Characterization of "Candidatus Liberibacter asiaticus" populations by double-locus analyses.

"Candidatus Liberibacter asiaticus" (CaLas) is associated with citrus Huanglongbing (HLB, yellow shoot disease), which is highly destructive to world citrus production. Understanding the relationships of CaLas isolates from different geographical regions is important for HLB research and development of disease management strategies. In this study, 301 CaLas isolates [85 Brazil, 132 China, and 84 U.S. (83 Florida and 1 California)] were collected, and genomic variations among them were evaluated based on the analyses of two genomic loci: trn1, characteristic of variable tandem repeat numbers (TRNs), and snp1, characteristic of single nucleotide polymorphisms (SNPs). Locus trn1 revealed the homogeneity of all Brazilian isolates, and locus snp1 revealed the homogeneity of most Florida isolates. When the two loci were analyzed simultaneously, i.e., double-locus (DL) analyses, CaLas isolates were clustered mostly according to geographical origins. DL genotype 1 included 97 % of the Chinese isolates, DL genotype 2 included all Brazilian isolates, and DL genotype 3 included 93 % of the U.S. isolates. DL analyses successfully revealed inter-continental overlapping or movement pattern of CaLas isolates. The isolate recently found in California belonged to Asiatic DL genotype 1.

Presymptomatic Fibrous Root Decline in Citrus Trees Caused by Huanglongbing and Potential Interaction with Phytophthora spp.

Huanglongbing (HLB), associated with 'Candidatus Liberibacter asiaticus', was first detected in Florida in late 2005 and is now widely distributed throughout the commercial citrus-growing regions. In recent seasons, concurrent with freeze and drought episodes, symptomatic HLB-infected trees were much more affected by the extremes of temperature and moisture than trees without HLB. Symptoms exhibited by the stressed trees were excessive leaf loss and premature fruit drop even when HLB-infected trees were managed with good nutritional and irrigation practices recommended to support health of HLB-affected trees. This stress intolerance may be due to a loss of fibrous roots. To assess root status of HLB-infected trees on 'Swingle' citrumelo rootstock (Citrus paradisi × Poncirus trifoliata), blocks of 2,307 3-year-old 'Hamlin' orange trees and 2,693 4-year-old 'Valencia' orange trees were surveyed visually and with a real-time polymerase chain reaction (PCR) assay to determine 'Ca. L. asiaticus' infection status. The incidence of 'Ca. L. asiaticus'-infected trees (presymptomatic: 'Ca. L. asiaticus'+, visually negative; and symptomatic: 'Ca. L. asiaticus'+, visually positive) trees was 89% for the Hamlin block and 88% for the Valencia block. 'Ca. L. asiaticus'+ trees had 30 and 37% lower fibrous root mass density for presymptomatic and symptomatic trees, respectively, compared with 'Ca. L. asiaticus'- trees. In a second survey, 10- to 25-year-old Valencia trees on Swingle citrumelo or 'Carrizo' citrange (C. sinensis (L.) × P. trifoliata) rootstock were sampled within 3 to 6 months after identification of visual HLB status as symptomatic ('Ca. L. asiaticus'+, visually positive) or nonsymptomatic ('Ca. L. asiaticus'-, visually negative) in orchards located in the central ridge, south-central, and southwest flatwoods. Pairs of HLB symptomatic and nonsymptomatic trees were evaluated for PCR status, fibrous root mass density, and Phytophthora nicotianae propagules in the rhizosphere soil. 'Ca. L. asiaticus'+ trees had 27 to 40% lower fibrous root mass density and, in one location, higher P. nicotianae per root but Phytophthora populations per cubic centimeter of soil were high on both 'Ca. L. asiaticus'+ and 'Ca. L. asiaticus'- trees. Fibrous root loss from HLB damage interacted with P. nicotianae depending on orchard location and time of year.

An improved regulatory sampling method for mapping and representing plant disease from a limited number of samples.

A key challenge for plant pathologists is to develop efficient methods to describe spatial patterns of disease spread accurately from a limited number of samples. Knowledge of disease spread is essential for informing and justifying plant disease management measures. A mechanistic modelling approach is adopted for disease mapping which is based on disease dispersal gradients and consideration of host pattern. The method is extended to provide measures of uncertainty for the estimates of disease at each host location. In addition, improvements have been made to increase computational efficiency by better initialising the disease status of unsampled hosts and speeding up the optimisation process of the model parameters. These improvements facilitate the practical use of the method by providing information on: (a) mechanisms of pathogen dispersal, (b) distance and pattern of disease spread, and (c) prediction of infection probabilities for unsampled hosts. Two data sets of disease observations, Huanglongbing (HLB) of citrus and strawberry powdery mildew, were used to evaluate the performance of the new method for disease mapping. The result showed that our method gave better estimates of precision for unsampled hosts, compared to both the original method and spatial interpolation. This enables decision makers to understand the spatial aspects of disease processes, and thus formulate regulatory actions accordingly to enhance disease control.

