 was later named
SunUp, the hybrid was made from the cross of the transgenic SunUp, and thenontransgenic Kapoho was named UH Rainbow (32).

Kapoho Field Trial
Approval was obtained in early 1995 and the field trial was set up in Kapoho in
October 1995, under the leadership of Ferreira. The trial was set up on the property of a farmer who had ceased growing papaya because of PRSV. One partof the trial consisted of replicated blocks to compare virus-resistance performances of SunUp and UH Rainbow, of cross-protected Kapoho (cross protectedwith PRSV HA 5-1), and of PRSV-tolerant lines that were being developed by
Ferreira and Francis Zee (of USDA Germplasm Repository, Hilo) and othertolerant lines developed in Thailand (V Prasartsee & D Gonsalves, unpublishedresults). Another part of the trial was established to simulate commercial conditions. A one-square acre solid block of UH Rainbow was planted adjacent to
the replicated blocks. Several rows of nontransgenic Sunrise were planted on
the perimeter of the replicated and solid blocks. An abandoned papaya fieldalongside the field plot was used as a primary source of the virus.
The results of the field trial clearly demonstrated the potential value of thetransgenic papaya for reclaiming papaya land in Puna (12; S. Ferreira, unpublished results). Except for three plants that showed infection at the beginningof the trial, none of the transgenic plants has become infected as of January
1998, 27 months after starting the trial. In stark contrast, 50% of the nontransgenic control plants within the experiment and in the border rows were infected
within four and a half months after transplanting; and all were infected byseven months. The growth differences between the transgenic and nontransgenic trees were remarkable; transgenic plants grew vigorously, with dark green
leaves and full fruit columns, whereas nontransgenic plants were stunted, withyellow and mosaic leaves and very sparse fruit columns. In the solid block,UH Rainbow averaged about 100,000 lbs of marketable fruit per acre/year,P1: PSA/DAT

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whereas nontransgenic plants averaged about 5000 lbs per acre/year. Althoughthese data were from only one trial, observations suggested that UH Rainbow
was a higher yielder than Kapoho. Also, the transgenic papaya performed muchbetter than the PRSV-tolerant lines and the cross-protected plants.
Despite their excellent resistance, would farmers accept these cultivars as an
acceptable substitute for Kapoho, a mainstay of the Hawaiian papaya industry
for several decades? The performance of UH Rainbow was especially criticalbecause it was targeted as the alternative to Kapoho. Taste, production, color,size, and packing and shipping qualities of Rainbow were analyzed. In addition
to tests by research personnel, field days were held to allow farmers, packers,
politicians, and University personnel to observe the field and fruit at the testsite. The consensus was that UH Rainbow is a more than adequate substitutefor Kapoho. The fruit is larger than Kapoho, but commercial packers do not
see this as a major impediment.
SunUp is a transgenic Sunset, and its properties mirror those of the latter.
Although Sunset and Sunrise are more adaptable to different environmental
conditions than Kapoho is, these cultivars were not widely grown on Hawaii
island. It will be interesting to see if SunUp increases the popularity of redfleshed cultivars, since it can be grown in areas where PRSV is the limiting
factor to economic production.

DEREGULATION AND COMMERCIALIZATION
OF TRANSGENIC PAPAYA
To develop a transgenic papaya with virus resistance and excellent fruit qualities represents one hurdle, but to get it deregulated and commercialized isanother. The areas of deregulation and commercialization have been in the
purvue of private companies who stand to benefit financially from developing
a product and moving it to the market. Hitherto, University personnel wererarely directly involved, until papaya. First, the ownership of the papaya wasplaced into the hands of the Papaya Administrative Committee (PAC), which
consisted of Hawaii papaya growers who had limited knowledge of these twoprocesses. And second, the researchers involved felt that academic testing of
the papaya was insufficient, and therefore the transgenic papaya was moved to
the growers in a timely manner. Given the circumstances and interests, the PAC
could be expected to follow up on the necessary steps to get the papaya to thecommercial stage.

