Internal Transcribed Spacer Primers Research Articles

Methodology comparison of environmental sediment fungal community analysis

Fungi play important roles in ecosystems. Analyzing fungal communities in environments has long been a challenge due to the large difference in compositions retrieved using different methods or sequencing regions, obscuring the true abundance and species information. Our study aimed to compare and determine more accurate approach for evaluating fungal populations in river sediment. To achieve this, different primer sets in the internal transcribed spacer (ITS) (ITS5/ITS1R, ITS1F/ITS2), 18S rRNA gene (0817F/1196R) for High-throughput sequencing (HTS), metagenomic shotgun sequencing (MS) directly from environmental samples, and HTS using ITS primers for the fungal samples collected from plate cultivation were used to characterize the fungal communities. We calculated diversity index and used FungalTraits to analyze methods preferences for fungal species. The study revealed that when analyzing the fungal species directly from environmental samples, amplification and sequencing of ITS region demonstrated more accuracy than MS and 18S rRNA gene sequencing methods, but displayed significant primer preference. Over 30 % fungal species from HTS after plate cultivation were not present in HTS from the environmental samples. NMDS analysis demonstrated significant disparities in species diversity among different methods, suggesting potential complementarity between them. Over 85% species identified by HTS using ITS primers belonged to filamentous fungi, while the MS mostly identified yeast (62%). Therefore, to get more accurate fungal community information in sediment, multiple methods were recommended by using cultivation, molecular biological methods dependent on PCR techniques like ITS1F/ITS2 primer for HTS and PCR independent method such as metagenomic shotgun sequencing techniques.

New Report of Stemphylium vesicarium (Wallr.) Simmons and Ulocladium alternariae (Cooke) Simmons as seed-borne fungi of wheat (Triticum aestivum L.) in Iraq

The objective of the present study is to determine fungal species Stemphylium vesicarium and Ulocladium alternariae associated with seeds of five wheat cultivars called Wefia (WAF), Eba 99 (EBA), Adena (ADN), Bohooth (BTH) and Mahmoudia (MHD). Those fungi were identified by using morphological and microscopic features as well as molecular sequencing. Molecular identification was carried out relying on internal transcribed spacer primers (ITS1 and ITS4). This is a new report recording S. vesicarium and U. alternariae as seed-borne fungi of wheat seeds in Iraq.

Development of PCR-based markers for the identification and detection of Lophodermella needle cast pathogens on Pinus contorta var. latifolia and P. flexilis

Morphological similarities and fastidious development of increasingly emerging fungal needle pathogens impede accurate disease diagnosis and early detection. This study analyzed the specificity and sensitivity of polymerase chain reaction (PCR)-based markers developed for emerging needle cast pathogens Lophodermella concolor and L. montivaga co-occurring on Pinus contorta var. latifolia, and Bifusella linearis and L. arcuata on P. flexilis. To design primers, we utilized sequences of the internal transcribed spacer (ITS) region and single-copy gene (RH_2175) of the TCP-1/cpn60 chaperonin family searched through genomes of related species. In addition to the DNA of target and non-target fungal species that were used for primer assays, environmental samples with next generation sequencing data were used to evaluate primer sensitivity. Direct amplification using ITS primer pairs generated 248–260 bp amplicons and successfully differentiated the needle pathogens used in this study. Nested amplification of single-copy gene RH_2175 primer pairs which produced 409–527 bp amplicons detected Rhytismataceae species and discriminated both Lophodermella pathogens on P. contorta var. latifolia, respectively. While ITS-based primers had higher sensitivity than the 2175-based primers, both primer sets for L. concolor and L. montivaga detected their respective pathogens in asymptomatic and symptomatic needles. These molecular tools can help monitor and assess needle diseases for forest management and phytosanitary regimes.

Genetic Diversity Analysis based on the Virulence, Physiology and Regional Variability in Different Isolates of Powdery Mildew in Pea.

Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen’s morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS (powdery mildew-internal transcribed region) primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations.

First Report of Corynespora cassiicola Causing Target Spot on Cotton (Gossypium hirsutum) in South India.

