Hyaloperonospora camelinae on Camelina sativa in Washington State: Detection, Seed Transmission, and Chemical Control

Camelina (Camelina sativa) plants with symptoms of downy mildew were obtained from three different locations in Washington State. Based on polymerase chain reaction (PCR) and sequencing of the internal transcribed spacer (ITS)1-5.8S-ITS2 region, the causal pathogen was identified as Hyaloperonospora camelinae. The PCR primers consistently amplified 699-bp bands from the infected plants but not from the asymptomatic plants. A comparison of the sequences with those in GenBank revealed 100% sequence similarity to H. camelinae. Growth and development of the H. camelinae was observed in different tissues using light microscopy and scanning electron microscopy (SEM). Light microscopic observation revealed the presence of oospores in the infected leaves and SEM revealed the presence of conidia and conidiophores on the seed surface. To determine whether H. camelinae is a seed-transmitted pathogen, seed collected from infected plants were planted in Sunshine professional growing mix maintained in a growth chamber. Disease symptoms were observed in 96% of the seedlings compared with 3% of the seedlings grown from seed from asymptomatic plants, which indicates that H. camelinae is a seed-transmitted pathogen. Seed treated with mefenoxam, a fungicide specific for Oomycetes, significantly reduced the incidence of the disease.

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First Report of Chino del Tomate and Pepper Hausteco Geminivurses in Greenhouse-Grown Tomato in Sonora, Mexico

Plant Disease ◽

10.1094/pdis.1999.83.4.396b ◽

1999 ◽

Vol 83 (4) ◽

pp. 396-396 ◽

Cited By ~ 3

Author(s):

A. M. Idris ◽

S. H. Lee ◽

J. K. Brown

Keyword(s):

Pcr Primers ◽

Quintana Roo ◽

Disease Symptoms ◽

Tomato Plants ◽

First Report ◽

Tomato Seedlings ◽

Cp Gene ◽

Biolistic Inoculation ◽

Flanking Sequences ◽

Polymerase Chain

Tomato plants grown in commercial greenhouses in Sonora, Mexico, developed either yellow mosaic, leaf curling, and stunting (phenotype 1; 20 to 35%) or chlorosis and a feathery appearance of leaves (phenotype 2; 15 to 25%) in December 1997 and again in October 1998. Polymerase chain reaction (PCR) with diagnostic primers (prAV324 and prAC889) that amplify an approximately 576-bp fragment of the coat protein (CP) gene of whitefly-transmitted geminiviruses indicated the presence of a begomovirus. Biolistic inoculation of tomato seedlings with RNase-treated extracts of three symptomatic tomato samples, each, resulted in reproduction of disease symptoms. With PCR primers prAV2644 and prAC1154 (1), the entire CP (approximately 776 bp) and its flanking sequences were amplified from extracts of symptomatic tomato, and amplicons were cloned and sequenced. Comparisons of a minimum of three CP gene sequences from each phenotype revealed the presence of at least two begomoviruses. The phenotype 1-associated CP gene shared 92.6, 86.2, and 85.3% identity with the CP sequence of chino del tomate (CdTV) [AF106936], tomato mottle, and abutilon mosaic geminiviruses, respectively. The CP sequence associated with phenotype 2 was 94.6, 77.1, and 77.1% identical to pepper hausteco (PHV) [X70418], bean golden mosaic-Guatemala (BGMV-GU), and Texas pepper (TPV) gem-iniviruses, respectively. Previously, CdTV was reported from tomato in Chiapas, Morelos, Sinaloa, and Tamaulipas, Mexico, while PHV has been identified in Guanajuato, Quintana Roo, Sinaloa, and Tamaulipas, Mexico, and in Texas (2). This is the first report of CdTV-like (>92% identity) and PHV-like (>94% identity) geminiviruses associated with greenhouse-grown tomatoes in Sonora, Mexico. References: (1) A. M. Idris and J. K. Brown. Phytopathology 88:648, 1998; (2) I. Torres-Pacheco et al. Phytopathology 86:1186, 1996.

