Light Microscopy and PCR Based Detection of the Causal Agent of Leaf Mosaic of Jute

Leaf mosaic transmitted by whitefly is a devastating disease of jute. It is thought to be caused by a virus belonging to begomovirus genus under geminivirus family. To confirm the identity of the causal agent, infected and healthy leaves were studied using light microscope and by using polymerase chain reaction (PCR) technique of DNA. The inclusion bodies were observed under light microscope as large, blue-violet, prominent inclusion bodies in the nucleus of the infected leaf tissues. In molecular detection technique DNA from infected and healthy plants was extracted and analyzed by polymerase chain reaction (PCR) using degenerate primers PALIv1978/PARIc496. PCR fragment of the expected size 1.2kb for the common region (CR) in the geminivirus were obtained from infected plants. DNA collected from healthy plant did not show any band during electrophoresis. Therefore, it can be concluded that leaf mosaic of jute is cause by a virus.DOI: http://dx.doi.org/10.3329/pa.v22i1-2.16463 Progress. Agric. 22(1 & 2): 19 - 25, 2011

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AN IMPROVED METHOD FOR INHIBITOR FREE NUCLEIC ACIDS FROM POLYPHENOL RICH LEAVES OF MUSA SPP

FLORA AND FAUNA ◽

10.33451/florafauna.v23i1pp25-36 ◽

2017 ◽

Vol 23 (1) ◽

Author(s):

N.NANDHA KUMAR ◽

K. SOURIANATHA SUNDARAM ◽

D. SUDHAKAR ◽

K.K. KUMAR

Keyword(s):

Polymerase Chain Reaction ◽

Quantitative Polymerase Chain Reaction ◽

Proteinase K ◽

Chain Reaction ◽

Banana Plant ◽

High Molecular Weight Dna ◽

Musa Spp ◽

Leaf Tissues ◽

Polymerase Chain

Excessive presence of polysaccharides, polyphenol and secondary metabolites in banana plant affects the quality of DNA and it leads to difficult in isolating good quality of DNA. An optimized modified CTAB protocol for the isolation of high quality and quantity of DNA obtained from banana leaf tissues has been developed. In this protocol a slight increased salt (NaCl) concentration (2.0M) was used in the extraction buffer. Polyvinylpyrrolidone (PVP) and Octanol were used for the removal of polyphenols and polymerase chain reaction (PCR) inhibitors. Proteins like various enzymes were degraded by Proteinase K and removed by centrifugation from plant extract during the isolation process resulting in pure genomic DNA, ready to use in downstream applications including PCR, quantitative polymerase chain reaction (qPCR), ligation, restriction and sequencing. This protocol yielded a high molecular weight DNA isolated from polyphenols rich leaves of Musa spp which was free from contamination and colour. The average yields of total DNA from leaf ranged from 917.4 to 1860.9 ng/ìL. This modified CTAB protocol reported here is less time consuming 4-5h, reproducible and can be used for a broad spectrum of plant species which have polyphenol and polysaccharide compounds.

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Quantification of Venturia inaequalis Growth in Malus × domestica with Quantitative Real-Time Polymerase Chain Reaction

Plant Disease ◽

10.1094/pdis-12-11-1058-re ◽

2012 ◽

Vol 96 (12) ◽

pp. 1791-1797 ◽

Cited By ~ 18

Author(s):

Michele Gusberti ◽

Andrea Patocchi ◽

Cesare Gessler ◽

Giovanni A. L. Broggini

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Malus Domestica ◽

Venturia Inaequalis ◽

Simultaneous Detection ◽

Scab Resistance ◽

Infected Leaf ◽

Chain Reaction ◽

Limit Of Quantification ◽

Polymerase Chain

A quantitative real-time polymerase chain reaction (qPCR) was developed and validated for quantification of Venturia inaequalis in infected leaf tissue of Malus × domestica. The method is based on dual-labeled hybridization probes, allowing simultaneous detection of host and pathogen DNA within one single reaction. Limit of quantification for the pathogen was 0.5 pg per reaction and, for the host, reached 5 pg per reaction. The fungal growth measured in four apple cultivars 2 weeks after inoculation significantly correlated with their different level of scab resistance and allowed the observation of ontogenic resistance. After sporulation on the youngest leaf, fungal biomass in susceptible ‘Gala’ was 118 times higher than in resistant ‘Florina’ and ‘Discovery’ while intermediate values were found with the intermediate susceptible ‘Milwa’. Correlation was also observed between severity classes obtained by visual scoring of symptoms and qPCR results. Moreover, qPCR demonstrated validity of the developed method as a disease severity forecast tool 10 days after the pathogen's inoculation and prior to the appearance of the symptoms. Applications of the methodology can include the quantification of scab resistance during breeding programs, evaluation of fungicide and biocontrol efficacy, and quantification of the fitness of different pathogenic strains.