Target spot with unusual symptoms was observed in upland cotton at Regional Agricultural Research Station, Lam, Guntur, Andhra Pradesh, India in 2017. Symptoms appeared in about 60-65 days after planting with minute pinhead size light orange to brick red spots that gradually expanded as circular to oval or irregular concentric spots that finally developed as target spot with yellow halo. These symptoms were also observed during 2019-20 and 2020-21 diagnostic field visits at Krishna zone of Andhra Pradesh, India. Frequent showers had intensified disease severity and defoliation of different cultivars (L 1060, RCH 659 BGII, NDLH-1938 and Jaadoo BG II) examined. Leaf bits containing healthy and diseased portions of the specimens were surface sterilized with 0.1% HgCl2 for a minute and rinsed in three changes of distilled water and were placed on PDA at 27±1˚C for pathogen development. Obtained pathogen was purified using single spore isolation technique. Purified colonies exhibited grey colour on top of culture plate and greyish brown on bottom of culture plate with flat, aerial round colonies having fibrous texture with dense mycelium. Conidiophore were indeterminate septate with intermediate branching. Enteroblastic conidiogenous cells produced conidia subhyaline that were solitary or in chains, straight to curved, 83.43±31.73 x 7.61±1.43 µm with 7.27±2.19 pseudospeta. Morphological characteristics of the isolated fungus were consistent with original description of Corynespora cassiicola on cotton (Jones 1961; Fulmer et al., 2012; Conner et al., 2013) which confirmed it as Corynespora morphologically. Pathogenicity test was conducted on popular hybrid Jaadoo BG II using three isolates Lam (CGGL 19001), Koniki (CPIK 19009) and Venkatapuram (CKMB 19011) obtained during 2019-20 survey. After 50 days of sowing conidial suspension containing 1x104 spores per ml was sprayed on healthy plants maintained in pots under protected conditions. Immediately after spraying, the plants were covered with poly propylene covers for 48 h to prevent cross contamination and to ensure humidity for pathogen establishment. Healthy plants sprayed with sterile water served as control. After seven days of incubation, inoculated plants developed brick red spots initially which later developed into target spot. Absence of symptoms in control plants indicated pathogenic nature of the fungus. Pathogen was re-isolated from diseased tissue to confirm its pathogenicity according to Koch's postulates. The identity of the pathogen was further examined using ITS primers. The DNA was isolated from 10 days old culture using CTAB method. Internal transcribed spacer (ITS) region of three isolates were amplified using ITS1 and ITS4 primers and obtained amplicon (550 bp) were used for sequencing. The sequenced data was deposited in NCBI and accession numbers were obtained as MZ314930, MZ314928 and MZ314927. BLAST search in GeneBank revealed 99% homology of CGGL 19001 isolate with sequences of C. cassiicola (accession number of MN393240) while CPIK 19009 and CKMB 19011 isolates showed 100% homology with C. cassiicola in cotton having accession numbers of MN393238, MN228955, MN733135, MN288934 and MN393238. Based on symptoms, fungal morphology, pathogenicity test and ITS sequences obtained, the fungus was identified as C. cassiicola the pathogen of cotton causing target spot disease. Earlier, Sarma and Nayudu (1970) isolated Corynespora from different host plants including cotton but could not prove its pathogenicity on cotton. Later target spot of cotton associated with C. cassiicola had been reported from Central India (Salunkhe et al., 2019) and this is the first report of C. cassiicola in cotton from South India. Hence, this report of C. cassiicola on cotton is the first report from Souh India. Identification of target spot pathogen is very important as it is emerging as a dominant foliar pathogen for which management strategies are to be worked out.

Phylogenetic relationship among taxa in the genus Adonis L. collected from Türkiye based on nrDNA internal transcribed spacer (ITS) markers.

