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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.

Three citrus hybrids, containing 50% to 75% sweet orange (Citrus sinensis) genome in their pedigrees and similar to sweet orange in fruit size, color, and taste, were tested for their potential to be classified as new “sweet orange” cultivars. ‘Hamlin’, ‘Midsweet’, and three other early to midseason sweet oranges, along with ‘Dancy’ tangerine (Citrus reticulata), a typical mandarin, were used for comparison. Fruit were picked on 23 Jan. 2014, 30 Dec. 2014, and 27 Jan. 2015. A total of 114 volatiles were detected and separated into seven groups by detection frequency: three groups with 43 volatile components did not show differences and thus contributed little information for classification of sweet orange vs. mandarin, and the remaining four groups with 71 volatiles contributed to distinctions between orange and mandarin. Among the hybrids, the pattern of volatile detection frequency for hybrid FF-1-74-52 was virtually identical to sweet orange, and cluster analysis agreed with the classification. The number of average peaks were 55 to 62 in sweet oranges, 67 in FF-1-74-52, and 17 to 37 in tangerine and other hybrids. Quantity analysis of individual volatiles and chemical classes indicated that FF-1-74-52 and sweet oranges were rich in total volatile abundance, and almost all chemical classes including mono and sesquiterpenes, aldehydes, alcohols, ketones, and esters. This was especially true for ethyl butanoate, which contributes a fruity top note, and valencene and all sesquiterpene hydrocarbons, which only contribute to citrus flavor indirectly through their contribution to headspace partitioning. Two other hybrids, FF-1-75-55 and FF-1-76-51, each had some similarity to sweet oranges in several chemicals and classes, but not in the overall volatile profile. All three sweet orange–like hybrids met the standards for mandarins and oranges in soluble solids content, titratable acidity (TA), and the ratio. The above volatile and nonvolatile flavor chemical profile comparisons strongly support a proposal to classify FF-1-74-52 as a “sweet orange” commercially, and all three hybrids were previously shown to be more similar to sweet orange in their volatile profile than is ‘Ambersweet’. ‘Ambersweet’ was a hybrid that was legally classified as a “sweet orange” in 1995 based on its volatile profile.

Huanglongbing {HLB [associated with Candidatus Liberibacter sp. (CLas)]} and asiatic citrus canker {ACC [causal organism Xanthomonas citri ssp. citri (XCC)]} are bacterial diseases that seriously threaten sustainability of the Florida citrus (Citrus sp.) industry. Sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) are highly susceptible to ACC and improvement through conventional breeding is a long-term process, making transgenic solutions attractive. No strong HLB resistance has been identified within cultivated citrus scion types: creation of transgenic citrus that would permit economic citrus production where HLB is endemic is a high priority. Little is known about the HLB pathosystem and thus broad-spectrum antimicrobial peptides (AMPs) have been the focus for current work, and identification of safe and effective transgenes is essential to our efforts. In vitro assessment of minimum inhibitory concentration (MIC) for 44 AMPs was conducted using Sinorhizobium meliloti and Agrobacterium tumefaciens as surrogates for the unculturable CLas because they are closely related alpha proteobacteria (class Alphaproteobacteria). XCC is also a gram-negative bacterium and was included in these analyses in anticipation that HLB and ACC resistance can be achieved with the same AMP transgene if expressed using non-tissue-specific promoters. Twenty AMPs from diverse sources were initially tested. AMPs with the lowest MICs included tachyplesin I from horseshoe crab (Tachypleus tridentatus), SMAP-29 from sheep (Ovis aries), D4E1 and D2A21 (which are synthetic AMPs derived through evaluation of critical amino acid residues in AMPs, overall peptide structure, and AMP effectiveness), the human (Homo sapiens) LL-37, and the honeybee (Apis mellifera) venom AMP melittin. These AMPs inhibited growth of all three test bacterial species at 1 μM or less. An additional 20 synthetic AMPs were designed based on structures of the most effective AMPs and seven of these showed effectiveness at 1 μM or less across all three test bacteria. Most AMPs were comparable in effectiveness across the three bacterial species, but some species × AMP interactions were observed. Hemolytic activity was assessed by exposure of porcine erythrocytes (from Sus scrofa) to the AMPs. Hemolysis from most AMPs was not significantly different from water, whereas melittin was highly hemolytic.