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Microwave miniprep of total genomic DNA from fungi, plants, protists and animals for PCR
Abstract
A rapid procedure for the isolation of total DNA from various eukaryotic organisms is described. This method uses a common microwave oven for the alteration of cellular walls and membranes. The procedure is rapid and can be completed in as little as 15 minutes. The method has been used to isolate DNA from reptiles, plants, slime molds and other protists, and this DNA has been used for successful amplification of ribosomal genes from these organisms. The procedure is a time-saving and cost-effective process that helps to eliminate several sources of contamination that can be found in other DNA isolation procedures.
... DNA was extracted from mycelium collected by scraping the surface of Petri dishes cultures of purified isolate. 100μl lysis buffer (50mM Tris-HCl pH 7.5, 50mM EDTA, 3% SDS and 1% 2-mercaptoethanol) was added and the nucleic acids were isolated according to the microwave mini-prep procedure described by Goodwin and Lee (1993). The final DNA pellet was supplemented into100μl TE buffer (10mM Tris-HCl pH 8.0, 0.1mM EDTA) and stored at -20 °C until used. ...
... The DNA Extraction was carried out using a commercial kit NucleoSpin Plant II (Macherey-Nagel Germany). From the mycelium collected by scraping the surface of the culture on a Petri dish of purified isolate, 100 μL lysis buffer were added and the nucleic acids were isolated using the microwave mini-preparation procedure described by (Goodwin and Lee 1993). The last DNA pellet was completed in a TE buffer and stored at −20 °C until use. ...
... DNA was extracted from mycelium collected by scraping the surface of Petri dishes cultures of purified isolate. One hundred microliters of lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM EDTA, 3% SDS and 1% 2mercaptoethanol) was added and the nucleic acids were isolated according to the microwave mini-prep procedure described by Goodwin and Lee (1993). The final DNA pellet was supplemented into 100 μl TE buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA) and stored at -20°C until used. ...
Le blé (Triticum spp.) est l'une des céréales les plus importantes au monde. Malheureusement, la plante de blé est la cible de plusieurs espèces du genre Fusarium. Ce genre provoque deux maladies graves : la pourriture du collet (FCR) et la brûlure de l'épi (FHB). Dans ce travail une enquête a été menée pour étudier les plus importantes espèces associées à cette maladie en Algérie, puis deux axes de recherche ont été abordés afin d’essayer de résoudre cette problématique. Cent dix-sept échantillons ont été collectés (2019), soixante-trois à partir du collet et
cinquante-quatre à partir des grains de blé dans plusieurs régions du nord-est Algérien. Les Fusarium spp. ont été identifiés au niveau de
l'espèce par le séquençage des régions de l'espaceur interne transcrit (ITS1) de l'ADNr. Aussi leurs traits de vie (taux de croissance et taux de
sporulation) ont été mesurés. Les tests de pathogénicité des isolats ont été réalisés par deux méthodes in vitro (test des tubes) et in vivo (test
des pots). Une souche hautement pathogène de Fusarium culmorum (FC11) a été utilisée dans trois essais de tolérance/ sensibilité afin d’évaluer la résistance variétale de huit cultivars (cv) de blé dur et neuf de blé tendre largement commercialisés en Algérie, plusieurs paramètres phénotypiques ont été mesurés. Le potentiel de bio-contrôle de 15 isolats de Trichoderma (T1 à T15), isolés de différents sols de la rhizosphère et d'écosystèmes algériens, a été évalué contre 4 souches de F. culmorum (FC11, FC2, FC4, et FC20), afin d’identifier une
nouvelle souche indigène ayant un fort potentiel de bio-contrôle contre la FCR et la FHB. Ce potentiel a été évalué par des tests in vitro (confrontation directe et indirecte) et confirmé par des tests in vivo. En outre des tests dans la serre et sur champs ont été réalisé afin de sélectionner un mode de traitement de bio-contrôle meilleur par l’usage de ces souches indigène. Un total de 34 isolats représentant 10 espèces de Fusarium a été obtenu. Les résultats ont montré l'existence de cinq espèces de Fusarium isolées des collets ;Fusarium culmorum (16), F. cerealis (3), F. acuminatum (2), F. graminearum (1) et F. oxysporum (1) ; et sept espèces isolées des grains; F. culmorum (3), F. incarnatum (3), F. graminearum (1), F. equiseti (1), F. asiaticum (1), F. fujikuroi (1) et F. brachygibbosum (1). Les souches de F. culmorum ont été dominantes et plus agressives, ainsi que F. graminearum et F. cerealis qui ont été agressives dans le test in vitro. Cependant, le reste des espèces a été plutôt saprophyte qu’agressif. Une corrélation significative a été enregistrée entre la vitesse de croissance et le taux de sporulation (r =0.35 P=0.012<0.05). Aussi une forte corrélation a été enregistrée entre le test des tubes et celui des pots (r =0.62, P = 0.007< 0.01), ce résultat démontre l’efficacité d’un simple test in vitro pour prédire l’agressivité de F. culmorum sur la FCR du blé. Les cv. Sétifis