A stochastic optimization method to estimate the spatial distribution of a pathogen from a sample.

Information on the spatial distribution of plant disease can be utilized to implement efficient and spatially targeted disease management interventions. We present a pathogen-generic method to estimate the spatial distribution of a plant pathogen using a stochastic optimization process which is epidemiologically motivated. Based on an initial sample, the method simulates the individual spread processes of a pathogen between patches of host to generate optimized spatial distribution maps. The method was tested on data sets of Huanglongbing of citrus and was compared with a kriging method from the field of geostatistics using the well-established kappa statistic to quantify map accuracy. Our method produced accurate maps of disease distribution with kappa values as high as 0.46 and was able to outperform the kriging method across a range of sample sizes based on the kappa statistic. As expected, map accuracy improved with sample size but there was a high amount of variation between different random sample placements (i.e., the spatial distribution of samples). This highlights the importance of sample placement on the ability to estimate the spatial distribution of a plant pathogen and we thus conclude that further research into sampling design and its effect on the ability to estimate disease distribution is necessary.

Guangdong and Florida populations of 'Candidatus Liberibacter asiaticus' distinguished by a genomic locus with short tandem repeats.

Huanglongbing (HLB) (yellow shoot disease) is a highly destructive disease that threatens citrus production worldwide. The disease was first observed in Guangdong, P.R. China, over 100 years ago, and was found in Florida, United States, in 2005. 'Candidatus Liberibacter asiaticus' has been associated with HLB in many citrus-growing regions around the world, including Guangdong and Florida. The global epidemiology of HLB, as well as management of the disease, relies on knowledge of 'Ca. L. asiaticus' populations in different geographical regions around the world. In this study, we identified a genetic marker containing small tandem repeats in the genome of 'Ca. L. asiaticus' and comparatively analyzed the tandem repeat numbers (TRNs) in 'Ca. L. asiaticus' populations from Guangdong and Florida. Analyses of TRNs showed that the bacterial population in Guangdong was different from that in Florida. The Guangdong population consisted predominately of strains with a TRN of 7 (TRN(7)) at a frequency of 47.6%. The Florida population consisted predominately of strains with a TRN of 5 (TRN(5)) at a frequency of 84.4%. TRNs ranged from 3 to 16. The apparent absence of TRNs of 9, 10, 11, and 12 separated the bacterial strains into two groups: TRNs < 10 (TRN(<10)) and TRNs > 10 (TRN(>10)). In Florida, TRN(<10) strains (103/109, or 94.5%) were widely distributed in all HLB-affected counties. TRN(>10) strains (6/109, or 5.5%) were found in central Florida. This is the first report documenting the differentiation of 'Ca. L. asiaticus' populations between Asia and North America and the possible presence of two differentially distributed genotypes of 'Ca. L. asiaticus' in Florida.

First Report of the Citrus Huanglongbing Associated Bacterium 'Candidatus Liberibacter asiaticus' from Sweet Orange, Mexican Lime, and Asian Citrus Psyllid in Belize.

