Management of Huanglongbing of Citrus: Lessons from São Paulo and Florida.

São Paulo, Brazil, and Florida, USA, were the two major orange production areas in the world until Huanglongbing (HLB) was discovered in São Paulo in 2004 and Florida in 2005. In the absence of resistant citrus varieties, HLB is the most destructive citrus disease known because of the lack of effective tools to reduce spread of the vector, Diaphorina citri (Asian citrus psyllid), and transmission of the associated pathogen, Candidatus Liberibacter asiaticus. In both countries, a three-pronged management approach was recommended and begun: planting only disease-free nursery trees, effective psyllid control, and removal of all symptomatic trees. In Brazil, these management procedures were continued and improved and resulted in relatively little overall loss of production. In contrast, in Florida the citrus industry has been devastated with annual production reduced by approximately 80%. This review compares and contrasts various cultural and pest management strategies that have been used to reduce infection by the pathogen and increase tolerance of HLB in the main orange-growing regions in the world.

Gene silencing of cathepsins B and L using CTV-based, plant-mediated RNAi interferes with ovarial development in Asian citrus psyllid (ACP), Diaphorina citri.

Diaphorina citri Kuwayama (Hemiptera: Liviidae) is a vector of the bacteria Candidatus Liberibacter americanus (CLam) and Candidatus Liberibacter asiaticus (CLas), which are phloem-restricted and associated with the most important and destructive worldwide citrus disease, Huanglongbing (HLB). Currently, no cure for HLB has been described. Therefore, measures have focused on reducing D. citri populations. In these insects, cathepsin B (DCcathB) and L (DCcathL) enzymes play an important role in digestion, and are involved in embryogenesis, immune defense, and ecdysis. In this study, we used a CTV-based vector to deliver dsRNA (CTV-dsRNA) into Citrus macrophylla plants targeting DCcathB and DCcathL genes in D. citri that fed on the phloem of these CTV-RNAi infected plants. Subsequently, we evaluated expression of DCcathB and DCcathL genes as well as the Vitellogenin (Vg) gene by RT-qPCR in D. citri fed on CTV-dsRNA occurring in plant phloem. It was found that a defective phenotype in D. citri females as a result of knockdown of DCcathB and DCcathL genes mediated by CTV dsRNA. These results showed that Psyllids fed on plants treated with the CTV-dsRNA exhibited downregulation of the Vg gene, one of the most important genes associated with embryogenic and female development, which was associated with dsRNA-mediated silencing of the two cathepsin genes. Based on our findings, a CTV-based strategy for delivering RNAi via plants that targets DCcathB and DCcathL genes may represent a suitable avenue for development of dsRNA-based tools to manage D. citri that limits the spread of HLB.

Crowdsourced Identification of Potential Target Genes for CTV Induced Gene Silencing for Controlling the Citrus Greening Vector Diaphorina citri.

Citrus Greening or Huanglongbing (HLB) is a disease of citrus, causing high reduction in citrus production and is transmitted by the Asian citrus psyllid Diaphorina citri Kuwayama vectoring a phloem-limited bacterium Candidatus Liberibacter sp. We report research results using crowdsourcing challenge strategy identifying potential gene targets in D. citri to control the insect using RNA interference (RNAi). From 63 submitted sequences, 43 were selected and tested by feeding them to D. citri using artificial diet assays. After feeding on artificial diet, the three most effective dsRNAs causing 30% mortality above control silenced genes expressing iron-sulfur cluster subunit of the mitochondrial electron transport chain complex (Rieske), heme iron-binding terminal oxidase enzyme (Cytochrome P450) and tetrahydrobiopterin (BH4) pathway enzyme (Pterin 4α-Carbinolamine Dehydratase). These sequences were cloned into a citrus phloem-limited virus (Citrus tristeza virus, CTV T36) expressing dsRNA against these target genes in citrus. The use of a viral mediated "para-transgenic" citrus plant system caused higher mortality to adult D. citri than what was observed using artificial diet, reaching 100% when detached citrus leaves with the engineered CTV expressing dsRNA were fed to adult D. citri. Using this approach, a virus-induced gene silencing (VIGS) can be used to test future transgenic cultivars before genetically engineering citrus. RNA Seq analysis after feeding D. citri CTV-RIE on infected leaves identified transcriptionally modified genes located upstream and downstream of the targeted RIE gene. These genes were annotated showing that many are associated with the primary function of the Rieske gene that was targeted by VIGS.

