scholarly journals Molecular identification of fungi associated with avocado (Persea americana Mill.) fruits

Agro-Science ◽  
2021 ◽  
Vol 20 (1) ◽  
pp. 80-86
Author(s):  
N.G. Iyanyi ◽  
A.E. Ataga ◽  
I.S. Rotimi ◽  
I. Blessing
Keyword(s):  
Pcr Amplification ◽  
Fungal Species ◽  
Persea Americana ◽  
Molecular Techniques ◽  
Control Measures ◽  
Universal Primer ◽  
Identification Of Fungi ◽  
Pcr Products ◽  
Fungal Organisms ◽  
Health Complications

Avocado (Persea americana Mill.) is grown for its nutritious fruit. However, the quantity and quality of these fruits are threatened by some fungal organisms which can cause health complications when it is consumed by man. DNA extraction provides a unique tool for identification of organisms. This study was conducted to isolate and identify fungal species associated with avocado fruit using both morphological and molecular techniques. Fungal species were isolated from Persea americana purchased from Choba market, Port Harcourt, Rivers State, Nigeria using Potato Dextrose Agar (PDA) as a growth medium. The morphology of isolated fungi on PDA were cotton-like blackish grey spots, white villous colonies, greyish powdery spores and black spores for isolates 1 to 4 respectively. Extraction of DNA from fungal isolates was carried out using Zymo Fungal/Bacteria DNA Miniprep Kit. PCR amplification of the ITS1-2 regions of isolates was carried out using fungal universal primer pair; ITS4 and ITS5.PCR amplification of the ITS1-2 gene sequences yielded amplicons between 537-580 base pairs. PCR products were sequenced and the sequencing result after BLAST search revealed the identity of the four fungal species as follows: Lasiodiplodia theobromae, Fusarium proliferatum, Penicillium sp. and Aspergillus niger. This study will promote the knowledge of specific fungal species associated with Persea americanna and help plant pathologists to proffer preventive and control measures to enhance fruit protection and yield quality.

scholarly journals Molecular characterisation of fungi associated with sewage-impacted soil

2021 ◽  
Vol 27 (2) ◽  
pp. 107-113
Author(s):  
N. G. Iyanyi ◽  
A. E. Ataga ◽  
E. A Obichi ◽  
S. C. Agbasoga
Keyword(s):  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Molecular Techniques ◽  
Septic Tank ◽  
Nucleotide Sequence Analysis ◽  
Molecular Method ◽  
Base Pairs ◽  
Universal Primer ◽  
Fungal Isolates

The decay of faecal matter from a septic system causes the arousal of fungi in the surrounding soil. These fungi can cause diseases if there is sewage spillage containing untreated or improperly treated wastewaters. Molecular techniques of identification of fungi have shown to be more dependable than traditional methods of identifying fungal species. This study was carried out to identify the fungal species associated with soil obtained from sewage-impacted soil near a septic tank using both traditional cultural techniques and molecular method. Fungi associated with the soil samples were isolated using serial dilution and Potato Dextrose Agar (PDA) method. Deoxyribonucleic Acid (DNA) was extracted from the pure cultures of fungal isolates using Quick DNA Fungal/Bacterial Miniprep kit. Polymerase Chain Reaction (PCR) amplification of internal transcribed spacer (ITS) region of the fungal isolates was carried out using universal primer pair; ITS4 and ITS5. The PCR products were sequenced and the sequences were blasted against National Centre for Biotechnology Information database. The result of the nucleotide sequence analysis revealed the identity of the isolates as Trichoderma harzanium with 580 base pairs and Aspergillus welwitschiae with 560 base pairs. Sequences of the isolates were aligned and compared with sequences on GenBank and a phylogenetic tree was constructed. The cultural method only aided in suggesting the suspected genera of the isolates while the molecular method was able to identify the isolates to the species level. This study will promote the knowledge of the fungal species associated with sewage-impacted soil and also aid researchers in proffering ways to enhance the prevention/control of diseases associated with sewage spill. Keyword: Septic tank, fungi, soil, phylogeny, sequencing

scholarly journals Molecular Characterization of Fungi Associated with Cowdung-impacted Soil

2020 ◽  
pp. 3819-3827
Author(s):  
Iyanyi N. G. ◽  
Ataga A. E. ◽  
Akinido C. E. ◽  
Udobong E.
Keyword(s):  
Disease Transmission ◽  
Pcr Amplification ◽  
Geotrichum Candidum ◽  
Fungal Species ◽  
Cow Dung ◽  
Base Pairs ◽  
Universal Primer ◽  
Absidia Corymbifera ◽  
Fungal Organisms

