Progress 10/01/04 to 09/30/05
Outputs
Xanthomonas axonopodis pv. citri (Xac) strain A(w), pathogenic on key lime trees, but not on grapefruit trees, produces a rapid necrosis (HR) after infiltration with high concentrations in grapefruit leaves. To investigate if an avirulence (avr) gene is involved with the rapid necrosis a cosmid library of A(w) Three clones eliciting HR in grapefruit leaves were conjugated into an Asiatic (A) citrus canker strain, which is pathogenic in grapefruit and key lime. Only one clone (799) when expressed in the A strain caused an HR in grapefruit. Furthermore, it did not cause an HR in key lime. PCR primers to the gene amplified DNA of all A(w) strains, but none of the A strains. When the gene was inactivated in the parent strain by marker exchange the mutant strain caused typical disease symptoms in grapefruit leaves. The HR reaction was restored by complementation of the mutant with the 799 subclone. Therefore, the avr gene appears to limit the host range of A(w) strains.
Citrus bacterial spot disease (CBS), incited by Xanthomonas axonopodis pv. citrumelo (Xacm), is being used as a model system for citrus canker. In disease control trials we evaluated the effect of bacteriophages on CBS disease development in a commercial citrus nursery. Two Xacm-specific clear-plaque-producing phages isolated from the nursery and a clear-plaque producing phage originating from an outside source were used. The local phages had narrower host ranges but were more durable on plant foliage than the foreign phage. Two blocks of Valencia orange and one block of Ray Ruby grapefruit were either not treated or sprayed twice weekly with the phage mixture. In the orange blocks phage treatment significantly reduced disease progress as measured by AUDPC, from 331 to 214 and from 324 to 233, whereas in the grapefruit the reduction was not significant although in three of six assessments phage significantly reduced CBS.
Impacts
This work will help in identifying strategies for controlling citrus bacterial canker
Publications
- B. Balogh, J. Jones, J. Dilley, and H. Yonce. 2005. The role of bacteriophages in suppression of citrus bacterial spot in a citrus nursery. Phytopathology 95:S6
- M. Rybak, and J. Jones. 2005 Identification of an avirulence gene in Xanthomonas axonopodis pv. citri strain A(w). Phytopathology 95:S91
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Progress 09/15/01 to 09/14/04
Outputs
Xanthomonas axonopodis pv. citri (Xac-A) strain Xac-Aw induces a hypersensitive reaction in grapefruit leaves and is virulent on Key Lime. A 1599 bp ORF was cloned and determined to be responsible for the HR and was designated avrGf1. When this gene was inactivated in Xac-Aw, typical citrus canker symptoms were produced in grapefruit. Another cosmid clone containing hrpX and hrpG resulted in HR in grapefruit leaves, when expressed in X. perforans, but did not in Xac-A. Subcloning showed that only hrpG was required for the HR. To determine what gene(s) in X. perforans was responsible for the HR, X. perforans cosmid clones were expressed in Xac-A containing an additional hrpG from X. perfornans. A cosmid containing a xopA homolog was identified. The xopA clone significantly reduced transconjugant Xac-A bacterial populations in grapefruit leaves compared to the wild-type Xac-A. The xopA region from X. perforans appears to be a host limiting factor. A second aspect of this
work focused on a biological control approach to use bacteriophages for reducing bacterial pathogen populations and disease severity on citrus. Bacteriophages isolated from citrus canker lesions in Florida and Argentina were evaluated based on plaque morphology, chloroform sensitivity, host range, genome size, DNA restriction profile and virion morphology. The phage isolates showed a lack of diversity. Mixtures of bacteriophages were evaluated for controlling citrus canker in greenhouse trials in Florida and in nursery trials in Argentina. Bacteriophages reduced citrus canker disease severity both in greenhouse and field trials. The level of control was inferior to chemical control with copper bactericides. The combination of bacteriophage and copper treatments did not result in increased control. Citrus canker field trials in Florida have been prohibited until recently, as the disease was under eradication. For this reason we evaluated the efficacy of phage treatment on a similar
bacterial citrus disease, citrus bacterial spot, incited by X. axonopodis pv. citrumelo. Bacteriophages reduced citrus bacterial spot severity. The level of 12 control was equal or inferior to chemical control with copper bactericides. The combination of bacteriophage and copper treatments did not result in increased control. In experiments monitoring the fate of bacteriophages on the citrus foliage following bacteriophage application, phage populations stayed steady on the foliage during nighttime but were drastically reduced within hours after sunrise. The rate of reduction varied among the phages. The ability of bacteriophages to multiply on the plant foliage in the presence of their bacterial host was investigated. Phages varied in their ability to multiply, and the ones that successfully increased in populations on the bacterial host on the leaf surface also reduced disease severity, whereas the ones that were unable to multiply in the target environment did not reduce disease
severity. In summary, bacteriophages show significant promise as part of an integrated management strategy for controlling citrus canker.
Impacts
Citrus canker is an emerging plant pathogen on citrus in Florida. Currently there are few strategies that can be employed to control bacterial diseases. Given that there are not many, biological control was pursued to determine if this is a viable alternative to standard chemical control. Bacteriophages showed significant promise as part of an integrated management strategy for controlling citrus canker. Therefore, if delivery systems and proper formulations could be developed for bacteriophage applications, this technology could be used as part of a management strategy. An avirulence gene was also identified in this study. The significance of this is that this is the first avirulence gene identified in the citrus canker bacterium that interacts with a resistance gene in citrus. Therefore, it is feasible that other resistance genes can be identified that could be used in a breeding program for control of citrus canker.
Publications
- Balogh, B., J. B. Jones, R. E. Stall, B. I. Canteros, and A. M. Gochez. 2004. Association of bacteriophages with citrus canker and their use for control. Phytopathology 94:S6.
- Dickstein, E., J. Jones, X. Sun, and D. Jones. 2005. Identification of citrus canker (Xanthomonas axonopodis pv. citri) and related strains using ribotyping and fatty acid analysis. Phytopathology 95:S25.
- Rybak,M., and J. Jones. 2005. Identification of an avirulence gene in Xanthomonas axonopodis pv. citri strain A(w). Phytopathology 95:S91
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Progress 10/01/01 to 10/01/02
Outputs
We are attempting to determine general ways that the citrus canker bacterium can mutate or change such that genes are lost or inactivated by one of several mechanisms or gained from other closely related bacteria by conjugation resulting in an altered host range. We propose to research both of these ways for causing changes in pathogenicity in Xanthomonas strains causing disease in citrus. We are currently determining if a gene or genes can be inactivated in order to affect host range and aggressiveness of a strain that causes typical canker on Mexican lime, but atypical symptoms on grapefruit. This is being done utilizing chemical mutagenesis. Currently, we have been unable to alter host specificity. We are also attempting to determine the effect of gain of genes on host range and aggressiveness by chromosomal transfer. We are currently modifying the strains by altering phenotypic characteristics and by introducing markers such that chromosomal transfer can more
easily be studied. Finally, we are attempting to determine the potential for using bacteriophages for controlling citrus canker in Florida. Strains of X. campestris pv. citri are being isolated from disease outbreaks in Florida and other regions in the world. These strains are being tested for sensitivity or resistance to the phages that have been obtained to date. If any new strain of X. c. citri is discovered to be resistant, then new phages (wild-type and/or h-mutant) that will lyse these bacteria will be isolated. This process is leading to the development of an effective phage bank for preparing h-mutant phage mixtures against most if not all strains of X. c. citri occurring throughout the United States.
Impacts
(N/A)
Publications
- No publications reported this period
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