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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.

In the process of recording and studying citrus diseases in the 20th century, citrus pathologists reported several suspected graft-transmissible disorders that were thought to be of virus or virus-like origin. While later work clarified and characterized most of these disorders, others were left unaddressed beyond their initial reports, and their status has remained unresolved for decades. For this reason, and for lack of a better term, such disorders are considered “phantoms”. In this work, our group performed an extensive literature review and communicated with renowned citrus pathologists and members of the International Organization of Citrus Virologists from around the world. Here we summarize and present in an organized manner the most up to date information for multiple phantom disorders, including disorders that have been subsequently characterized as a result of research efforts of the past 20 years. This review article could act as a reference point for citrus pathologists, regulatory agencies, and industry to clear up any confusion regarding citrus phantom disorders.

Plant-infecting viruses utilize various strategies involving multiple viral and host factors to achieve successful systemic infections of their compatible hosts. Lettuce infectious yellows virus (LIYV), genus Crinivirus, family Closteroviridae, has long, filamentous flexuous virions and causes phloem-limited infections in its plant hosts. The LIYV-encoded P26 is a distinct non-virion protein that shows no similarities to proteins in current databases: it induces plasmalemma deposits over plasmadesmata (PD) pit fields and is speculated to have roles in LIYV virion transport within infected plants. In this study, P26 was demonstrated to be a PD-localized protein, and its biological significance was tested in planta by mutagenesis analysis. An LIYV P26 knockout mutant (P26X) showed viral RNA replication and virion formation in inoculated leaves of Nicotiana benthamiana plants, but failed to give systemic infection. Confirmation by using a modified green fluorescent protein (GFP)-tagged LIYV P26X showed GFP accumulation only in infiltrated leaf tissues, while wild-type LIYV GFP readily spread systemically in the phloem. Attempts to rescue P26X by complementation in trans were negative. However a translocated LIYV P26 gene in the LIYV genome rescued systemic infection, but P26 orthologs from other criniviruses did not. Mutagenesis in planta assays showed that deletions in P26, as well as 2 of 11 specific alanine-scanning mutants, abolished the ability to systemically infect N. benthamianaIMPORTANCE Plant viruses encode specific proteins that facilitate their ability to establish multicellular/systemic infections in their host plants. Relatively little is known of the transport mechanisms for plant viruses whose infections are phloem limited, including those of the family Closteroviridae. These viruses have complex, long filamentous virions that spread through the phloem. Lettuce infectious yellows virus (LIYV) encodes a non-virion protein, P26, which forms plasmalemma deposits over plasmodesmata pit fields, and LIYV virions are consistently found attached to those deposits. Here we demonstrate that P26 is a unique movement protein required for LIYV systemic infection in plants. LIYV P26 shows no sequence similarities to other proteins, but other criniviruses encode P26 orthologs. However, these failed to complement movement of LIYV P26 mutants.

Due to the error-prone nature of viral RNA-dependent RNA polymerases, the replication of RNA viruses results in a diversity of viral genomes harboring point mutations, deletions, insertions, and genome rearrangements. Citrus tristeza virus (CTV), a causal agent of diseases of economically important citrus species, shows intrinsic genetic stability. While the virus appears to have some mechanism that limits the accumulation of single-nucleotide variants, the production of defective viral genomes (DVGs) during virus infection has been reported for certain variants of CTV. The intra-host diversity generated during plant infection with variant T36 (CTV-T36) remains unclear. To address this, we analyzed the RNA species accumulated in the initially infected and systemic leaves of Nicotiana benthamiana plants inoculated with an infectious cDNA clone of CTV-T36, which warranted that infection was initiated by a known, well-defined sequence variant of the virus. CTV-T36 limited the accumulation of single-nucleotide mutants during infection. With that, four types of DVGs—deletions, insertions, and copy- and snap-backs—were found in all the samples, with deletions and insertions being the most common types. Hot-spots across the genome for DVG recombination and short direct sequence repeats suggest that sequence complementarity could mediate DVG formation. In conclusion, our study illustrates the formation of diverse DVGs during CTV-T36 infection. To the best of our knowledge, this is the first study that has analyzed the genetic variability and recombination of a well-defined sequence variant of CTV in an herbaceous host. [ABSTRACT FROM AUTHOR]

The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.