Deregulation
The task of securing deregulation of papaya by APHIS, Food and Drug
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up by Richard Manshardt and our lab. APHIS was largely concerned with thepotential risk of transgenic papaya on the environment. Two main risks wereof heteroencapsidation of the incoming virus with coat protein produced by
the transgenic papaya and of recombination of the transgene with incoming
viruses. The former might allow nonvectored viruses to become vector transmissible, whereas the latter might result in the creation of novel viruses. A thirdconcern—that escape of the transgenic genes to wild relatives might make therelatives more weedy or even make papaya more weedy because of resistance to
PRSV—was of no consequence since there are no papaya relatives in the wildin Hawaii, nor is papaya considered a weed there, even in areas were there is no
PRSV. In November 1996, transgenic line 55-1 and another recently tested line
63-1 (50) and their derivatives were deregulated by APHIS (47). This action
greatly increased the efficiency of the ongoing field trial because fruit no longer
had to be buried at the test site, which allowed us to sample and send fruit tovarious laboratories and to the packing house without undue constraints.
According to the EPA, the coat protein transgenes are a pesticide because
they confer resistance to plant viruses. A pesticide is subjected to tolerance
levels in the plant. In the permit application, we petitioned for an exemption
from tolerance levels of the coat protein produced by the transgenic plant. We
contended that the pesticide (the coat protein gene) was already present in many
fruits consumed by the public, since much of the papaya eaten in the tropics isfrom PRSV-infected plants. In fact, we had earlier used cross protection (thedeliberate infection of papaya with a mild strain of PRSV) to control PRSV.
Fruit from these trees was sold to consumers. Furthermore, there is no evidence
to date that the coat protein of PRSV or other plant viruses is allergenic or
detrimental to human health in any way. Finally, measured amounts of coatprotein in transgenic plants were much lower than those of infected plants. An
exemption from tolerance to lines 55-1 and 63-1 was granted in August 1997.
The FDA is concerned with food safety of transgenic products. This agency
follows a consultative process whereby the investigators submit an application
with data and statements corroborating that the product is not harmful to humanhealth. Several aspects of the transgenic papaya were considered: the concentration range of some important vitamins, including vitamin C; the presence of
GUS and nptII genes; and whether transgenic papaya had abnormally high concentrations of benzyl isothiocyanate. This latter compound has been reported
in papaya (48). FDA approval was granted in September 1997.

Commercialization
In the United States, a transgenic product cannot legally be commercialized
unless it is fully deregulated and until licenses are obtained for the use of
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the product or that have been used to develop the product. The processes inquestion were the gene gun and parasite-derived resistance, in particular, coatprotein–mediated protection. The components were translational enhancement
leader sequences and genes (nptII, GUS, and coat protein). This crucial hurdle
involved legal and financial considerations beyond our means and expertise.
These tasks were taken up by the industry’s PAC and its legal counsel, Michael
Goldman.
Several factors favored the PAC efforts to obtain licenses: (a) Although the
Hawaiian papaya industry is important to the state, its annual worth is relatively
small (annual farm gate value of $17 million); (b) the license holders were notactively working toward developing virus-resistant transgenic papaya; (c) thetransgenic papaya was urgently needed to rescue Hawaii’s papaya industry;and (d ) licensing the transgenic papaya in a timely manner would demonstrate
goodwill in trying to help a distressed industry. Detracting from the the possible
success of the PAC in obtaining a license was the expense, the relatively small
size of the industry, and the extra keen public scrutiny that focuses on transgenic
products. Failure by growers or shippers to follow the license agreements could
be a source of embarrassment to all involved. Nevertheless, license agreements
were obtained from all parties in April 1998, allowing the commercial cultivation of the papaya or its derivatives in Hawaii only. Fruits can be sold outside
Hawaii, provided that the importing state or country allows the importation andsale of transgenic papaya. Fruit derived from the licensed transgenic papaya
grown outside of Hawaii cannot be sold commercially.
Even before license agreements had been obtained, the PAC had commissioned the Hawaii Agricultural Research Center to produce UH Rainbow seeds
so that they would be available when needed. The seeds are being produced on
Kauai, where PRSV is not present. Seeds sufficient for 1000 acres are scheduled for distribution in May 1998. Growers should therefore be able to startplanting and thus reclaiming abandoned lands in Puna after mid-1998.

