Transgenic Citrange troyer rootstocks overexpressing antimicrobial potato Snakin-1 show reduced citrus canker disease symptoms.

Citrus canker is a major disease caused by Xanthomonas citri pv. citri. Snakin-1 is an antimicrobial peptide, which was previously shown to be effective against different bacterial and fungal diseases in potato, wheat and lettuce when expressed in transgenic plants. We generated transgenic Citrange Troyer citrus rootstocks constitutively expressing this peptide and 5 different transgenic lines were challenged against virulent X. citri isolates. Challenge assays conducted in vitro using detached leaves and in planta by infiltration revealed a significant reduction of the number and size of canker lesions in some of the transgenic lines.

Management of Citrus Canker in Argentina, a Success Story.

Citrus canker is an important bacterial disease of citrus in several regions of the world. Strains of Xanthomonas citri type-A (Xc-A) group are the primary pathogen where citrus canker occurs. After Xc-A entered the Northeast of Argentina in 1974, the disease spread rapidly from 1977 to 1980 and then slowed down and remained moving at slow pace until 1990 when it became endemic. Citrus canker was detected in Northwest Argentina in 2002. This paper presents the main steps in the fight of the disease and the management strategies that have been used to control citrus canker at this time. We think the process might be usefull to other countries with the same situation. Results from more than 40 years of research in Northeast (NE) Argentina indicate that we are at the limit of favorable environment for the disease. The severity of citrus canker is greatly affected by the environment and El Niño Southern Oscillation (ENSO) phenomenon which causes cyclic fluctuations on the disease intensity in the NE region. Weather-based logistic regression models adjusted to quantify disease levels in field conditions showed that the environmental effect was strongly modulated by the distance from a windbreak. Production of healthy fruits in citrus canker endemic areas is possible knowing the dynamics of the disease. A voluntary Integrated Plan to Reduce the Risk of Canker has been in place since 1994 and it allows growers to export unsymptomatic, uninfested fresh fruit to countries which are free of the disease and require healthy, pathogen free fruits. The experience from Argentina can be replicated in other countries after appropriate trials.

Adaptation of genetically monomorphic bacteria: evolution of copper resistance through multiple horizontal gene transfers of complex and versatile mobile genetic elements.

Copper-based antimicrobial compounds are widely used to control plant bacterial pathogens. Pathogens have adapted in response to this selective pressure. Xanthomonas citri pv. citri, a major citrus pathogen causing Asiatic citrus canker, was first reported to carry plasmid-encoded copper resistance in Argentina. This phenotype was conferred by the copLAB gene system. The emergence of resistant strains has since been reported in Réunion and Martinique. Using microsatellite-based genotyping and copLAB PCR, we demonstrated that the genetic structure of the copper-resistant strains from these three regions was made up of two distant clusters and varied for the detection of copLAB amplicons. In order to investigate this pattern more closely, we sequenced six copper-resistant X. citri pv. citri strains from Argentina, Martinique and Réunion, together with reference copper-resistant Xanthomonas and Stenotrophomonas strains using long-read sequencing technology. Genes involved in copper resistance were found to be strain dependent with the novel identification in X. citri pv. citri of copABCD and a cus heavy metal efflux resistance-nodulation-division system. The genes providing the adaptive trait were part of a mobile genetic element similar to Tn3-like transposons and included in a conjugative plasmid. This indicates the system's great versatility. The mining of all available bacterial genomes suggested that, within the bacterial community, the spread of copper resistance associated with mobile elements and their plasmid environments was primarily restricted to the Xanthomonadaceae family.

Control of Citrus Canker and Citrus Bacterial Spot with Bacteriophages.

Bacteriophages, alone or in combination with copper bactericides, were evaluated for managing Asiatic citrus canker and citrus bacterial spot incited by Xanthomonas axonopodis pathovars citri and citrumelo, respectively. In a set of five greenhouse experiments, phage treatment provided consistent control of citrus canker, causing an average of 59% reduction in disease severity. However, treatment with phage was ineffective if applied with skim milk, a protective formulation, which increases phage residual activity. In nursery settings, phage treatment also reduced disease but was less effective than copper-mancozeb, a chemical bactericide. The integration of phage and copper-mancozeb resulted in equal or less control than copper-mancozeb application alone. Phage treatments were evaluated in a commercial citrus nursery for reducing citrus bacterial spot caused by natural inoculum. Phage treatment provided significant disease reduction on moderately sensitive Valencia oranges in two trials (48 and 35%); however, on the highly susceptible grapefruit host it was ineffective. In an experimental citrus nursery, phage treatment provided significant control of citrus bacterial spot caused by a phage-sensitive strain, but was equally or less effective than copper-mancozeb. The combination of phage and copper-mancozeb did not increase control compared with copper-mancozeb alone.

Resistance to Citrus Canker Caused by Xanthomonas axonopodis pv. citri in an Accession of Citrus sp. Similar to Grapefruit.

Citrus canker is caused by Xanthomonas axonopodis pv. citri. All grapefruit (Citrus paradisi Macf.) cultivars are highly susceptible to the A-group strains of Xanthomonas axonopodis pv. citri. A type of grapefruit (Citrus sp.) called Dalan Dan (DD) has shown high resistance in the field in Misiones, Argentina where it is widely planted. The DD is Accession No. NC678 in the Campinas Citrus Germplasm Collection (Centro APTA Citrus Sylvio Moreira, Cordeiropolis, SP, Brazil) and Accession No. CCC-618 Dalan Dan in the Argentina Citrus Germplasm Collection at INTA (Concordia, Entre Ríos). Molecular studies completed in Brazil have shown high similarities among grapefruit varieties and DD (1). We report here the characterization of quantitative resistance to citrus canker in DD. Quantitative resistance in citrus is expressed as lower number of lesions per leaf area (3). Lesions per square centimeter were calculated after 3 to 4 weeks of two sets of inoculation of three inoculum levels (5 × 102, 2.5 × 103, and 5 × 103 cells per ml) in leaves of developing leaf flushes. The period of greatest susceptibility in DD was 15 to 23 days for the three inoculum levels; 1.35 ± 0.34 to 0.01 ± 0.01, 4.98 ± 0.91 to 0.91 ± 0.48, and 9.85 ± 2.17 to 1.91 ± 1.37 lesions per cm2, respectively. By contrast, the period of greatest susceptibility in grapefruit cv. Duncan was 15 to 29 days for the same three inoculum levels; 2.78 ± 0.46 to 1.22 ± 0.39, 17.29 ± 2.79 to 5.55 ± 0.93, and 22.87 ± 3.27 to 11.95 ± 3.55 lesions per cm2, respectively. The results shown here suggest that the DD citrus accession is susceptible to citrus canker for a very short time during the development of leaf flushes, which results in very high resistance in the field. This citrus germplasm material could be valuable as a source of resistance to improve other varieties or as a replacement of grapefruit due to the similar fruit and juice qualities. References: (1) M. J. Corazza-Nunes et al. Euphytica 126:169, 2002. (2) M. J. Corazza-Nunes et al. Summa Phytopathol. 32:322, 2006. (3) R. E. Stall et al. Phytopathology 72:1097, 1982.