Polymerase chain reaction (PCR) with the pathogen-specific primers and electron microscopy are the two techniques of choice that have been used for detection and identification of Candidatus Liberibacter spp. in the Huanglongbing (HLB)-affected citrus. Due to the low population and uneven distribution of Liberibacter in the diseased citrus trees finding the bacteria with transmission electron microscopy has been a challenge. Work with samples from HLB-affected citrus during the past 5 years has resulted in certain guidelines for the selection of plant tissues that have led to success in visualization of the bacteria. With PCR-positive field citrus trees, the best source is phloem tissue from seed coats of developing seeds in young fruit supported by leaves with blotchy mottle symptoms. These half mature seeds have been shown to contain large numbers of intact bacteria. However, when working with young potted trees there are usually no fruit to sample. In these trees we had some success in finding bacteria in petioles or midveins of PCR-positive, young developing leaves (2/3 to fully expanded, but not hardened) sampled above leaves showing blotchy mottle symptoms. However, we have found larger numbers of bacteria in roots of the potted diseased trees. We have experienced the most success in sampling root tissue in areas where the phloem has just completed differentiation, in PCR-positive small pioneer and fibrous roots showing primary and secondary growth.

Citrus tristeza virus (CTV) isolates collected from citrus germplasm, dooryard and field trees in California from 1914 have been maintained in planta under quarantine in the Citrus Clonal Protection Program (CCPP), Riverside, California. This collection, therefore, represents populations of CTV isolates obtained over time and space in California. To determine CTV genetic diversity in this context, genotypes of CTV isolates from the CCPP collection were characterized using multiple molecular markers (MMM). Genotypes T30, VT, and T36 were found at high frequencies with T30 and T30+VT genotypes being the most abundant. The MMM analysis did not identify T3 and B165/T68 genotypes; however, biological and phylogenetic analysis suggested some relationships of CCPP CTV isolates with these two genotypes. Phylogenetic analysis of the CTV coat protein (CP) gene sequences classified the tested isolates into seven distinct clades. Five clades were in association with the standard CTV genotypes T30, T36, T3, VT, and B165/T68. The remaining two identified clades were not related to any standard CTV genotypes. Spatiotemporal analysis indicated a trend of reduced genotype and phylogenetic diversity as well as virulence from southern California (SC) at early (1907-1957) in comparison to that of central California (CC) isolates collected from later (1957-2009) time periods. CTV biological characterization also indicated a reduced number and less virulent stem pitting (SP) CTV isolates compared to seedling yellows isolates introduced to California. This data provides a historical insight of the introduction, movement, and genetic diversity of CTV in California and provides genetic and biological information useful for CTV quarantine, eradication, and disease management strategies such as CTV-SP cross protection.

Long noncoding RNAs (IncRNAs) are commonly defined as transcripts that lack protein-coding capacity and are longer than 200 nucleotides. Since the emergence of next-generation sequencing technologies in this century, thousands of IncRNAs have been identified from nearly all living organisms. Notably, various pathogens also express their own IncRNAs in host cells during infection. In plants, many IncRNAs exhibit dynamic expression patterns in response to environmental stimuli, including pathogen attacks. In contrast to well-established methods in identifying such IncRNAs, the current understanding of IncRNAs' functional mechanisms is in its infancy. Some IncRNAs serve as precursors for generating small RNAs or serve as target mimics to sequester functional small RNAs, which have been extensively reviewed in the literature. This review focuses on the emerging evidence supporting that certain IncRNAs function as negative or positive regulators of plant immunity. A common theme is that those regulations rely on specific interactions between IncRNAs and key regulatory proteins. Viroids as single-stranded circular noncoding RNAs provide a handle to investigate how RNA local motifs render interaction specificity between IncRNAs and regulatory proteins. [ABSTRACT FROM AUTHOR]

