scholarly journals Methods for detection of Phytophthora fragariae var. rubi on raspberry

2009 ◽  
Vol 24 (3) ◽  
pp. 177-184 ◽  
Author(s):  
Mirjana Koprivica ◽  
Ivana Dulic-Markovic ◽  
Radivoje Jevtic ◽  
Dave Cooke
Keyword(s):  
Nested Pcr ◽  
Isoamyl Alcohol ◽  
French Bean ◽  
Causal Agent ◽  
Universal Primers ◽  
Pcr Product ◽  
Extraction Buffer ◽  
Phytophthora Fragariae ◽  
Separation Columns ◽  
Crop Research

Phytophthora fragariae var. rubi (Wilcox & Duncan), a causal agent of raspberry root rot, is a serious soil-borne pathogen listed by EPPO as an A2 quarantine pest. Root samples were collected from badly diseased raspberry plants showing a variety of characteristic and often dramatic symptoms during surveys carried out in western Serbia in 2002. Identification of the causal agent was performed in collaboration work with the Scottish Crop Research Institute (S.C.R.I.), Dundee, UK. Necrotic roots were plated on selective French bean agar (incorporating ampicilin, ryfamicin, bavistin and hymexasol). Detection of isolates was based on cultural and morphological features compared with referent cultures. DNA was extracted directly from the sampled roots using extraction buffer (200 mM Tris-HCl pH 8.5, 250 mM NaCl, 25 mM EDTA, 0.5% SDS), purified by multi spin separation columns [Thistle Scientific (Axygen)] or in 24:1 mixture of chlorophorm-isoamyl alcohol and amplified by nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round). Diluted DNA extracts were also amplified by conventional PCR with modified 'universal' Phytophthora primers (ITS 6, ITS 7 and ITS 8, Cooke et al., 2000) and digested with Msp1. Digestion patterns of the universal primers PCR product from infected roots matched those of Scottish strains. P. fragariae var. rubi occurred on 8 out of 14 sites. Our results indicate that nested PCR (ITS 4 and DC 6 for first round, DC 1 and DC 5 for second round) or digestion of the 'universal' Phytophthora primers PCR product for detection of P. fragariae var. rubi are more sensitive and less time-consuming and therefore recommended for use.

Molecular detection of equine infectious anemia virus in clinically normal, seronegative horses in an endemic area of Mexico

2021 ◽  
pp. 104063872110061
Author(s):  
César I. Romo-Sáenz ◽  
Patricia Tamez-Guerra ◽  
Aymee Olivas-Holguin ◽  
Yareellys Ramos-Zayas ◽  
Nelson Obregón-Macías ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Equine Infectious Anemia Virus ◽  
Antibody Detection ◽  
Economic Losses ◽  
Clinically Normal ◽  
Pcr Product ◽  
Surveillance Programs ◽  
Equine Infectious Anemia ◽  
Anemia Virus ◽  
Equine Industry

Equine infectious anemia (EIA) is a highly infectious disease in members of the Equidae family, caused by equine infectious anemia virus (EIAV). The disease severity ranges from subclinical to acute or chronic, and causes significant economic losses in the equine industry worldwide. Serologic tests for detection of EIAV infection have some concerns given the prolonged seroconversion time. Therefore, molecular methods are needed to improve surveillance programs for this disease. We attempted detection of EIAV in 6 clinical and 42 non-clinical horses in Nuevo Leon State, Mexico, using the agar gel immunodiffusion (AGID) test for antibody detection, and nested and hemi-nested PCR for detection of proviral DNA. We found that 6 of 6, 5 of 6, and 6 of 6 clinical horses were positive by AGID, nested PCR, and hemi-nested PCR, respectively, whereas 0 of 42, 1 of 42, and 9 of 42 non-clinical horses were positive by these tests, respectively. BLAST analysis of the 203-bp 5′-LTR/ tat segment of PCR product revealed 83–93% identity with EIAV isolates in GenBank and reference strains from other countries. By phylogenetic analysis, our Mexican samples were grouped in a different clade than other sequences reported worldwide, indicating that the LRT/ tat region represents an important target for the detection of non-clinical horses.