The citrus industries of North and South America are endangered by Huanglongbing (HLB), also known as citrus greening, a devastating disease associated with 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus', two species of fastidious phloem-limited bacteria spread by the Asian citrus psyllid (ACP), Diaphorina citri, Kuwayama. The first reports of HLB from the Americas were from Brazil in 2004 followed by Florida in 2005 (3). The ACP was found in Belize in 2005 (S. Williams, personal communication) and is now present throughout Central America. On the basis of the report that the HLB-associated bacteria can be easily detected in the ACP vector (4), an initial sampling of ACP from 67 locations was collected in February 2009 from trees in the Belize, Corozal, Orange Walk, Stann Creek, and Toledo Districts of Belize, and shipped in 95% ethanol to Riverside, CA for analysis. DNA was extracted from lots containing three to five psyllids from each of the 67 samples with Fast DNA kits (MP Biomedicals, Solon, OH) and analyzed by multiplex qPCR for 'Ca. L. asiaticus' and 'Ca. L. americanus' with a Stratagene MX3005P thermocycler with primers and Taqman probes to detect the 16sRNA gene of 'Ca. L. asiaticus' or 'Ca. L. americanus' and a psyllid gene, wingless, as an internal control target (4). Nine of the sixty-seven psyllid extractions were clearly positive for 'Ca. L. asiaticus' with cycle threshold values of 24 to 29. 'Ca. L. americanus' was not detected in any of the samples. From the districts previously sampled for ACP, leaves and fruit peduncles were collected from Citrus sinensis and C. aurantifolia plants showing HLB symptoms of asymmetrical leaf mottle and lopsided fruit with aborted seeds. DNA extracted from 10 of the 12 plant samples with a Qiagen Plant DNeasy kit (Qiagen Inc., Valencia, CA) was positive for 'Ca. L. asiaticus' with the qPCR procedure of Li et al (3). The presence of 'Ca. L. asiaticus' in the positive plant and ACP samples was corroborated by amplification, cloning, and sequencing of a 1,168-bp region of the 16S rRNA gene (2) with SpeedSTAR HS DNA polymerase (TaKaRa Bio Inc., Shiga, Japan). Consensus sequences obtained from three clones each from psyllids (Accession No. GQ502291) and plants (Accession No. GU061003) showed >99% identity to corresponding regions of 'Ca. L. asiaticus' in GenBank. The presence of 'Ca. L. asiaticus' was further indicated by amplification of a 227-bp fragment from the same 10 positive plant samples using primers for the 'Ca. L. asiaticus' preprotein translocase subunit SecE gene (nucleotides 31418 to 31644 of the genomic DNA) (1). Presence of trees with HLB symptoms and the detection of the associated 'Ca. L. asiaticus' confirm the disease in the Cayo, Corozal, Stann Creek, and Toledo districts in Belize. Analyses of psyllids from limited surveys conducted from 2006 to 2008 had not detected 'Ca. L. asiaticus' or 'Ca. L. americanus'. Confirmation of HLB in Belize has significant implications to the citrus industries in Central America. References: (1) T. H. Hung et al. J. Phytopathol. 147:599, 1999. (2) S. Jagoueix et al. Mol. Cell. Probes 10:43, 1996. (3) W. Li et al. J. Microbiol. Methods 66:104, 2006. (4) K. L. Manjunath et al. Phytopathology 98:387, 2008.

Bidens mottle virus and Apium virus Y Identified in Ammi majus in Florida.

Ammi majus (bishop's weed), a member of the Apiaceae, is grown from seed for cut flowers in South Florida. In March 2005, plants were found to be showing virus-like symptoms including mosaic, vein clearing, and leaf rugosity (3) that rendered their flowers unmarketable. Inclusion morphology in epidermal strips from these infected plants indicated the presence of one or more potyviruses. This was confirmed by ELISA with commercially available antiserum for potyvirus identification (Agdia, Elkhart, IN). Clover yellow vein virus (ClYVV) was identified by sequencing and confirmed with specific antiserum (4). However, ClYVV was not identified in all potyvirus-infected samples from 2005, indicating the presence of one or more additional potyviruses. Bidens mottle virus (BiMoV) was subsequently identified in one of three potyvirus-infected samples by immunodiffusion tests using specific antiserum for BiMoV (Department of Plant Pathology, University of Florida), cylindrical inclusion morphology in epidermal strips, host range data, and sequencing of cloned reverse transcription (RT)-PCR products from degenerate potyvirus primers (2). Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence (GenBank Accession No. EU255631) were 95 and 98% identical, respectively, to a Florida isolate of BiMoV recently reported from tropical soda apple (1). Similar virus-like symptoms were again observed in A. majus in January 2007 and persisted through March. ELISA testing again indicated the presence of a potyvirus. However, neither ClYVV nor BiMoV were identified in the initial 2007 samples. Instead, sequence analysis of the cloned RT-PCR products amplified with degenerate potyvirus primers (2) from seven potyvirus-infected samples collected on two dates in January and one each in February and March revealed the presence of Apium virus Y (ApVY). The 3' terminal portion of the genome (GenBank Accession No. EU255632) was found to be 90 to 91% identical to ApVY sequences in GenBank at the nucleotide level. Deduced amino acid sequences of the NIb and CP regions of these RT-PCR products were 96 and 95% identical, respectively, to ApVY sequences in GenBank. One of these seven ApVY-infected samples (collected in March 2007) was determined to be coinfected with BiMoV by sequence analysis of the cloned RT-PCR products. Six clones were sequenced. Three were determined to be ApVY as indicated above. Nucleotide and deduced amino acid sequences of a partial polyprotein gene sequence from the other three clones were 95 and 97% identical, respectively, to the 2005 A. majus BiMoV isolate. Although ClYVV and BiMoV have previously been reported in other hosts in Florida, to the best of our knowledge, this is the first report of BiMoV and ApVY in A. majus anywhere and the first report of ApVY in North America. References: (1.) C. A. Baker et al. Plant Dis. 91:905, 2007. (2.) A. Gibbs and A. J. Mackenzie. J. Virol. Methods 63:9, 1997. (3.) M. S. Irey et al. (Abstr.) Phytopathology (suppl.)95:S46, 2005. (4.) M. S. Irey et al. Plant Dis. 90:380, 2006.

