2003 Christmas, made by Mike Melzer
Plant diseases caused by viruses result in farm losses of millions of dollars annually in Hawaii and the Pacific. Banana bunchy top virus (BBTV), Citrus tristeza virus (CTV), and Pineapple mealybug wilt associated virus (PMWaV) are examples of viruses that presently limit production of banana, citrus, and pineapple in Hawaii and other Pacific Islands, respectively. My research program addresses the first goal of the Hawaii Plan of Work: "An agricultural system that is highly competitive in the global economy"; and focuses on the Program 1.1 and 1.2 of the Hawaii POW: "Diversified agriculture" and "Fundamental plant and animal sciences". The goal of my research program is to develop new knowledge on virus-plant and virus-vector interactions and to use this knowledge to resolve virus problems of economic crops in Hawaii. Our research use biotechnology and other approaches to increase production, efficiency, and profitability of diversified agricultural industries while protecting the environment; and to incorporate research-based technology to reduce losses due to virus diseases.
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Biotech and Organic Farming
Banana bunchy top virus
Banana bunchy top virus (BBTV) is the most devastating virus disease of bananas in many areas of Asia, Africa, and the South Pacific. In 1989, BBTV was first identified in Hawaii at a farm near Punaluu, Oahu. Since 1989, it has been found in many farm and residential areas on the island of Oahu. BBTV was later found on the islands of Hawaii and Kauai in 1995 and 1997, respectively. The virus is transmitted by the banana aphid (Pentalonia nigronervosa) in a persistent manner. New growth from infected plants is stunted and distorted, resulting in a characteristic "bunchy top" appearance. No fruit is produced on infected plants, and a high incidence of BBTV in production areas can cause complete yield loss. Specific objectives of this project are: (1) to develop rapid, sensitive, and reliable assays for detection of BBTV in plants and insects; (2) to characterize the genome organization and vector transmission of BBTV; (3) to develop resistant transgenic banana plants to control BBTV; and (4) to develop assays to detect other banana viruses in Hawaii.
Detection We have previously developed enzyme-linked immunosorbent assay (ELISA), dot-blot hybridization assay (dot-blot), and polymerase chain reaction (PCR) assay to detect BBTV. BBTV-specific clones were used in dot-blot hybridization assays using radioactive and nonradioactive probes. PCR was used to detect BBTV from single aphid vectors. More recently, the BBTV coat protein gene was cloned into an in vitro expression vector and a large amount of BBTV coat protein was obtained. BBTV-specific antibodies were produced in rabbits. These antibodies work well in ELISA assays and have been used in ELISA to detect BBTV infections for banana growers and HDOA BBTV eradication projects in Hawaii. We also helped Dr. Fred Brooks establish a BBTV detection system in American Samoa using our antibodies.
Characterization Three BBTV isolates from Hawaii, China, and the Philippines were characterized. Three single-stranded DNA (ssDNA) components were cloned, sequenced, and compared. We found that the movement gene sequences of all isolates are very similar. However, based on sequence information of the coat protein and Rep genes, the Hawaii BBTV isolate is more closely related to the Australian BBTV isolate, and the isolates from China and the Philippines are more closely related to the Taiwanese BBTV isolates. These results support the idea of separating BBTV isolates into two groups, the Asia group and the Pacific group, as suggested by James Dale. Resistant transgenic banana plants developed with genes from one group may, therefore, not be resistant to infection by the other BBTV group. Thus, we also cloned the coat protein, movement, and Rep genes from the Chinese and Philippine BBTV isolates for use in transformation studies to develop transgenic plants with broad and durable resistance.
An efficient system was developed to screen BBTV-resistant transgenic banana plants in greenhouse experiments. Previously, we obtained 100% BBTV infection in banana plants when five viruliferous aphids were given two days inoculation feeding time. We have found that BBTV is acquired by banana aphids within 4 hours and is transmitted within 15 minutes of feeding. Ten viruliferous banana aphids were used to inoculate each banana plant with inoculation feeding times of one week. The inoculated banana plants were kept in the greenhouse (23°C) for several months to allow symptoms to develop.