Citrus tristeza virus P33 Protein is Required for Efficient Transmission by the Aphid Aphis (Toxoptera) citricidus (Kirkaldy).

Plant viruses are threatening many valuable crops, and Citrus tristeza virus (CTV) is considered one of the most economically important plant viruses. CTV has destroyed millions of citrus trees in many regions of the world. Consequently, understanding of the transmission mechanism of CTV by its main vector, the brown citrus aphid, Aphis (Toxoptera) citricidus (Kirkaldy), may lead to better control strategies for CTV. The objective of this study was to understand the CTV-vector relationship by exploring the influence of viral genetic diversity on virus transmission. We built several infectious clones with different 5'-proximal ends from different CTV strains and assessed their transmission by the brown citrus aphid. Replacement of the 5'- end of the T36 isolate with that of the T30 strain (poorly transmitted) did not increase the transmission rate of T36, whereas replacement with that of the T68-1 isolate (highly transmitted) increased the transmission rate of T36 from 1.5 to 23%. Finally, substitution of p33 gene of the T36 strain with that of T68 increased the transmission rate from 1.5% to 17.8%. Although the underlying mechanisms that regulate the CTV transmission process by aphids have been explored in many ways, the roles of specific viral proteins are still not explicit. Our findings will improve our understanding of the transmission mechanisms of CTV by its aphid vector and may lead to the development of control strategies that interfere with its transmission by vector.

Development of infectious cDNA clones of Grapevine leafroll-associated virus 3 and analyses of the 5' non-translated region for replication and virion formation.

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3'-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5' non-translated region (5' NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5' NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5'-terminus were indispensable for replication, compared to downstream variable portion of the 5' NTR. Some of the functional mutations in the 5' NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.

Acquisition, Replication and Inoculation of Candidatus Liberibacter asiaticus following Various Acquisition Periods on Huanglongbing-Infected Citrus by Nymphs and Adults of the Asian Citrus Psyllid.

The Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae), is the primary vector of Candidatus Liberibacter asiaticus (Las) implicated as causative agent of citrus huanglongbing (citrus greening), currently the most serious citrus disease worldwide. Las is transmitted by D. citri in a persistent-circulative manner, but the question of replication of this bacterium in its psyllid vector has not been resolved. Thus, we studied the effects of the acquisition access period (AAP) by nymphs and adults of D. citri on Las acquisition, multiplication and inoculation/transmission. D. citri nymphs or adults (previously non-exposed to Las) were caged on Las-infected citrus plants for an AAP of 1, 7 or 14 days. These 'Las-exposed' psyllids were then transferred weekly to healthy citrus or orange jasmine plants, and sampled via quantitative polymerase chain reaction (qPCR) analysis 1-42 days post-first access to diseased plants (padp); all tested nymphs became adults 7-14 days padp. Our results indicate that following 1 or 7 day AAP as nymphs 49-59% of Las-exposed psyllids became Las-infected (qPCR-positive), whereas only 8-29% of the psyllids were infected following 1-14 day AAP as adults. Q-PCR analysis also indicated that Las titer in the Las-exposed psyllids (relative to that of the psyllid S20 ribosomal protein gene) was: 1) significantly higher, and increasing at a faster rate, following Las acquisition as nymphs compared to that following Las acquisition as adults; 2) higher as post-acquisition time of psyllids on healthy plants increased reaching a peak at 14-28 days padp for nymphs and 21-35 days padp for adults, with Las titer decreasing or fluctuating after that; 3) higher with longer AAP on infected plants, especially with acquisition as adults. Our results strongly suggest that Las multiplies in both nymphs and adults of D. citri but attains much higher levels in a shorter period of time post-acquisition when acquired by nymphs than when acquired by adults, and that adults may require longer access to infected plants compared to nymphs for Las to reach higher levels in the vector. However, under the conditions of our experiments, only D. citri that had access to infected plants as nymphs were able to inoculate Las into healthy citrus seedlings or excised leaves. The higher probability of Las inoculation into citrus by psyllids when they have acquired this bacterium from infected plants during the nymphal rather than the adult stage, as reported by us and others, has significant implications in the epidemiology and control of this economically important citrus disease.