Dung is the undigested remains of food taken in by herbivores. It is a combination of faeces and urine at a ratio of 3:1. It mostly consists of cellulose, hemicelluloses and lignin. Cow dung harbours several microorganisms, including various species of fungi. The aim of this study was to isolate and characterize the fungal organisms associated with cow dung-impacted soil using both traditional cultural techniques and molecular method. DNA extraction was carried out using Zymo Quick DNA Fungal/Bacterial Mini prep kit. The Polymerase Chain Reaction (PCR) amplification of the Internal Transcribed Spacer (ITS) genes, using the universal primer pair; ITS4 and ITS5, generated amplicon sizes of 372 and 834 base pairs. The amplicons were sequenced using Sanger sequencing and the isolates were identified as Lichtheimia ramosa and Galactomyces pseudocandidus. Phylogenetic analysis showed the relationship that exists between the fungal species and other closely-related species in GenBank such as: Aspergillus amstelodami, Absidia corymbifera, Mycocladus corymbiferus and Geotrichum candidum. This study has provided information on some of the fungal organisms harboured by cowdung-impacted soil which will help predict the possibility for disease transmission to plants or humans through cow dung.

scholarly journals Fast and reliable PCR/sequencing/RFLP assay for identification of fungi in onychomycoses

2006 ◽  
Vol 55 (9) ◽  
pp. 1211-1216 ◽  
Author(s):  
Michel Monod ◽  
Olympia Bontems ◽  
Christophe Zaugg ◽  
Barbara Léchenne ◽  
Marina Fratti ◽  
...  
Keyword(s):  
Rflp Analysis ◽  
Fungal Species ◽  
Universal Primers ◽  
Cure Rate ◽  
Routine Identification ◽  
Identification Of Fungi ◽  
Pcr Products ◽  
Nail Sample ◽  
Fungal Dna ◽  
Rflp Assay

Fusarium spp. and other non-dermatophyte fungi are repeatedly isolated from abnormal nails. To investigate whether these fungi are the aetiological agents of infection or simply transient contaminants, a PCR/sequencing/RFLP assay was developed for direct and routine identification of the infecting fungi in onychomycosis. Fungal DNA was readily extracted using a commercial kit after dissolving nail fragments in a Na2S solution. Amplification of part of the 28S rDNA by PCR was performed with universal primers and the fungal species were identified by sequencing. The PCR/sequencing results were comparable with microbiological identification from the same nail sample. In addition to dermatophytes, Fusarium spp. and other less frequently isolated non-dermatophyte fungi were identified as single fungal agents in onychomycosis. Moreover, mixed infections were clearly demonstrated in 10 % of cases by RFLP analysis of PCR products. Identification of infectious agents could be obtained in 2 days, whilst results from fungal cultures take 1–3 weeks. Rapid and reliable molecular identification of the infectious fungus expedites the choice of appropriate antifungal therapy, thereby improving the cure rate of onychomycosis.

Polymerase Chain Reaction–Restriction Fragment Length Polymorphism Analysis of a Short Fragment of the Cytochrome b Gene for Identification of Flatfish Species

1998 ◽  
Vol 61 (12) ◽  
pp. 1684-1685 ◽  
Author(s):  
ANA CÉSPEDES ◽  
TERESA GARCÍA ◽  
ESTHER CARRERA ◽  
ISABEL GONZÁLEZ ◽  
BERNABÉ SANZ ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Cytochrome B ◽  
Pcr Amplification ◽  
Cytochrome B Gene ◽  
Polymorphism Analysis ◽  
Chain Reaction ◽  
Greenland Halibut ◽  
Universal Primer ◽  
Pcr Products ◽  
Polymerase Chain

Restriction site analysis of polymerase chain reaction (PCR) products from a conserved region of the cytochrome b gene has been used for the specific identification of sole (Solea solea), European plaice (Pleuronectes platessa), flounder (Platichthys flesus), and Greenland halibut (Reinhardtius hippoglossoides). PCR amplification of the cytochrome b gene using a universal primer together with a primer specifically designed as a part of this study produced a 201-bp fragment in all species analyzed. Digestions of the PCR products with Sau3Al, BsmAl, Rsal, and Mn/l endonucleases, followed by agarose gel electrophoresis of the digested PCR products, yielded specific profiles that enabled direct identification of each species analyzed.

scholarly journals Isolation and Molecular Identification of some Fungi Associated with Jatropha curcas (L.)

