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 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 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 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 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 Molecular Detection of Apiosporina morbosa, Causal Agent of Black Knot in Prunus virginiana

Plant Disease ◽  
2005 ◽  
Vol 89 (8) ◽  
pp. 815-821 ◽  
Author(s):  
J. X. Zhang ◽  
W. G. D. Fernando ◽  
W. R. Remphrey
Keyword(s):  
Pcr Amplification ◽  
High Sensitivity ◽  
Its Region ◽  
Fungal Species ◽  
Causal Agent ◽  
Diseased Plant ◽  
Pcr Assay ◽  
Single Spore ◽  
Prunus Virginiana ◽  
Pure Cultures

A specific and sensitive polymerase chain reaction (PCR) assay was developed to detect Apiosporina morbosa, the causal agent of black knot disease on chokecherry, Prunus virginiana (including the cultivar ‘Shubert Select’). A pair of A. morbosa-specific forward and reverse primers (AMF and AMR) was designed from the internal transcribed spacer (ITS) regions of A. morbosa, preamplified by universal ITS primers ITS1 and ITS4, and compared with the ITS region sequences of Fusarium, Alternaria, Phoma, and Cladosporium species associated with black knots. The primers were tested for their specificity to A. morbosa detection in the PCR assays using DNA derived from 64 pure cultures, including 42 single-spore isolates of A. morbosa and 22 isolates of other fungi, as well as healthy and diseased plant branches collected from the field. A product of ~400 bp was amplified from DNA of all isolates belonging to A. morbosa. No product was amplified from DNA of other fungal species, confirming the specificity of the newly designed primers. Within plant tissues, the pathogen was detected at further distances from the edges of knots on thicker branches bearing larger knots compared with thinner branches bearing smaller knots. The PCR assay has shown high sensitivity, needing only 100 fg of the A. morbosa DNA for a reliable PCR amplification with the AMF and AMR primers.

scholarly journals Botryosphaeria Tree Fungal Pathogens and Their Diversity

2021 ◽  
Vol 10 (1) ◽  
pp. ACCEPTED
Author(s):  
Wendu A. Darge ◽  
Samuel S. Woldemariam
Keyword(s):  
Fungal Pathogens ◽  
Global Climate ◽  
Its Region ◽  
Fungal Species ◽  
Molecular Techniques ◽  
Nutrient Deficiencies ◽  
Morphological Findings ◽  
Optimal Health ◽  
Molecular Phylogenetic ◽  
Host Tree Species

The genus Botryosphaeria identified in 1863 as saprophytes of dead tissue of woody plants have been described as pathogens of economically important plantation trees in agriculture and native forests. The genus is a species-rich, worldwide distributed occurring on diverse host ranges. Species of the Botryosphaeria are reported as the pathogens of many plantation trees, including species of Acacia, Eucalyptus, and Pinus causing canker and rapid dieback diseases which often end up in death. Botryosphaeria fungal pathogens have cross pathogenicity on different host tree species which enables them important and focus area of research. The taxonomy of Botryosphaeria spp. have been under research, identification of these fungi has generally been based on morphological features of the anamorph that usually seen under the microscope. Characters that are used to classify genera in the Botryosphaeria have mostly relied on the macroscopic features of the ascospores and the conidial features. Currently, molecular techniques such as DNA sequencing involving amplification of ITS region are important for exact identification of the genera to species level. Recent molecular, phylogenetic and morphological findings showed that order Botryosphaeriales is diverse consisting nine families and 33 genera with 23 genera only in the family Botryosphaeriaceae. Botryosphaeria spp. are naturally endophytes associated with tree plants known to cause monocyclic or polycyclic diseases resulting in polyetic epidemics. The factor that makes plants more prone to Botryosphaeria fungal species is assumed to be stress or wounding associated with the host plants. Global climate change driven drought is an important factor that initiate stress resulting in nutrient deficiencies. Botryosphaeria fungal tree diseases can be best managed by ensuring plants are in optimal health through appropriate integration of cultural, silvicultural and fungicidal applications to effectively prevent and control the diseases.

scholarly journals First Report of Cane Blight on Blackberry Caused by Diaporthe eres in Croatia

