Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime.

Citrus huanglongbing (HLB) is a devastating disease associated with Candidatus Liberibacter asiaticus spp. (CLas), a bacterium restricted to the sieve tube system of the phloem that is transmitted by the psyllid vector, Diaphorina citri. In this study, the human antimicrobial peptides, lysozyme and ß-defensin 2, were targeted to the vascular tissue of Mexican lime (Citrus x aurantifolia [Christm.] Swingle) by fusion to a phloem-restricted protein. Localized expression was achieved, via Agrobacterium tumefaciens-mediated transformation of the stem, which led to protein expression and mobilization within the vascular tissue of heterotrophic tissues. HLB-infected plants were monitored for 360 days. Lower bacteria titers were observed in plants expressing either ß-defensin 2, lysozyme, or the combination thereof, and these plants had increased photosynthesis, compared to untreated control trees. Thus, targeting of antimicrobial proteins to the vascular tissue was effective in decreasing CLas titer, and alleviating citrus greening symptoms. Based on these findings, this strategy could be used to effectively treat plants that are already infected with bacterial pathogens that reside in the phloem translocation stream.

NMR-Based Metabolomic Analysis of Huanglongbing-Asymptomatic and -Symptomatic Citrus Trees.

Huanglongbing (HLB) is one of the most severe diseases that affects citrus trees worldwide and is associated with the yet uncultured bacteria Candidatus Liberibacter spp. To assess the metabolomic differences between HLB-asymptomatic and -symptomatic tissues, extracts from leaf and root samples taken from a uniform 6-year-old commercial orchard of Valencia trees were subjected to nuclear magnetic resonance (NMR) and chemometrics. The results show that the symptomatic trees had higher sucrose content in their leaves and no variation in their roots. In addition, proline betaine and malate were detected in smaller amounts in the HLB-affected symptomatic leaves. The changes in metabolic processes of the plant in response to HLB are corroborated by the relationship between the bacterial levels and the metabolic profiles.

Molecular characterization of 'Candidatus Liberibacter' species/strains causing huanglongbing disease of citrus in Kenya

This study was undertaken to characterize the alpha subgroup of the proteobacteria causing the huanglongbing (HLB) disease of citrus from three different ecological zones of Kenya namely the Lower highlands (LH2, LH3, 1800-1900 m above sea level); Upper midlands (UM3, UM4, 1390-1475m), Lower midlands (LM5, LM4, LM3 of 1290-1340-1390m), by isolation and sequencing DNA encoding the L10 and L12 ribosomal proteins and the intergenic region. A 7I6-basepair DNA fragment was amplified and sequenced and consisted of 536 basepairs of DNA encoding the L10 protein, 44 basepairs of DNA intergenic region and 136 basepairs of DNA that partially encodes the L12 protein. Sequences of rpL10/L12 protein genes from Kenyan strains were 98 percent and 81 percent similar to the South African 'Candidatus Liberibacter africanus strain Nelspruit' and the Asian 'Candidatus Liberibacter asiaticus' strains, respectively. The intergenic rDNA sequence of Kenyan strain from UM and LM showed 84 percent similarity with 'Candidatus L. africanus strain Nelspruit' and 50 percent similarity with 'Candidatus L. asiaticus' strain. However, the LH strain had an 11- basepairs deletion, while the LM4 had a 5-basepair deletion in the intergenic region compared to 'Candidatus L. africanus strain Nelspruit'. The L10 amino acid sequence was 100 percent homologous among HLB bacteria obtained from the agro-ecological zones in Kenya and the L10 protein sequence was also homologus to 'Candidatus L. africanus strain Nelspruit'. Nevertheless, the L10 amino acid sequence of 'Candidatus L. asiaticus' and the 'Candidatus L. africanus subsp. capensis' differed from the Kenyan strains by 18.36 percent and 11.82 percent, respectively. Phylogenetic analysis of both the L10/L12 rDNA sequences and the L10 amino acid sequences clustered the Kenyan strains of the 'Candidatus Liberibacter' species with members of alpha subdivision of proteobacteria.