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

Plant Disease ◽  
2006 ◽  
Vol 90 (7) ◽  
pp. 973-973 ◽  
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.

scholarly journals First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman

Plant Disease ◽  
2011 ◽  
Vol 95 (3) ◽  
pp. 360-360 ◽  
Author(s):  
A. M. Al-Subhi ◽  
N. A. Al-Saady ◽  
A. J. Khan ◽  
M. L. Deadman
Keyword(s):  
Nested Pcr ◽  
Solanum Melongena ◽  
Positive Control ◽  
Rrna Gene ◽  
Direct Pcr ◽  
First Report ◽  
Pcr Product ◽  
Pcr Products ◽  
Broom Disease ◽  
Phytoplasma Infection

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman. References: (1) A. J. Khan et al. Phytopathology 92:1038, 2002. (2) M. A. Saghai-Maroof et al. Proc. Natl. Acad. Sci. USA, 81:8014, 1984.

scholarly journals First Report of Colletotrichum boninense Causing Anthracnose on Pepper in China

Plant Disease ◽  
2013 ◽  
Vol 97 (1) ◽  
pp. 138-138 ◽  
Author(s):  
Y. Z. Diao ◽  
J. R. Fan ◽  
Z. W. Wang ◽  
X. L. Liu
Keyword(s):  
Internal Transcribed Spacer ◽  
Severe Disease ◽  
Causal Agent ◽  
Its Sequences ◽  
Universal Primers ◽  
Pathogenicity Test ◽  
Conidial Suspension ◽  
Single Spore ◽  
First Report ◽  
Species Specific

Anthracnose, caused by Colletotrichum spp., is a severe disease and results in large losses in pepper (Capsicum frutescens) production in China (4). Colletotrichum boninense is one of the Colletotrichum species in pepper in China. In August 2011, anthracnose symptoms (circular, sunken lesions with orange to black spore masses) were observed on pepper fruits in De-Yang, Sichuan Province, China. Three single-spore isolates (SC-6-1, SC-6-2, SC-6-3) were obtained from the infected fruits. A 5-mm diameter plug was transferred to potato dextrose agar (PDA); the isolates formed colonies with white margins and circular, dull orange centers. The conidia were cylindrical, obtuse at both ends, and 10.5 to 12.6 × 4.1 to 5.0 μm. The colonies grew rapidly at 25 to 28°C, and the average colony diameter was 51 to 52 mm after 5 days on PDA at 25°C. Based upon these characters, the causal agent was identified as C. boninense. To confirm the identity of the isolates, the internal transcribed spacer (ITS) regions were amplified with the ITS1/ITS4 universal primers (1). The internal transcribed spacer (ITS) sequences (Accession No. JQ926743) of the causal fungus shared 99 to 100% homology with ITS sequences of C. boninense in GenBank (Accession Nos. FN566865 and EU822801). The identity of the causal agent as C. boninense was also confirmed by species-specific primers (Col1/ITS4) (2). In a pathogenicity test, five detached ripe pepper fruits were inoculated with 1 μl of a conidial suspension (106 conidia/mL) or five fruits with 1 μl of sterile water were kept as control. After 7 days in a moist chamber at 25°C, typical anthracnose symptoms had developed on the five inoculated fruits but not on control fruits. C. boninense was reisolated from the lesions, and which was confirmed by morphology and molecular methods as before. There have reports of C. boninense infecting many species of plants, including pepper (3). To our knowledge, this is the first report of C. boninense causing anthracnose on pepper in China. References: (1) A. K. Lucia et al. Phytopathology 93:581, 2002. (2) S. A. Pileggi et al. Can. J. Microbiol. 55:1081, 2009. (3) H. J. Tozze et al. Plant Dis. 93:106, 2009. (4) M. L. Zhang. J. Anhui Agri. Sci. 2:21, 2000.