WORLDWIDE CONTROL OF PRSV
Now that transgenic papaya appears to be a practical way to control PRSV in
Hawaii, what about worldwide control of PRSV with transgenic papaya? The
constraints of the license agreement with the germplasm of line 55-1 or theirderivatives (e.g. SunUp and UH Rainbow) make it unlikely that line 55-1 will
be made available for commercial use outside Hawaii. Moreover, our resultswith hemizygous plants of line 55-1 showed these plants to be susceptible to
PRSV isolates outside Hawaii. These observations suggested that transgenic
papaya with coat protein genes specific to targeted PRSV isolates would need
to be developed for transgenic papaya to effectively control PRSV worldwide.

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However, recent results show that transgenic papaya with resistance to a broader
range of PRSV strains can be developed.

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Homozygous Line 55-1 is Resistant to PRSV Isolates
Outside Hawaii
Further infectivity studies on line 55-1 by Tennant gave interesting results
showing that resistance was markedly affected by combinations of coat protein transgene dosage (hemizygous vs homozygous), developmental stage ofpapaya seedlings, and coat protein gene sequence homology of PRSV isolates(49, 50). The results demonstrated that hemizygous plants (e.g. UH Rainbow)
are quite susceptible even to some of the Hawaiian isolates when inoculated at
a very small seedling stage but are resistant when inoculated at later stages. But
as noted earlier, these hemizygous plants are susceptible to a number of isolates
originating outside of Hawaii. Strikingly, 55-1 plants that were homozygous forthe coat protein gene (i.e. SunUp) were resistant to a larger range of the isolates(except for the Thailand isolate), regardless of plant age. And, large plants ofhomozygous 55-1 that were inoculated with PRSV-Thailand showed resistanceor long delays prior to symptom expression. These observations suggested that
resistance of line 55-1 was being governed by RNA-mediated protection (4, 26).
Others had clearly shown with other transgenic plants that RNA-mediated protection was affected by gene dosage (38, 46), sequence differences between
the transgene and the attacking virus (4, 27), and developmental stage of theplant (38). The underlying mechanism of RNA-mediated protection is likely
to be posttranscriptional gene silencing; a phenomenon in which the transgene
transcript is degraded following apparently normal levels of transcription in
the nucleus (9). In fact, nuclear run-off experiments with isolated nucleic acidfrom line 55-1 confirmed that the coat protein transgene was posttranscriptionally silenced (49). The narrow protection of the hemizygous line 55-1 and UH
Rainbow and the concomitant broader protection of the homozygous SunUp
now had an explanation.

Other Transgenic Papaya Lines Show Broader ResistanceOur results so far had been obtained from a single line of transgenic papaya.
Could transgenic resistance be repeated with other lines and other genes? Preliminary results from other laboratories clearly show that transgenic papaya
will be useful for controlling PRSV. Another transgenic line (designated 63-1),
generated from the same transformation experiments that resulted in line 55-1,
also shows high resistance under field conditions (tested in a replicated plot in
the Kapoho field trial). Additionally, this line has broader resistance to PRSV
isolates than line 55-1 has (49, 50). We have subsequently produced numerous
transgenic plants with the nontranslatable coat protein gene of PRSV HA; aP1: PSA/DAT

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number of the transgenic plants were resistant to isolates outside Hawaii (19).
These results clearly show that resistance is RNA-mediated and that R0 plants
with broader resistance than line 55-1 can be obtained. Yeh’s laboratory has
also developed transgenic plants with the coat protein gene of PRSV from
Taiwan; some of these are resistant to PRSV strains from Taiwan, Thailand,and Hawaii (62). New transgenic lines expressing coat protein genes (translatable or nontranslatable) of PRSV isolates from Brazil, Jamaica, and Thailand(D Gonsalves, unpublished results) and Australia (J Dale, personal communication) have been produced. Recently, transgenic papaya with the Nib gene of
PRSV HA 5-1 has been produced (M Fitch, personal communication).

