Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: We have generated 52 transgenic Mexican lime lines containing a transgene that targets the coat protein, p20, p23, and 3-untranslated regions of Citrus tristeza virus. These transgenes were introduced into the Mexican lime genome a variety of configurations such as sense, antisense, inverted repeat, and stacked. A subset of these lines has been assessed by PCR and Southern hybridization to confirm integration of the transgene. All lines assayed appeared to harbor between 1 and 6 copies of the transgene. These lines have been evaluated in the greenhouse for resistance to CTV by exposure to viruliferous brown citrus aphids (Toxoptera citricida). In addition, we have removed the nopaline synthase terminator sequence from the transformation construct described above. We have transformed Mexican lime with this construct and have regenerated approximately 70 shoots. Twenty-five of these shoots were successfully micrografted in vitro to promote rapid growth. We have obtained antisera raised against p12, a citrus-blight-associated protein, from Dr. K.S. Derrick in Florida and have performed serological assays on healthy trees and those that failed the water injection assay. In general, our results with the antisera in Western blot analysis confirmed the high expression of p12 in trees that also failed the water injection assay, and support the diagnoses of CB in these trees. However, we have also identified some trees that failed the water injection assay, but were p12 negative, as well as trees that passed the water injection assay, but showed elevated p12 expression. We have been growing the various rootstocks for the field trial, which will be continued. While surveying Hawaii citrus trees, we discovered a tree with unusual symptoms on its foliage and young bark tissue. These symptoms resembled that of citrus leprosis, a viral disease of great economic importance in South and Central America. Tissues from this tree reacted with an antibody raised against the citrus leprosis virus, CiLV-C. RT-PCR using CiLV-C-specific primers, however, was negative. The CiLV-C genome is bipartite with 9 and 5 kb RNAs. Double-stranded RNAs of 8 kbp and 3 kbp were present in the Hawaii tree, indicating this is a distinct virus. We have fully sequenced the 8 kbp dsRNA and have found it is distantly related to the 9 kb RNA of CiLV-C, further indicating this is a distinct virus. Transmission electron microscopy of symptomatic foliar tissue has revealed the presence of short, bacilliform particles approximately 30x50 nm in size. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In Hawai'i, the citrus industry belongs in the diversified agriculture sector which is becoming increasingly important to the states economy as it is helping to offset losses in production and acreage of pineapple and sugarcane. Citriculture has the potential to further strengthen diversified agriculture in Hawai'i, as approximately 11,000 metric tonnes of citrus are imported into the state each year, while <200 metric tonnes are produced locally. Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. As no serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and abroad. Citrus Blight (CB) is a wilt disease of major importance for citrus grown in tropical and subtropical regions. CB was first reported in Hawaii in 1991 and has also become a major hindrance to the local citrus industry. In recent years, thousands of new citrus trees have been planted across Hawaii, mostly on citrange rootstocks. These rootstocks are commonly used in California nurseries from which many Hawaii growers have obtained planting materials for new orchards. The fact that these are two of the most sensitive rootstocks for CB does not affect California citrus orchards, as CB is not a problem in California. However, in Hawaii this has the potential to be devastating to our developing citrus industry. The long-term goal of this research is to help growers identify and manage CB, re-establish a viable citrus industry in Hawaii, and establish appropriate importation guidelines for citrus into Hawaii and the Pacific Island regions that do not currently have CB. Citrus leprosis is an economically important viral disease of citrus in the Americas. The disease occurs in many South and Central American countries, and is a major hindrance to citrus production in regions of Brazil where $75 million USD are spent annually for its control. In the early 1900's, citrus leprosis was responsible for heavy losses in Florida's citrus industry. The disease, however, began to subside by 1930, and was not observed in recent surveys of Florida and Texas citrus groves. Citrus leprosis is moving up through Central America and again poses a threat to citrus production in the continental United States. Citrus research programs such as the California Citrus Research Board have identified citrus leprosis as an "Item of Interest" in the security of California citrus production. Future study of related viruses, such as the one discovered in Hawaii, will be of great value to combating this disease.
