Antibacterial FANA oligonucleotides as a novel approach for managing the Huanglongbing pathosystem.

Candidatus Liberibacter asiaticus (CLas), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri, is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2'-deoxy-2'-fluoro-D-arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a CLas-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a CLas essential gene. CLas relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a CLas-specific FANA ASO into infected Citrus trees significantly reduced CLas titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Peptide conjugated morpholinos for management of the huanglongbing pathosystem.

BACKGROUND: 'Candidatus Liberibacter asiaticus' (CLas) is the causal agent of the devastating citrus disease Huanglongbing (HLB) and is transmitted by the insect vector Diaphorina citri (Hemiptera: Liviidae). A potential approach for treating CLas infection is the use of synthetic nucleic acid-like oligomers to silence bacterial gene expression. Peptide conjugated morpholinos (PPMOs) targeting essential genes in CLas and the psyllid vector's endosymbiotic bacteria, Wolbachia (-Diaphorina, wDi), were evaluated using in vitro and in vivo assays. RESULTS: Expression of the wDi gyrA gene was significantly reduced following incubation of wDi cells with PPMOs. In addition, the viability of isolated wDi cells was greatly reduced when treated with PPMOs as compared to untreated cells. Feeding D. citri adults with a complementary PPMO (CLgyrA-14) showed significantly reduced (70% lower) expression of the CLas gyrA gene. CLas relative density was significantly lower in the psyllids fed with CLgyrA-14, when compared to untreated insects. Psyllids that were treated with CLgyrA-14 were less successful in transmitting the pathogen into uninfected plants, compared to untreated insects. CONCLUSION: The expression of essential genes in the D. citri symbiont, wDi and the HLB pathogen were suppressed in response to PPMO treatments. This study demonstrates the potential of PPMOs as a novel strategy for management of bacterial pathogens of fruit trees, such as HLB. © 2020 Society of Chemical Industry.

GUT GENES ASSOCIATED WITH THE PERITROPHIC MATRIX IN Reticulitermes flavipes (Blattodea: Rhinotermitidae): IDENTIFICATION AND CHARACTERIZATION.

The peritrophic matrix (PM) is an acellular structure that lines the gut of most insects. It is an attractive target for pest management strategies because of its close involvement in digestive processes and role as a barrier against pathogens and toxins. The purpose of this study was to identify and characterize the genes that translate for principal components of the Reticulitermes flavipes PM. Genes encoding a gut chitin synthase (CHS), two proteins with peritrophin-A domains, and a chitin deacetylase were identified from an R. flavipes symbiont-free gut cDNA library, a pyrosequencing study of termite lignocellulose digestion, and a metatranscriptomic analysis of R. flavipes fed on agricultural biomass. Quantitative expression analysis of the identified genes, in the termite digestive tract, revealed that the transcripts coding for a CHS (RfCHSB) and the proteins with peritrophin-A domains (RfPPAD1 and RfPPAD2) were predominantly expressed in the midgut, suggesting an association with the PM. The peritrophin identity of the RfPPAD2 gene was confirmed by immunodetection of its translated peptide in the midgut and PM. The discovery and characterization of PM components of R. flavipes provides a basis for further investigation of the viability of this structure as a target for candidate termiticides.

Antifeedant effect of commercial chemicals and plant extracts against Schistocerca americana (Orthoptera: Acrididae) and Diaprepes abbreviatus (Coleoptera: Curculionidae).

BACKGROUND: The deterrent effect of seven commercial compounds against Schistocerca americana (Drury) nymphs and Diaprepes abbreviatus L. adults, two important citrus pests, was investigated. Extracts of three Florida local plants were also tested as deterrents. Antifeedant activity was assayed using a leaf-disk bioassay in choice and no-choice tests. The residual activity of the commercial compounds that significantly deterred the insects was assayed by applying them to foliage of citrus plants exposed to three time intervals of sunlight. RESULTS: Sabadilla, azadirachtin and ryanodine effectively deterred S. americana, whereas rotenone, sabadilla and ryanodine reduced the feeding activity of D. abbreviatus. Methanol and methylene chloride extracts of Ceratiola ericoides Michaux deterred D. abbreviatus, but only methylene chloride extract dissuaded S. americana. Methanol extract of Ardisia crenata Sims functioned as a feeding deterrent against both S. americana and D. abbreviatus, but only the latter species was deterred by methylene chloride extract of A. crenata. Extracts of Illicium parviflorum Michaux ex Ventetat only dissuaded the insects in choice bioassays. Rapid loss of effectiveness was observed under field conditions. Sabadilla was the only compound that maintained its antifeedant properties in the field, but only against S. americana. CONCLUSION: Based on their deterrency, some of the commercial products and plant extracts have potential for use as substitute crop protectants against the pest species tested.