Genus Adonis L. contain approximately 40 annual and perennial species, which are widely distributed in the temperate zones of Asia and Europe, and less frequently in southwestern Asia, northern Africa and the Mediterranean region. The aim of the study was to evaluate the phylogenetic relationship among Adonis taxa collected from Türkiye based on nrDNA Internal transcribed spacer (ITS) markers. Samples of 64 individual genotypes from 21 populations of 10 Adonis taxa were collected from different regions of the country during vegetation period between 2014 and 2018. ITS1, ITS4, P16 and P25 primers within ITS technique was used to genotype the plant materials. Then, genotypic data was used to estimate magnitude and organization of infraspecific variation in different populations of Adonis. About 600bp DNA sequences were obtained from each 64 Adonis genotypes belonging to 21 different populations. The dendrogram obtained from Adonis taxa and out-group sequences had two large main groups. While the out-group species were placed in the first large main group, the sect. Consiligo (perennial) and sect. Adonis (annuals) were placed in different sub-groups of the second large main group. Genetic similarity among Adonis taxa varied between A. microcarpa and A. dentata (98.46%). Principal component analysis indicated that two important components in Adonis taxa genotypes. The expected heterozygosity ranged from 0.0252 (sub-population A) to 0.3460 (sub-population C), with an average of 0.1154. In addition, population differentiation measurements (Fst) ranged from 0.0025 (sub-population C) to 0.9016 (sub-population A) with a relatively high average 0.6601. Present analyses revealed that phylogenetic classification (grouping) of Adonis taxa largely depended on morphological structure and present ITS primers were quite efficient in putting forth the genetic diversity of such species. The results of this study suggested that ITS markers could be used in the identification of genetic diversity among the Adonis taxa. The results obtained from molecular data can be used to explore the genetic variation pattern, population structure, and the evolutionary history of genus Adonis in the future.

Composition of Rhizosphere Microbial Communities Associated With Healthy and Verticillium Wilt Diseased Cotton Plants.

Rhizosphere microbial communities are known to be related to plant health; using such an association for crop management requires a better understanding of this relationship. We investigated rhizosphere microbiomes associated with Verticillium wilt symptoms in two cotton cultivars. Microbial communities were profiled by amplicon sequencing, with the total bacterial and fungal DNA quantified by quantitative polymerase chain reaction based on the respective 16S and internal transcribed spacer primers. Although the level of V. dahliae inoculum was higher in the rhizosphere of diseased plants than in the healthy plants, such a difference explained only a small proportion of variation in wilt severities. Compared to healthy plants, the diseased plants had much higher total fungal/bacterial biomass ratio, as represented by quantified total fungal or bacterial DNA. The variability in the fungal/bacterial biomass ratio was much smaller than variability in either fungal or bacterial total biomass among samples within diseased or healthy plants. Diseased plants generally had lower bacterial alpha diversity in their rhizosphere, but such differences in the fungal alpha diversity depended on cultivars. There were large differences in both fungal and bacterial communities between diseased and healthy plants. Many rhizosphere microbial groups differed in their abundance between healthy and diseased plants. There was a decrease in arbuscular mycorrhizal fungi and an increase in several plant pathogen and saprophyte guilds in diseased plants. These findings suggested that V. dahliae infection of roots led to considerable changes in rhizosphere microbial communities, with large increases in saprophytic fungi and reduction in bacterial community.

First Report of Alternaria alternata Causing Leaf Spot on Beta vulgaris in North Dakota, USA

Sugar beet is an economically important crop which is contributing 55% of the total sugar in the USA. In June 2018, irregular dark brown somewhat circular spots were observed on sugar beet leaves in Hickson, North Dakota. The symptoms covered approximately 5% on the lower leaves. Symptomatic leaf tissue were excised from the junction of diseased and healthy tissue. Small pieces (5 mm²) were surface sterilized with 10% sodium hypochlorite for 1 min, rinsed thrice with sterile distilled water, air dried and transferred to Potato Dextrose Agar (PDA), and incubated at 24°C with a 12-h photoperiod for 5 days. Dark-green velvety colony appeared in two weeks. Three isolates were developed by the single spore isolation technique. Conidia were club-shaped, two to four transverse septa, and pale brown, without any beak, often in chains (4 to 8 conidia) and or solitary. The dimension of conidia varied from 25-40x7-14 μm [1,2]. Based on the morphological characters, the fungus was tentatively identified as Alternaria species. Genomic Deoxyribonucleic Acids (gDNAs) were extracted from the culture generated from a single spore using Qiagen kit. ITS4/ITS5 were used to amplify the fragments of the Internal Transcribed Spacer (ITS) region. The amplified PCR products were cleaned and sent for Sanger sequencing by GenScript (GenScript, Piscataway, NJ). The sequences from GenScript were congruence to the reference sequence ID MT126620.1. The entire sequences were deposited at NCBI (GenBank accession nos. MK441717). Koch postulates were followed by spraying conidia suspension (5×105 conidia/ml) to 8-week age of 20 plants of and kept in humidity chamber at 28-30 °C, 80-85 % RH. Mock-inoculated seeds were also sown as a control. Three weeks of post inoculation, the similar irregular dark brown symptoms observed in twelve plants. No symptoms were found in the mock. The experiment was conducted twice. The fungus was reisolated from the diseased leaf tissue, as described above. Macroscopic and microscopic analysis indicated the similar dark-green colony and morphology, respectively. Molecular detection performed using the same ITS primers and sent for Sanger sequencing by GenScript, this study further confirmed that the isolate was similar to A. alternata [3]. Another close species of Alternaria was recently reported in sugar beet to cause leaf spot in Minnesota [4,5]. To our best knowledge, this is the first report of A. alternata causing leaf spot on sugar beet in North Dakota.

