scholarly journals Molecular Characterization of Stolbur Group Subgroup E (16SrXII-E) Phytoplasma Associated with Potatoes in China

Plant Disease ◽  
2012 ◽  
Vol 96 (9) ◽  
pp. 1372-1372 ◽  
Author(s):  
M. Cheng ◽  
J. Dong ◽  
L. Zhang ◽  
P. J. Laski ◽  
Z. Zhang ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
Inner Mongolia ◽  
Disease Incidence ◽  
Restriction Enzymes ◽  
Potato Disease ◽  
Growing Seasons ◽  
Pcr Product ◽  
Disease Surveys

Phytoplasmas have been reported from more than 70 plant species in China, most of which are from woody plants and very few are from potatoes (Solanum tuberosum). During the growing seasons of 2005 through 2011, potato disease surveys were conducted in seed and commercial fields in Yunnan Province and Inner Mongolia. Potato plants displayed symptoms of curled, yellowish and purplish leaves, shortened internodes, aerial tuber formation, and few small malformed underground tubers. Although the location of the fields surveyed each year varied, the disease seems to have become increasingly prevalent. In Yunnan, disease incidence was 5 to 24% in 2005 and 15 to 100% in 2010 and 2011. In Inner Mongolia, disease incidence in seed potato fields was 5 to 15% in 2006 and 25 to 50% in 2011. Total DNA was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instruction. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.25-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, HhaI, HpaI, KpnI, MseI, and TaqI. Comparing the RFLP patterns of samples with previously published phytoplasma strains, the phytoplasmas matched patterns of the stolbur group, subgroup E (16SrXII-E) (1). In addition, the PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (2). The nested PCR product was cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska Fairbanks and GENEWIZ (South Plainfield, NJ). Nucleotide sequences (GenBank Accession No. EU293841) were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to ‘Candidatus Phytoplasma fragariae’ with RFLP patterns identical to group 16SrXII-E. To our knowledge, this is the first molecular characterization of the stolbur group phytoplasmas associated with potato disease in China. The potato is becoming increasingly important in China. The impacts of stolbur on potato yield losses, disease distributions, and insect vectors are 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.

scholarly journals First Report of Clover Proliferation Group Phytoplasmas (16SrVI-A) Associated with Purple Top Diseased Potatoes (Solanum tuberosum) in China

Plant Disease ◽  
2011 ◽  
Vol 95 (7) ◽  
pp. 871-871 ◽  
Author(s):  
M. Cheng ◽  
J. Dong ◽  
P. J. Laski ◽  
Z. Zhang ◽  
J. H. McBeath
Keyword(s):  
Nested Pcr ◽  
Disease Incidence ◽  
Restriction Enzymes ◽  
Potato Production ◽  
First Report ◽  
Growing Seasons ◽  
Pcr Product ◽  
Disease Surveys ◽  
Production Areas ◽  
Inner Mongolia Autonomous Region

Phytoplasma diseases on potatoes are not well understood and have gone largely undetected in China. During the growing seasons of 2005 through 2010, potato disease surveys were conducted in seed and commercial fields in Yunnan Province. Samples were also harvested from three seed potato production areas in the Inner Mongolia Autonomous Region in 2007 and 2010. Disease incidence in these fields ranged from 15 to 85%. Plants displayed symptoms of branch proliferation, aerial tuber formation, upward rolling yellowish and purplish apical leaves, and extremely short stolen or chain tubers (irregular-shaped tubers). Total DNA from 250 samples was extracted from the leaves, stems, and roots of symptomatic and asymptomatic plants. A nested PCR was performed by using primer pair P1/P7 followed by R16F2n/R16R2 to detect the presence of phytoplasmas (1,3). An approximate 1.2-kb PCR product was amplified from symptomatic plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting the 1.2-kb amplicon singly with restriction enzymes AluI, BfaI, MseI, HhaI, HinfI, HpaII, KpnI, RsaI, and TaqI. The RFLP patterns of 120 of the 250 samples matched patterns of the clover proliferation (CP) group (16SrVI) subgroup A (16SrVI-A) phytoplasma (1). In addition, the nested PCR product of P1A/P7A (2) following P1/P7 amplification was cloned and sequenced (GenBank Accession No. HQ609490). Nucleotide sequences were analyzed by iPhyClassifier software (4), confirming the relationship of this phytoplasma to ‘Candidatus Phytoplasma trifolii’ with RFLP patterns identical to group 16SrVI-A. To our knowledge, this is the first report of the CP group phytoplasmas associated with purple top diseased potatoes in China. 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.

