Huanglongbing disease of citrus, associated with infection by the bacterium ‘Candidatus Liberibacter asiaticus’ (LAS), has spread rapidly in the US since 2005. Attempts to culture LAS in vitro have not yielded a consistently reproducible culture method; therefore, obtaining knowledge about the infection process is difficult. To determine conditions which sustain LAS viability, LAS inoculum obtained from seeds of fruit from infected pomelo trees (Citrus grandis ‘Mato Buntan’) was added to different media, and cell viability was monitored for several weeks using quantitative polymerase chain reaction (qPCR) in conjunction with ethidium monoazide (EMA). Among media tested, King’s B (K) did not support viability of LAS cells, while grapefruit juice (G) allowed LAS cells to survive in vitro for ~20 days. In media that sustained LAS viability, a reproducible biofilm-like substance was formed over time at the air-liquid interface of culture flasks and glass slides inserted in cultures. Fluorescence in situ hybridization (FISH) showed the biofilm contains aggregates of LAS cells, which was confirmed by qPCR. 16S rDNA libraries of the biofilm samples have been constructed and will be sequenced via Illumina next-generation sequencing to determine their bacterial composition. To elucidate why juice-based media prolongs LAS viability, the elemental nutrient compositions of the media and the biofilm were analyzed via inductively coupled plasma optical emission spectrometry (ICP-OES). Compositions were compared, and specific elements, such as potassium and calcium, were more abundant in media that sustain LAS cell viability. Results will contribute to future development of a culture medium for LAS.

The equivalent of US$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 reverse-transcription polymerase chain reaction with primers specific for CiLV-C and by electron microscope analyses confirmed the susceptibility of these plants to CiLV-C.

The potential of viroid infection to dwarf citrus growing in intensive plantings is well established.How viroids exert this dwarfing effect is not known, but one possibility involves limiting the size of the root system.As part of an ongoing effort to develop Citrus viroid III (CVd-III) for use with rootstocks other than trifoliate orange or its hybrids, we have studied the effects of viroid infection on root development under greenhouse conditions using three rootstock/scion combinations; i.e., rooted Etrog citron cuttings, trifoliate orange seedlings, and young Valencia orange/trifoliate orange grafted trees.Groups of 10 young Etrog cuttings growing under greenhouse conditions were slash-inoculated with CVd-IIIb RNA transcripts and then observed for up to 12 mo with periodic cutbacks.Three months post-inoculation, the viroid-infected Etrog plants were significantly shorter than the uninoculated controls.By 6 mo post-inoculation, the inhibitory effect of CVd-IIIb on root dry weight had also become statistically significant.Between 6 and 12 mo, effects on root weight and development continued to intensify.Graft inoculation of trifoliate orange seedlings or Valencia scions growing on trifoliate orange rootstocks with either CVd-IIIa or CVd-IIIb resulted in a similar (though not statistically significant) inhibition of root dry weight accumulation over an 18 mo period.

Author(s): Gottwald, T. R.; Hall, D. G.; Beattie, G. A. C.; Ichinose, K.; Nguyen, M. C.; Le, Q. D.; Bar-Joseph, M.; Lapointe, S.; Stover, E.; Parker, P. E.; McCollum, G.; Hilf, M. E.