scholarly journals Detection of Lawsonia intracellularis in a dog with inflammatory bowel disease using nested PCR and serology

2012 ◽  
Vol 48 (No. 4) ◽  
pp. 108-112 ◽  
Author(s):  
K. Tomanová ◽  
J. Klimeš ◽  
J. Smola ◽  
R. Husník
Keyword(s):  
Inflammatory Bowel Disease ◽  
Bowel Disease ◽  
Nested Pcr ◽  
Clinical Symptoms ◽  
Chronic Diarrhoea ◽  
Lawsonia Intracellularis ◽  
Nested Polymerase Chain Reaction ◽  
Pcr Product ◽  
Laboratory Examinations ◽  
Inflammatory Bowel

A nested polymerase chain reaction (PCR) assay and serological examinations were used to detect the presence of Lawsonia intracellularis in a two and a half years old German smooth-coated cocker spaniel with clinical symptoms of chronic diarrhoea and histologically proven inflammatory bowel disease. Fourteen rectal swabs taken over a period of two weeks and eight biopsy specimens taken over a period of six months were used for laboratory examinations. Using the nested PCR, the DNA of L. intracellularis was found in a total of 2 cases, i.e. one rectal swab and one biopsy specimen of the duodenum six months later. The species specificity of the nested PCR product was confirmed by sequencing. The presence of specific IgG antibodies against L. intracellularis was demonstrated by the IFAT in five samples of blood serum taken over a period of seven months.

scholarly journals Detection of Arbuscular Mycorrhizal Fungi in the Roots of Strawberry Plants Fertilized with Organic Bioproducts

2012 ◽  
Vol 77 (1) ◽  
pp. 17-27 ◽  
Author(s):  
Anna Lisek ◽  
Lidia Sas Paszt ◽  
Beata Sumorok
Keyword(s):  
Plant Growth ◽  
Arbuscular Mycorrhizal Fungi ◽  
Nested Pcr ◽  
Mycorrhizal Fungi ◽  
Arbuscular Mycorrhizal ◽  
Am Fungi ◽  
Universal Primers ◽  
Mineral Fertilizers ◽  
Reaction Products ◽  
The Impact

Summary In organic farming, mineral fertilizers are replaced by various preparations to stimulate plant growth and development. Introduction of new biopreparations into horticultural production requires an assessment of their effects on the growth and yielding of plants. Among the important indicators of the impact on plants of beneficial microorganisms contained in bioproducts is determination of their effectiveness in stimulating the growth and yielding of plants. Moreover, confirmation of the presence of arbuscular mycorrhizal (AM) fungi in the roots and plant growth promoting rhizobacteria (PGPR) in the rhizosphere is also necessary. In addition to conventional methods, molecular biology techniques are increasingly used to allow detection and identification of AM fungi in plant roots. The aim of this study was identification and initial taxonomic classification of AM fungi in the roots of ‘Elkat’ strawberry plants fertilized with various biopreparations using the technique of nested PCR. Tests were performed on DNA obtained from the roots of ‘Elkat’ strawberry plants: not fertilized, treated with 10 different biopreparations, or fertilized with NPK. Amplification of the large subunit of ribosomal gene (LSU rDNA) was carried out using universal primers, and then, in the nested PCR reaction, primers specific for the fungi of the genera Glomus, Acaulospora, and Scutellospora were used. Colonization of strawberry roots by arbuscular mycorrhizal fungi was determined on the basis of the presence of DNA fragments of a size corresponding to the types of the fungi tested for. As a result of the analyses, the most reaction products characterizing AM fungi were found in the roots of plants treated with the preparation Florovit Eko. The least fragments characteristic of AM fungi were detected in the roots of plants fertilized with NPK, which confirms the negative impact of mineral fertilizers on the occurrence of mycorrhizal fungi in the roots of strawberry plants. The roots of plants fertilized with Tytanit differed from the control plants by the presence of one of the clusters of fungi of the genus Glomus and by the absence of a cluster of fungi of the genus Scutellospora. In the roots of plants treated with other biopreparations there were reaction products indicating the presence of fungi of the genera Glomus, Scutellospora and Acaulospora, like in the roots of the control plants. The results will be used to assess the suitability of microbiologically enriched biopreparations in horticultural production.