Asymptomatic spread of huanglongbing and implications for disease control.

Huanglongbing (HLB) is a bacterial infection of citrus trees transmitted by the Asian citrus psyllid Diaphorina citri. Mitigation of HLB has focused on spraying of insecticides to reduce the psyllid population and removal of trees when they first show symptoms of the disease. These interventions have been only marginally effective, because symptoms of HLB do not appear on leaves for months to years after initial infection. Limited knowledge about disease spread during the asymptomatic phase is exemplified by the heretofore unknown length of time from initial infection of newly developing cluster of young leaves, called flush, by adult psyllids until the flush become infectious. We present experimental evidence showing that young flush become infectious within 15 d after receiving an inoculum of Candidatus Liberibacter asiaticus (bacteria). Using this critical fact, we specify a microsimulation model of asymptomatic disease spread and intensity in a grove of citrus trees. We apply a range of psyllid introduction scenarios to show that entire groves can become infected with up to 12,000 psyllids per tree in less than 1 y, before most of the trees show any symptoms. We also show that intervention strategies that reduce the psyllid population by 75% during the flushing periods can delay infection of a full grove, and thereby reduce the amount of insecticide used throughout a year. This result implies that psyllid surveillance and control, using a variety of recently available technologies, should be used from the initial detection of invasion and throughout the asymptomatic period.

Citrus tristeza virus: making an ally from an enemy.

Virus diseases of perennial trees and vines have characteristics not amenable to study using small model annual plants. Unique disease symptoms such as graft incompatibilities and stem pitting cause considerable crop losses. Also, viruses in these long-living plants tend to accumulate complex populations of viruses and strains. Considerable progress has been made in understanding the biology and genetics of Citrus tristeza virus (CTV) and in developing it into a tool for crop protection and improvement. The diseases in tree and vine crops have commonalities for which CTV can be used to develop a baseline. The purpose of this review is to provide a necessary background of systems and reagents developed for CTV that can be used for continued progress in this area and to point out the value of the CTV-citrus system in answering important questions on plant-virus interactions and developing new methods for controlling plant diseases.

Citrus tristeza virus-based RNAi in citrus plants induces gene silencing in Diaphorina citri, a phloem-sap sucking insect vector of citrus greening disease (Huanglongbing).

A transient expression vector based on Citrus tristeza virus (CTV) is unusually stable. Because of its stability it is being considered for use in the field to control Huanglongbing (HLB), which is caused by Candidatus Liberibacter asiaticus (CLas) and vectored by Asian citrus psyllid, Diaphorina citri. In the absence of effective control strategies for CLas, emphasis has been on control of D. citri. Coincident cohabitation in phloem tissue by CLas, D. citri and CTV was exploited to develop a novel method to mitigate HLB through RNA interference (RNAi). Since CTV has three RNA silencing suppressors, it was not known if CTV-based vector could induce RNAi in citrus. Yet, expression of sequences targeting citrus phytoene desaturase gene by CTV-RNAi resulted in photo-bleaching phenotype. CTV-RNAi vector, engineered with truncated abnormal wing disc (Awd) gene of D. citri, induced altered Awd expression when silencing triggers ingested by feeding D. citri nymphs. Decreased Awd in nymphs resulted in malformed-wing phenotype in adults and increased adult mortality. This impaired ability of D. citri to fly would potentially limit the successful vectoring of CLas bacteria between citrus trees in the grove. CTV-RNAi vector would be relevant for fast-track screening of candidate sequences for RNAi-mediated pest control.

