Journal of Citrus Pathology

Currently, oxytetracycline and streptomycin are being applied to citrus groves in Florida for the control of citrus greening disease caused by the unculturable bacterium Candidatus Liberibacter asiaticus. Here, the closest cultured relative, L. crescens, was used to estimate the frequency of spontaneous antimicrobial resistance of Liberibacter spp. Results yielded thirteen streptomycin and zero oxytetracycline mutants after exposing 13 billion cells to the antimicrobials. These low rates, alongside the restrictive habitats of the vector and pathogen, suggest resistance may develop very slowly, if at all. Thus, the treatments will likely remain useful long enough before pathogen-resistant or -tolerant genotypes are deployed.

Citrus tristeza virus (CTV) causes several disease syndromes in different citrus hosts; namely quick decline, seedling yellows and stem pitting. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The formation of stem pits has previously been linked to the ability of the virus to colonize xylem tissue outside of its typical phloem limitation, thereby disrupting normal xylem development. The nature of this compromised tissue has not been fully elucidated. In this study, stem pits were characterized at the molecular anatomical level using a combination of techniques to better understand the characteristics of the xylem and phloem tissues impacted by severe pitting. Biological staining was used to visualize CTV-induced stem pitting and was complemented with a novel technology that has not previously been used to study CTV-induced stem pitting, namely serial block-face scanning electron microscopy (SBF-SEM). This proof-of-concept study yielded new insights into the morphology of stem pitting-affected tissue. The utility of SBF-SEM for stem pitting characterization was also demonstrated and an optimized protocol for its application on hard, woody material is presented.

Two proteins of the citrus tatter leaf virus (CTLV), a strain of the apple stem grooving virus (ASGV), capable of inducing citrus bud union disorders on commercially important trifoliate and citrange rootstocks, were identified as viral suppressors of RNA silencing (VSR). Both the coat protein (CP) and the movement protein (MP) suppressed RNA silencing in GFP-transgenic Nicotiana benthamiana 16c plants in agrobacterium-mediated co-infiltration assays; the MP acted as a local VSR, while the CP acted as a systemic VSR. When the potato virus X (PVX) infectious vector harbored either the CTLV CP or MP gene, viral infection and symptom development were promoted in N. benthamiana. Deletions of amino acids in the CP sequence or the MP sequence resulted in failure to promote PVX infections as well as suppression of silencing in agrobacterium-mediated co-infiltration assays. Mass spectrometry-based immunoprecipitation proteomics showed that neither the CTLV CP nor the MP interacts with cellular components directly involved in host antiviral RNA silencing pathways. RNA immunoprecipitation (RIP) and RNA-protein pull-down assays indicated that the CTLV MP interacts with double-stranded RNA (dsRNA) presumably through a protein complex or proteins containing RNA binding domains. It is possible that the MP prevents dsRNA cleavage through this mechanism, leading to suppression of host antiviral RNA silencing. These findings confirmed that CTLV uses VSRs as part of its overall strategy to overcome host antiviral defenses and are indicative of the ability of ASGV and CTLV to infect a wide range of hosts including different species of woody and herbaceous plants.

This brief report expands upon the original review article published in Journal of Citrus Pathology in 2023 on citrus “phantom” disorders of presumed virus and virus-like etiology and addresses five additional disorders: citrus seed-borne virus disorder in New Zealand, bergamot vein yellowing in Greece, bergamot gummosis in Italy, bud knot in Italy, and a disorder resembling citrus crinkly leaf in Cuba. Each disorder is characterized by distinct symptoms and transmission patterns yet remains unresolved in terms of causative agents or conditions. By providing comprehensive information on these phantom citrus disorders, this report aims to serve as an additional reference for the citrus research community, industry stakeholders, and regulatory offices.