Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae

The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3′ poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5′ and 541 nt at 3′ termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98–99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate

Nucleotide sequence and genome organization of Dweet mottle virus and its relationship to members of the family Betaflexiviridae

The nucleotide sequence of Dweet mottle virus (DMV) was determined and compared to sequences of members of the families Alphaflexiviridae and Betaflexiviridae. The DMV genome has 8,747 nucleotides (nt) excluding the 3′ poly-(A) tail. DMV genomic RNA contains three putative open reading frames (ORFs) and untranslated regions of 73 nt at the 5′ and 541 nt at 3′ termini. ORF1 potentially encoding a 227.48-kDa polyprotein, which has methyltransferase, oxygenase, endopeptidase, helicase, and RNA-dependent RNA polymerase (RdRP) domains. ORF2 encodes a movement protein of 40.25 kDa, while ORF3 encodes a coat protein of 40.69 kDa. Protein database searches showed 98–99% matches of DMV ORFs with citrus leaf blotch virus (CLBV) sequences. Phylogenetic analysis based on the RdRP core domain revealed that DMV is closely related to CLBV as a member of the genus Citrivirus. DMV did not satisfy the molecular criteria for demarcation of an independent species within the genus Citrivirus, family Betaflexiviridae, and hence, DMV can be considered a CLBV isolate

Longevity of Imidacloprid Soil Drench on Citrus Nursery Stock for Sale at Retail Stores in Florida

The Florida psyllid testing project (Manjunath et al. 2008, Halbert et al. 2012) showed that about 10% of regulatory samples of Diaphorina citri Kuwayama collected by Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS/DPI) inspectors from plants for sale in Florida were positive for Candidatus Liberibacter asiaticus (Las). Most of the commercial nurseries that produce the plants do not have psyllids or Las, so the most likely source of contamination is the retail venues themselves. If this is the case, great benefit could be achieved by preventing psyllid infestation in retail stores. Florida has a requirement that citrus plants for sale be treated with an imidacloprid-based soil drench (ISD). Producers are required to tag the plant with the date of treatment. The treatment expires in six months, but our data indicate that three months probably is more realistic. In 2009, there was an increase in plants infested with psyllids 30 days post-ISD treatment. In later years, this increase was not so pronounced or did not exist, suggesting that growers are getting better control

Longevity of Imidacloprid Soil Drench on Citrus Nursery Stock for Sale at Retail Stores in Florida

The Florida psyllid testing project (Manjunath et al. 2008, Halbert et al. 2012) showed that about 10% of regulatory samples of Diaphorina citri Kuwayama collected by Florida Department of Agriculture and Consumer Services, Division of Plant Industry (FDACS/DPI) inspectors from plants for sale in Florida were positive for Candidatus Liberibacter asiaticus (Las). Most of the commercial nurseries that produce the plants do not have psyllids or Las, so the most likely source of contamination is the retail venues themselves. If this is the case, great benefit could be achieved by preventing psyllid infestation in retail stores. Florida has a requirement that citrus plants for sale be treated with an imidacloprid-based soil drench (ISD). Producers are required to tag the plant with the date of treatment. The treatment expires in six months, but our data indicate that three months probably is more realistic. In 2009, there was an increase in plants infested with psyllids 30 days post-ISD treatment. In later years, this increase was not so pronounced or did not exist, suggesting that growers are getting better control

PHYLOGENETIC RELATIONSHIPS AMONG SPECIES OF PROTOTHACA FROM PANAMABASED ON CYTOCHROME C OXIDASE I (COI) SEQUENCES.

ABSTRACT: The genus Protothaca (Bivalvia: Veneridae) represents a commercially important group of clams with a widedistribution in the Pacific coast of America and Asia. According to The Integrated Taxonomic Information System (ITIS),there are nine accepted species (http://www.itis.gov/); nevertheless the number of recognized species varies based on thesources revised. In this study, a molecular approach was used to phylogenetically analyze five species belonging to the genusProtothaca. A fragment of 550 bp from the mitochondrial cytochrome oxidase I gene was analyzed from P. asperrima, P.columbiensis, P. grata, P. mcgyntyi and P. jedoensis. The COI sequences revealed seven haplotypes for P. asperrima, four forP. columbiensis and two for P. grata. Bayesian analysis showed two major well supported clusters consisting of (1) genus-Protothaca clade and (2) a clade with rest of the members of Veneridae. All the species of Protothaca clustered together withthe exception of P. jedoensis. Intraspecific genetic differences in P. asperrima were very high at 17-18 %; P. columbiensisand P. grata specimens had differences in the range of 0,18-0.36 % and 0,92 % respectively. Interestingly, Protothacaasperrima haplotypes A and H showed the highest level of intraspecific genetic distances. Considering the traditionalcontroversies in classification, the level of COI divergence in P. asperrima haplotypes and the differences observed in othergenera within the family Veneridae, we propose a revision of the species including elevating the rank of the two distinctclusters of P. asperrima to closely related species status. Key words: Genetic difference, COI, mtDNA, P. asperrima, Veneridae. RESUMEN: El género Protothaca (Bivalvia:Veneridae) representa un grupo de almejas comercialmente importantescon una amplia distribución en las costas del Pacífico de América y Asia. De acuerdo con el Sistema Integrado de InformaciónTaxonomica (ITIS), hay nueve especies aceptadas; sin embargo el número de especies reconocidas varía en función de lasfuentes revisadas. La taxonomía actual del género se basa en la morfología de la concha y la clasificación sigue siendocontrovertida. En este estudio, se utilizó un enfoque molecular para analizar filogenéticamente cinco especies pertenecientesal género Protothaca. Se analizó un fragmento de 550 pb del gen mitocondrial de citocromo oxidasa I de P. asperrima, P.columbiensis, P. grata, P. mcgyntyi y P. jedoensis. Las secuencias de COI revelaron siete haplotipos de P. asperrima, cuatropara P. columbiensis y dos para P. grata. El análisis bayesiano mostró dos grupos principales, bien soportados consistentes en(1) clado del género-Protothaca y (2) un clado con el resto de los miembros de Veneridae. Todas las especies de Protothacaagruparon juntas con la excepción de P. jedoensis. La diferencia genética intraespecífica en P. asperrima fué bastante altaentre 17-18%; Las diferencias entre especímenes de P. columbiensis y P. grata estuvieron en el rango de 0.18-0.36% y 0.92%respectivamente. Curiosamente, los haplotipos A y H de Protothaca asperrima mostraron el nivel más alto de distanciasgenéticas intraespecífica. Teniendo en cuenta las controversias tradicionales en la clasificación, el nivel de divergencia COI enlos haplotipos de P. asperrima y las diferencias observadas en otros géneros dentro de la familia Veneridae, proponemos unarevisión de las especies, incluyendo la elevación del rango de los dos grupos diferentes de P. asperrima al estatus de especiesestrechamente relacionadas. Palabras clave: Diferencia genética, COI, mtDNA. P. aspérrima, Venerida