Molecular characterization of Candidatus Phytoplasma aurantifolia isolate infecting chickpea (Cicer arietinum) in Dharwad, Karnataka

2019 ◽  
Author(s):  
Gurupada Balol ◽  
C Channakeshava ◽  
M S Patil
Keyword(s):  
16S Rdna ◽  
Molecular Characterization ◽  
Cicer Arietinum ◽  
Nested Pcr ◽  
Rdna Sequences ◽  
Pcr Product ◽  
16S Rdna Sequences ◽  
Axillary Proliferation ◽  
Pale Green

Chickpea plants showing phytoplasma symptoms were observed in the research plots at University of Agricultural Sciences, Dharwad, Karnataka, India. The symptoms included phyllody, pale green leaves, bushy appearance and excessive axillary proliferation. The causal agent of the phyllody disease was identified based on symptoms, amplification of 16S rDNA of the phytoplasma by nested PCR with primers P1/P7 and R16F2n/R16R2 and 1,800 bp and 1,200 bp size products were amplified in first round PCR and nested-PCR respectively. The PCR product was sequenced and compared with the reference phytoplasma sequences collected from the database (NCBI). 16S rDNA sequences of Dharwad chickpea phytoplasma shared the highest nucleotide identity of (>98%) with Periwinkle phyllody16SrII-E (EU096500). This study indicated the association of ‘Candidatus Phytoplasma aurantifolia’ the 16SrII-E group infecting chickpea from Northern Karnataka.

scholarly journals First Report of Cercospora apii, Causal Agent of Cercospora Early Blight of Celery, in Serbia

Plant Disease ◽  
2014 ◽  
Vol 98 (8) ◽  
pp. 1157-1157 ◽  
Author(s):  
A. Milosavljević ◽  
E. Pfaf-Dolovac ◽  
M. Mitrović ◽  
J. Jović ◽  
I. Toševski ◽  
...  
Keyword(s):  
Early Blight ◽  
Molecular Data ◽  
Causal Agent ◽  
Apium Graveolens ◽  
Universal Primers ◽  
Internal Transcribed Spacer Region ◽  
First Report ◽  
Molecular Features ◽  
Sterile Needle ◽  
Histone H3 Gene