Genetic Engineering Initially, we transformed banana cultivars 'Williams' and 'Grand Nain' using Agrobacterium-mediated transformation systems. Approximately 300 putative transgenic banana plants were obtained from transformation experiments using a meristem system with different BBTV genes. All non-transgenic control banana plants developed typical symptoms about 1 month after inoculation. Most of the putative transgenic banana plants developed typical BBTV symptoms within 1 month; several plants showed a 2&endash;3 week delay in symptom development. Seven BBTV-resistant transgenic banana plants were obtained and characterized. BBTV symptom development was delayed for as long as 1 year after challenge inoculation. However, these plants were chimeric, and eventually they all developed BBTV symptoms. Our results demonstrated that pathogen-derived resistance technology can be used to control BBTV and that the gene constructs we used can produce BBTV-resistant plants, but we need to modify the transformation system to obtain true transgenic plants that are not chimeric.
We recently developed a more efficient system to transform and regenerate transgenic banana plants. Secondary somatic embryogenesis culture was established using immature male flower buds of banana cultivar 'Apple' (Musa spp. AAB group). An average of 75% to 85% of the secondary somatic embryos regenerated plantlets. We have also used this regeneration system to develop procedures for efficient production of 'Apple' banana transgenic plants using microprojectile bombardment-mediated transformation. The bombardment conditions were optimized, and fully transformed banana plants have been obtained. Our banana transformation and regeneration system is different from that patented by Zeneca Inc. entitled "Method of genetically transforming banana plants" (International Application Number: PCT/US98/14661). First, our system is much more efficient, reliable, reproducible, robust, time-saving, and economical. Second, we use secondary somatic embryogenesis, and they use a banana cell suspension approach. Third, we use microprojectile bombardment; they use Agrobacterium-mediated technology. In 2001, we therefore filed a patent application entitled "efficient"Efficient regeneration and transformation of banana using secondary somatic embryogenesis via microprojectile bombardment." Currently, we are using our new system to produce transgenic banana plants resistant to BBTV. The most promising BBTV-gene construct is being used in this new transformation system to develop large numbers of true transgenic plants so that reliably BBTV-resistant banana plants can be produced for the banana growers in Hawaii. Hundreds of transgenic banana plants have been produced and are being evaluated for resistance to BBTV infection.
Other Banana Viruses Banana streak badnavirus (BSV) has become a very important banana virus worldwide, because of its potential to cause severe diseases and to move in Musa germplasm to other countries. Banana bract mosaic virus (BBrMV), found in several countries in Asia, is also a potentially important banana virus. It is a potyvirus transmitted by aphids. Cucumber mosaic virus (CMV) causes chlorosis, mosaic, and heart rot in bananas and has been found in most banana-growing areas of the world. We have developed ELISA and PCR assays to detect BSV, BBrMV, and CMV for quarantine and prevention studies.
Value, Quality, and Impact
Bananas are planted on 1,150 acres in the state of Hawaii and generate $10.4 million in revenue. Banana bunchy top virus poses a threat to the banana industry in Hawaii. The diagnostic assays developed in this study for the detection of BBTV are sensitive, rapid, and reliable, and they enabled us to help banana growers in Hawaii by indexing their stocks of banana plants prior to propagating large numbers of plants in tissue culture. We characterized the molecular biology and vector transmission of BBTV and established a foundation for the development of control strategies for BBTV. We developed various BBTV gene constructs and transformed banana plants to develop resistant transgenic plants. We also developed an aphid transmission system for evaluation of transgenic plants for resistance to BBTV infection. The successful production of resistant transgenic banana plants may provide an environmentally safe and durable approach for control of BBTV, reduce the use of insecticides, and help banana growers achieve sustainable agriculture. The establishment of a system for transformation and regeneration of bananas in Hawaii is a significant achievement and has broad applications for the control of other banana diseases and the improvement of banana quality. For example, black Sigatoka disease and various nematode diseases limit banana production worldwide.