DNA barcoding of Pericopsis mooniana from two different populations in Indonesia based on rDNA ITS (Internal Transcribed Spacer)

A DNA barcoding is a taxonomy method that provide fast and acurate species identification, thus it can be applied to anticipate when a species can not be identified morphologically. This study aimed to assess molecular characteristics of Pericopsis mooniana from two wild populations in Indonesia using a barcoding marker rDNA ITS (Internal Transcribed Spacer) as additional information for establishing conservation strategy of this species. Leaf samples of P. mooniana were collected from natural forests at South East Sulawesi and South Kalimantan, and a germplasm orchard at Purwodadi Botanic Garden. Molecular characteristic was evaluated based on a pair of ITS primers that produce between 634-641 bp nucleotide. The sequence data were analyzed by Bioedit version 7.0.5.3 and Mega 7 software. Results showed that P. mooniana from South East Sulawesi and South Kalimantan were clustered together, while P. mooniana from Purwodadi Botanic Garden were separated with only one base difference from those two population. In conclusion, ITS barcoding in this study shows a diffences between P. mooniana collected from South East Sulawesi and South Kalimantan with P. mooniana that originally from Papua. A further study using more barcoding marker and more population could be approached to obtain more information on molecular characters of P. mooniana populations that will useful for establishing conservation and tree breeding program of this species.

Molecular Characterisation and Identification of Three Mushrooms Found in the Niger Delta Region

Mushrooms are a group of fungi that are diverse and in the Niger Delta region; there are various species, some of which share similar morphological features. Over the years, mushrooms have been put to several important uses ranging from food, nutraceuticals, feed for livestock, and more recently bioremediation, hence, the need for an accurate approach of identification is eminent. This study aims to identify three mushroom samples with the potential for crude oil degradation using molecular tools. The molecular identification of the mushrooms was carried out with the ITS (Internal Transcribed Spacer) region to analyze their genetic diversity. The three mushroom samples were identified as Pleurotus ostreatus, Pleurotus floridanus and Lentinus squarrosulus. The blast results showed 86.0%, 97.9%, and 88.4% for Pleurotus ostreatus, Pleurotus floridanus and Lentinus squarrosulus respectively for sequence similarity. The samples were assigned unique accession numbers on GenBank The use of molecular characterization gives reliable results to the species level. The results from this study have increased the database of Pleurotus and Lentinus DNA and serve as a basis for the identification of unidentified species by comparing their PCR amplified sequences with ITS primers.

Diversity and Functions of Endophytic Fungi Associated with Roots and Leaves of Stipa purpurea in an Alpine Steppe at Qinghai-Tibet Plateau.

Stipa purpurea is a unique and dominant herbaceous plant species in the alpine steppe and meadows on the Qinghai-Tibet Plateau (QTP). In this work, we analyzed the composition and diversity of the culturable endophytic fungi in S. purpurea according to morphological and molecular identification. Then, we investigated the bioactivities of these fungi against plant pathogenic fungi and 1- aminocyclopropane-1-carboxylate deaminase (ACCD) deaminase activities. A total of 323 fungal isolates were first isolated from S. purpurea, and 33 fungal taxa were identified by internal transcribed spacer primers and grouped into Ascomycota. The diversity of endophytic fungi in S. purpurea was significantly higher in roots as compared to leaves. In addition, more than 40% of the endophytic fungi carried the gene encoding for the ACCD gene. The antibiosis assay demonstrated that 29, 35, 28, 37 and 34 isolates (43.9, 53.1, 42.4, 56.1, and 51.5%) were antagonistic to five plant pathogenic fungi, respectively. Our study provided the first assessment of the diversity of culturedepending endophytic fungi of S. purpurea, demonstrated the potential application of ACCD activity and antifungal activities with potential benefits to the host plant, and contributed to high biomass production and adaptation of S. purpurea to an adverse environment.