Publications
- Melzer, MJ, Borth, WB, Sether, DM, Ferreira, S, Gonsalves, D, Hu, JS. 2010. Genetic diversity and evidence for recent modular recombination in Hawaiian Citrus tristeza virus. Virus Genes 40:111-118.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Anthurium mosaic: In 2003, a disease with viral-like symptoms appeared at an anthurium farm on the island of Hawaii that supplies the cut flower market. The anthuriums were established about two years prior to the onset of the disease. Diseased anthurium plants collected from this farm exhibited mosaic, mottling, and vein-clearing symptoms on leaves, which along with the spathe, were often distorted with irregular margins, rendering the plants unsuitable for sale. We have isolated three distinct dsRNAs ranging from 3.0-3.5 kbp in size, from symptomatic anthurium plants. These dsRNA segments were completely sequenced and found to belong to a virus that is most closely related to members of the mycovirus family Chrysoviridae. An RT-PCR assay was developed to detect this putative virus. Ti Ringspot: In early 2009, ti farmers on the windward side of the island of Oahu reported unusual symptoms on their ti plants. Symptoms were small (3-15mm) ringspots present primarily on the basal 2/3 of the leaf. These viral-like symptoms have not been previously reported from ti. Symptomatic plants were widespread at affected farms, and according to the farmers, the disease spread quickly throughout the fields. Agarose gel electrophoresis revealed that high molecular weight (15-20 kbp) dsRNA was present in both types of tissue. The dsRNAs were isolated from the gel and used as templates for molecular cloning. We sequenced over 300 clones from the dsRNA libraries that were generated. Analysis of the sequence data revealed that at least four previously unreported species of closteroviruses were present in the ti samples. Tomato yellow leaf curl virus: Tomato yellow leaf curl is perhaps the most destructive viral disease of tomato (Lycopersicon esculentum) with losses of up to 100% in the tropics and subtropics. The disease is caused by Tomato yellow leaf curl virus (TYLCV) (sensu lato), a complex of circular monopartite ssDNA viruses of the genus Begomovirus, Family Geminiviridae. On tomato, TYLCV inhibits leaflet growth, causing yellowing and upward curling of the leaf margin. Flowers are aborted, and the entire plant will develop a stunted appearance. Whiteflies (Bemisia tabaci) are the natural vector of TYLCV which transmit the virus in a persistent, circulative manner. In October 2009, a home gardener in Kahului, Maui reported tomatoes with symptoms similar to tomato yellow leaf curl to the University of Hawaii Agricultural Diagnostic Service Center (ADSC) in Manoa. Samples were sent through the ADSC to our laboratory where we isolated DNA from symptomatic tissues. PCR was performed using degenerate primers PAR1c715 and PAL1v1978. An amplicon of expected size (~1.5 kilobases) was generated, cloned, and sequenced. The sequence data revealed the tomato plants were infected with a strain of TYLCV that was most similar (>98% identical) to strains of TYLCV from Japan (Tochigi and Tomigusuku) and China (ZJHZ12, Sh10, and ZJ8). This represents the first known case of TYLCV in Hawaii. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In Hawaii, diversified agriculture is becoming increasingly important to Hawaii's economy as it is helping to offset losses in production and acreage of two of Hawaii's main agricultural products, pineapple and sugarcane. Acreage devoted to diversified agriculture has steadily increased since 1970 and record high sales of $456 million in 2006 have been achieved (HASS 2009). Anthurium and ti leaves belong to the floriculture and nursery product sector of Hawaii agriculture, which was valued at $98.6 million in 2008, down 10% the 2007 value of $108.7 million (HASS 2009). Cut anthurium flowers are the 5th most valuable crop in this sector, with $3.74 million sold in 2008, which was down 21% from the 2007 value of $4.53 million (HASS 2009). Cut ti leaves had sales of $376,000 in 2008, down from $387,000 in 2007. The decline in revenue of these two crops is probably due to diminished production in 2008, which were down 19% and 12% for cut anthurium and ti leaves, respectively. Hawaii tomato growers farmed 740 acres and generated $9.9 million in 2007, representing about 27% of the total farm revenue for vegetable and melon crops in Hawaii (HASS 2009). This represents a 264% increase in acreage and 267% increase in revenues over a 10 year span. This dramatic increase can be attributed to the popularity of locally-grown field and greenhouse tomatoes in Hawaii's supermarkets and food industries. Early-picked tomatoes grown elsewhere and ripen on their trip to Hawaii cannot compare to the high quality, freshly-picked specimens grown in the islands.