Pineapple mealybug wilt associated virus
Mealybug wilt of pineapple (MWP) is a devastating disease found in all the pineapple-growing regions of the world. The disease is characterized by severe leaf tip dieback, downward curling of the leaf margins, and reddening and wilting of the leaves that can lead to total collapse of the plant. The etiology of MWP has long been in question. Early researchers hypothesized that a latent transmissible factor such as a virus was involved. A long, flexuous rod-shaped closterovirus has been isolated from MWP symptomatic plants in Hawaii. Subsequently, closterovirus particles have been detected from both MWP symptomatic and asymptomatic pineapple plants worldwide. Specific objectives of this project include: (1) to develop rapid detection assays for the closterovirus; (2) to study the cause (etiology) of MWP; (3) to examine the epidemiology of the virus; and (4) to develop alternative, environmentally sound strategies to manage MWP.
Virus diversity We found recently that the closterovirus particles, referred to as Pineapple mealybug wilt associated virus (PMWaV), are actually a complex of at least two different viruses. They are currently designated as PMWaV-1 and PMWaV-2. Genomic characteristics of these viruses place them within the Closteroviridae family in the proposed genus Ampelovirus. Based on phylogenetic analyses of partial sequences, PMWaV-1 and PMWaV-2 share approximately 50% homology. PMWaV-2 is most closely related to grapevine leafroll associated closterovirus-3 (GLRaV-3), sharing 64% to 72% homology across four open reading frames. In RT-PCR studies with degenerate primers made from conserved sequences of the PMWaVs and closely related closteroviruses, we found that a third PMWaV, designated PMWaV-3, is present in pineapple accessions maintained at the USDA-ARS National Clonal Germplasm Repository in Hilo, HI. PMWaV-3 has also been detected in some MWP symptomatic plants maintained in a UH Manoa greenhouse.
Virus detection and distribution Monoclonal antibodies (MAb) for PMWaV-1 and PMWaV-2 have been produced in my lab to detect and differentiate the two viruses. These PMWaV-specific MAbs were used to develop tissue blot immunoassays (TBIA), which provide reliable and appropriate methods for mass testing of pineapple from Hawaii and around the world. Tens of thousands of plants from Hawaii and around the world have been screened for PMWaVs with TBIA. Both viruses are found worldwide in MWP symptomatic and asymptomatic pineapple. In field selections of Smooth Cayenne grown in Hawaii, incidence of PMWaV-1 varies from 15% to 100%, depending on the selection; PMWaV-2 incidence varies from 0 to 20%. Virus incidence in the newer low-acid hybrids varies from 0 to 16% for PMWaV-1 and 0 to 12% for PMWaV-2. We have also developed an RT-PCR assay, which can detect and differentiate PMWaV-1, PMWaV-2, and PMWaV-3. This assay is extremely sensitive and suitable for screening young plants in tissue cultures, which are too small to be screened with TBIA. Based on TBIA and RT-PCR results, PMWaV-2 infection is consistently found in association with MWP, whereas PMWaV-1 is not.
Virus transmission We have demonstrated that PMWaV-1 and PMWaV-2 are both mealybug-transmitted viruses. The pink pineapple mealybug, Dysmicoccus brevipes (Cockerell), which tends to feed near the base of the plant, and the gray pineapple mealybug, D. neobrevipes Beardsley, which is more common on aerial portions of the plant, cause serious problems in Hawaii, and both are competent vectors of PMWaVs. Mealybugs are not born viruliferous; they must first feed on a PMWaV-infected pineapple plant to acquire the virus. The mealybugs have been shown to acquire and transmit virus during all stages of active feeding. We also challenged a wide assortment of vegetation growing in or near pineapple fields with viruliferous mealybugs and screened for PMWaVs with TBIA and RT-PCR assays to assess the possibility of those plants being alternate hosts for the viruses. No alternate hosts of the viruses were identified although several grass species were found to be hosts of pineapple mealybugs. Based on these findings, acquisition sources appear to be limited to pineapple (Ananas and Pseudoananas species). The PMWaV-infected pineapple plants that occur throughout a typical plantation field appear to be the sole virus-acquisition source for mealybugs, which subsequently transmit the virus or viruses to other pineapple plants.
