Mark E. Hilf

Sequences of Citrus Tristeza Virus Separated in Time and Space Are Essentially Identical

ABSTRACT The first Citrus tristeza virus (CTV) genomes completely sequenced (19.3-kb positive-sense RNA), from four biologically distinct isolates, are unexpectedly divergent in nucleotide sequence (up to 60% divergence). Understanding of whether these large sequence differences resulted from recent evolution is important for the design of disease management strategies, particularly the use of genetically engineered mild (essentially symptomless)-strain cross protection and RNA-mediated transgenic resistance. The complete sequence of a mild isolate (T30) which has been endemic in Florida for about a century was found to be nearly identical to the genomic sequence of a mild isolate (T385) from Spain. Moreover, samples of sequences of other isolates from distinct geographic locations, maintained in different citrus hosts and also separated in time (B252 from Taiwan, B272 from Colombia, and B354 from California), were nearly identical to the T30 sequence. The sequence differences between these isolates were within or near the range of variability of the T30 population. A possible explanation for these results is that the parents of isolates T30, T385, B252, B272, and B354 have a common origin, probably Asia, and have changed little since they were dispersed throughout the world by the movement of citrus. Considering that the nucleotide divergence among the other known CTV genomes is much greater than those expected for strains of the same virus, the remarkable similarity of these five isolates indicates a high degree of evolutionary stasis in some CTV populations.

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.

Infection with Strains of Citrus Tristeza Virus Does Not Exclude Superinfection by Other Strains of the Virus

ABSTRACT 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.

Persistent Infection and Promiscuous Recombination of Multiple Genotypes of an RNA Virus within a Single Host Generate Extensive Diversity

Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses.

Presence of P1b and absence of HC-Pro in Squash vein yellowing virus suggests a general feature of the genus Ipomovirus in the family Potyviridae

The genus Ipomovirus is one of six currently recognized genera in the family Potyviridae. The complete nucleotide sequence of Squash vein yellowing virus (SqVYV), a putative ipomovirus recently described in Florida, has been determined. The 9836 nt SqVYV genomic RNA [excluding the poly(A) tail] has one large open reading frame encoding a single polyprotein of 3172 amino acids, typical of the genome organization for most members in the family Potyviridae. The 10 mature proteins predicted to be derived from the SqVYV polyprotein include P1a and P1b but no HC-Pro, similar to Cucumber vein yellowing virus (CVYV) but different from Sweet potato mild mottle virus (SPMMV), both recognized members of the genus Ipomovirus. Phylogenetic analysis of these proteins supports classification of SqVYV as a novel species within the genus Ipomovirus. However, the similar genome organization strategy of SqVYV and CVYV, which differs from that of SPMMV, indicates that the taxonomy of the genus Ipomovirus needs to be re-examined and a new genus created within the family Potyviridae to accommodate the observed discrepancies in ipomovirus genome organization.

Metabolic Interplay between the Asian Citrus Psyllid and Its Profftella Symbiont: An Achilles' Heel of the Citrus Greening Insect Vector.

‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including ‘Candidatus Profftella armatura’, are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.

Transmission of Citrus leprosis virus C by Brevipalpus phoenicis (Geijskes) to alternative host plants found in citrus orchards.

The equivalent of US

Validation of ‘Variable Number of Tandem Repeat’-Based Approach for Examination of ‘Candidatus Liberibacter asiaticus’ Diversity and Its Applications for the Analysis of the Pathogen Populations in the Areas of Recent Introduction

75 million is spent each year in Brazil to control Brevipalpus phoenicis, a mite vector of Citrus leprosis virus C (CiLV-C). In this study, we investigated the possibility that hedgerows and windbreaks normally found in citrus orchards could host CiLV-C. Mites confined by an adhesive barrier were reared on sweet orange fruit with leprosis symptoms then were transferred to leaves of Hibiscus rosa-sinensis, Malvaviscus arboreus, Grevilea robusta, Bixa orellana, and Citrus sinensis. Ninety days post infestation, the descendant mites were transferred to Pera sweet orange plants to verify the transmissibility of the virus back to citrus. Nonviruliferous mites which had no feeding access to diseased tissue were used as controls. Local chlorotic or necrotic spots and ringspots, symptoms of leprosis disease, appeared in most plants tested. Results generated by reversetranscription polymerase chain reaction with primers specific for CiLV-C and by electron microscope analyses confirmed the susceptibility of these plants to CiLV-C.

Dramatic Change in Citrus tristeza virus Populations in the Dominican Republic

Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of ‘Candidatus Liberibacter’ among which ‘Ca. Liberibacter asiaticus’ (CLas) has most wide distribution. Recently, a number of studies identified different regions in the CLas genome with variable number of tandem repeats (VNTRs) that could be used for examination of CLas diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using CLas populations from Florida. Our results showed that the numbers of tandem repeats in the four loci tested display very distinguishable “signature profiles” for the two Florida-type CLas haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of CLas populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of CLas. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.

Effects of temperature, storage period and the number of individuals on the detection of the false spider mite Cardinium endosymbiont

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and has been an important concern for the citrus industry in the Dominican Republic. Earlier studies documented widespread distribution of mild isolates of the T30 genotype, which caused no disease in the infected trees, and a low incidence of isolates of the VT and T3 genotypes, which were associated with economically damaging decline and stem-pitting symptoms in sweet orange and Persian lime, the two major citrus varieties grown in the Dominican Republic. In light of the dramatic increase in the number of severely diseased citrus trees throughout the country over the last decade, suggesting that field populations of CTV have changed, we examined the CTV pathosystem in the Dominican Republic to assess the dynamics of virus populations. In this work, we characterized the molecular composition of 163 CTV isolates from different citrus-growing regions. Our data demonstrate a dramatic change in CTV populations, with the VT genotype now widely disseminated throughout the different regions and with the presence of two new virus genotypes, T36 and RB. Multiple infections of trees resulted in development of complex virus populations composed of different genotypes.