Publications
- Sether, D. M., Borth, W. B., Melzer, M. J., and Hu, J. S. 2009 Spatial and temporal incidences of Pineapple mealybug wilt associated viruses in pineapple planting blocks. Plant Disease. (in press)
- Hu, J.S., Sether, D.M., Melzer, M.J., Subere, C.V., Cheah, K., Chen, Y., Qi L., Borth, W., Wang, I.C., Nagai, C. and Wang, M.L. 2009. Characterization and management of pineapple mealybug wilt associated viruses. Acta Hort. (ISHS) 822:185-190.
- Sether, D. M., Melzer, M.J., Borth, W.B., and Hu, J.S. 2009. Genome organization and phylogenetic relationship of Pineapple mealybug wilt associated virus-3 with other family Closteroviridae members. Virus Genes 38:414-420.
- Wang, M.L., Uruu G., Xiong, L.W., He, X. L., Nagai, C., Cheah, K. T., Hu, J. S., Nan, G.L., Sipes, B.S., Atkinson, H.J., Moore, P. H., Rohrbach, K.G., Paull, R.E. 2009. Production of transgenic pineapple plants via adventitious bud regeneration. In Vitro Plant, Cellular and Developmental Biology 45:112-121.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Citrus is a major fruit crop in many tropical and subtropical regions. In 2006, an estimated 102 million tons of citrus fruit were harvested worldwide. We have generated transgenic Mexican lime (Citrus aurantifolia) plants containing a synthetic sequence that is ~95% identical to the coat protein (CP) gene of known strains of CTV from Hawaii and around the world. This synthetic sequence was introduced in sense, anti-sense, and inverted-repeat configurations. We have also transformed Mexican lime with an additional transformation construct that targets the three post-transcriptional gene silencing (PTGS) suppressors of CTV. This construct features partial synthetic CP, p20, and p23 genes, as well as the 3' UTR which controls their transcription. These transgenic plants have been allowed to grow in the greenhouse for approximately 6 months to attain a size suitable for the virus challenge bioassay. Fifty-three lines were selected for the virus challenge bioassay, as well as six control plants: three wild-type, and three of a line transformed only with an empty vector. Citrus blight (CB) is a wilt disease of major importance for citrus grown in tropical and subtropical regions. CB is also a major hindrance to the local citrus industry in Hawaii. We have tested a number of declining citrus trees from different sites with a water injection assay, which is a very useful field diagnosis. Tree afflicted with CB will take up very little water through the xylem. In our preliminary survey, water uptake varied from 10 ml in 16 seconds for healthy-appearing citrus to 0.5 ml in 30 seconds for trees with severe blight symptoms, suggesting that those symptomatic trees were afflicted with CB. We have obtained the p12 antisera from Dr. K.S. Derrick in Florida and have performed serological assays on healthy trees and those that failed the water injection assay. In general, our results with the antisera confirmed the high expression of p12 in trees that also failed the water injection assay, and support the diagnoses of CB in these trees. However, we have also identified some trees that failed the water injection assay, but were p12 negative, as well as trees that passed the water injection assay, but showed elevated p12 expression. It is unclear if these results were due to the sectorial nature of CB or the presence of another disease with similarities to CB in Hawaii We have selected 10 rootstocks to evaluate for their tolerance to CB, and acclimatization to Hawaiian soils and growing conditions. There have been two substitutions in the rootstock trial; initially we proposed to use Smooth Flat Seville orange and Volkamer lemon, due to their popularity as rootstocks in Florida. More recent data, however, suggests these two rootstocks are in decline, and have been supplanted in popularity by Kuharske and X-639 citranges. As such, in our trial we will use these two more popular rootstocks. We are currently acquiring seeds of the ten rootstocks, and plan to install the experiment in 2009. We have selected a site on the island of Molokai that will be suitable for this experiment. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