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 Molecular Characterization of ‘Candidatus Phytoplasma asteris’ Associated with Aster Yellows-Diseased Potatoes in China

Plant Disease ◽  
2011 ◽  
Vol 95 (6) ◽  
pp. 777-777 ◽  
Author(s):  
M. Cheng ◽  
J. Dong ◽  
P. J. Laski ◽  
Z. Zhang ◽  
J. H. McBeath
Keyword(s):  
Nested Pcr ◽  
Inner Mongolia ◽  
Restriction Enzymes ◽  
Molecular Characteristics ◽  
Reference Pattern ◽  
Aster Yellows ◽  
Potato Plants ◽  
Group I ◽  
Pcr Product ◽  
Aster Yellows Phytoplasma

Potatoes (Solanum tuberosum) are one of the most important crops in China following rice, wheat, and corn. Aster yellows phytoplasma appeared to be widespread in China; it was found to cause diseases on alfalfa, oranges, peaches, periwinkles, bamboo (1), and cactus (4). However, scant information of this pathogen on potatoes is available except for a few short reports published during the 1950s. During the potato disease surveys conducted from 2005 to 2010 in Yunnan and Inner Mongolia, 10 to 35% of potato plants exhibited symptoms of yellowing or purpling of apical leaves, with the top leaves rolling inward and aerial tubers formation. Total DNA was extracted from midveins of leaves and roots of 125 diseased and asymptomatic plants with a DNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A nested PCR was carried out with the first round primer pair P1/P7 followed by the second round primer pair R16F2n/R16R2 (2,3). A PCR product of approximately 1.2 kb was amplified from diseased plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns were analyzed by digesting a 1.2-kb product using restriction enzymes AluI, BfaI, BstUI, HhaI, HpaI, KpnI, MseI, and RsaI. Comparing the RFLP patterns with previously published phytoplasma strains (2), aster yellows phytoplasma found on potato plants in Yunnan and Inner Mongolia belong to group I, subgroup B (16SrI-B). The PCR product from P1/P7, diluted 1:30, was amplified by using primer pair P1A/P7A (3) and P1A/16S-SR (3). The nested-PCR products from P1A/P7A and P1A/16S-SR were cloned into pCR8/GW/TOPO vector (Invitrogen, Carlsbad, CA) and sequenced by the Core Lab of the University of Alaska–Fairbanks and GENEWIZ (South Plainfield, NJ). The nucleotide sequence (GenBank Accession No. HQ599228) was analyzed by iPhyClassifier software and had 99.53% sequence identity to the reference strain (GenBank Accession No. M30790) for ‘Candidatus Phytoplasma asteris’. The RFLP similarity is 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 revealing the molecular characteristics of a phytoplasma associated with aster yellows-diseased potatoes in China. References: (1) H. Cai et al. Plant Prot. 31:38, 2005. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) I.-M. Lee et al. Int. J. Syst. Evol. Microbiol. 54:337, 2004. (4) W. Wei et al. Plant Dis. 91:461, 2007.

scholarly journals Molecular Typing ofNeisseria gonorrhoeaeIsolates by Opa-Typing and Ribotyping in New Delhi, India