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GB Virus C/Hepatitis G Virus Isolates in Japanese Haemophiliacs and their Origins

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615181 ◽

1998 ◽

Vol 80 (08) ◽

pp. 242-245 ◽

Cited By ~ 12

Author(s):

Yoshihide Fukuda ◽

Tetsuo Hayakawa ◽

Junki Takamatsu ◽

Hidehiko Saito ◽

Hiroaki Okamoto ◽

...

Keyword(s):

Sequence Similarity ◽

West African ◽

Blood Products ◽

Hepatitis G Virus ◽

Gb Virus C ◽

Route Of Transmission ◽

Polymerase Chain ◽

Hepatitis G ◽

Virus C ◽

Virus Isolates

SummaryJapanese haemophiliacs have been at high risk for infection with parenterally-transmissible viruses through the use of blood products, especially imported ones. Recently, novel transfusion-transmissible virus, GB virus C (GBV-C)/hepatitis G virus (HGV) were isolated. We investigated the origin and route of transmission of GBV-C/HGV isolates in haemophiliacs in Japan. GBV-C/HGV RNA was measured by nested reverse transcription polymerase chain reaction in 91 Japanese haemophiliacs. Phylogenetic analysis and genotypic grouping of GBV-C/HGV isolates in Japanese haemophiliacs were performed based on sequences in the 5’ untranslated region, and the characteristics were compared with those of reported isolates. GBV-C/HGV infection was present in 19 of 91 haemophiliacs (20.9%). Sequence analysis showed that 15 of the 19 isolates (78.9%) showed sequence similarity to a group in which mainly West African isolates have been reported. The other 4 isolates (21.1%) showed sequence similarity to Asian isolates. None of the GBV-C/HGV isolates showed sequences similar to those generally found in isolates from USA and Europe. The majority of GBV-C/HGV isolates found in Japanese haemophiliacs who are considered to have been infected by imported blood products were similar to those detected in West Africa.

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A strategy to detect and isolate an intron-containing gene in the presence of multiple processed pseudogenes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.17.6691 ◽

1989 ◽

Vol 86 (17) ◽

pp. 6691-6695 ◽

Cited By ~ 19

Author(s):

B Davies ◽

S Feo ◽

E Heard ◽

M Fried

Keyword(s):

Ribosomal Protein ◽

Recombinant Dna ◽

Single Gene ◽

Ribosomal Protein Gene ◽

Pcr Primers ◽

Pcr Product ◽

Dna Libraries ◽

Processed Pseudogenes ◽

Polymerase Chain ◽

Genomic Dna Sequence

We have devised a strategy that utilizes the polymerase chain reaction (PCR) for the detection and isolation of intron-containing genes in the presence of an abundance of processed pseudogenes. The method depends on the genomic DNA sequence between the PCR primers spanning at least one intron in the gene of interest, resulting in the generation of a larger intron-containing PCR product in addition to the smaller PCR product amplified from the intronless pseudogenes. A unique intron probe isolated from the larger PCR product is used for the detection of intron-containing clones from recombinant DNA libraries that also contain pseudogene clones. This method has been used successfully for the selective isolation of an intron-containing rat L19 ribosomal protein gene in the presence of multiple pseudogenes. Analysis of a number of mammalian ribosomal protein multigene families by PCR indicates that they all contain only a single gene with introns.