Exploring the limits of vector construction based on Citrus tristeza virus.

We examined the limits of manipulation of the Citrus tristeza virus (CTV) genome for expressing foreign genes in plants. We previously created a vector with a foreign gene cassette inserted between the major and minor coat protein genes, which is position 6 from the 3' terminus. Yet, this virus has 10 3'-genes with several other potential locations for expression of foreign genes. Since genes positioned closer to the 3' terminus tend to be expressed in greater amounts, there were opportunities for producing greater amounts of foreign protein. We found that the virus tolerated insertions of an extra gene in most positions within the 3' region of the genome with substantially increased levels of gene product produced throughout citrus trees. CTV was amazingly tolerant to manipulation resulting in a suite of stable transient expression vectors, each with advantages for specific uses and sizes of foreign genes in citrus trees.

A personal history of virus-based vector construction.

The ability to express foreign genes or to silence endogenous genes in plants has revolutionized both basic and applied plant biology. Virus-based expression systems, in which the foreign mRNA is greatly amplified by virus replication, can produce very high levels of proteins or peptides in leaves and other tissues. Vectors have been available for about 25 years. They are commonplace as laboratory tools, but their initial commercial expectations have not been met for numerous reasons. Yet, applications of viral vectors are still evolving. This chapter focuses on our laboratory's involvement in developing virus-based vectors in plants. We created the first 'add-a-gene' vectors that were capable of replication and movement throughout plants. These vectors were based on tobacco mosaic virus. Through the evolution of several prototypes, stable vectors were developed that produced relatively large amounts of product in plants. Recently, we created similar vectors for citrus trees based on citrus tristeza virus. Even though the citrus vectors were created as laboratory tools for improving the crop, circumstances have changed the applications to protection and therapy of trees in the field.

Virus-based transient expression vectors for woody crops: a new frontier for vector design and use.

Virus-based expression vectors are commonplace tools for the production of proteins or the induction of RNA silencing in herbaceous plants. This review considers a completely different set of uses for viral vectors in perennial fruit and nut crops, which can be productive for periods of up to 100 years. Viral vectors could be used in the field to modify existing plants. Furthermore, with continually emerging pathogens and pests, viral vectors could express genes to protect the plants or even to treat plants after they become infected. As technologies develop during the life span of these crops, viral vectors can be used for adding new genes as an alternative to pushing up the crop and replanting with transgenic plants. Another value of virus-based vectors is that they add nothing permanently to the environment. This requires that effective and stable viral vectors be developed for specific crops from endemic viruses. Studies using viruses from perennial hosts suggest that these objectives could be accomplished.

Enhancement or attenuation of disease by deletion of genes from Citrus tristeza virus.