2021 ◽  
Vol 37 (2) ◽  
pp. 103-112
Author(s):  
N.G Iyany ◽  
A.E Ataga
Keyword(s):  
Jatropha Curcas ◽  
Rhizopus Oryzae ◽  
Fungal Species ◽  
Molecular Techniques ◽  
Accurate Identification ◽  
Universal Primer ◽  
Aspergillus Tamarii ◽  
Jatropha Curcas L ◽  
Fungal Isolates ◽  
Aspergillus Sp

Jatropha curcas is a plant of great economic importance that experiences high incidence of fungal attack. Misidentification of the fungal species is bound to occur with the use of traditional cultural methods where organisms are identified morphologically and/or microscopically. This study was carried out to isolate and identify the fungi associated with Jatropha curcas (L.) using both traditional/ cultural techniques and molecular methods. The fungi were isolated from diseased leaves and stems of J. curcas using both Standard Blotter and Potato Dextrose Agar (PDA) methods. DNA was extracted from the fungal isolates using Zymo Fungal/Bacteria DNA MiniPrep Kit. Amplification of the Internal Transcribed Spacer (ITS) regions of the fungal isolates was carried out using fungi universal primer pairs for ITS4 and ITS5. The amplicons were sequenced and the isolates were identified as Penicillium brevicompactum, Aspergillus sp., Botryosphaeria rhodina, Aspergillus nomius, Aspergillus tamarii, Rhizopus oryzae, Penicillium citrinum and Fusarium solani. Phylogenetic analysis was carried out to know the relationship between the isolates and other closely-related species in GenBank. Jatropha curcas is colonized by many fungal species some of which may be pathogenic to the plant, and molecular techniques pose the best alternative for accurate identification of these organisms. Keywords: Jatropha curcas, fungi, polymerase chain reaction, phylogeny, sequencing

scholarly journals A Case of DisseminatedMycobacterium aviumInfection in a Dog in Greece

2014 ◽  
Vol 2014 ◽  
pp. 1-3 ◽  
Author(s):  
V. Kontos ◽  
E. I. Papadogiannakis ◽  
G. Mantziaras ◽  
M. Styliara ◽  
S. Kanavaki
Keyword(s):  
Lymph Node ◽  
Lymph Nodes ◽  
Mycobacterium Avium ◽  
Fine Needle Aspiration ◽  
Bacterial Culture ◽  
Pcr Amplification ◽  
Needle Aspiration ◽  
Molecular Techniques ◽  
Fine Needle ◽  
Pcr Products

A Basset Hound dog was presented with anorexia, fever, diarrhea, significant level of splenomegaly, and enlargement of mesenteric and superficial lymph nodes. Cytology of fine-needle-aspiration material, obtained from popliteal lymph node, revealed macrophages with intracytoplasmic, nonstaining, slender, rod-like structures, indicative of mycobacteria. Bacterial culture of lymph node aspirated material produced a colony which by means of molecular techniques (PCR amplification and hybridization of PCR products) was subsequently identified asMycobacterium avium. This is the first report of disseminatedM. aviuminfection in a dog in Greece.

scholarly journals Morpho-molecular identification and first report of Fusarium equiseti in causing chilli wilt from Kashmir (Northern Himalayas)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ammarah Hami ◽  
Rovidha S. Rasool ◽  
Nisar A. Khan ◽  
Sheikh Mansoor ◽  
Mudasir A. Mir ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Pcr Amplification ◽  
Its Region ◽  
Fungal Species ◽  
Pathogenicity Test ◽  
Kashmir Valley ◽  
Fusarium Equiseti ◽  
Infected Root ◽  
Fusarium Sp ◽  
High Degree

AbstractChilli (Capsicum annuum L.) is one of the most significant vegetable and spice crop. Wilt caused by Fusarium Sp. has emerged as a serious problem in chilli production. Internal transcribed spacer (ITS) region is widely used as a DNA barcoding marker to characterize the diversity and composition of Fusarium communities. ITS regions are heavily used in both molecular methods and ecological studies of fungi, because of its high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. In the present study we focused on morphological and molecular characterization of pathogen causing chilli wilt. Chilli plants were collected from four districts of Kashmir valley of Himalayan region. Pathogens were isolated from infected root and stem of the plants. Isolated pathogens were subjected to DNA extraction and PCR amplification. The amplified product was sequenced and three different wilt causing fungal isolates were obtained which are reported in the current investigation. In addition to Fusarium oxysporum and Fusarium solani, a new fungal species was found in association with the chilli wilt in Kashmir valley viz., Fusarium equiseti that has never been reported before from this region. The studies were confirmed by pathogenicity test and re-confirmation by DNA barcoding.

scholarly journals Presence of plasmid-mediated quinolone resistance (PMQR) genes in non-typhoidal Salmonella strains with reduced susceptibility to fluoroquinolones isolated from human salmonellosis in Gyeonggi-do, South Korea from 2016 to 2019

Gut Pathogens ◽  
2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Sohyun Lee ◽  
Nanjoo Park ◽  
Sujung Yun ◽  
Eunseon Hur ◽  
Jiwon Song ◽  
...  
Keyword(s):  
South Korea ◽  
Pcr Amplification ◽  
Public Health Problem ◽  
Test Method ◽  
Quinolone Resistance ◽  
Human Salmonellosis ◽  
Pcr Products ◽  
Antimicrobial Susceptibility Tests ◽  
Susceptibility Tests ◽  
Sequence Types