Improved methods for genome sequencing of Liberibacters by BAC library-based metagenomics approach

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented

Improved methods for genome sequencing of Liberibacters by BAC library-based metagenomics approach

Liberibacters have not yet been successfully cultured; their minimal genomes carry multiple copies of several genes. Sequences identical to phage genomes have been found in many Liberibacters. Available evidences suggest that the Liberibacter genomes are adapting rapidly in different hosts and environments. Characterization of genomes of rapidly changing unculturable organisms can be challenging. We have used a model system based on Candidatus Liberibacter psyllaurous associated with tomato “psyllid yellows” (Hansen et al., 2008) to develop methodologies using alternate techniques for sequencing metagenomes. We have constructed a BAC library from infected tomato psyllids (Bactericera cockerelli). The library consists of 57,600 clones arrayed in 150 plates each with 384 wells. DNA from individual clones were pooled for screening purposes. Initial identification of clones with Liberibacter sequences were conducted based on 16s ribosomal sequences, and contiguous clones were characterized by end sequencing and identified as containing Liberibacter genome fragments. Screening of additional clones from the library was based on probes developed on such sequences. A total of 245 clones with Liberibacter genome fragments have been identified. A total of 63 bar-coded BAC clones were sequenced by using Roche 454 technology. BAC clones from this library contain large inserts (average size 70 kb). Similarities and differences with other well characterized genomes of Liberibacters (Duan et al., 2009, Lin et al., 2011) will be presented

Home Detection Kit for Candidatus Liberibacter asiaticus (LAS) Associated with Citrus Huanglongbing from Psyllids

Management of citrus huanglongbing (HLB) requires rapid detection of infected psyllids and trees in an orchard. Detection of HLB associated bacteria (LAS) can be done using psyllids since detection in infected trees is usually delayed (Manjunath et al., 2008). We have developed an easy to use, rapid and affordable detection kit for grower use for testing psyllids for LAS at a reasonable price for initial investment and an operating cost of about $2 per sample.. Eight psyllid samples can be simultaneously tested within 45 minutes. The psyllid DNA extraction and detection of LAS are conducted using a SmartDART™ unit which is operated by software installed on any android device for visualizing real time results. The test results can be e-mailed for both storage and analysis.  The DNA prepared can be stored refrigerated and sent to a laboratory for validation. No other equipment (even pipets) is required for the test. The detection system was validated using a large number LAS isolates from many citrus varieties, from different countries; the results were comparable to that of traditional real time PCR data. Development of methods for multiplex detection of the pathogen and the host DNA from both psyllids and plant host are in progress. We believe the detection system will be useful for growers in intra-orchard management, for extension workers, nurserymen, and in areas where the disease has become endemic as well as in those areas where the disease has been recently introduced

Home Detection Kit for Candidatus Liberibacter asiaticus (LAS) Associated with Citrus Huanglongbing from Psyllids

Management of citrus huanglongbing (HLB) requires rapid detection of infected psyllids and trees in an orchard. Detection of HLB associated bacteria (LAS) can be done using psyllids since detection in infected trees is usually delayed (Manjunath et al., 2008). We have developed an easy to use, rapid and affordable detection kit for grower use for testing psyllids for LAS at a reasonable price for initial investment and an operating cost of about $2 per sample.. Eight psyllid samples can be simultaneously tested within 45 minutes. The psyllid DNA extraction and detection of LAS are conducted using a SmartDART™ unit which is operated by software installed on any android device for visualizing real time results. The test results can be e-mailed for both storage and analysis.  The DNA prepared can be stored refrigerated and sent to a laboratory for validation. No other equipment (even pipets) is required for the test. The detection system was validated using a large number LAS isolates from many citrus varieties, from different countries; the results were comparable to that of traditional real time PCR data. Development of methods for multiplex detection of the pathogen and the host DNA from both psyllids and plant host are in progress. We believe the detection system will be useful for growers in intra-orchard management, for extension workers, nurserymen, and in areas where the disease has become endemic as well as in those areas where the disease has been recently introduced