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Detection ofErwinia amylovorain plant material using novel polymerase chain reaction (PCR) primers

New Zealand Journal of Crop and Horticultural Science ◽

10.1080/01140671.2001.9514158 ◽

2001 ◽

Vol 29 (1) ◽

pp. 35-43 ◽

Cited By ~ 34

Author(s):

R. K. Taylor ◽

P. J. Guilford ◽

R. G. Clark ◽

C. N. Hale ◽

R. L. S. Forster

Keyword(s):

Polymerase Chain Reaction ◽

Plant Material ◽

Pcr Primers ◽

Chain Reaction ◽

Polymerase Chain

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A New ‘Candidatus Liberibacter’ Species Associated with Diseases of Solanaceous Crops

Plant Disease ◽

10.1094/pdis-93-3-0208 ◽

2009 ◽

Vol 93 (3) ◽

pp. 208-214 ◽

Cited By ~ 168

Author(s):

Lia W. Liefting ◽

Paul W. Sutherland ◽

Lisa I. Ward ◽

Kerry L. Paice ◽

Bevan S. Weir ◽

...

Keyword(s):

16S Rrna ◽

Pcr Primers ◽

23S Rrna ◽

Rrna Gene ◽

High Identity ◽

Specific Pcr ◽

Transmission Electron ◽

Candidatus Liberibacter ◽

Polymerase Chain ◽

Specific Pcr Primer

A new disease of glasshouse-grown tomato and pepper in New Zealand has resulted in plant decline and yield loss. Affected plants are characterized by spiky, chlorotic apical growth, curling or cupping of the leaves, and overall stunting. Transmission electron microscopy revealed the presence of phloem-limited bacterium-like organisms in symptomatic plants. The strategy used to identify the bacterium involved using specific prokaryote polymerase chain reaction (PCR) primers in combination with universal 16S rRNA primers. Sequence analysis of the 16S rRNA gene, the 16S/23S rRNA spacer region, and the rplKAJL-rpoBC operon revealed that the bacterium shared high identity with ‘Candidatus Liberibacter’ species. Phylogenetic analysis showed that the bacterium is distinct from the three citrus liberibacter species previously described and has been named ‘Candidatus Liberibacter solanacearum’. This is the first report of a liberibacter naturally infecting a host outside the Rutaceae family. A specific PCR primer pair was developed for its detection.

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First Report of a Group 16SrII Phytoplasma Infecting Chickpea in Oman

Plant Disease ◽

10.1094/pd-90-0973c ◽

2006 ◽

Vol 90 (7) ◽

pp. 973-973 ◽

Cited By ~ 8

Author(s):

N. A. Al-Saady ◽

A. M. Al-Subhi ◽

A. Al-Nabhani ◽

A. J. Khan

Keyword(s):

Nested Pcr ◽

Animal Feed ◽

Restriction Enzymes ◽

Universal Primers ◽

Polymorphism Analysis ◽

Disease Symptoms ◽

First Report ◽

Pcr Products ◽

Polymerase Chain ◽

Group 2

Chickpea (Cicer arietinum), locally known as “Dungo”, is grown for legume and animal feed mainly in the interior region of Oman. During February 2006, survey samples of chickpea leaves from plants showing yellows disease symptoms that included phyllody and little leaf were collected from the Nizwa Region (175 km south of Muscat). Total nucleic acid was extracted from asymptomatic and symptomatic chickpea leaves using a cetyltrimethylammoniumbromide method with modifications (3). All leaf samples from eight symptomatic plants consistently tested positive using a polymerase chain reaction assay (PCR) with phytoplasma universal primers (P1/P7) that amplify a 1.8-kb phytoplasma rDNA product and followed by nested PCR with R16F2n/R16R2 primers yielding a product of 1.2 kb (2). No PCR products were evident when DNA extracted from healthy plants was used as template. Restriction fragment length polymorphism analysis of nested PCR products by separate digestion with Tru9I, HaeIII, HpaII, AluI, TaqI, HhaI, and RsaI restriction enzymes revealed that a phytoplasma belonging to group 16SrII peanut witches'-broom group (2) was associated with chickpea phyllody and little leaf disease in Oman. Restriction profiles of chickpea phytoplasma were identical with those of alfalfa witches'-broom phytoplasma, a known subgroup 16SrII-B strain (3). To our knowledge, this is the first report of phytoplasma infecting chickpea crops in Oman. References: (1) A. J. Khan et al. Phytopathology, 92:1038, 2002. (2). I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998 (3) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA. 81:8014, 1984.