Summary: Stem pitting is a common virus‐induced disease phenotype that tremendously impacts growth of perennial woody plants. How stem pitting develops in the infected trees remains unclear.Here, we assessed the development of stem pits upon infection of citrus by Citrus tristeza virus (CTV), which has been regarded as 'phloem‐limited'. By taking advantage of a highly susceptible virus host – Citrus macrophylla – and a CTV isolate lacking a viral effector – the p33 protein, the development pattern of stem pitting was revealed via time‐course observations and histological analyses.The stem pits result from the virus‐colonized nonlignified 'gumming' malformations which are initiated by virus invasion into multiple spatially separated tissue layers – protophloem, metaphloem, and, unexpectedly, metaxylem. Notably, invasion of CTV into the unspecialized metaxylem cells interrupted the differentiation of the xylem tracheary elements. With the radial spread of CTV into the adjacent cells towards the stem periphery, the clusters of virus‐colonized immature metaxylem cells extended in size, merging, at a certain stage, with virus‐bearing cells in the protophloem and metaphloem layers.Collectively, our data provide a new insight into the process of the stem pitting development and the role of the xylem tissue in the virus pathogenicity. [ABSTRACT FROM AUTHOR]

Summary: To defend against pathogens, plants have developed a complex immune system, which recognizes the pathogen effectors and mounts defence responses. In this study, the p33 protein of Citrus tristeza virus (CTV), a viral membrane‐associated effector, was used as a molecular bait to explore virus interactions with host immunity. We discovered that Citrus macrophylla miraculin‐like protein 2 (CmMLP2), a member of the soybean Kunitz‐type trypsin inhibitor family, targets the viral p33 protein. The expression of CmMLP2 was up‐regulated by p33 in the citrus phloem‐associated cells. Knock‐down of the MLP2 expression in citrus plants resulted in a higher virus accumulation, while the overexpression of CmMLP2 reduced the infectivity of CTV in the plant hosts. Further investigation revealed that, on the one hand, binding of CmMLP2 interrupts the cellular distribution of p33 whose proper function is necessary for the effective virus movement throughout the host. On the other hand, the ability of CmMLP2 to reorganize the endomembrane system, amalgamating the endoplasmic reticulum and the Golgi apparatus, induces cellular stress and accumulation of the reactive oxygen species, which inhibits the replication of CTV. Altogether, our data suggest that CmMLP2 employs a two‐way strategy in defence against CTV infection. [ABSTRACT FROM AUTHOR]

Citrus tristeza virus (CTV) is a causal agent of diseases that have challenged the global citrus production for more than a century. The disease named ‘tristeza’, which means ‘sadness’ in Portuguese and Spanish, has spread by aphids and vegetative propagation, resulting in costly pandemics that reshaped the world citrus production by forcing the adaption of disease‐tolerant rootstocks. Furthermore, management of the second major CTV‐induced disease, stem pitting, became a unique example of the use of cross‐protection on a large nationwide scale, allowing many citrus growing regions to control the disease that could not be managed through horticultural practices. The information gathered in this review commemorates a hundred years of research on the virus and the respective diseases, which began with classical horticultural approaches and advanced toward the cutting‐edge molecular biology studies. In this regard, it is proper to mention that CTV research greatly benefited from close international collaboration between research institutions and scientists from the affected and nonaffected citrus areas. Moreover, despite the considerable initial losses caused by tristeza, the world's fresh fruit and juice concentrate citrus industries reemerged as highly productive following the research advancements, a situation that changed throughout Florida and Brazil in the past two decades or so with the outbreak of the devastating citrus greening (Huanglongbing) bacterial disease. This review encompasses past and recent advances in the CTV research positioning the citrus‐CTV pathosystem as a pivotal model system for investigating virus interactions with perennial woody hosts. The review will also serve as an updated version of the respective section on CTV in the Description of Plant Viruses that the Association of Applied Biologists manages. We dedicated this review to the 85th Birthday celebration of Prof. Moshe Bar‐Joseph, a world‐renowned plant pathologist whose half‐a‐century‐long career devoted to citrus diseases yielded many important, pioneering discoveries on CTV and other closteroviruses. Many of those are highlighted in this review. Prof. Bar‐Joseph retired from the Volcani Center, Agricultural Research Organization in Israel several years ago, yet he continues to be actively engaged in research and provides his expertise to citrus pathologists around the world in the present days. [ABSTRACT FROM AUTHOR]