In August 2008, unusual symptoms were observed in Mexican lime trees (Citrus aurantifolia (Christm.) Swing) in the municipality of Luperón, province of Puerto Plata on the north coast of the Dominican Republic. Symptoms observed in young and old trees included blotchy mottle on leaves, healthy-appearing larger branches with smaller side branches that displayed chlorotic leaves, abscised and lopsided fruit, and branch dieback, all symptoms similar to those of citrus huanglongbing associated with 'Candidatus Liberibacter' spp. (1). Symptoms were observed in an area of ~100 ha surrounding Luperón, where Mexican lime trees were grown as seedlings and no commercial plantings of other citrus were present. Symptomatic leaves were collected from 16 trees in September 2008, and DNA was extracted from petioles and midveins with a DNeasy kit (Qiagen, Gaithersburg, MD) or with chloroform/isoamyl alcohol (24:1). Real-time PCR with the16S rDNA primer/probe set specific to 'Ca. Liberibacter asiaticus' and performed as described (2) gave Ct values comparable with the positive control for five samples. Conventional PCR with the forward (5'-tcgagcgcgtatgcaatacg-3') and reverse (5'-ctacctttttctacgggataacgc-3') primers used in real-time PCR (2) amplified a 75-bp product from these five samples. Eleven to twelve clones were sequenced from each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ489643-FJ489647) showed 98% (e.g., EU921622, EU921618) to 100% identity (e.g., FJ236554, FJ263702) with 16S rDNA sequences of 'Ca. Liberibacter asiaticus'. A larger 912-bp portion of the 16S rDNA gene was amplified from each sample by conventional PCR with sense (5'-gagcctaccaaggctacgat-3') and antisense (5'-gcgttatcccgtagaaaaaggtag-3') primers designed from the sequence of 'Ca. Liberibacter asiaticus' strain LJZ-4730 (GenBank Accession No. FJ263700). Nine to twelve clones were sequenced for each sample and BLAST analysis of a consensus sequence for each sample (GenBank Accession Nos. FJ811891-FJ811895) showed 100% identity to 'Ca. Liberibacter asiaticus' (DQ471900, DQ4719010) from Florida and Brazil, respectively. To confirm this identification, 693 bp of the outer membrane protein gene (omp) were amplified from each of the five positive samples by conventional PCR using sense (5'-gtgattctgagggtgagcg-3') and antisense (5'-cgaactcactgagaactgatc-3') primers designed from nucleotides 15 to 33 and 687 to 707, respectively, of the omp gene from 'Ca. Liberibacter asiaticus' strain MZ (GenBank Accession No. EF580135). The consensus sequences from 12 clones from each sample (GenBank Accession Nos. FJ489638-FJ489642) showed 99% (e.g., FJ236564, AY842429) to 100% (e.g., FJ236566, EF580135) nucleotide identity and the predicted translation product showed 100% amino acid identity (e.g., AAX47433, AAX47431) with the omp gene from 'Ca. Liberibacter asiaticus'. These results confirm the presence of and to our knowledge, this is the first report of 'Ca. Liberibacter asiaticus' in the Dominican Republic. Huanglongbing is a destructive disease of citrus (1) and its spread is expected to adversely affect citrus production in the Dominican Republic with subsequent negative effects predicted for the employment of thousands of people. References: (1) J. M. Bové, J. Plant Pathol. 88:7, 2006. (2) W. Li et al. J. Microbiol. Methods 66:104, 2006.