et Akhamokh ont montré les niveaux de tolérance les plus intéressants parmi les cultivars de blé dur et tendre testés, respectivement. Dans cette étude, nous avons conclu qu’un simple test in vitro sur boite de Pétri rapide, facile et stable peut être utilisé pour prédire la résistance variétale de différents cultivars du blé contre l'infection initiale des graines par F. culmorum. Les résultats du bio-contrôle par Trichoderma in
vitro ont montré une inhibition significative de la croissance mycélienne des espèces de F. culmorum par rapport au contrôle. L'isolat T14 a été sélectionné pour le bio-contrôle dans les tests in vivo. Des expériences en tube et en pot contre F. culmorum (FC2) ont montré que T14 a diminué la sévérité de la FCR avec 50 et 63,63% de réduction, respectivement. L'infection par FHB a été significativement réduite par T14 dans tous les cultivars de blé dur testés. L'antagoniste T14 a été caractérisé sur le plan moléculaire, en utilisant le facteur d'élongation de la traduction 1-alpha (TEF1-α) et ITS1. Les résultats ont identifié le T14 comme étant Trichoderma afroharzianum avec des numéros d'accession attribués par NCBI GenBank comme MW171248 et MW159753. Cette étude met en évidence que F. culmorum est l'espèce
dominante associée à la FCR et la FHB en Algérie. De plus, il s'agit du premier rapport concernant l'identification de F. incarnatum, F.
fujikuroi, F. cerealis,F. asiaticum, F. oxysporum, F. acuminatum et F. brachygibbosum du blé en Algérie. Aussi cette étude a mis en évidence la complexité des tests de résistance contre la FCR et FHB, et a démontré la nécessité d'utiliser autant de protocoles de dépistage de la résistance dans la mesure du possible. Trichoderma afroharzianum, évalué pour la première fois en Algérie en tant qu'agent de bio-contrôle, est une approche de bio-contrôle prometteuse contre la fusariose. En outre le traitement de bio-contrôle avec graines enrobées avant semis a
montré une efficacité plus importante en comparaison avec les autres modes de traitement.
... To simplify and speed up an Lxx diagnostic, a fieldapplicable reagent-free heat-induced DNA isolation method for efficient release of Lxx DNA may be assessed, bypassing multistep processing, and using commercial kits. Indeed, high temperatures are known to degrade microorganism cell walls, releasing nuclear content (Goodwin and Lee 1993;Lou et al. 1993;Strus 1997;Jose and Brahmadathan 2006;Merk et al. 2006;Umer et al. 2021). Specifically, Jose and Brahmadathan (2006) found 94°C for 2 min sufficient for bacterial cell wall denaturation. ...
... To validate the LAMP assay, qPCR experiments were devised to detect the targeted intergenic spacer (IGS) region using synthetic targets, bacterial cell samples, and sugarcane sap samples collected from the field. RSD detection from sugarcane xylem sap samples was done using two conventional qPCR methods: (i) Using directly expressed xylem sap samples in the PCR reactions as a template (Goodwin and Lee 1993;Ghai et al. 2014), or (ii) Using a commercial kit to extract Lxx DNA from the sugarcane sap samples before using as a template. The qPCR analyses were conducted following the 2X SensiFAST SYBER No-ROX Master Mix manual (New England Biolabs, USA). ...
Context
Ratoon stunting disease (RSD), caused by Leifsonia xyli subsp. xyli (Lxx), poses a significant economic threat to sugarcane (Saccharum hybrid) worldwide. RSD is hard to manage due to its elusive visible symptomology and disease rating of cultivars is subjective.
Aims
We aimed to develop a sensitive, rapid, and quantitative Lxx diagnostic method able to correlate Lxx titre and disease resistance rating of sugarcane cultivars.
Methods
A Lxx diagnostic method was developed using heat lysis-based reagent-free DNA isolation from xylem sap followed by loop-mediated isothermal amplification (LAMP)-based colorimetric and fluorescence quantification within a single microcentrifuge tube. Bacterial titre was then statistically correlated with industry-agreed disease resistance ratings for key sugarcane cultivars.
Key results
The diagnostic was highly sensitive (1 cell/μL) and reproducible (%s.d. = <5%, for n = 3), and showed excellent linear dynamic range (i.e. 10 pM−1 aM or 10⁷−10⁰ copies/μL, r = 0.99) for quantitative Lxx detection. LAMP quantifications were completely concordant with quantitative polymerase chain reaction quantification from the same samples. Additionally, a strong correlation was determined between the detected quantitative bacterial titres and known cultivar disease resistance ratings (r = 0.82, n = 10, P < 0.001).
