Management of Stem Pitting Diseases Caused by Citrus Tristeza Virus

NON-TECHNICAL SUMMARY: Citrus tristeza virus (CTV) continues to be a threat to the citrus industries in the mainland U.S., because there are many isolates that if introduced would destroy the economic viability of our industries. CTV presently is a major constraint to citrus production in Hawaii. Isolates of CTV cause two different disease syndromes. Decline isolates kill citrus varieties on the sour orange rootstock, which unfortunately is the most desirable rootstock for many areas. Isolates that cause stem-pitting diseases, which result in reduced growth and production of small, unmarketable fruit, regardless of rootstock, present the greatest threat potentially eliminating the commercial production of sweet oranges or grapefruit. Lime production in the U.S. already is limited by existing lime stem-pitting isolates of CTV. The probability is high that severe stem-pitting isolates will be introduced into the U.S., by accident, by bioterrorism, or by emerging from a `hidden' component of an existing population. In that event, we need to be capable of managing the disease. Cross protection, the immunization of trees with mild strains to prevent induction of disease by severe strains, is a strategy that is complementary to development of resistant trees to manage CTV-induced diseases. We propose to engineer isolates of CTV that will effectively protect trees against stem-pitting diseases.

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APPROACH: Citrus tristeza virus (CTV) continues to be a threat to the citrus industries in the mainland U.S., because there are many isolates that if introduced would destroy the economic viability of our industries. CTV presently is a major constraint to citrus production in Hawaii. Isolates of CTV cause two different disease syndromes. Decline isolates kill citrus varieties on the sour orange rootstock, which unfortunately is the most desirable rootstock for many areas. Isolates that cause stem-pitting diseases, which result in reduced growth and production of small, unmarketable fruit, regardless of rootstock, present the greatest threat potentially eliminating the commercial production of sweet oranges or grapefruit. Lime production in the U.S. already is limited by existing lime stem-pitting isolates of CTV. The probability is high that severe stem-pitting isolates will be introduced into the U.S., by accident, by bioterrorism, or by emerging from a `hidden' component of an existing population. In that event, we need to be capable of managing the disease. Cross protection, the immunization of trees with mild strains to prevent induction of disease by severe strains, is a strategy that is complementary to development of resistant trees to manage CTV-induced diseases. We propose to engineer isolates of CTV that will effectively protect trees against disease. We have an ongoing research program on CTV that has focused on decline isolates that are problematic in Florida. Using procedures proposed here we already have identified viral gene products involved in production of disease symptoms by the Florida decline strain and have disarmed it to a mild phenotype. This isolate is under tests to protect trees from the decline isolate. This is a proposal to develop similar information concerning stem-pitting isolates of CTV - information that will allow an effective defense in the event that severe stem-pitting isolates of CTV are introduced into the U.S.
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PROGRESS: 2004/09 TO 2006/09<BR>
This project was to examine how Citrus tristeza virus (CTV) induces stem pits in citrus that caused poor vigor, small fruit, and lack of productivity. We approached this problem by examining how CTV interacted with citrus tissues that develop into stem pits and viral gene expression that induces development of stem pits. Induction of stem pits by CTV is associated with movement of the virus. We are examining movement of CTV in citrus trees by examining the distribution of a green fluorescent protein (GFP)-marked CTV construct, and it appears that movement of the virus in its large woody plant host is remarkably different from that of well-studied viruses in herbaceous host. Most of the replication of CTV occurs in phloem-associated cells adjacent to sieve elements: companion cells, phloem parenchyma, and probably immature sieve elements. There appears to be one movement mechanism required for movement from sieve elements to these phloem-associated cells and a second mechanism that allows limited cell-to-cell movement. Most infected cells occur in clusters of phloem-associated cells and the development of stem pits appears to be associated with the cell-to-cell movement of the virus. A surprising observation that we made was that deletion of genes from CTV induced stronger stem pitting than occurred with the full-length wild-type parent. CTV T36 induces moderate stem pitting in C. macrophylla and Mexican lime. However, deletion of the p33 ORF, p18 ORF, or combinations of them induced greatly increased stem pitting in both hosts. Thus, stem-pitting determinants do not appear to be simple nucleotide or amino acid changes that induce disease. It is possible that the deletions affect the levels of expression of some viral genes, perhaps RNA silencing suppressors, and this could be the cause of the increase or decrease in stem pitting. To localize viral sequences involved in stem pitting, we examined hybrids between CTV T36, which does not induce stem pitting in sweet orange or grapefruit, and our most severe stem-pitting isolate, T68, which induces stem pitting in both. The initial objective is to substitute 3' sequences of T68 into T36. The chimeric viruses replicate and move like wild type CTV isolates in our test hosts. T68 and T36 are different strains differing in sequence through the 3' region of the genomes by approximately 10%. The hybrids were assayed for sweet orange and grapefruit stem pitting. The previous results suggested that CTV genes involved in suppression of silencing might affect the induction of stem pits. However, all of the T68 genes involved in suppression of silencing in different combinations were substituted into T36 with no changes in stem pitting. Thus, the induction of stem pits appears to be a more complex phenomenon. This work is being continued.
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IMPACT: 2004/09 TO 2006/09<BR>
The objective is to develop methods to manage stem pitting diseases caused by Citrus tristeza virus.