Brevipalpus phoenicis mites are vectors of Citrus leprosis virus (CiLV), the causal agent of leprosis, an important viral disease of citrus. Brevipalpus individuals harbor an endosymbiont bacterium belonging to the phylum Bacteroidetes , genus Cardinium , which causes feminization in these mites. We determined the prevalence of these endosymbionts in B. phoenicis mite populations from different citrus production areas in southeastern Brazil. PCR was performed with primers specific for the amplification of a fragment within the 16S rDNA gene of the endosymbiont. All B. phoenicis populations tested yielded bands of the expected size when analyzed by agarose gel electrophoresis and analysis of the sequence of the amplified DNA confirmed the presence and identity of the endosymbiont in B. phoenicis populations from different citrus production areas. Brevipalpus phoenicis (Acari: Tenuipalpidae) mites are known to transmit Citrus leprosis virus (CiLV), the causal agent of leprosis, which is considered the most important viral disease of citrus in Brazil due to the severe symptoms it induces and the high cost involved in controlling the mite vector. The presence of an endosymbiont bacterium belonging to the (phylum Bacteroidetes , genus Cardinium ) was recently reported in a B. phoenicis population from coffee trees in São Paulo, Brazil (2, 4). Several studies have shown that this endosymbiotic bacterium is able to induce a number of reproductive effects in its hosts such as cytoplasmic incompatibility, partheno-genesis and fecundity enhancement in arthropod species. In some cases, e.g. in B. phoenicis , the bacterium causes feminization of the mites (2). Here, we report the first effort to determine the prevalence of this endosymbiont in B. phoenicis mite populations from different citrus production areas of Brazil. The populations of B. phoenicis were collected from 10 different citrus production areas in the southeastern region of Brazil: Piracicaba, Cordeirópolis, Conchal, Matão, Gavião Peixoto, Barretos, Onda Verde, Bauru, and Monte Alegre do Sul, São Paulo State, and from Teresópolis, Rio de Janeiro State. All populations were maintained at the Entomology Laboratory at the Citriculture Center, Cordeirópolis, SP, Brazil. Total DNA was extracted as described by Weeks et al. (1) and suspended in 12 μl of sterile distilled water. For polymerase chain reaction (PCR) amplification analysis of mite populations, a region within the 16S rDNA was amplified using primers specific for this sequence in CFB bacteria (2). All PCR reactions contained 4 μl of template DNA, 2.5 μl of 10 × PCR buffer, 0.9 μl of 50 mM MgCl 2 , 0.5 μl of a 10-mM dNTP mixture, 5 pmoles of each primer and 0.2 μl of Taq DNA polymerase (5 U/μl) in a total volume of 25 μl. Amplifications were 420 Sixteenth IOCV Conference, 2005—Short Communications performed with an initial denaturation at 95°C for 5 min, followed by 30 cycles at 95°C for 30 s, 55°C for 45 s, 72°C for 30s, with a final extension cycle at 72°C for 5 min. Aliquots of PCR products were analyzed by electrophoresis in 1% agarose in TAE buffer. All B. phoenicis populations tested yielded bands of the expected size (832 bp) and sequence analysis of the PCR products from two populations confirmed they were amplified from the correct region of the 16S rDNA gene of the endosymbiont. This data confirmed the identity and the presence of the endosymbiont in B. phoenicis populations collected from citrus in the southeastern region of Brazil. Weeks et al. (3) showed that CFB also infects a significant number of arthropod species, and may be horizontally transmitted. Consequently, other false spider mite populations from citrus are being tested for the presence of the bacterium and more sequencing is in progress in order to assess the genetic variability of this organism using both 16S rDNA and the internal transcribed spacer (ITS) region. In B. phoenicis is not clear whether the endosymbiont has any effect besides feminization of the mites, but studies are being carried out to determine if the endosymbiont plays a role in CiLV acquisition and transmission to citrus plants.