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Detection of Subgroup III Geminivirus Isolates in Leaf Extracts by Degenerate Primers and Polymerase Chain Reaction

Phytopathology ◽

10.1094/phyto-86-1288 ◽

1996 ◽

Vol 86 (12) ◽

pp. 1288 ◽

Cited By ~ 179

Author(s):

S. Wyatt

Keyword(s):

Polymerase Chain Reaction ◽

Chain Reaction ◽

Leaf Extracts ◽

Degenerate Primers ◽

Polymerase Chain

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Deconstructing the Polymerase Chain Reaction II: an improved workflow and effects on artifact formation and primer degeneracy

PeerJ ◽

10.7717/peerj.7121 ◽

2019 ◽

Vol 7 ◽

pp. e7121

Author(s):

Ankur Naqib ◽

Silvana Poggi ◽

Stefan J. Green

Keyword(s):

Polymerase Chain Reaction ◽

Community Structure ◽

Microbial Community ◽

Annealing Temperature ◽

Degenerate Primer ◽

Chain Reaction ◽

Degenerate Primers ◽

Polymerase Chain ◽

Dna Template ◽

Microbial Dna

Polymerase chain reaction (PCR) amplification of complex microbial genomic DNA templates with degenerate primers can lead to distortion of the underlying community structure due to inefficient primer-template interactions leading to bias. We previously described a method of deconstructed PCR (“PEX PCR”) to separate linear copying and exponential amplification stages of PCR to reduce PCR bias. In this manuscript, we describe an improved deconstructed PCR (“DePCR”) protocol separating linear and exponential stages of PCR and allowing higher throughput of sample processing. We demonstrate that the new protocol shares the same benefits of the original and show that the protocol dramatically and significantly decreases the formation of chimeric sequences during PCR. By employing PCR with annealing temperature gradients, we further show that there is a strong negative correlation between annealing temperature and the evenness of primer utilization in a complex pool of degenerate primers. Shifting primer utilization patterns mirrored shifts in observed microbial community structure in a complex microbial DNA template. We further employed the DePCR method to amplify the same microbial DNA template independently with each primer variant from a degenerate primer pool. The non-degenerate primers generated a broad range of observed microbial communities, but some were highly similar to communities observed with degenerate primer pools. The same experiment conducted with standard PCR led to consistently divergent observed microbial community structure. The DePCR method is simple to perform, is limited to PCR mixes and cleanup steps, and is recommended for reactions in which degenerate primer pools are used or when mismatches between primers and template are possible.

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Molecular Identification of Bacterial Pathogen Infecting Coconut Leaf Beetle

Buletin Palma ◽

10.21082/bp.v16n2.2015.147-153 ◽

2016 ◽

Vol 16 (2) ◽

pp. 147

Author(s):

JELFINA C. ALOUW ◽

DIANA NOVIANTI ◽

MELDY L.A. HOSANG

Keyword(s):

Polymerase Chain Reaction ◽

16S Rrna ◽

Serratia Marcescens ◽

Molecular Identification ◽

Ribosomal Rna ◽

Causal Agent ◽

16S Ribosomal Rna ◽

Font Size ◽

Chain Reaction ◽

Polymerase Chain

ABSTRACT Many species of microorganisms can cause diseases and mortality of insect pests. Accurate detection and identification of the entomophatogens are essential for development of biological control agent to the pest. Brontispa longissima, a serious and invasive pest of coconut, was infected by bacterium causing mortality of the larvae and pupae in coconut field. Objective of the research was to identify bacterium as a causal agent of the field-infected B. longissima using molecular technique. Research was conducted between April and August 2011. Molecular identification using polymerase chain reaction (PCR) amplification of 16s ribosomal RNA of the infected larvae and sequencing of the gene showed that Serratia marcescens is the causal agent of the disease.Keywords: Brontispa longissima, coconut, 16s rRNA, Serratia marcescens. Identifikasi Molekular Bakteri Pathogen yang Menginfeksi Hama Daun Kelapa Brontispa longissima(Coleoptera:Chrysomelidae)ABSTRAK Banyak mikroorganisme dapat menimbulkan penyakit pada serangga hama. Deteksi dan identifikasi yang akurat dari pathogen penyebab penyakit pada serangga (entomopathogen) hama merupakan tahap yang penting dalam pengembangan pengendalian biologi untuk hama tersebut. Brontispa longissima sebagai hama penting dan bersifat invasif pada tanaman kelapa diinfeksi oleh sejenis bakteri yang menyebabkan kematian larva dan pupa dari serangga tersebut di lapangan. Penelitian ini bertujuan untuk mengidentifikasi organisme penyebab penyakit pada hama B. longissima dengan menggunakan teknik molekuler. Penelitian dilaksanakan pada bulan April sampai dengan Agustus 2011. Identifikasi bakteri dilakukan dengan mengamplifikasi 16s ribosomal RNA dari larva yang terinfeksi dengan menggunakan PCR (polymerase chain reaction). Hasil analisis sekuens nukleotida 16s ribosomal RNA dari larva yang terinfeksi menunjukkan bahwa Serratia marcescens adalah bakteri penyebab dari penyakit tersebut.Kata kunci: Brontispa longissima, kelapa, 16s rRNA, Serratia marcescens.

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Low incidence of peach latent mosaic viroid in peach mother blocks in Serbia

Pesticidi i fitomedicina ◽

10.2298/pif1402109j ◽

2014 ◽

Vol 29 (2) ◽

pp. 109-114

Author(s):

Darko Jevremovic ◽

Svetlana Paunovic

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcription ◽

Mosaic Disease ◽

Causal Agent ◽

Rt Pcr ◽

Chain Reaction ◽

Polymerase Chain ◽

Low Incidence

Peach latent mosaic viroid (PLMVd) is the causal agent of peach latent mosaic disease that is common on peaches and nectarines worldwide. Most of the isolates do not cause any symptoms on the foliage and the disease may be latent for years. A survey to investigate the presence of PLMVd in selected peach mother blocks in 9 Serbian districts was carried out in 2011 through 2013. A total of 315 trees/samples originating from 43 mother blocks, representing 35 peach and nectarine varieties and 2 rootstocks, were tested by Reverse Transcription - Polymerase Chain Reaction (RT-PCR). PLMVd was detected in 13 samples (4.13%) belonging to 7 varieties and one vineyard peach rootstock. Infected samples were found in 7 mother blocks from 3 districts. Our results indicated a low incidence of PLMVd in the analyzed peach mother blocks.

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