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Development of AFLP-derived, functionally specific markers for environmental persistence studies of fungal strains

Canadian Journal of Microbiology ◽

10.1139/w05-140 ◽

2006 ◽

Vol 52 (5) ◽

pp. 451-461 ◽

Cited By ~ 8

Author(s):

S S Hynes ◽

O Chaudhry ◽

M A Providenti ◽

M L Smith

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Genetically Modified Organisms ◽

Quantitative Polymerase Chain Reaction ◽

Pcr Primers ◽

Chain Reaction ◽

Environmental Persistence ◽

Fungal Strains ◽

Target Dna ◽

Polymerase Chain

The ability to rapidly identify and quantify a microbial strain in a complex environmental sample has widespread applications in ecology, epidemiology, and industry. In this study, we describe a rapid method to obtain functionally specific genetic markers that can be used in conjunction with standard or real-time polymerase chain reaction (PCR) to determine the abundance of target fungal strains in selected environmental samples. The method involves sequencing of randomly cloned AFLP (amplified fragment length polymorphism) products from the target organism and the design of PCR primers internal to the AFLP fragments. The strain-specific markers were used to determine the fate of three industrially relevant fungi, Aspergillus niger, Aspergillus oryzae, and Chaetomium globosum, during a 4 month soil microcosm experiment. The persistence of each of the three fungal strains inoculated separately into intact soil microcosms was determined by PCR analyses of DNA directly extracted from soil. Presence and absence data based on standard PCR and quantification of the target DNA by real-time PCR showed that all three strains declined after inoculation (~14-, 32-, and 4-fold for A. niger, A. oryzae, and C. globosum, respectively) but remained detectable at the end of the experiment, suggesting that these strains would survive for extended periods if released into nature.Key words: Canada domestic substances list (DSL), Canadian Environmental Protection Act (CEPA), genetically modified organisms (GMO), quantitative polymerase chain reaction (qPCR).

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RT-PCR microlocalization of mRNAs for calbindin D28k and vitamin D receptor in the murine nephron

AJP Renal Physiology ◽

10.1152/ajprenal.1996.270.4.f677 ◽

1996 ◽

Vol 270 (4) ◽

pp. F677-F681 ◽

Cited By ~ 2

Author(s):

L. Liu ◽

A. Khastgir ◽

J. M. McCauley ◽

S. T. Dunn ◽

J. H. Morrissey ◽

...

Keyword(s):

Vitamin D ◽

Vitamin D Receptor ◽

Calcium Binding ◽

Spatial Relationship ◽

Pcr Primers ◽

Rt Pcr ◽

Calbindin D28k ◽

Actin Mrna ◽

Polymerase Chain ◽

Convoluted Tubules

The spatial relationship between vitamin D receptor (VDR) and calbindin D28k [calcium binding protein D28k (CaBP-D28k)] gene expression within the murine kidney was studied by localizing their mRNAs in discrete nephron structures using reverse transcription-polymerase chain reaction (RT-PCR). Primers for beta-actin mRNA were used as a control for the presence of tissue during RT-PCR for CaBP-D28k mRNA. mRNA for CaBP-D28k was found only in distal convoluted tubules (DCTs), connecting tubules (CNTs), and cortical collecting ducts (CCDs). In contrast, VDR mRNA was detected in glomeruli, S2 proximal convoluted tubules, cortical thick ascending limbs of Henle's loop, DCTs, CNTs, and initial CCDs. The presence of both VDR and CaBP-D28k mRNA in DCTs, CNTs, and CCDs is consistent with the hypothesis that cacitriol acts via the VDR to stimulate CaBP-D28k synthesis. Conversely, the presence of VDR mRNA in other parts of the nephron suggests that calcitriol has genomically mediated actions within the kidney in addition to stimulation of CaBP-D28k synthesis.