Plant Disease ◽  
2011 ◽  
Vol 95 (5) ◽  
pp. 612-612 ◽  
Author(s):  
K. Vrandecic ◽  
D. Jurkovic ◽  
J. Cosic ◽  
J. Postic ◽  
L. Riccioni
Keyword(s):  
Morphological Characteristics ◽  
Its Region ◽  
Aluminum Foil ◽  
Fungal Species ◽  
First Report ◽  
5.8S Rdna ◽  
Potted Plants ◽  
Conidial State ◽  
Fungal Isolates ◽  
And Control

A cane disease of blackberry (Rubus sp.) cv. Thornfree was observed in May and June 2010 in two growing regions in the eastern part of Slavonia in Croatia. Symptoms consisted of bleached areas between and around cane nodes with some canes showing wilt symptoms. Infected areas were covered with numerous, black pycnidia immersed in the epidermal tissue. Disease occurrence in orchards growing cv. Thornfree ranged between 1 and 15%. Thirty disease samples were collected, disinfected (1 min in 70% ethanol and 2 min in 1% NaOCl), and placed in a moist chamber for 4 days. Fungal sporulating structures were then picked off and placed on potato dextrose agar (PDA). Fungal isolates obtained were identified as a Phomopsis sp., the conidial state of Diaporthe (3), on the basis of cultural and morphological characteristics. Alpha conidia were unicellular, hyaline, fusiform, sometimes tapering toward one or both ends, biguttulate (sometimes with several guttules), and 5.2 to 9.7 × 1.4 to 2.7 μm (average 6.5 × 2.1 μm). Beta conidia were hyaline, aseptate, filiform, hamate, and 16.6 to 28.2 × 0.5 to 1.5 μm (average 24.0 × 1.1 μm). The teleomorph was not observed. Biomolecular analysis was performed to identify the fungal species by sequencing the internal transcribed spacer (ITS) region spanning ITS 1, 5.8S rDNA, and ITS 2 of two isolates (Phk1 and Phk2). The amplified product was sequenced (GenLab-Enea, Rome, Italy) and a BLAST search of the NCBI nucleotide database was performed. Sequences from Phk1 and Phk2 (GenBank Accession Nos. HQ533144 and HQ533143, respectively) were identical to authentic and vouchered Diaporthe eres Nitschke (GenBank DQ491514, BPI 748435, and CBS 109767) ITS sequences in GenBank. Fungal isolates for pathogenicity tests were grown on PDA at 25°C for 7 days (12 h light/dark regimen). Inoculations were made on 30 to 40 cm long green shoots of potted plants of the blackberry cv. Thornfree. One-centimeter long wounds were made with a sterile scalpel and mycelia of D. eres were placed in the wounds. Inoculation sites were covered with a piece of wet cotton wool and aluminum foil to retain moisture. Three replications of 10 plants each were inoculated and these plus 10 control plants (inoculated with plugs of PDA only) were maintained in a growth chamber at 25°C. After 25 days, lesions developed on all 30 inoculated plants that averaged 15 mm long and control plants remained symptomless. D. eres was reisolated from inoculated plants, thus completing Koch's postulates. Phomopsis spp. have previously been reported on blackberry canes in Serbia (1) and Yugoslavia (2,4), however, to our knowledge, this is the first report of the occurrence of D. eres (anamorph P. oblonga) on blackberry in Croatia. References: (1) M. Arsenijevic. Biljni Lekar 34:117, 2006. (2) M. Muntanola-Cvetkovic et al. Zast. Bilja 36:325, 1985. (3) B. C. Sutton. Page 569 in: The Coelomycetes. CMI, Kew, Surrey, UK, 1980. (4) M. Veselic et al. Zast. Bilja 49:76, 1998.

scholarly journals Molecular Depiction of Thirteen Indian Toxic Plants with ITS Markers

2020 ◽  
Vol 2 (2) ◽  
pp. 179-189
Author(s):  
Kiran Kumari ◽  
Saurabh Bhargava ◽  
Rajvinder Singh
Keyword(s):  
Plant Species ◽  
Forensic Toxicology ◽  
Its Region ◽  
Molecular Techniques ◽  
Reference Sequence ◽  
Morphological Identification ◽  
Molecular Technique ◽  
Accurate Identification ◽  
Universal Primer ◽  
Toxic Plants