Molecular Characterization of Fusarium oxysporum Causing Chickpea Wilt by Internal Transcribed Spacer (ITS) Marker

Fusarium wilt caused by Fusarium oxysporum f. sp. ciceris is one of the serious disease causes tremendous loss to crop throughout the world and has assumed serious proportions in the recent years. Wilt affected chickpea plant roots samples were collected from forty eight different regions of Maharashtra. The collected samples were characterized by designed ITS primers. The Fusarium oxysporum f. sp. ciceris (Foc) showed about 302 bp amplicon when subjected to PCR with these primers. Further the amplified product was digested with five restriction enzymes viz. AluI, EcoRI, HaeIII, MboI and MspI and restriction fragment length polymorphism (RFLP) pattern were analysed. A dendrogram derived from ITS-RFLP analysis of the rDNA region divided the Foc isolates into four clusters. The maximum genetic distance 0.75 exhibited by five Foc isolates viz. Foc-30, Foc-36, Foc-24, Foc-25 and Foc-48 belonging to Shahartakli, Shelur, Nashik, Satara, Kolhapur regions respectively and found to be more diverse among the other Foc isolates. Whereas isolate no. Foc-20, Foc-21 and Foc-8 from Ambajogai, Karjat and Rahata shown minimum genetic distance (0.13). This showed the genetic variability among the Foc isolates collected from different regions of Maharashtra.

Primer and Database Choice Affect Fungal Functional but Not Biological Diversity Findings in a National Soil Survey

The internal transcribed spacer (ITS) region is the accepted DNA barcode of fungi. Its use has led to a step-change in the assessment and characterisation of fungal communities from environmental samples by precluding the need to isolate, culture, and identify individuals. However, certain functionally important groups, such as the arbuscular mycorrhizas (Glomeromycetes), are better characterised by alternative markers such as the 18S rRNA region. Previous use of an ITS primer set in a nationwide metabarcoding soil biodiversity survey revealed that fungal richness declined along a gradient of productivity and management intensity. Here, we wanted to discern whether this trend was also present in data generated from universal 18S primers. Furthermore, we wanted to extend this comparison to include measures of functional diversity and establish trends with soil types and soil organic matter (SOM) content. Over the 413 individual sites examined (arable, grassland, woodland, moorland, heathland), we found congruent trends of total fungal richness and β-diversity across land uses, SOM class and soil type with both ITS and 18S primer sets. A total of 24 fungal classes were shared between datasets, in addition to 15 unique to ITS1 and 12 unique to 18S. However, using FUNGUILD, divergent trends of functional group richness became apparent, especially for symbiotrophic fungi, likely driven by an increased detection rate of Glomeromycetes in the 18S dataset. The disparate trends were also apparent when richness and β-diversity were compared to soil properties. Additionally, we found SOM class to be a more meaningful variable than soil type biodiversity analyses because organic matter was calculated for each sample whereas soil type was assigned from a national soil map. We advocate that a combination of fungal primers should be used in large-scale soil biodiversity surveys to capture important groups that can be underrepresented by universal barcodes. Utilising such an approach can prevent the oversight of ubiquitous but poorly described species as well as critically important functional groups.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens.

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

First Report of Ophiosphaerella agrostidis Infecting Creeping Bentgrass in Wisconsin