CTV and its most effective vector, the brown citrus aphid (BrCA), have been present in Hawaii for at least 50 years. No effective control strategies have ever been employed to control CTV or the BrCA in Hawaii, resulting in high disease incidence and diversity of the virus. The development of citrus resistant to the many Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and worldwide. Many of the strains that are found in these regions are also present in Hawaii, therefore plants that are resistant to CTV infections in Hawaii have a high probability that they will also be resistant at other locations in the world where citrus is cultivated. CTV-resistant citrus would spark the development of a very strong citrus industry in Hawaii, and further enhance diversified agriculture in Hawaii. Although Hawaii may not become a citrus exporter in the near future, a strong citrus industry would greatly reduce our reliance on imported citrus fruit which accounts for up to 90% of the citrus sold in local markets. About 90% of the citrus trees planted in Hawaii over the last 5 years are on either C-35 or Carizzo citrange rootstocks. These rootstocks are commonly used in California nurseries from which many Hawaii growers have obtained planting materials for new orchids. The fact that these are two of the most sensitive rootstocks for CB does not affect California citrus orchards, as CB is not a problem in California. However, in Hawaii this has the potential to be devastating to our developing citrus industry. Indeed, the effects are already being noticed on farms where these trees are more than 5 years old and CB losses are already high. This research complements our current work on Citrus tristeza virus, and contributes to making citrus a significant component of diversified agriculture in Hawaii. However, in regions where CB is severe, 5-10% of trees are lost to the disease every year. Many consider CB to be the most economically serious disease of citrus in Florida where approximately 650,000 to 900,000 trees are lost to CB annually. In Sao Paulo, Brazil, an estimated 10 million trees are lost every year. In the Letsitele region of South Africa, researchers have estimated that CB would cost growers over $30 million (USD) from 1996 to 2000. As such, any insights into the etiology and pathogenesis-related gene expression associated with CB afflicted trees, and the development of practical strategies to manage CB obtained from this work would not only benefit Hawaii's citrus industry, but also other citrus-producing regions in the Pacific Region and the continental USA that are affected by CB.
Publications
- Melzer, M. J, Mauch, H., Borth, W., Ferreira, S., Sether, D., Gonsalves, D., Pena, L., and Hu, J. Development of transgenic Mexican lime plants for resistance to Citrus tristeza virus using post-transcriptional gene silencing. First International Symposium on Biotechnology of Fruit Species. ISHS, Dresden, Germany Sept. 1-5, 2008.
- Jiang, B., Hong, N., Wang, G.P., Hu, J.S., Zhang, J.K., Wang, C.X., Liu, Y., Fan, X.D. 2008. Characterization of Citrus tristeza virus strains from southern China based on analysis of restriction patterns and sequences of their coat protein genes. Virus Genes 37:185-192.
- Melzer M., Hilf, M., L. Pena, H. Mauch, W. Borth, F. Zee, S. Garnsey, S. Ferreira, D. Sether, D. Gonsalves, and J. S. Hu 2008. Molecular diversity and management of Citrus tristeza virus in Hawaii. 11th Citrus International Congress. Wuhan, China October 27-30, 2008.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: We have previously obtained seeds from 6 transgenic lines of Mexican lime that are putatively CTV-resistant from Dr. Leandro Pena at the Instituto Valenciano de Investigaciones Agrarias in Valencia, Spain. Micro-satellite analysis was used to distinguish nucellar (hemizygous) and zygotic (homozygous) seedlings in the transgenic lines and a wild-type control. A nucellar seedling from each of the six transgenic lines and the wild-type control was propagated onto sixteen C-35 citrange rootstocks and grown in the greenhouse. These grafted plants have now attained a suitable size for field evaluation. We have also received the required permits from the Hawaii Department of Agriculture and USDA-APHIS allowing the field trial to commence. We have sequenced over 100 coat protein genes from Hawaiian CTV. This, as well as coat protein gene sequence data of CTV strains from around the world obtained from GenBank, were used to generate a synthetic transgene that was ~95% identical to
all of these genes. We have generated multiple lines of transgenic Mexican lime plants containing this synthetic gene in sense, anti-sense, and inverted-repeat configurations. PCR and genomic Southern analysis have confirmed the transgenic nature of these plants, and we are continuing to generate additional lines for each of the transgene configurations. Several of the transgenic lines have been propagated by bud-grafting for virus-resistance bioassays.
PARTICIPANTS: Michael Melzer at the University of Hawaii
TARGET AUDIENCES: citrus growers in Hawaii and plant virologists
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. As no serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, which has not been self-sufficient in citrus for decades, but also other citrus-producing regions in the US and abroad. Many of the strains that are found in these regions are also present in Hawaii, making plants resistant in Hawaii potentially resistant elsewhere.