Stem pitting is a common virus-induced disease of perennial woody plants induced by a range of different viruses. The phenotype results from sporadic areas of the stem in which normal xylem and phloem development is prevented during growth of stems. These alterations interfere with carbohydrate transport, resulting in reduced plant growth and yield. Citrus tristeza virus (CTV), a phloem-limited closterovirus, induces economically important stem-pitting diseases of citrus. CTV has three nonconserved genes (p33, p18, and p13) that are not related to genes of other viruses and that are not required for systemic infection of some species of citrus, which allowed us to examine the effect of deletions of these genes on symptom phenotypes. In the most susceptible experimental host, Citrus macrophylla, the full-length virus causes only very mild stem-pitting symptoms. Surprisingly, we found that certain deletion combinations (p33 and p18 and/or p13) induced greatly increased stem-pitting symptoms, while other combinations (p13 or p13 plus p18) resulted in reduced stem pitting. These results suggest that the stem-pitting phenotype, which is one of more economically important disease phenotypes, can result not from a specific sequence or protein but from a balance between the expression of different viral genes. Unexpectedly, using green fluorescent protein-tagged full-length virus and deletion mutants (CTV9Δp33 and CTV9Δp33Δp18Δp13), the virus was found at pitted areas in abnormal locations outside the normal ring of phloem. Thus, increased stem pitting was associated not only with a prevention of xylem production but also with a proliferation of cells that supported viral replication, suggesting that at random areas of stems the virus can elicit changes in cellular differentiation and development.

A plant virus evolved by acquiring multiple nonconserved genes to extend its host range.

Viruses have evolved as combinations of genes whose products interact with cellular components to produce progeny virus throughout the plants. Some viral genes, particularly those that are involved in replication and assembly, tend to be relatively conserved, whereas other genes that have evolved for interactions with the specific host for movement and to counter host-defense systems tend to be less conserved. Closteroviridae encode 1-5 nonconserved ORFs. Citrus tristeza virus (CTV), a Closterovirus, possesses nonconserved p33, p18, and p13 genes that are expendable for systemic infection of the two laboratory hosts, Citrus macrophylla and Mexican lime. In this study, we show that the extended host range of CTV requires these nonconserved genes. The p33 gene was required to systemically infect sour orange and lemon trees, whereas either the p33 or the p18 gene was sufficient for systemic infection of grapefruit trees and the p33 or the p13 gene was sufficient for systemic infection of calamondin plants. Thus, these three genes are required for systemic infection of the full host range of CTV, but different genes were specific for different hosts. Remarkably, either of two genes was sufficient for infection of some citrus hybrids. These findings suggest that CTV acquired multiple nonconserved genes (p33, p18, and p13) and, as a result, gained the ability to interact with multiple hosts, thus extending its host range during the course of evolution. These results greatly extend the complexity of known virus-plant interactions.

Agroinoculation of Citrus tristeza virus causes systemic infection and symptoms in the presumed nonhost Nicotiana benthamiana.

Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.

3'-coterminal subgenomic RNAs and putative cis-acting elements of Grapevine leafroll-associated virus 3 reveals 'unique' features of gene expression strategy in the genus Ampelovirus.

BACKGROUND: The family Closteroviridae comprises genera with monopartite genomes, Closterovirus and Ampelovirus, and with bipartite and tripartite genomes, Crinivirus. By contrast to closteroviruses in the genera Closterovirus and Crinivirus, much less is known about the molecular biology of viruses in the genus Ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums. RESULTS: The gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution. CONCLUSIONS: The results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.

Heterologous minor coat proteins of Citrus tristeza virus strains affect encapsidation, but the coexpression of HSP70h and p61 restores encapsidation to wild-type levels.

The long flexuous bipolar virions of Citrus tristeza virus (CTV), a Closterovirus, are encapsidated with two capsid proteins at opposite ends: the minor coat protein (CPm) encapsidates the 5' 630 nts of the genomic RNA and the major coat protein encapsidates the remainder of the genome. In this study, we found encapsidation of CTV CPm in the absence of other assembly-related proteins is highly specific in contrast to most plant viruses that allow virion assembly by a range of heterologous coat proteins. Heterologous CPms with 95-96% amino acid identity from related strains in CTV-CPm, a replicon with CPm as the only assembly-related ORF, either failed to initiate encapsidation or reduced encapsidation substantially. Substitution of subsets of amino acids revealed that the amino acids that differ between positions 121 and 180 of the VT strain, and 61 and 120 of the T3 strain were involved in specific encapsidation. We further mapped the specific encapsidation to a single amino acid: mutation of methionine(165) to threonine (VT type) or serine(105) to proline (T3 type) in CTV-CPm failed to form nucleocapsids. However, the heterologous CPm in combination with both HSP70h and p61 proteins, but not HSP70h or p61 alone, encapsidated at wild-type levels, suggesting that specific encapsidation by CPm was mitigated by the combination of HSP70h and p61. Thus, in addition to the previously described functions of HSP70h and p61 of greatly enhanced virion formation and restriction of CPm encapsidation to the 5' 630 nts of the genomic RNA, these proteins facilitate encapsidation by heterologous CPms.