2009 ◽  
Vol 2009 ◽  
pp. 1-6 ◽  
Author(s):  
Pejvak Khaki ◽  
Preena Bhalla ◽  
Ahmad Mir Fayaz ◽  
Sohiela Moradi Bidhendi ◽  
Majid Esmailzadeh ◽  
...  
Keyword(s):  
Molecular Typing ◽  
Preventive Measures ◽  
Restriction Enzymes ◽  
New Delhi ◽  
Pcr Product ◽  
Sexual Contacts ◽  
High Level ◽  
Epidemiological Characteristics ◽  
Epidemiological Characterization

Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics ofN. gonorrhoeaeisolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes,AvaII andHincII, and opa-typing withTaqI andHpaII for epidemiological characterization of gonococcal population. Ribotyping withAvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed withHincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed withTaqI-digested PCR product while opa-typing withHpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9%) between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains.

scholarly journals Molecular Characterization ofCryptosporidiumspp. in Wild Rodents of Southwestern Iran Using 18s rRNA Gene Nested-PCR-RFLP and Sequencing Techniques

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Jasem Saki ◽  
Masoud Foroutan-Rad ◽  
Reza Asadpouri
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
18S Rrna ◽  
Restriction Enzymes ◽  
18S Rrna Gene ◽  
Rrna Gene ◽  
Wild Rodents ◽  
Nested Polymerase Chain Reaction ◽  
Pcr Rflp ◽  
Southwest Of Iran

Background. Rodents could act as reservoir forCryptosporidiumspp. speciallyC. parvum, a zoonotic agent responsible for human infections. Since there is no information aboutCryptosporidiuminfection in rodents of Ahvaz city, southwest of Iran, hence, this survey was performed to determine the prevalence and molecular characterization ofCryptosporidiumspp. in this region.Materials and Methods. One hundred rodents were trapped from different regions of Ahvaz city. Intestine contents and fecal specimens of rodents were studied using both microscopy examination to identify oocyst and nested-polymerase chain reaction (PCR) technique for 18s rRNA gene detection. Eventually restriction fragment length polymorphism (RFLP) method usingSspIandVspIrestriction enzymes was carried out to genotype the species and then obtained results were sequenced.Results. Three out of 100 samples were diagnosed as positive and overall prevalence ofCryptosporidiumspp. was 3% using both modified Ziehl-Neelsen staining under light microscope and nested-PCR (830 bp) methods. Afterwards, PCR-RFLP was performed on positive samples andC. parvumpattern was identified. Finally PCR-RFLP findings were sequenced and presence ofC. parvumwas confirmed again.Conclusions. Our study showed rodents could be potential reservoir forC. parvum. So an integrated program for control and combat with them should be adopted and continued.

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.

scholarly journals First Report on the Detection and Molecular Characterization of Bovine Herpesvirus 1 from a Clinical case of Infectious Bovine Rhinotracheitis in Pakistan

2021 ◽  
Vol 41 (01) ◽  
pp. 160-162
Author(s):  
Aayesha Riaz
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
Bovine Herpesvirus ◽  
Nasal Discharge ◽  
Infectious Bovine Rhinotracheitis ◽  
Bovine Herpesvirus 1 ◽  
Nasal Swabs ◽  
Herpesvirus 1 ◽  
First Time

Bovine herpesvirus 1 (BHV-1) is a noteworthy reason for many Cattle/Buffalo diseases. Infectious bovine rhinotracheitis (IBR) is one of the diseases which are caused by the BHV-1. In the present study a cow which was suspected of IBR was examined. The animal was suffering from fever and respiratory distress along with rhinitis (red nose), in appetence, and dyspnea. The nasal mucosa and muzzle were distinctly inflamed, with nasal discharge. DNA from blood samples and nasal swabs were subjected to nested PCR using glycoprotein B gene (gB) Primers. The samples were found positive for BHV-1 gB gene. Sequencing and phylogenetic analysis revealed close similarities with other BHV-1 gB gene sequences. The accession numbers assigned to this pioneer sequences in GenBank are MT449510 (BHV-1-Pak 1) and MT449511 (BHV-1-Pak 2). In this study, we reported for the first time the detection of DNA of BHV-1 through nested PCR assay and molecular characterization of BHV-1 gB gene in Pakistan. This study will be useful in further diagnoses of BHV-1 in Pakistan and in development of BHV-1 vaccine to reduce economical losses due to IBR

scholarly journals Prevalence and Molecular Characterization of Giardia duodenalis in Calves in Turkey