Dead spot is a disease of golf course putting greens and is caused by the pathogen Ophiosphaerella agrostidis Dernoeden et al. (syn. O. agrostis) (Câmara et al. 2000). The disease is found on creeping bentgrass (Agrostis stolonifera L.) and hybrid bermudagrass (Cynodon dactylon [L.] Pers. × C. transvaalensis Burtt-Davy) in the eastern and southern United States as well as Canada (Kaminski and Dernoeden 2002; Kaminski and Hsiang 2006; Krausz et al. 2001). In June 2017, disease symptoms resembling dead spot were observed on a golf course in Wisconsin. Small (<3 cm) necrotic spots first appeared approximately 10 months after establishment of a 50/50 blend of ‘007’ and ‘SR1119’ creeping bentgrass on sand-based putting greens. The disease became more severe during the summer months with as many as 10 spots per square foot and spots up to 4 cm in diameter. The disease was less severe on greens infected in 2017, but new symptoms appeared in 2018 on greens that were seeded during the summer of 2017. Dark brown ectotrophic hyphae were observed on bentgrass stolons, and pseudothecia were observed embedded within necrotic tissue. A single fungal morphotype was consistently isolated from leaves, stem bases, and stolons, and the colonies were rose-quartz color when cultured on potato dextrose agar. To demonstrate pathogenicity, ‘Penn A-1’ creeping bentgrass seedlings were grown for 4 to 6 weeks in 2.5-cm Cone-tainers in an autoclaved sand-based growing medium with a mechanical analysis of 95% sand, 4% silt, and 1% clay. Inoculum was prepared by placing mycelia from a hyphal-tipped isolate on an autoclaved mix of seed of tall fescue (Festuca arundinacea Schreb.) and wheat (Triticum aestivum L.) germ (50% [vol/vol]) and grown at 20°C for 10 days. The inoculum (5 g) was set at the surface of the sand and surrounding the stem bases of the creeping bentgrass in the center of each Cone-tainer (n = 6). A noninfested inoculum and noninoculated grass each served as a nontreated control. The grass plants grown in individual Cone-tainers were enclosed in clear plastic bags to increase relative humidity and incubated at 20°C under 14 h of light per day from fluorescent lights. Pathogenicity tests were conducted twice. No symptoms developed in either of the nontreated control treatments. After 7 days, tissue along the periphery of each inoculation point became covered by pink mycelia, and newly infected leaves appeared tan or brownish-red. Reisolation of the pathogen from symptomatic tissue produced fungal colonies similar in color, morphology, and growth rate to the original isolate. The internal transcribed spacer (ITS) regions of DNA from the original and reisolated fungus were amplified using ITS primer pair ITS1/ITS4. Sequencing of the amplified region resulted in a 408-bp sequence (GenBank accession no. MK561423) matching 100% identity with GenBank accession numbers AF191548.1 and MF351996.1. Based on field symptoms, morphological characteristics, pathogenicity tests, and ITS sequences, the pathogen was identified as O. agrostidis. To our knowledge, this is the first report of dead spot on creeping bentgrass in Wisconsin.

Opportunistic pathogenic fungi isolated from feces of feral pigeons in Mafikeng, North West Province of South Africa.

Background and Aim:Pigeon feces are increasingly being implicated in the spread of bacterial pathogens such as Escherichia coli, Campylobacter, Salmonella, Listeria, and Chlamydia. Fungi are rarely investigated except for Cryptococcus that has emerged as an important pathogen in old people and immunosuppressed patients. This study investigated fungi in pigeon feces collected from Mafikeng, the North West Province of South Africa.Materials and Methods:Freshly dropped feces were collected and enriched in phosphate-buffered saline overnight at 48°C and then subcultured on Sabouraud’s dextrose agar and incubated at 48°C for 2 weeks observing any fungal growth from day 2. The growths were picked up, DNA extracted, and polymerase chain reaction was done using the internal transcribed spacer primers.Results:Fungi isolated included: Aspergillus (Aspergillus tubingensis), Cryptococcus (Cryptococcus albidus and Cryptococcus randhawai), Fusarium spp., and Rhodotorula (Rhodotorula mucilaginosa and Rhodotorula kratochvilovae). Most of these isolates are known opportunistic pathogens and have been isolated in clinical conditions elsewhere. Other isolates such as Graphium dubautiae, Myrmecridium schulzeri, Naganishia albida, Paecilomyces lilacinus, and Zygopleurage zygospora were not found to be of any human health significance.Conclusion:We, therefore, concluded that the presence of these opportunistic pathogens is a significant human health risk, especially in the face of the HIV/AIDS pandemic that results in immunosuppression.

Development of specific SNP molecular marker from Thistle using DNA sequences of ITS region

Thistle is a perennial plant that is widely used for medicinal purposes. Information on the genetic diversity of thistle populations are great important for their conservation and germ plasmic utilization. Although thistle is an important medicinal plant species registered in South Korea, no molecular markers are currently available to distinguish them from other similar species from different countries. In this study, we developed single nucleotide polymorphism (SNP) markers derived from the nuclear ribosomal DNA internal transcribed spacer (ITS) regions of genomic sequences to identify distinct Korean-specific thistle species via an amplification refractory mutation system (ARMS)-PCR and high resolution melting (HRM) curve analyses. We performed molecular authentication of four different kinds of thistle species from different regions using DNA sequences in the ITS intergenic region. We also developed a quantitative PCR assay using species-specific ITS primers, which allowed us to estimate the ratio of Korean-specific thistle species using varying ratios of mixed genomic DNA templates from the two species. The SNP markers developed in this study are useful for rapidly identifying specific thistle species from different countries.