The pathogenicity determinant of Citrus tristeza virus causing the seedling yellows syndrome maps at the 3'-terminal region of the viral genome.

Citrus tristeza virus (CTV) (genus Closterovirus, family Closteroviridae) causes some of the more important viral diseases of citrus worldwide. The ability to map disease-inducing determinants of CTV is needed to develop better diagnostic and disease control procedures. A distinctive phenotype of some isolates of CTV is the ability to induce seedling yellows (SY) in sour orange, lemon and grapefruit seedlings. In Florida, the decline isolate of CTV, T36, induces SY, whereas a widely distributed mild isolate, T30, does not. To delimit the viral sequences associated with the SY syndrome, we created a number of T36/T30 hybrids by substituting T30 sequences into different regions of the 3' half of the genome of an infectious cDNA of T36. Eleven T36/T30 hybrids replicated in Nicotiana benthamiana protoplasts. Five of these hybrids formed viable virions that were mechanically transmitted to Citrus macrophylla, a permissive host for CTV. All induced systemic infections, similar to that of the parental T36 clone. Tissues from these C. macrophylla source plants were then used to graft inoculate sour orange and grapefruit seedlings. Inoculation with three of the T30/T36 hybrid constructs induced SY symptoms identical to those of T36; however, two hybrids with T30 substitutions in the p23-3' nontranslated region (NTR) (nucleotides 18 394-19 296) failed to induce SY. Sour orange seedlings infected with a recombinant non-SY p23-3' NTR hybrid also remained symptomless when challenged with the parental virus (T36), demonstrating the potential feasibility of using engineered constructs of CTV to mitigate disease.

Infection with strains of Citrus tristeza virus does not exclude superinfection by other strains of the virus.

Superinfection exclusion or homologous interference, a phenomenon in which a primary viral infection prevents a secondary infection with the same or closely related virus, has been observed commonly for viruses in various systems, including viruses of bacteria, plants, and animals. With plant viruses, homologous interference initially was used as a test of virus relatedness to define whether two virus isolates were "strains" of the same virus or represented different viruses, and subsequently purposeful infection with a mild isolate was implemented as a protective measure against isolates of the virus causing severe disease. In this study we examined superinfection exclusion of Citrus tristeza virus (CTV), a positive-sense RNA closterovirus. Thirteen naturally occurring isolates of CTV representing five different virus strains and a set of isolates originated from virus constructs engineered based on an infectious cDNA clone of T36 isolate of CTV, including hybrids containing sequences from different isolates, were examined for their ability to prevent superinfection by another isolate of the virus. We show that superinfection exclusion occurred only between isolates of the same strain and not between isolates of different strains. When isolates of the same strain were used for sequential plant inoculation, the primary infection provided complete exclusion of the challenge isolate, whereas isolates from heterologous strains appeared to have no effect on replication, movement or systemic infection by the challenge virus. Surprisingly, substitution of extended cognate sequences from isolates of the T68 or T30 strains into T36 did not confer the ability of resulting hybrid viruses to exclude superinfection by those donor strains. Overall, these results do not appear to be explained by mechanisms proposed previously for other viruses. Moreover, these observations bring an understanding of some previously unexplained fundamental features of CTV biology and, most importantly, build a foundation for the strategy of selecting mild isolates that would efficiently exclude severe virus isolates as a practical means to control CTV diseases.