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.

Candidate gene markers for Candidatus Liberibacter asiaticus for detecting citrus greening disease.

Citrus Huanglongbing (HLB) also known as citrus greening is one of the most devastating diseases of citrus worldwide. The disease is caused by Candidatus Liberibacter bacterium, vectored by the psyllid Diaphorina citri Kuwayama and Trioza erytreae Del Guercio. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection. The aim of this study was to develop effective gene-specific primer pairs for polymerase chain reaction based method for quick screening of HLB disease. Thirty-two different gene-specific primer pairs, across the Ca. Liberibacter asiaticus genome, were successfully developed. The possibility of these primer pairs for cross-genome amplification across 'Ca. Liberibacter africanus' and 'Ca. Liberibacter americanus' were tested. The applicability of these primer pairs for detection and differentiation of Ca Liberibacter spp. is discussed.

Development of rapid, sensitive and non-radioactive tissue-blot diagnostic method for the detection of citrus greening.

Citrus huanglongbing (HLB or citrus greening) is one of the most devastating diseases of citrus worldwide. The disease is caused by Gram-negative, phloem-limited α-proteobacterium, 'Candidatus Liberibacter asiaticus', vectored by the psyllid, Diaphorina citri Kuwayama. Citrus plants infected by the HLB bacterium may not show visible symptoms sometimes for years following infection and non-uniform distribution within the tree makes the detection of the pathogen very difficult. Efficient management of HLB disease requires rapid and sensitive detection early in the infection followed by eradication of the source of pathogen and the vector. The polymerase chain reaction (PCR) based method is most commonly employed for screening the infected/suspected HLB plants and psyllids. This is time consuming, cumbersome and not practical for screening large number of samples in the field. To overcome this, we developed a simple, sensitive, non-radioactive, tissue-blot diagnostic method for early detection and screening of HLB disease. Digoxigenin labeled molecular probes specific to 'Ca. L. asiaticus' nucleotide sequences have been developed and used for the detection of the pathogen of the HLB disease. The copy number of the target genes was also assessed using real-time PCR experiments and the optimized real-time PCR protocol allowed positive 'Ca. L. asiaticus' detection in citrus samples infected with 'Ca. L. asiaticus' bacterium.

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.

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.

Examination of the responses of different genotypes of citrus to huanglongbing (citrus greening) under different conditions.

ABSTRACT Citrus Huanglongbing (HLB) is one of the most devastating diseases of citrus worldwide. The causal agent of HLB in Florida is thought to be 'Candidatus Liberibacter asiaticus'. In this work, we examined the responses of 30 different genotypes of citrus to Florida isolates of 'Ca. L. asiaticus' under controlled conditions in the greenhouse or growth room. Although 'Ca. L. asiaticus' was able to multiply in all of the plants, a wide range of responses was observed among different hosts. Based on the symptoms developed and the ability of plants to continue growth, the different genotypes were grouped into four categories: sensitive, which exhibited severe chlorosis on leaves, greatly reduced growth, and eventual death; moderately tolerant, which exhibited some scattered distinct symptoms but little or no growth reduction and no plant death; tolerant, which exhibited very minimal symptoms; and genotypes, which exhibited variable reactions. Interestingly, although 'Ca. L. asiaticus' was unevenly distributed within each particular plant, comparison of titers of the bacterium in different citrus genotypes revealed that most accumulated similar levels of 'Ca. L. asiaticus', demonstrating that there is no strict correlation between bacterial titer and severity of disease. Incubation of infected plants in the growth room with continuous light greatly affected symptoms production by reducing the time before distinctive symptoms developed and significantly increasing severity of chlorosis of leaves of all citrus genotypes. These results provide additional evidence of the correlation between disruption of phloem translocation of carbohydrates during HLB infection and the appearance of chlorotic symptoms in leaves of infected trees. We also examined interaction between 'Ca. L. asiaticus' and Citrus tristeza virus, which usually occurs in trees that become infected with HLB, and found no synergistic effect of the two pathogens. We trust that observations reported here will provide reagents for further examination of the 'Ca. L. asiaticus'-citrus interaction to advance the understanding of how 'Ca. L. asiaticus' causes disease and to develop methods or trees to overcome the disease.

Molecular characterization of Citrus tatter leaf virus historically associated with Meyer lemon trees: complete genome sequence and development of biologically active in vitro transcripts.