Plant identification is an overwhelming task due to different biological attributes and great diversity in plant species. In the absence of physical markers, molecular techniques have become useful for the identification of species of origin of medicinal plant seeds, pastes, and formulations of suspected plants. The ITS region of nuclear rRNA was amplified from thirteen different toxic plant species by using universal primer ITS 1 & 4. Nucleotide sequences of all selected plants were submitted in NCBI and accession numbers were acquired. The results of this study give accurate identification of thirteen plant species and proved the ITS region of 18s-26s nuclear ribosome to be an important tool for phylogenetic analysis and species identification of plants. The sequence was aligned with top matched reference sequence and presented in Clustal Omega software for making a phylogenetic neighbour tree. The significance of these findings is paramount in forensic toxicology scenarios especially when fragmentary plant material is found in the stomach/intestine and its morphological identification becomes impossible. In these circumstances, the PCR based molecular technique surely plays a significant role in solving complicated forensic cases.

scholarly journals Development of high-resolution melting PCR (HRM-PCR) assay to identify native fungal species associated with the wheat endosphere

2020 ◽  
Vol 61 (4) ◽  
pp. 629-635
Author(s):  
Tomasz Cłapa ◽  
Katarzyna Mikołajczak ◽  
Lidia Błaszczyk ◽  
Dorota Narożna
Keyword(s):  
High Resolution ◽  
Large Scale ◽  
High Resolution Melting ◽  
Low Cost ◽  
Fungal Species ◽  
Molecular Techniques ◽  
Pcr Assay ◽  
Fungal Isolates ◽  
Large Scale Screening

Abstract Understanding the complexity and biodiversity of fungal communities associated with the wheat endosphere can facilitate the identification of novel strains that might be beneficial to the host plant. However, the differentiation and taxonomic classification of the endosphere-associated fungi with respect to various cultivars and plant organs are challenging, time-consuming, and expensive, even with the use of molecular techniques. In the present work, we describe a fast, simple, and low-cost method based on high-resolution melting PCR (HRM-PCR) for the identification and differentiation of wheat endogenous fungal isolates. Using this approach, we differentiated 28 fungal isolates, which belonged to five different genera, namely Alternaria, Penicillium, Epicoccum, Fusarium, and Trichoderma. Furthermore, the results of the study revealed that this method can allow large-scale screening of cultured samples.

scholarly journals Molecular Characterization of Fungi Associated with Dump Site Soil

2021 ◽  
pp. 19-30
Author(s):  
N. G. Ogbuji ◽  
A. E. Ataga ◽  
P. M. Tari-Ukuta ◽  
C. J. Olisedeme
Keyword(s):  
Aspergillus Fumigatus ◽  
Trichoderma Harzianum ◽  
Its Region ◽  
Molecular Techniques ◽  
Nucleotide Sequence Analysis ◽  
Genetic Constitution ◽  
Species Identity ◽  
Dump Site ◽  
Aspergillus Flavipes ◽  
Port Harcourt

Aims: A study was conducted to identify fungal species isolated from dumpsite soil in University of Port Harcourt using molecular techniques. Methodology: Molecular methods for determining the species of a fungus based on the amplification and sequencing of the internal subscribed spacer (ITS) region of the fungal rRNA operon using molecular markers was applied. Soil sample was collected from a dumpsite in the University of Port Harcourt, Rivers State, Nigeria. Isolation of fungi associated with the dumpsite soil was carried out using spread plate method. Fungal genomic DNA was extracted using Quick-DNA Fungal/Bacterial Miniprep kit. The ITS1-2 gene of the isolates was amplified by Polymerase Chain Reaction (PCR) using the primer pair; ITS4 and ITS5. Results: The sequences of the amplified ITS region were blasted against known sequences on the National Centre for Biotechnology Information (NCBI) database. Nucleotide sequence analysis revealed the species identity of the fungal isolates to be: Aspergillus fumigatus, Trichoderma harzianum, Aspergillus felis, Aspergillus templicola, Aspergillus flavipes, Aspergillus fumigatus and Cunninghamella binariae. Phylogenetic analysis was carried out to ascertain the relationship between the isolates and other closely-related isolates on GenBank. Isolates 2 (Trichoderma harzianum) and 7 (Cunninghamella binariae), 3 (Aspergillus felis) and 6 (Aspergillus fumigatus), and 4 (Aspergillus templicola) and 5 (Aspergillus flavipes) were found to be more closely related to each other. Conclusion: The molecular techniques employed successfully identified the organisms to the species level as these techniques are based on the genetic constitution of organisms. The result obtained from this study will complement the information on the fungal organisms associated with dumpsite soil.

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