Resistance of 55-1 is Coat Protein GeneSequence Dependent
RNA-mediated protection clearly suggests that resistance is dependent on thehomology of the transgene and the incoming virus. If the sequences of the coatprotein transgene are different enough from the coat protein of the incoming
virus, the transgenic plant would be susceptible to that virus. Indeed, observations suggest that the resistance of homozygous line 55-1 is overcome by PRSV
isolates with coat protein genes with around 90% identity to the transgene. For
example, PRSV strains from Thailand and Taiwan have around 90% identity
to the transgene and can overcome the resistance of SunUp (3, 56). Can wedetermine the minimal difference in coat protein sequence identity that will
overcome resistance of SunUp?
A major breakthrough in Yeh’s laboratory should enable us to understandthe question on sequence homology and resistance that is posed above. His
laboratory recently developed infectious transcripts of PRSV HA (6) and other
PRSV isolates (SD Yeh, personal communication). Furthermore, he and his coworkers constructed a recombinant hybrid virus (PRSV HA-YKcp) containing
the entire genome of PRSV HA except that the coat protein gene (plus about
70 nucleotides of the NIb and the untranslatable 30 region) was exchanged
with that of PRSV-YK from Taiwan (SD Yeh, unpublished data). Workers in
our laboratory have inoculated UH Rainbow (hemizygous for PRSV HA 5-1
coat protein transgene) and SunUp (homozygous for PRSV HA 5-1 coat proteintransgene) with PRSV HA, PRSV YK, and the hybrid virus (PRSV HA-YKcp).
Preliminary greenhouse experiments have shown that UH Rainbow and SunUp
are resistant to PRSV HA, but susceptible to PRSV YK and PRSV HA-YKcp.
These results suggest that differences in the coat protein gene of the attacking
virus and the transgene are critical factors that affect resistance in line 55-1. It
will be interesting to make virus hybrids with chimeric coat proteins and checktheir ability to overcome the resistance to line 55-1.

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Technology Transfer to Control PRSV Worldwide
Developing countries produce 98% of the world’s papaya crop (31). Can thesecountries capitalize on the technology to develop virus-resistant transgenic
papaya that are horticulturally acceptable locally? Our laboratory at Cornell
University is committed to helping other countries obtain this needed technology. Thus far, we have focused on developing a cost-efficient and time-efficienttechnology transfer program, without the intervention of a large foreign aid
donor. The program combines training with collaborative, goal-specific research. The goal is to develop the transgenic plants and conduct the initial
screening for virus resistance within 18 months to 2 years. Typically, arrangements are made in collaboration with a government agency from the nation
requesting the technology. One example is a program with Thailand’s Department of Agriculture to develop transgenic papaya for the villages of Northeast
Thailand. The coat protein gene of PRSV from Thailand was engineered in
our laboratory; a Thai scientist, Nonglak Sarindu, was then sent to our laboratory to transform their local papaya cultivars. Regenerated plants were rooted
and established in the greenhouse, and then Sarindu conducted the initial testing for resistance. After 20 months in Geneva, New York, Sarindu returned
home with promising transgenic plants and embryo cultures. The plants arefruiting in a cage house at a government station in Northeast Thailand. Evaluation of the transgenic plants is continuing in the greenhouse, and eventually they will be tested in the field. Sarindu was trained in all the skills necessary to develop the transgenic papaya and to subsequently advise in thecontinuing process for dealing with transgenic products. Our primary goal
was to develop the transgenic papaya within the targeted time period of ayear and a half. Training was important but subsidiary since it could be addressed through hands-on work on the project. The scientist was responsible
for completing the application to the regulatory agency for approval to import the transgenic papaya and to test them in the home country. Funding forthe project was covered by the foreign government; included were transportation and living expenses for the scientist and a fee to cover the cost of
supplies.
Proper technology transfer involves the continuation of the project in thehome country. This phase often fails because the developed product is of only
academic value and thus there is no pressing need to push to a practical end or
there is no commitment to work through the necessary bureaucratic procedures.
A well-planned project must objectively anticipate potential impediments toprevent the project from reaching its goal. Perhaps the best guarantee is to havea project that is feasible and yet of demonstrably high importance. Development of PRSV-resistant plants fits this category. This method of technology

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transfer can serve as a model system for dealing with other transgenic products
at all levels.
The program has worked well so far. Transgenic papaya targeted for Jamaica,
Brazil, and Thailand has been developed and shown resistance to local strains of
PRSV. A program is also ongoing with Venezuela. However, the success of thisapproach will be determined four to five years from now, when the transgenic
plants are actually in the field and, it is hoped, helping to control PRSV.