Publications
- No publications reported this period
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Progress 10/01/05 to 09/30/06
Outputs
Previously we had sequenced over 100 coat protein genes from Hawaiian CTV. This, as well as coat protein gene sequence data of CTV strains from around the world obtained from GenBank, was used to generate a synthetic transgene that was 95% identical to all of these genes. We have generated multiple lines of transgenic Mexican lime plants containing this synthetic gene in sense, anti-sense, and inverted-repeat configurations. PCR and genomic Southern analysis have confirmed the transgenic nature of these plants, and we are continuing to generate additional lines for each of the transgene configurations. Several of the transgenic lines are growing well in the greenhouse, and will be propagated by bud-grafting for virus-resistance bioassays by the end of 2006. We have been working with citrus and the citrus industry in Hawaii for six years, with most of our efforts focused on Citrus tristeza virus (CTV). During this work we have also observed trees in decline which were
not infected with CTV. These declining trees have the classic symptoms of Citrus Blight (CB), and therefore we have tested a number of these declining citrus trees from different sites with a water injection assay that is commonly used for CB diagnosis. This preliminary survey indicated CB was prevalent in Hawaii. A more comprehensive survey conducted at a declining commercial citrus orchard in Mountain View, Hawaii indicated CB was at least partly responsible for the unthrifty citrus trees. We have obtained antisera raised against p12, a CB-associated protein, from Dr. K.S. Derrick in Florida and have performed serological assays on healthy trees and those that failed the water injection assay. In general, our results with the antisera in Western blot analysis confirmed the high expression of p12 in trees that also failed the water injection assay, and support the diagnoses of CB in these trees. However, we have also identified some trees that failed the water injection assay, but
were p12 negative, as well as trees that passed the water injection assay, but showed elevated p12 expression. It is unclear if these results were due to the sectorial nature of CB or the presence of another disease with similarities to CB in Hawaii. We have also designed primers for reverse-transcription PCR detection of an Ideaovirus sequence reported from citrus that may have a role in CB. PCR products of the expected size have been obtained and submitted for sequencing to confirm the presence of Ideaovirus-like sequences in citrus with CB. Partial purifications of flexuous rod-shaped particles that have been associated with CB in Florida are also underway.
Impacts
Citrus tristeza virus (CTV) and its most effective vector, the brown citrus aphid (BrCA) have been present in Hawaii for at least 50 years. As no serious control strategies have ever been employed to control CTV or the BrCA, resulting in high incidence and diversity of the virus. As such, Hawaii is the ideal location to test plants putatively resistant to CTV that have been developed in Hawaii, the US mainland, or elsewhere. The development of citrus resistant to Hawaiian CTV strains would be beneficial to not only Hawaii, but also other citrus-producing regions in the US and abroad. Citrus Blight (CB) is a wilt disease of major importance for citrus grown in tropical and subtropical regions. CB was first reported in Hawaii in 1991 and has also become a major hindrance to the local citrus industryThe fact that these are two of the most sensitive rootstocks for CB does not affect California citrus orchards, as CB is not a problem in California. However, in Hawaii this
has the potential to be devastating to our developing citrus industry. Indeed, the effects are already being noticed on farms where these trees are more than 5 years old and CB losses are already high. As such, this potentially lucrative industry is at risk due to the high susceptibility of this rootstock to CB. The long-term goal of this research is to help growers identify and manage CB, re-establish a viable citrus industry in Hawaii, and establish appropriate importation guidelines for citrus into Hawaii and the Pacific Island regions that do not currently have CB.
Publications
- Melzer, M. .J., Borth, W.B., Zee, F., Hilf, M.E., Garnsey, S.M., and Hu, J.S. 2006. Incidence and diversity of Citrus tristeza virus in the Hawaii. Phytopathology 96: 77.
- Melzer, M. .J., Borth, W.B., Zee, F., Hilf, M.E., Garnsey, S.M., and Hu, J.S. 2006. Incidence, distribution, and diversity of Citrus tristeza virus in the Hawaiian islands. In: Proc. 16th Conf. IOCV pages 179-186.
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