Citrus tristeza virus (CTV), the causal agent of tristeza quick decline and stem pitting in citrus, and its most effective vector, the brown citrus aphid (BrCA) ( Toxoptera citricida Kirk.), have been present together in Hawaii for over 50 yr. In this study the incidence, diversity, and population structure of Hawaiian CTV strains were examined. Citrus tissue samples collected on Kauai (8 sites, 91 trees), Oahu (13, 86), Molokai (4, 12), Maui (10, 87), and Hawaii (15, 129) were subjected to RT-PCR for the CTV coat protein gene as well as tissue blot immunoassays (TBIAs) using two CTV antibodies. Samples testing positive for either RT-PCR or TBIA were subjected to further RT-PCR using primers specific to CTV genotypes T3, T30, T36, and VT. The incidence of CTV on Kauai, Oahu, Molokai, Maui, and Hawaii was 59, 87, 58, 63, and 83% respectively, with an overall incidence of 74% (298/405). Strains with similarity to T3, T30, T36, and VT genotypes were identified in these samples, although their distribution was not uniform throughout the islands. Most infections, however, were composed of CTV strains that did not resemble any of these genotypes. The coat protein (CP) sequence was determined for over 100 Hawaiian CTV strains. Phylogenetic analysis using these sequences as well as CP sequences of CTV strains from other parts of the world indicate that Hawaii has previously uncharacterized strains of the virus. Stem pitting, an economically important disease of citrus caused by Citrus tristeza virus (CTV) was first observed in Hawaii in 1952, although the disease was already widespread at the time (4). With the most effective vector, the brown citrus aphid ( Toxoptera citricida Kirk.), abundant in the islands (9), it is likely that CTV was rapidly disseminated after its arrival. This disease has reduced Hawaii’s citrus industry to its present day status of small farms and backyard plantings of mostly CTV-tolerant citrus. As citrus is being revisited as a crop to vitalize Hawaii’s agriculture industry, control of CTV must be undertaken. In order to develop a sound management strategy for CTV in Hawaii, we undertook a comprehensive study using molecular and serological techniques to determine the incidence, distribution, and diversity of CTV in the Hawaiian Islands. MATERIALS AND METHODS Citrus samples. Four hundred and five citrus trees were sampled from 50 sites on the islands of Kauai, Oahu, Molokai, Maui, and Hawaii (Fig. 1). These sites ranged from groves with >500 trees to single backyard plants. Young stem segments were collected from both vigorous and unthrifty trees at each site when possible. These samples were kept on ice in the field and were then stored at -20°C until analyzed. Serological analysis. Tissueblot immunological assays (TBIAs) based on the method described by Hu et al. (7), were used to detect CTV in the citrus samples. Two antiCTV rabbit polyclonal antisera were used in these assays. The first (1212) was raised against purified virions of an Australian isolate of CTV. The second (kindly provided by the late D. J. Gumpf), designated CCPP, was raised against an in vitro -expressed 180 Sixteenth IOCV Conference, 2005—Citrus Tristeza Virus CTV CP and is used by the California Citrus Clonal Protection Program for CTV detection. In order to screen each sample with multiple CTV antibodies, each stem segment was recut 0.5-1.0 mm above the previous cut and pressed successively onto separate membranes. Molecular analysis. Approximately 100 mg of bark tissue was ground in liquid nitrogen and total nucleic acids (TNAs) were extracted as described by Stewart and Via (11), except that DIECA was omitted from the extraction buffer. Firststrand cDNA synthesis was performed using 2 μl of TNAs, random hexamers, and MMLV-RT (Promega, Madison, WI) following the manufacturer’s instructions. A PCR assay using the T36CP primer set was then used to detect CTV in these samples (6). Samples testing positive by either TBIA or the T36CP primer set were further characterized with 10 additional primer sets that amplified the 5’-UTR, K17, and POL markers of CTV genotypes T30, T36, and VT, and the K17 marker of the T3 genotype as previously described (5, 6). For all PCR assays, 1 μl of the cDNA reaction was used as template in a 20 μl reaction containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl 2 , 10 pmol of each CTV primer, 200 μM of each dNTP, 1 U of Taq DNA polymerase, overlaid with mineral oil. The amplification protocol consisted of one 5 min cycle at 94°C; 40 cycles at 94°C for 60 s, 56°C for 60 s, and 72°C for 60 s; ending with one 7 min cycle at 72°C. Amplification products were resolved in a 1% (w/v) agarose gel stained with ethidium bromide and viewed under UV light. Sequence variation in Hawaiian CTV. Sequence variation in the Hawaiian CTV population was assessed for each island using the coat protein gene (CP) of CTV isolates. A CTV-positive sample from each site on an island was selected, Fig. 1. Map of the major Hawaiian Islands. Noted are the sampling site locations ( ), the number of trees sampled, and the percentage of samples testing positive for Citrus tristeza virus. The shaded islands of Niihau, Lanai, and Kahoolawe (from left to right) were not included in the survey. Sixteenth IOCV Conference, 2005—Citrus Tristeza Virus 181 and l μl of the cDNA reaction (from above) for each of these samples was pooled. For example, on the island of Kauai we visited 8 sites and therefore the pooled Kauai cDNAs came from 8 CTV-positive samples. The pooled cDNA reactions were then used as template in PCR using the T36CP primer set as described above with only 30 amplification cycles. The resulting PCR product was ligated into pGEM-T Easy (Promega). Between 21 and 24 individual clones from each of the islands of Kauai, Oahu, Maui, and Hawaii, and 10 individual clones from Molokai were sequenced on an ABI377 cycle sequencer (PE Applied Biosystems, Foster City, CA). Plasmids were sequenced on one or both strands, depending on the signal strength of the electrophoretogram. In an exceptional case where sequencing of both strands could not resolve a base, the base was assigned the most common nucleotide at that position as determined by comparison to the other sequences. Polymorphic positions in these ‘Hawaiian’ CTV CP sequences were compared with those of ‘exotic’ CTV CP sequences from California (strain SY568, GenBank accession number AF001623), Florida (T30, AF260651; T36, U1603), Israel (VT, U56902), Japan (NUagA, AB046398), Mexico (BC15-1, AF342894; QR2753-1, AF342893), Portugal (13C, AF184113; 19-21, AF184114), South Korea (SK1, AF339088; SK2, AF249279), and Spain (B35, L12175; T385, Y18420) using the Pretty function of SeqWeb 1.2 (GCG, University of Wisconsin, WI). Primer sequences were excluded from the comparison. Nucleotide sequence alignments of the CTV CP genes were generated by neighbor joining using ClustalX (1.81) and visualized by TreeView (1.6.6).