SOCIOECONOMIC EFFECT OF TRANSGENIC PAPAYATransgenic papaya—SunUp and UH Rainbow—is the first virus-resistant fruit
crop to be commercialized in the United States. Over all, it is only the second crop with virus-resistance to be commercialized, next to Asgrow Seed
Companys Freedom II squash. For the papaya, results so far hold promise ofreviving the Hawaiian papaya industry. Despite extensive documentation of the
impact of PRSV on the industry, especially since 1992, few, if any, studies havemeasured the impact, both sociologically and economically, of a transgenic
crop on a local population. Thus, the scenario in Hawaii provides an excellent
opportunity to objectively determine what impact a transgenic product has on
an industry and its supporting farmers. We hope to measure this impact.

FINAL THOUGHTS
I have attempted to describe a series of events over a number of years that haveled up to what appears to be a likely control of PRSV, at least in Hawaii. Proofof success in controlling PRSV in papaya will only be determined in the next
several years, as the transgenic papaya is widely planted in Hawaii and as moretransgenic papaya are produced and tested worldwide. Work on controlling
PRSV should also lead to advancements in fundamental aspects of PDR, crossprotection, and management of plant viruses with the use of transgenic plants.
Finally, the transgenic papaya will, in my view, allow for the timely assessmentof the socioeconomic effect of a transgenic product on agriculture, especiallyas it relates to farmers in developing countries.
ACKNOWLEDGMENTS
I especially thank my wife Carol Gonsalves for her encouragement throughout
the years and for critically reading the manuscript. Many people have played apart in the research on controlling PRSV. To all of them, I am grateful.
Visit the Annual Reviews home page at
http://www.AnnualReviews.org.

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CONTENTS
One Phytopathologist's Growth Through IPM to Plant Health: The Key toApproaching Genetic Yield Potential, J. Artie Browning
KATHERINE ESAU, 1898–1997, Jennifer A. Thorsch, Ray F. Evert
Diversity Among Xanthomonads Pathogenic on Pepper and Tomato, J. B.
Jones, R. E. Stall, H. Bouzar
Current Concepts of Active Defense in Plants, Steven W. Hutcheson
Cypress Canker: A Pandemic in Progress, Antonio Graniti
Application of Mating Type Gene Technology to Problems in Fungal
Biology, B. Gillian Turgeon
Biology and Molecular Biology of Viruses in the Genus Tenuivirus,
Bryce W. Falk, James H. Tsai
Developing Sustainable Systems for Nematode Management, Kenneth R.
Barker, Stephen R. Koenning
Homoserine Lactone-Mediated Gene Regulation in Plant-AssociatedBacteria, Leland S. Pierson III, Derek W. Wood, Elizabeth A. Pierson
Management of Fire Blight: A Case Study in Microbial Ecology, K. B.
Johnson, V. O. Stockwell
Recombination in Magnaporthe grisea , Robert S. Zeigler
Root-Knot Nematode Resistance Genes in Tomato and Their Potential for
Future Use, Valerie M. Williamson
Satellite Viruses of Tobamoviruses, J. Allan Dodds
Function of Root Border Cells in Plant Health: Pioneers in the
Rhizosphere, M. C. Hawes, L. A. Brigham, F. Wen, H. H. Woo, Y. Zhu
Genetics and Physiology of Aflatoxin Biosynthesis, G. A. Payne, M. P.
Brown
Type III Protein Secretion Systems in Plant and Animal Pathogenic
Bacteria, Sheng Yang He
Programmed Cell Death in Plant Disease: The Purpose and Promise of
Cellular Suicide, D. G. Gilchrist
Control of Papaya Ringspot Virus in Papaya: A Case Study, Dennis
Gonsalves
Root Cortex--The Final Frontier for the Biocontrol of Root-Rot with
Fungal Antagonists: A Case Study on A Sterile Red Fungus, K.
Sivasithamparam
Systemic Resistance Induced by Rhizosphere Bacteria, L. C. van Loon, P.
A. H. M. Bakker, C. M. J. Pieterse
The Impact of Reduced Tillage on Soilborne Plant Pathogens, W. W.
Bockus, J. P. Shroyer

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