Yellow leaf of sugarcane is caused by at least three different genotypes of sugarcane yellow leaf virus, one of which predominates on the Island of Réunion.

The genetic diversity of sugarcane yellow leaf virus (SCYLV) was analyzed with 43 virus isolates from Réunion Island and 17 isolates from world-wide locations. We attempted to amplify by reverse-transcription polymerase chain reaction (RT-PCR), clone, and sequence four different fragments covering 72% of the genome of these virus isolates. The number of amplified isolates and useful sequence information varied according to each fragment, whereas an amplicon was obtained with diagnostic primers for 59 out of 60 isolates (98%). Phylogenetic analyses of the sequences determined here and additional sequences of 11 other SCYLV isolates available from GenBank showed that SCYLV isolates were distributed in different phylogenetic groups or belonged to single genotypes. The majority of isolates from Réunion Island were grouped in phylogenetic clusters that did not contain any isolates from other origins. The complete six ORFs (5612 bp) of five SCYLV isolates (two from Réunion Island, one from Brazil, one from China, and one from Peru) were amplified, cloned, and sequenced. The existence of at least three distinct genotypes of SCYLV was shown by phylogenetic analysis of the sequences of these isolates and additional published sequences of three SCYLV isolates (GenBank accessions). The biological significance of these genotypes and of the origin of the distinct lineage of SCYLV in Réunion Island remains to be determined.

Clover yellow vein virus Identified in Ammi majus in Florida.

Ammi majus L., a member of the Apiaceae and also known as large bullwort, false Queen Anne's lace, or bishop's-weed, is frequently used in the floral trade to add a lacey look to floral bouquets. A. majus is native to the Mediterranean Region but it is cultivated in major growing areas including Holland, Israel, the United Kingdom, and the United States. During March 2005, virus-like symptoms including mosaic, generalized chlorosis, vein clearing, interveinal chlorosis, and leaf rugosity were observed in nearly all field-grown A. majus plants at two locations in Martin County, Florida. Inclusion body morphology suggested the presence of one or more potyviruses in the symptomatic plants. Potyvirus infection was confirmed in 11 symptomatic plants using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Agdia, Elkhart, IN). Nucleotide and deduced amino acid sequences of a 1,625-bp region of one of the reverse transcription-polymerase chain reaction products amplified with degenerate potyvirus primers (1) from total RNA of symptomatic plants (GenBank Accession No. DQ333346) were 96 to 97% and 93 to 99% identical, respectively, to Clover yellow vein virus (ClYVV) sequences in GenBank. All symptomatic plants tested were potyvirus positive using ELISA, but only a subset was infected with ClYVV suggesting that the field symptoms were the result of infection with additional potyviruses, all of which are likely aphid transmitted. Although several potyviruses have been reported from A. majus (2), to our knowledge, this represents the first report of ClYVV infection. References: (1) A. Gibbs and A. Mackenzie. J. Virol. Methods 63:9, 1997. (2) P. Van Dijk and L. Bos. Neth. J. Plant Pathol. 95(Suppl.) 2:1, 1989.

Detection of Sugarcane yellow leaf virus in Quarantine and Production of Virus-free Sugarcane by Apical Meristem Culture.

Sugarcane yellow leaf virus (SCYLV) was detected for the first time in 1996 in the Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD) sugarcane quarantine at Montpellier by reverse transcription-polymerase chain reaction (RT-PCR) in varieties from Brazil, Florida, Mauritius, and Réunion. Between 1997 and 2000, the virus was found by RT-PCR and/or tissue-blot immunoassay (TBIA) in additional varieties from Barbados, Cuba, Guadeloupe, Indonesia, Malaysia, Philippines, Puerto Rico, and Taiwan, suggesting a worldwide distribution of the pathogen. An excellent correlation was observed between results obtained for the two diagnostic techniques. However, even though only a few false negative results were obtained by either technique, both are now used to detect SCYLV in CIRAD's sugarcane quarantine in Montpellier. The pathogen was detected by TBIA or RT-PCR in all leaves of sugarcane foliage, but the highest percentage of infected vascular bundles was found in the top leaves. The long hot water treatment (soaking of cuttings in water at 25°C for 2 days and then at 50°C for 3 h) was ineffective in eliminating SCYLV from infected plants. Sugarcane varieties from various origins were grown in vitro by apical bud culture and apical meristem culture, and the latter proved to be the most effective method for producing SCYLV-free plants.