Etiology and impact We have recently demonstrated that both PMWaV-2 and mealybug feeding are necessary factors for the induction of MWP in both containerized pineapple plants and in a large field study. Mealybug feeding in the absence of PMWaV-2 or PMWaV-2 infection in the absence of mealybug feeding did not result in MWP. Nor did the presence of mealybug feeding and PMWaV-1 without PMWaV-2 result in MWP. In a large field study, the average fresh fruit weight from plants that developed MWP symptoms was 35% lower than yields from PMWaV-free plants exposed to mealybugs. We have also found correlations between infection with PMWaV-1 and significant reduction of yield in ratoon crops.
PMWaV infection on precocious flowering.Precocious flowering has been such a limiting factor for pineapple production that the Hawaii pineapple industry ranks it as a high priority problem. Asynchronous fruit maturation resulting in a broad ripening period is commercially unacceptable because out-of-cycle or precocious fruit ripening complicates harvest date predictions. Extra harvest labor is also required because the fruits are scattered and lodged fruit is difficult to locate. If precocious fruit are left unharvested, they will be overripe when the majority of the fruit in the field are scheduled for harvesting. We recently found that fruit from PMWaV-1 infected plants had a significantly broader ripening period than PMWaV-1-free plants. The use of PMWaV-free plants might therefore be a new approach for managing precocious flowering.
Epidemiology Recently we found that MWP-symptomatic plants and PMWaV-2-infected plants were significantly aggregated within rows of the same bed but not between adjacent rows of different beds when plants were inoculated with mealybugs under field conditions. Spread of PMWaV-2 and concomitant development of MWP was also aggregated within rows indicating that PMWaV-2 was spreading within the field. This finding provides strong evidence that plants in the immediate proximity of PMWaV-2-infected plants have a significantly higher probability of developing MWP and has implications for control strategies.
Virus elimination PMWaV-free pineapple plants were recovered from PMWaV-1-infected crowns through the use of apical and lateral bud propagation. This tissue culture technique was developed in collaboration with Dr. Francis Zee at the USDA-ARS National Clonal Germplasm Repository in Hilo, Hawaii, and can be used to eliminate PMWaV from infected pineapple crowns. This technique will provide a means for recovering PMWaV-free plants from pineapple selections that have a high PMWaV incidence, as in one case, 100%. Virus elimination is also useful for hybrids or selections that are currently being propagated in tissue culture if the mother or stock plants are infected with PMWaV.
Transgenic plants development Our recent research shows that PMWaV-2 and mealybug feeding are both needed for development of MWP symptoms in pineapple. Our goal is to develop PMWaV-resistant pineapple plants for control of MWP. Various gene constructs that have been developed using RNA-mediated resistance technology have been used to transform pineapple plants. Some of the putatively transformed pineapple plants have been tested twice in bioassays in the greenhouse for virus resistance. Some of these plants have no MWP symptoms and are PMWaV-negative. Most of the other transformed and all non-transformed control plants have developed virus infection and MWP symptoms after challenge. Our system to evaluate transformed pineapple plants for virus resistance in greenhouse conditions is efficient and accurate. We will use our system to optimize pineapple transformation and regeneration systems further and to produce transgenic pineapple plants with virus-resistance, nematode-resistance, and flowering-control genes.
Value, Quality, and Impact
Pineapple is the largest agricultural commodity in Hawaii. The farm value of the 2000 pineapple crop is estimated at $101.5 million. Currently, the Hawaii pineapple industry utilizes 20,700 acres and produces 354,000 tons of fruit. Although MWP has been studied for more than 90 years, the etiology of this disorder has been in question. We are the first to characterize the closteoviruses, to develop reliable assays to detect the viruses, and to identify factors involved in disease development. The information will be useful in developing alternative environmentally sound strategies to manage MWP.