PCR detection of Colletotrichum gloeosporioides in Psidium guajava

Colletotrichum gloeosporionides is the causative agent of a major disease affecting Psidium guajava fruits from fructescence to post-harvest. A polymerase chain reaction (PCR)-based method for the detection of C. gloeosporioides was developed in this study. A set of two primers (CGF and CGR) specific for C. gloeosporioides were designed based on the sequences of the ribosomal internal transcribed spacer (ITS) regions. The specificity of primers was analyzed by the absence of amplified product with DNA of tested isolates. The designed primers amplified a single product of ~390 bp with DNA extracted from isolates of C. gloeosporioides, and did not amplify DNA from several Colletotrichum species and other different fungal genera. The sensitivity of conventional PCR was 10 pg purified genomic DNA in 25 μL reaction. A nested PCR system was established to combine specific and universal ITS primers (ITS1/ITS4), and this enhanced the sensitivity to 1 fg of DNA per 25 μL reaction, representing a 10,000-fold improvement over conventional PCR. The optimised reaction system could detect C. gloeosporionides successfully from diseased plant tissue, infected soil and water. The developed system can detect C. gloeosporioides during the early stages of disease onset and provide evidence to guide management and decision-making.

Comparing reliability of skin microfilarial load with first internal transcribed spacer primers DNA amplification in monitoring onchocerciasis treatment control

Treatment compliance is a serious challenge to community-directed treatment intervention (CDTI) targeting onchocerciasis eradication. Isolated cases of active infections could jeopardise decision to stop mass drug distribution. Onchocerciasis co-endemicity with lymphatic filariasis in the study-area informed the use of primers capable of differentiating sympatric infections in a single assay. The study-population was hitherto found to be negative for presence of skin microfilaria. DNA extracted from skin snips of patients (n=63) comprised of males, n=21 and females, n=42 were analysed using 18S first internal transcribed spacer (ITS1) in polymerase chain reaction (PCR) assay. Amplified DNA sequences were subjected to basic local alignment search tool. Spectrophotometric analysis of DNA concentrations ranged between 1.5- and 24.5 Nano gram (ng) and the two positive controls (adult worm DNA extract) were 10.5 and 105 ng at 260 nanometer (nm) wave length. The 260/ 280 nm ratios were between 1.44 and 1.68. Four samples (6.35%), two males and two females, and the two positive controls showed PCR amplification products. Two of the positive samples indicated double bands in an agarose gel electrophoregram with molecular weight of 350 and 500 base pairs (bp), while two had single band of 350bp, which is within the expected Mw range of 344 bp for Onchocerca volvulus. The double bands may not be due to unspecific amplification. Moreover, the DNA sequence of a sample had 69.1% homology with control sequence but not with O. volvulus sequences available in the GenBank database. This study underscored the need to assess effectiveness of CDTI with highly sensitive test and not depend on microscopy of emerged microfilaria from skin snips. Whether the DNA was from dead or living microfilaria remained a conjecture. Keywords : Skin microfilaria; DNA sequences.

Molecular Identification of Fungal Contamination in Date Palm Tissue Cultures.

Fungal contamination of in vitro cultures of date palm (Phoenix dactylifera L.) is the major constraint to their initiation and maintenance. Different molecular approaches have been applied successfully to analyze both inter- and intraspecific variation among fungal species as well as determine their identity. This chapter describes step-by-step procedures of molecular identification of fungal contaminants by internal transcribed spacer (ITS) products of the most common fungal contaminants of date palm tissue culture. To begin with, samples of genera Alternaria, Aspergillus, Cladosporium, Epicoccum, and Penicillium were collected to isolate each fungal genus and extraction of genomic DNA. Polymerase chain reactions were accomplished by ITS primers (ITS1 and ITS4) for each fungal contaminant as well as for sequencing. Subsequently, they are analyzed by Basic Local Alignment Search Tool (BLAST) search of ITS sequence to reveal the identity of each individual fungal contaminant species. The molecular identification herein is a rapid and reliable procedure to identify date palm fungal contaminants which is very important in their control and treatment.