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Evaluation of Olive as a Host of Xylella fastidiosa and Associated Sharpshooter Vectors

Plant Disease ◽

10.1094/pdis-01-14-0014-re ◽

2014 ◽

Vol 98 (9) ◽

pp. 1186-1193 ◽

Cited By ~ 44

Author(s):

Rodrigo Krugner ◽

Mark S. Sisterson ◽

Jianchi Chen ◽

Drake C. Stenger ◽

Marshall W. Johnson

Keyword(s):

Control Method ◽

Xylella Fastidiosa ◽

Primary Control ◽

Homalodisca Vitripennis ◽

Insecticide Treatment ◽

Disease Symptoms ◽

Leaf Scorch ◽

Polymerase Chain ◽

Low Efficiency ◽

Branch Dieback

Olive (Olea europaea) trees exhibiting leaf scorch or branch dieback symptoms in California were surveyed for the xylem-limited, fastidious bacterium Xylella fastidiosa. Only approximately 17% of diseased trees tested positive for X. fastidiosa by polymerase chain reaction, and disease symptoms could not be attributed to X. fastidiosa infection of olive in greenhouse pathogenicity assays. Six strains of X. fastidiosa were isolated from olive in Southern California. Molecular assays identified strains recovered from olive as belonging to X. fastidiosa subsp. multiplex. Pathogenicity testing of olive strains on grapevine and almond confirmed that X. fastidiosa strains isolated from olive yield disease phenotypes on almond and grapevine typical of those expected for subsp. multiplex. Mechanical inoculation of X. fastidiosa olive strains to olive resulted in infection at low efficiency but infections remained asymptomatic and tended to be self-limiting. Vector transmission assays demonstrated that glassy-winged sharpshooter (Homalodisca vitripennis) could transmit strains of both subspp. multiplex and fastidiosa to olive at low efficiency. Insect trapping data indicated that two vectors of X. fastidiosa, glassy-winged sharpshooter and green sharpshooter (Draeculacephala minerva), were active in olive orchards. Collectively, the data indicate that X. fastidiosa did not cause olive leaf scorch or branch dieback but olive may contribute to the epidemiology of X. fastidiosa-elicited diseases in California. Olive may serve as an alternative, albeit suboptimal, host of X. fastidiosa. Olive also may be a refuge where sharpshooter vectors evade intensive areawide insecticide treatment of citrus, the primary control method used in California to limit glassy-winged sharpshooter populations and, indirectly, epidemics of Pierce's disease of grapevine.

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Evaluation of Resistance to Rhabdocline Needlecast in Douglas Fir Variety Shuswap, with Quantitative Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto-100-4-0337 ◽

2010 ◽

Vol 100 (4) ◽

pp. 337-344 ◽

Cited By ~ 8

Author(s):

M. Catal ◽

G. C. Adams ◽

D. W. Fulbright

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Douglas Fir ◽

Chain Reaction ◽

Disease Symptoms ◽

Seed Sources ◽

Release Times ◽

Spore Release ◽

Target Dna ◽

Polymerase Chain

A quantitative polymerase chain reaction assay was developed that could detect DNA of Rhabdocline pseudotsugae and R. oblonga among DNA of Douglas fir needles to a limit as low as three copies of target DNA. Differential infection rates of two varieties (seed sources) of Douglas fir interplanted in a field were studied in relation to staggered bud breaks. Infection of Douglas fir var. San Isabel corresponded to ascospore release times for Rhabdocline spp., whereas infection of var. Shuswap Lake did not occur throughout the spore release period during 2 years of study, despite abundant inoculum and adequate moisture during bud break. Rhabdocline spp. DNA was never detected in Shuswap Lake and disease symptoms were not observed in any year. We provide evidence that Shuswap Lake is resistant and probably immune to Rhabdocline spp. infection and Rhabdocline needlecast under Michigan conditions.

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