Citrus tristeza virus
Citrus is a major fruit crop in many tropical and subtropical regions. In 1997/98, an estimated 90 million tons of citrus fruit were harvested worldwide. However, Citrus tristeza virus (CTV) is a major factor limiting citrus production throughout the world. In many citrus-growing regions, including South and Central America, the Caribbean Basin, southern Europe, South Africa, California, and Israel, CTV has been responsible for the loss of millions of mature trees at a cost of billions of dollars. CTV is transmitted most efficiently by the brown citrus aphid (BrCA). In the United States, the BrCA occurs in Hawaii and has been found recently in Florida. It threatens to become a serious problem for the citrus-growing regions of Texas, Arizona, and California. Specific objectives of this project include: (1) to study the incidence and distribution of CTV strains in Hawaii; (2) to select potential candidate CTV strains for use in cross-protection; (3) to produce CTV-resistant citrus trees using a pathogen-derived resistance approach, and (4) to establish a field screening facility to screen CTV-resistant transgenic citrus plants produced by other labs.
We have developed assays to detect and differentiate CTV strains by ELISA and PCR. Polyclonal and monoclonal antibodies and primers were provided by Steve Garnsey and Mark Hilf. In the past year, nearly 400 citrus trees have been sampled at 46 sites across the state of Hawaii and evaluated for CTV infection by PCR using Mark Hilf's approach and by TBIA using monoclonal antibodies. Overall, nearly 75% of these trees tested positive for CTV by either PCR or TBIA. The CTV incidences were found to be 59% on Kauai, 87% on Oahu, 63% on Maui, and 83% on Hawaii. Using this approach, we have found that CTV strains T30, T36, VT, and T3-like are present on all the Hawaiian islands. T30 and T3-like strains are most prevalent on Oahu. Strains similar to VT are common on all islands in Hawaii. Some of the CTV isolates, as identified by CTV-specific polyclonal antibodies, were negative in PCR tests with all primers available. These results showed that there are strains of CTV present in Hawaii that have not been characterized and may not be present on the continental United States. We have initiated collaborative efforts with Dennis Gonsalves at Cornell University to develop "synthetic" gene constructs to develop broad and durable resistance to this important citrus virus disease.
Value, Quality, and Impact
Within the continental United States, citrus is a high-value crop in California, Texas, Arizona, and Florida, where the industry has controlled CTV by use of certified CTV-free budwood, quarantine, and removal of infected trees. Severe CTV strains are present to a limited extent in these areas but are apparently not transmitted efficiently by the aphid vectors that occur in these states. In Florida, however, the most efficient vector of CTV, the brown citrus aphid (BrCA), Toxoptera citricida, has recently become established and poses a great threat to the citrus industry there. Wherever this vector has become established, the incidence of infection by severe CTV strains has increased. Due to the presence of the BrCA in Central America and Florida, the citrus industies in California, Arizona, and Texas are at risk for increased incidence of severe citrus tristeza disease. CTV is also the major factor limiting production of citrus in Hawaii. CTV occurs almost everywhere in Hawaii where citrus is grown. The virus is present in backyard plantings and in most of the commercial citrus nursery stocks. Citrus growers have repeatedly failed to grow lime successfully on Molokai and Hawaii because of CTV.
Because such diverse CTV isolates, and perhaps others that are not yet well-characterized, are already present in Hawaii, we plan to produce CTV-resistant transgenic citrus plants using gene constructs from different CTV strains. In addition, we plan to make Hawaii a center for field testing of citrus engineered for CTV resistance. Such a center would also be valuable for the testing and selection of CTV isolates to cross-protect against severe stem pitting isolates of CTV. Several laboratories in the United States are working on various approaches, including coat protein-mediated resistance, to develop CTV-resistant transgenic citrus. However, testing of these plants against a wide range of CTV isolates is problematic. The establishment of a field testing site in Hawaii would allow the evaluation of trees for CTV-resistance against a wide range of CTV isolates in the presence of the most efficient vector (BrCA). This information could be used by many groups involved in CTV research and would greatly hasten the development of CTV-resistant stocks for use by the citrus industry in the United States and around the world.