Celery (Apium graveolens var. dulce) is a very important vegetable crop intensively cultivated in eastern and southern Serbia. During a field survey in August and September 2012, we observed symptoms similar to those of Cercospora early blight in eastern Serbia, with some of the affected fields showing up to 80% disease severity. The lesions on leaves were amphigenous, subcircular to angular and more or less confluent. Lesions enlarged and merged with age, followed by the development of necrotic area causing a continuous deterioration of the plant. Conidiophores arising from the stromata formed dense fascicles, sometimes appearing solitary, brown at the base, paler toward the apex, simple, straight to slightly curved, and rarely geniculate (dimensions 40 to 90 × 5 to 8 μm). Conidia were solitary, hyaline, at first cylindro-obclavate then acicular to acicular-obclavate, straight to slightly curved, subacute to obtuse at the apex, while truncated and thickened at the base (dimensions 45 to 160 × 4 to 5 μm), 5 to 13 septate. Based on the morphological features, we identified the pathogen as Cercospora apii Fresen. (2). In order to obtain monosporic isolates of the fungus, single conidia were cultivated on potato dextrose agar (PDA). To confirm the pathogenicity of the isolates, 5 mm-diameter mycelial plugs from the PDA plates were placed upside down on the adaxial leaf surface of 2-week-old celery seedlings of cv. Yuta. Control plants were inoculated with a sterile PDA plug. Three leaves per plant were disinfected with 70% ethanol, epidermis was scratched with a sterile needle to promote the infection, and inoculated. A total of 12 plants were inoculated with the mycelial plugs and 12 were used as control plants. Inoculated and control plants were kept in a moist chamber for 48 h and then transferred to a greenhouse at 25 ± 2°C. After 2 weeks, the first necrotic spots appeared on inoculated leaves, similar to the symptoms manifested in the field, while control plants remained symptomless. The pathogen was re-isolated and its identity was verified based on morphological and molecular features. To confirm the pathogen's identity, three isolates (CAC4-1, CAC24, and CAC30) were subjected to molecular identification based on the internal transcribed spacer region (ITS) using the ITS1/ITS4 universal primers (5), a partial calmodulin gene (CAL) using CAL-228F/CAL2Rd primers (1,4), and partial histone H3 gene (H3) using CYLH3F/CYLH3R primers (3). Sequences of the amplified regions were deposited in GenBank under accessions KJ210596 to KJ210604. The BLAST analyses of the ITS sequences revealed 100% identity with several Cercospora species (e.g., C. apii [JX143532], C. beticola [JX143556], and C. zebrina [KC172066]), while sequences of CAL and H3 showed 100% identity solely with sequences of C. apii (JX142794 and JX142548). Based on combined morphological and molecular data, the pathogen infecting celery was identified as C. apii, which to our knowledge represents the first report of the presence of the causal agent of Cercospora early blight disease in Serbia. References: (1) I. Carbone and L.M. Kohn. Mycologia 91:553, 1999. (2) P. W. Crous and U. Braun. CBS Biodivers. Ser. 1:1, 2003. (3) P. W. Crous et al. Stud. Mycol. 50:415, 2004. (4) J. Z. Groenewald. Stud. Mycol. 75:115, 2013. (5) T. J. White et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, Inc., San Diego, CA, 1990.

scholarly journals First Report of Aster Yellows (16SrI-B) Associated with Potatoes in Alaska

Plant Disease ◽  
2011 ◽  
Vol 95 (6) ◽  
pp. 767-767
Author(s):  
J. H. McBeath ◽  
P. J. Laski ◽  
M. Cheng
Keyword(s):  
Nested Pcr ◽  
Disease Transmission ◽  
Sequence Data ◽  
Restriction Enzymes ◽  
Reference Pattern ◽  
Aster Yellows ◽  
First Report ◽  
Group I ◽  
Pcr Product ◽  
Pcr Products

During a disease survey conducted in 2009 in Alaska, one potato plant (Solanum tuberosum) with symptoms characteristic of aster yellows, such as apical leaves rolling inward, leaves turning yellow or purple, and presence of aerial tubers, was found in a commercial field. Total DNA was extracted from leaves, stems, and roots of the symptomatic and symptomless plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the instructions of the manufacturer. A nested PCR was carried out with the first round primer pair P1/P7 followed by second round primer pair R16F2n/R16R2 (1,3). An approximate 1.2-kb PCR product was amplified from the symptomatic plant, but not symptomless plants. The PCR products from R16F2n/R16R2 were digested using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. The restriction fragment length polymorphism (RFLP) patterns were compared with those from known phytoplasma strains (1) and they matched the patterns for aster yellows subgroup B (16SrI-B). After P1/P7 amplification, the nested PCR product of primer pair P1A/16S-SR (2) was purified with a MiniElute Gel Extraction kit (Qiagen), sequenced by GENEWIZ (South Plainfield, NJ), and the sequence data analyzed by iPhyClassifier software (4). The results indicated that the sequence (GenBank Accession No. HQ599231) had 99.65% similarity to ‘Candidatus Phytoplasma asteris’ reference strain (GenBank Accession No. M30790). The RFLP similarity was identical (coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank Accession No. NC 005303). To our knowledge, this is the first report on the molecular identification of aster yellows phytoplasma associated with potatoes in Alaska. The source of the phytoplasma and pathway of disease transmission is currently under investigation. References: (1) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (2) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (3) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996. (4) Y. Zhao et al. Int. J. Syst. Evol. Microbiol. 59:2582, 2009.

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