AbstractNon-typhoidal salmonellosis remains a pressing public health problem worldwide. Quinolones, particularly fluoroquinolones, are widely used to treat various infections, including non-typhoidal salmonellosis, which can be a serious illness. The emergence of fluoroquinolone-resistant Salmonella has resulted in treatment failure and high mortality rates. In this study, we estimated the presence of plasmid-mediated quinolone resistance (PMQR) genes in Salmonella enterica isolated from human salmonellosis patients in South Korea from 2016 to 2019. We evaluated the association of these genes with fluoroquinolone susceptibility. Antimicrobial susceptibility tests for Salmonella isolates were performed using the Vitek II system, and the minimum inhibitory concentrations (MIC) of ciprofloxacin and levofloxacin were determined using the E-test method. Plasmid-mediated quinolone resistance (PMQR) genes were detected by PCR amplification and quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes were analyzed following Sanger sequencing of the PCR products. Thirty-four Salmonella strains with reduced susceptibility to fluoroquinolones (ciprofloxacin MIC ≥ 0.125 µg/mL and levofloxacin MIC ≥ 0.25 µg/mL) were selected from 208 human clinical Salmonella isolates. Among them, 22 Salmonella strains harbored one PMQR gene (qnrA, qnrB, or qnrS), and three Salmonella strains carried two PMQR genes (qnrS and aac(6′)-Ib-cr or qnrA and qnrB). qnrS was the most common PMQR gene. Serotyping revealed that Salmonella 4,[5]12:i:- (32.4%, 11/34) and Salmonella Typhimurium (29.4%, 10/34) were the two most predominant serovars, and Multi-locus sequence typing (MLST) showed that ST19 and ST34 were the most frequent sequence types. In conclusion, qnr gene-positive Salmonella 4,[5],12:i:- and Salmonella Typhimurium were the main serovars responsible for reduced susceptibility to fluoroquinolones. Therefore, our findings suggest that PMQR-positive Salmonella strains, which can be isolated from various samples including human, food, and the environment, should be carefully monitored.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

2021 ◽  
pp. 030098582199156
Author(s):  
Alexandra N. Myers ◽  
Unity Jeffery ◽  
Zachary G. Seyler ◽  
Sara D. Lawhon ◽  
Aline Rodrigues Hoffmann
Keyword(s):  
Polymerase Chain Reaction ◽  
Diagnostic Accuracy ◽  
Molecular Techniques ◽  
Fungal Culture ◽  
Chain Reaction ◽  
Identification Of Fungi ◽  
Fungal Dna ◽  
Polymerase Chain ◽  
Panfungal Pcr ◽  
Pcr Targeting

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Two main clusters within Trypanosoma cruzi zymodeme 3 are defined by distinct regions of the ribosomal RNA cistron

Parasitology ◽  
2002 ◽  
Vol 124 (2) ◽  
pp. 177-184 ◽  
Author(s):  
M. B. A. MENDONÇA ◽  
N. S. NEHME ◽  
S. S. SANTOS ◽  
E. CUPOLILLO ◽  
N. VARGAS ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Ribosomal Rna ◽  
Southern Blotting ◽  
Pcr Amplification ◽  
Rrna Gene ◽  
Pcr Products ◽  
Phylogenetic Lineages ◽  
Definition Of ◽  
Ribosomal Cistron ◽  
Panstrongylus Geniculatus

Trypanosoma cruzi is currently classified into 2 major phylogenetic lineages, T. cruzi I and II, that correlate with the formerly described zymodeme 1 and 2, respectively. Another isoenzymic group (zymodeme 3–Z3) was also described. In this study, we analysed the genetic diversity among Z3 isolates of the Brazilian Amazon by restriction fragment length polymorphism of the intergenic transcribed spacers (ITSs) of the ribosomal RNA cistron and the size of the divergent domain D7 of the 24Sα rRNA gene. DNAs from 12 T. cruzi Z3 isolates obtained from humans (2), Panstrongylus geniculatus (1), and Rhodnius brethesi (9) were submitted to PCR amplification of the ITSs plus the 5·8S rDNA. The PCR products were digested with 4 distinct endonucleases and the profiles analysed by a numerical methodology. The phenetic dendrogram revealed a clear dichotomy in the Z3 group, defining 2 groups that were named Z3-A and Z3-B. Dimorphism was also found in the band sizes of the amplified D7 divergent domain of the 24Sα rDNA, which showed a perfect correlation with the ITSs clustering. The organization of the ribosomal cistron was investigated by Southern blotting and shown to be conserved in the genome of the 2 Z3 groups. This study shows that the rDNA cistron allows the definition of 2 distinct subclusters in Z3 isolates.

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