Author(s): Freitas-Astua, J.; Locali, E. C.; Antonioli-Luizon, R.; Kitajima, E. W.; Hilf, M. E.; Gottwald, T. R.; Machado, M. A.

Symptoms induced by 266 isolates of Citrus tristeza virus (CTV) obtained from 30 countries were evaluated in graft-inoculated Mexican lime, sour orange, Madam vinous sweet orange and Duncan grapefruit seedlings, and sweet orange plants budded on sour orange rootstock.Sequential tests were conducted for 16 yr in a USDA-ARS quarantine facility at Beltsville, MD using consistent protocols.Two standard isolates were included in each test for reference.The isolates tested varied markedly in their ability to induce symptoms in specific indicators as well as in the severity of symptoms.The different patterns of symptoms observed in the five indicators suggested that seedling yellows, the decline syndrome in sweet orange grafted on sour orange, stem pitting in sweet orange, and stem pitting in grapefruit are independent expressions of CTV pathogenicity and may occur in various combinations.Although some general associations between some symptoms and CTV genotypes and reaction to the selective monoclonal antibody MCA13 were observed, absolute correlations were not established.

Author(s): Novelli, V. M.; Freitas-Astua, J.; Astua-Monge, G.; Carvalho, S. A.; Locali, E. C.; Rodrigues, V.; Arrivabem, F.; Hilf, M. E.; Gottwald, T. R.; Machado, M. A.

Close planting of viroid-infected citrus growing on trifoliate orange or trifoliate hybrid rootstocks has been shown to increase fruit yield and lower production costs.We would like to adapt Citrus viroid III (CVd-III) for use with other rootstocks and, toward that end, have examined the effect of introducing sequence changes in the lower portion of its central conserved region on replication and symptom expression in Etrog citron.Mutations at positions 173-174 and 179-181 appeared to be essentially neutral, having little or no effect on foliar symptoms.The effects of deletions affecting positions 53-54 in the pathogenicity domain were more complex.Like the corresponding portion of Potato spindle tuber viroid (PSTVd), the central conserved region of CVd-III contains a putative loop E motif.Unlike PSTVd, however, mutagenesis of CVd-III positions 189-191 failed to yield any stable sequence variants.Results from UV cross-linking experiments indicate that there are significant differences between the three-dimensional structures of the loop E motif in these two viroids.Understanding the effects of these structural differences on viroid replication may allow us to generate CVd-III variants having altered host range and dwarfing properties.