Examination of the effect of aphid vector population composition on the spatial dynamics of citrus tristeza virus spread by stochastic modeling.

ABSTRACT Aphid vector species population composition is known to affect the spatial patterns of citrus tristeza virus (CTV) and the changes in these patterns over time. However, the biological processes that are associated with virus spread have not been well defined. The spatiotemporal dynamics of CTV were examined using data collected from research plots in the Dominican Republic and Costa Rica, where the brown citrus aphid (BCA), Toxoptera citricida, was the predominant species, and in Florida, where the BCA was absent and the melon aphid, Aphis gossypii, was the predominant vector. Data were analyzed using a spatiotemporal stochastic model for disease spread, and parameter values were evaluated using Markov chain Monte Carlo stochastic integration methods. Where the melon aphid was the dominant species, the model parameter likelihood values supported the hypothesis that the disease was spread through a combination of random background transmission (transmission originating from inoculum sources outside the plot) and a local interaction (transmission from inoculum sources within the plot) operating over short distances. Conversely, when BCA was present, results often suggested a local short-range transmission interaction that was not restricted to nearest-neighbor interactions and that the presence of background infection was not necessary to explain the observations.

First Report of Sugarcane Yellow Leaf Virus in the French West Indies.

Unusually severe leaf yellowing symptoms, similar to those described for yellow leaf syndrome (1), have been observed in several sugarcane clones in Guadeloupe since 1994, and since 1997 in Martinique. Leaf samples exhibiting various types of yellowing were taken from five different sugarcane clones, and analyzed by immunosorbent electron microscopy. Spherical particles, 24 to 28 nm in diameter and characteristic of luteoviruses, were found in two of five samples. The two infected samples showed yellowing on the underside of the midrib and one had a pinkish coloration on the upper side. The presence of sugarcane yellow leaf virus (ScYLV), the causal agent of sugarcane yellow leaf disease, was confirmed by reverse transcription-polymerase chain reaction (2) in these two samples and in 36 of 184 sugarcane clones bred in Guadeloupe and sent to Cirad's quarantine station in Montpellier, France. Following these observations, surveys were undertaken with a tissue blot enzyme immunoassay to analyze the distribution of ScYLV in sugarcane clones in the French West Indies. The midrib base of the first visible dewlap leaf was used to detect the presence of the virus in the phloem. In a first survey, clones of various origins worldwide were taken from germplasm collections. Two to three leaf samples per clone were analyzed from 78 clones in a collection in Guadeloupe and from 36 in a collection in Mar-tinique. Fifty of the 114 clones were infected by ScYLV, and ScYLV was detected in 21 of the 32 clones exhibiting severe leaf yellowing (score 3 or higher on a 1 to 5 scale). In a second survey, 19 leaf samples were taken from each of 53 clones from plants produced by Cirad's breeding program in Guadeloupe. The virus was detected in at least one sample for 25 of these 53 clones. ScYLV incidence in commercial fields was tested in Martinique in the variety B5992, which constitutes 57% of the cultivated area. Twenty leaves from different stools were sampled in six different fields, five of which had ScYLV-infected plants. The percentage of virus-infected stalks ranged from 0 to 90% whereas the percentage of stalks showing symptoms ranged from 50 to 100%. ScYLV appears widespread in the French West Indies, perhaps because a vector (Melanaphis sacchari) exists in Martinique and Guadeloupe. However, ScYLV was not found in all symptomatic plants, indicating that even if this luteovirus is a causal agent of leaf yellowing in the French West Indies, there may be other causal agents as well. References: (1) J. C. Comstock et al. Sugar J. 3:33, 1994. (2) J. C. Comstock et al. Sugar Cane 4:21, 1998.

Comparison and classification of dental arch forms of Indian and Chinese subjects with normal occlusions.

To compare the dental arch forms of Indian and Chinese subjects, 30 untreated Indian and 30 untreated Chinese adults with normal occlusion and symmetrical arches were examined. The arches were classified as narrow, wide, mid, pointed and flat, according to the method developed by Monique Raberin etal., from Lyon, France. For the sample examined the Chinese population was found to have significantly wider arches compared to the Indian population.