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3' end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wild-type virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22 degrees C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5' positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction.

Three genes of Citrus tristeza virus are dispensable for infection and movement throughout some varieties of citrus trees.

Citrus tristeza virus (CTV), a member of the Closteroviridae, possesses a 19.3-kb positive-stranded RNA genome that is organized into twelve open reading frames (ORFs). The CTV genome contains two sets of conserved genes, which are characteristic of this virus group, the replication gene block (ORF 1a and 1b) and the quintuple gene block (p6, HSP70 h, p61, CPm, and CP). With the exception of the p6 gene, they are required for replication and virion assembly. CTV contains five additional genes, p33, p18, p13, p20 and p23, in the 3' half of the genome, some of which (p33, p18 and p13) are not conserved among other members of this virus group, and have been proposed to have evolved for specific interactions with the citrus host. In the present study, the requirements for systemic infection of citrus trees of p33, p6, p18, p13 and p20 were examined. Viral mutants with a deletion in the p6 or the p20 ORF failed to infect citrus plants systemically, suggesting their possible roles in virus translocation/systemic infection. However, we found that deletions within the p33, p18 or p13 ORF individually resulted in no significant loss of ability of the virus to infect, multiply, and spread throughout citrus trees. Furthermore, deletions in the p33, p18 and p13 genes in all possible combinations including deletions in all three genes allowed the virus to systemically invade citrus trees. Green fluorescent protein-tagged CTV variants with deletions in the p33 ORF or the p33, p18 and p13 ORFs demonstrated that the movement and distribution of these deletion mutants were similar to that of the wild-type virus.

Genetic Marker Analysis of a Global Collection of Isolates of Citrus tristeza virus: Characterization and Distribution of CTV Genotypes and Association with Symptoms.

ABSTRACT Genetic markers amplified from three noncontiguous regions by sequence specific primers designed from the partial or complete genome sequences of Citrus tristeza virus (CTV) isolates T3, T30, T36, and VT were used to assess genetic relatedness of 372 isolates in an international collection. Eighty-five isolates were judged similar to the T3 isolate, 81 to T30, 11 to T36, and 89 to VT. Fifty-one isolates were mixed infections by two or more identifiable viral genotypes, and 55 isolates could not be assigned unequivocally to a group defined by marker patterns. Maximum parsimony analysis of aligned marker sequences supported the grouping of isolates on the basis of marker patterns only. Specific disease symptoms induced in select citrus host plants were shared across molecular groups, although symptoms were least severe among isolates grouped by markers with the T30 isolate and were most severe among isolates grouped by markers with the T3 isolate. Isolates assigned the same genotype showed variable symptoms and symptom severity. A classification strategy for CTV isolates is proposed that combines genetic marker patterns and nucleotide sequence data.

Serological Differentiation of the Citrus Tristeza Virus Isolates Causing Stem Pitting in Sweet Orange.

Citrus tristeza virus (CTV) complex comprises a number of isolates or strains producing several economically important disease syndromes in commercial Citrus spp. The stem pitting syndrome is the most important, and causes substantial losses in many citrus-producing regions of the world. In an attempt to develop a serological tool to rapidly differentiate stem pitting isolates of CTV, we evaluated many combinations of trapping and detecting antibodies in an indirect double-antibody sandwich (I-DAS) enzyme-linked immunosorbent assay (ELISA). Two combinations of trapping and detecting antibodies were found suitable for differentiating stem pitting isolates in extracts of infected sweet orange plants. One used a polyclonal serum raised against bacterially expressed CTV coat protein (CP) for trapping and a conformational monoclonal antibody 3E10 for detection, and the other used two polyclonal antisera generated against bacterially expressed CTV CP. Seventy-six CTV isolates from 20 countries, including 35 that cause stem pitting in sweet orange plants, were analyzed in I-DAS-ELISA using different combinations of polyclonal and monoclonal antibodies for trapping and as intermediate detecting antibodies. The ELISA format developed produces a strong positive signal for CTV isolates that cause stem pitting in sweet orange plants and a negative ELISA signal for CTV isolates that do not cause stem pitting. When combined with data on a universal ELISA format, i.e., reacting with a broad range of CTV isolates, these selective ELISA formats allowed reliable serological differentiation of CTV isolates that caused stem pitting in infected sweet orange plants.