2017 ◽  
Vol 45 (1) ◽  
pp. 6
Author(s):  
Mehmet Gultekin ◽  
Kerem Ural ◽  
Nuram Aysul ◽  
Adnam Ayan ◽  
Canberk Balikci ◽  
...  
Keyword(s):  
Molecular Characterization ◽  
Nested Pcr ◽  
Giardia Duodenalis ◽  
Pcr Assay ◽  
Giardia Infection ◽  
Western Turkey ◽  
Aegean Region ◽  
Animal Populations ◽  
Faecal Samples

Background: Giardia duodenalis (G. duodenalis) is an ubiquitous, flagellated intestinal protozoan with major public health significance worldwide. Limited data are available on the epidemiology of G. duodenalis in dairy cattle from Turkey. Determining the zoonotic potential of the Giardia infection requires molecular characterization. The aim of the present study was to investigate the prevalence and to molecularly characterize G. duodenalis in calves less than three months of age in Aydin, Aegean region of Turkey.Materials, Methods & Results: The study was conducted on different dairy farms in the south-western part of the Turkey, Aegean Region, Aydin. A total of 198 Holstein Friesian calves less than three months of age, of both sexes were enrolled into the study. Faecal samples from each calf were collected manually from the rectum using a disposable latex glove. The consistency of collected samples was recorded as diarrhoeic or non-diarrhoeic.  Diagnosis of G. duodenalis infection was made microscopically by detection of cysts in the faecal samples. One hundred and sixteen (58.5%) of the 198 faecal samples were diarrheic. Giardia cysts were found in 27 (23.28%) of the diarrheic samples and in 8 (9.76%) of nondiarrheic samples (P < 0.05). The overall prevalence of giardiosis in calves was determined as 17.67%. The prevalence of Giardia genotypes was identified by DNA sequence analysis of the beta-giardin gene for every PCR positive sample. The beta-giardin nested PCR assay was revealed assemblage A and sub-genotype A3 was detected in all of 35 samples (100%).Discussion: The highest prevalence of Giardia infection in calves is reported at the age between 1 and 6 months, and the prevalence shows decreased rate from the age of 6 months. The present study was conducted in Aydin, a province of south-western Turkey in the Aegean Region, and the overall prevalence from a total of 198 dairy calves was 17.67%. The prevalence rate in calves with diarrhoea was higher and reached up to 23.28%, whereas it was 9.76% in non-diarrhoeic calves. A prevalence study with molecular characterization of G. duodenalis isolates in cattle has not yet been reported from Turkey. Molecular studies have shown that mostly assemblage E predominates in cattle, but recent studies denoted that assemblage A is increasingly being detected and might be more widespread than expected before. In the present study, Giardia positive samples identified with a beta-giardin nested PCR assay. The sub-genotype A3 was identified in all samples. The same sub-genotype was identified in human and dog samples from different countries. Furthermore, sub-genotype A3 was found in humans and dogs from Turkey. In this context, results of the present study suggested an important role of calves as potential reservoirs of human infections in Turkey. In conclusion, epidemiological data revealed that G. duodenalis infection is frequent in calves with diarrhoea in Aydin, Turkey. The presence of the potentially zoonotic sub-genotype A3 and high prevalence of Giardia infection in diarrheic calves indicated the importance of treatment and necessary preventative measures. Further studies in human and animal populations living in this region are warranted regarding the zoonotic epidemiology of Giardia duodenalis.

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