Publications

3992

AbstractBACKGROUNDHuanglongbing (HLB) is a systemic disease of citrus caused by the bacterial pathogen Candidatus Liberibacter asiaticus (CLas) that limits citrus production worldwide. CLas is an obligate bacterial pathogen that multiplies in citrus trees and in the insect vector, the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. There is no cure for HLB currently and broad‐spectrum antibiotics represent one possible therapeutic against disease symptoms. Single‐stranded nucleic acid analogs, 2′‐deoxy‐2’‐Fluoro‐β‐D‐arabinonucleic antisense oligonucleotides (FANA ASOs), can modulate gene expression by enzymatic degradation or steric blocking of an RNA target. FANA ASOs recognize and bind to specific RNA forms, including mRNA, miRNA and long noncoding RNA, through complementary base‐pairing.RESULTSInjection of oxytetracycline (OTC) into mature citrus trees with HLB ameliorated symptoms of disease, increased fruit yield and quality of juice as compared with that produced by noninjected controls. Injection of trees with FANA ASOs reduced CLas abundance but did not improve fruit yield and quality above control levels at the injection dosage tested. Reduced pathogen relative abundance following OTC injection was coincident with lower CLas acquisition and inoculation by laboratory deployed and wild‐type D. citri collected from the field, respectively. However, injections of FANA ASOs did not consistently reduce CLas transmission as compared with noninjected controls.CONCLUSIONSTrunk injection of OTC may be useful in management of HLB by reducing CLas infection in trees and disrupting transmission. However, more research is needed to verify the potential usefulness of FANA ASOs. Optimizing FANA ASO dosage in trees and exploring the potential of multiplex FANA ASOs that simultaneously target multiple mRNAs could possibly enhance their efficacy against CLas. © 2024 Society of Chemical Industry.

The occurrence of ‘Candidatus Liberibacter’ spp. and ‘Ca. Phytoplasma’ spp. associated with blotchy mottle symptoms poses challenges to huanglongbing (HLB) diagnosis using molecular techniques. The ability to detect multiple targets simultaneously and specifically is a key aspect met by quantitative PCR (qPCR). A set of primers and hydrolysis probes useful in either single or multiplex reactions for the detection and quantification of HLB-associated bacteria were developed. Sequences from conserved genes of the ribosomal proteins for Liberibacter and phytoplasma circumvent the lack of specificity and cross-reactivity problems related to 16Sr DNA gene amplification, allowing precise and specific detection of HLB-associated bacteria in citrus and in the Liberibacter vector, Diaphorina citri. The triplex reaction exhibited high quality and precision as a robust tool for quantifying ‘Ca. L. asiaticus’ (CLas), ‘Ca. L. americanus’ (CLam), and 16Sr IX phytoplasma. Triplex qPCR showed consistent results and comparable sensitivity to the ribonuclease reductase test, although quantification cycle (Cq) values were higher when compared with 16SrDNA qPCR. Detection tests using field samples indicate that the qPCR triplex can identify HLB-associated bacteria in samples with varying levels of symptoms, ranging from typical to asymptomatic. Assessment of field samples from growers indicated more than 78.6% had Cq lower than 35.0, below the cutoff established for qPCR reactions used in this work. qPCR triplex is a safe, specific, and sufficiently sensitive technique for detecting CLas, CLam, and 16Sr IX phytoplasma simultaneously, in both citrus and D. citri samples. Its application is of importance in assisting growers in making decisions for HLB management.

AbstractBACKGROUNDThe brown marmorated stink bug Halyomorpha halys has become an invasive insect pest of many crops. A promising control strategy to manage the proliferation of H. halys is based on the suppression of its obligate and vertically transmitted uncultivated symbiotic bacterium Candidatus Pantoea carbekii through surface‐sterilization of H. halys eggs. Indeed, the application of antimicrobial formulations on the eggs of H. halys could cause mortality of endosymbiont and consequently of newly emerged nymphs.In this study, a microbial live/dead assay was applied directly on H. halys eggs to evaluate Ca. P. carbekii loss of viability after treatments with seven commercial formulations including fungicides (copper hydroxide, sulphur, sweet orange essential oil) and plant biostimulants (flavonoids and chestnut tannin extract) compared with two disinfectants for civil and industrial use (sodium hypochlorite/hydrated sodium/tetraborate decahydrate and peracetic acid/hydrogen peroxide). Impact of mode of application was also evaluated, as surface treatment of egg masses was performed through spraying and dipping in laboratory conditions. Antimicrobial activity data were finally complemented with observations of egg hatching and vitality of the nymphs.RESULTSThe optimization of live/dead staining is useful for evaluating Ca. P. carbekii mortality directly on eggs, providing a rapid and reliable culture‐independent approach. Sodium hypochlorite, copper, sulphur, tannins and sweet orange essential oil showed an antimicrobial effect against Ca. P. carbekii and a H. halys egg hatching reduction and nymph's vitality.CONCLUSIONSThe antimicrobial and insecticidal effects of these commercial products should be further studied to assess their in‐field efficiency as well as the impact of these substances on non‐target organisms. The approach followed in this study could be considered a robust pipeline of analyses to evaluate the effectiveness of antimicrobial eco‐friendly compounds in symbiotic control of H. halys. © 2024 Society of Chemical Industry.