Novel Plantibodies Show Promise to Protect Citrus from Greening Disease

Candidatus Liberibacter asiaticus (CLas), the bacteria responsible for citrus greening disease [huanglongbing (HLB)], has become a worldwide threat to citrus (Citrus sp.) production. HLB has proven difficult to study and treat because of the complex interactions between CLas, the citrus host, and insect vectors. We have selected for single chain fragment variable (scFv) antibodies from a specialized bacteriophage library for binding activity against CLas proteins InvA and TolC. Portions of each protein were chosen as antigens based on predicted binding availability and theorized necessary functions in pathogenicity. Binding affinity for individual scFv-expressing clones was confirmed by phage enzyme-linked immunosorbent assay (ELISA). The scFv sequences were stably transformed under the control of a tandem Cauliflower mosaic virus 35S (CaMV 2x35S) promoter by Agrobacterium tumefacien–mediated transformation into ‘Carrizo’ citrange (Citrus sinensis × Poncirus trifoliate), a citrus rootstock cultivar. Replicated plants of single transformations were inoculated by infestation with CLas positive asian citrus psyllid (Diaphorina citri), a CLas vector. Inoculation and disease progression was monitored through quantitative real-time polymerase chain reaction. Inoculated transgenic plants showed significantly reduced CLas titer compared with wild types. A subpopulation of transgenic plants displayed no measurable surviving bacteria after 12 months. Interestingly, individual replicated plants from the same transgenic events strongly segregated into two populations by resistance phenotype: a minority that were indistinguishable from wild-type plants and a majority that were highly resistant. Our results are the first step in developing a novel protection strategy for HLB.

Annotation of chitin biosynthesis genes in Diaphorina citri, the Asian citrus psyllid

The polysaccharide chitin is critical for the formation of many insect structures, including the exoskeleton, and is required for normal development. Here we report the annotation of three genes from the chitin synthesis pathway in the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), the vector of Huanglongbing (citrus greening disease). Most insects have two chitin synthase (CHS) genes but, like other hemipterans, D. citri has only one. In contrast, D. citri is unusual among insects in having two UDP-N-acetylglucosamine pyrophosphorylase (UAP) genes. One of the D. citri UAP genes is broadly expressed, while the other is expressed predominantly in males. Our work helps pave the way for potential utilization of these genes as pest control targets to reduce the spread of Huanglongbing.

Effect of Sunlight on the Efficacy of Commercial Antibiotics Used in Agriculture

Antibiotic stewardship is of paramount importance to limit the emergence of antibiotic-resistant bacteria in not only hospital settings, but also in animal husbandry, aquaculture, and agricultural sectors. Currently, large quantities of antibiotics are applied to treat agricultural diseases like citrus greening disease (CGD). The two commonly used antibiotics approved for this purpose are streptomycin and oxytetracycline. Although investigations are ongoing to understand how efficient this process is to control the spread of CGD, to our knowledge, there have been no studies that evaluate the effect of environmental factors such as sunlight on the efficacy of the above-mentioned antibiotics. We conducted a simple disc-diffusion assay to study the efficacy of streptomycin and oxytetracycline after exposure to sunlight for 7- or 14-day periods using Escherichia coli and Bacillus subtilis as the representative strains of Gram-negative and Gram-positive organisms, respectively. Freshly prepared discs and discs stored in the dark for 7 or 14 days served as our controls. We show that the antibiotic potential of oxytetracycline exposed to sunlight dramatically decreases over the course of 14 days against both E. coli and B. subtilis. However, the effectiveness of streptomycin was only moderately impacted by sunlight. It is important to note that antibiotics that last longer in the environment may play a deleterious role in the rise and spread of antibiotic-resistant bacteria. Further studies are needed to substantively analyze the safety and efficacy of antibiotics used for broader environmental applications.

Characterization of chitin deacetylase genes in the Diaphorina citri genome

Chitin deacetylases (CDAs) are one of the least understood components of insect chitin metabolism. The partial deacetylation of chitin polymers appears to be important for the proper formation of higher order chitin structures, such as long fibers and bundles, that contribute to the integrity of the insect exoskeleton and other structures. Some CDAs may also play a role in bacterial defense. Here we report the characterization of four CDA genes from the Asian citrus psyllid, Diaphorina citri, laying the groundwork for future study of these genes. D. citri is the vector for Candidatus Liberibacter asiaticus (CLas), which is responsible for the global outbreak of Huanglongbing (citrus greening) disease. The manual annotation was done as part of a collaborative community annotation project (https://citrusgreening.org/annotation/index).