Conclusion
The novel LAMP-based Lxx diagnostic was validated as a fast, simple, and relatively cost-effective means of RSD resistance rating, making it a reliable contribution towards RSD management.
Implications
The development of this diagnostic tool provides a practical solution for accurately measuring Lxx titre and assessing disease resistance in sugarcane plants, aiding in effective risk management of RSD spread, and mitigating its economic impact on sugarcane crops worldwide.
... Symptoms were described for the first time on H. macrophylla of French origin in Argentina (Rivera et al., 2004). Since then, the disease has also been reported on other Hydrangea species worldwide , such as H. paniculata in Italy (Garibaldi et al., 2017). ...
... DNA of the 34 Botrytis spp. and the 11 antagonistic isolates was extracted as described by Goodwin and Lee (1993) and PCR amplification of the ITS1-5.8S-ITS2 region of rDNA was conducted with ITS1 and ITS4 universal primers (White et al., 1990). ...
... After 14 d of cultivation, the colonies were preliminarily classified based on colony pattern morphology, and 10% of the strains were selected from each group for molecular identification. Total DNA was extracted using a modified method published by Goodwin and Lee [29]. This involved adding an appropriate amount of hyphae to a lysis solution and freezing the hyphae at −20 • C. The hyphae were then crushed and kept at 65 • C for 30 min. ...
... After 14 d of cultivation, the colonies were preliminarily classified based on colony pattern morphology, and 10% of the strains were selected from each group for molecular identification. Total DNA was extracted using a modified method published by Goodwin and Lee [29]. This involved adding an appropriate amount of hyphae to a lysis solution and freezing the hyphae at −20 °C. ...
The endophytic fungal diversity of Cirsium kawakamii, a herb indigenous to Taiwan, was analyzed in this study. In addition, some fungal isolates were evaluated for the risk they pose as plant pathogens. In total, 1836 endophytic fungi were isolated from C. kawakamii from Hehuanjian, Puli Township, and Tatachia. They were classified into 2 phyla, 8 classes, 40 families, and 68 genera. Colletotrichum, Fusarium, Phomopsis, and Xylaria, (Ascomycota, Sordariomycetes) were the dominant genera. The genus accumulation curve (based on the bootstrap estimator) was non-asymptotic, with estimated richness significantly exceeding the richness captured by our sampling to date. Considering the collection time, the data indicated significant differences in the proportions of the C. kawakamii endophyte genus from Hehuanjan, Puli Township (across two seasons), and Tatachia. The Shannon and Gini–Simpson indices revealed variations in diversity, with C. kawakamii endophytes (Puli Township in winter) significantly reducing alpha diversity compared with other seasons and locations. Meanwhile, the Gini–Simpson index suggested that there were no significant differences in richness among the four sampling sites. The PCA results unveiled distinct community structures across different locations and seasons, explaining 46.73% of the total variation in fungal community composition significantly affected diversity and richness. In addition, a considerable number of Fusarium isolates exhibited harmful properties towards wheat, potatoes, and apples. It is postulated that these fungi belong to the Fusarium tricinctum species complex (FTSC).
... Genomic DNA was extracted from fungal mycelia grown on PDA for 5-7 days, using lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM EDTA, 3% SDS, 1% 2-mercaptoethanol) and the miniprep (microwave) method described in Goodwin and Lee (1993). For accurate identification of the isolates, DNA amplification of portions of the glyceraldehyde-3-phosphate dehydrogenase (gpd), translation elongation factor 1-alpha (tef1), and RNA polymerase second largest subunit (rpb2) genes was performed using the primer pairs gpd1-gpd2 (Berbee et al. 1999), EF1-728F/EF1-986R (Carbone and Kohn 1999), and RPB2-5F/ RPB2-7cR (Liu et al. 1999), respectively. ...
Alternaria species have often been reported as plant pathogens and are commonly isolated from diseased plant hosts. During an investigation of this genus in Algeria, seven Embellisia-like isolates were collected from Apiaceae. Phylogenetic analysis using sequences at four loci, the internal transcribed spacer of the rDNA region (ITS), glyceraldehyde-3-phosphate dehydrogenase (gpd), translation elongation factor 1-alpha (tef1), and RNA polymerase second largest subunit (rpb2), revealed that these isolates grouped into three sections, namely Embellisia, Embellisioides, and Eureka. Four isolates had significant differences with their closest species and were determined to be new species, namely Alternaria longiformis and A. radicicolaspp. nov. The three other isolates of section Eureka were identified as A. eureka and A. hungarica, the latter species being described as a new record in Algeria. Detailed descriptions of new species are provided based on colony color, aspect, diameter, conidial size, septa, sporulation patterns and compared with other relevant Alternaria species within same sections. All these species were weakly pathogenic on carrot, coriander, and fennel under greenhouse experiments. Apiaceae may constitute a reservoir of Alternaria species that could represent potential pathogens for other plant families.
... Genomic DNA extraction of mycelia from seven-day-old fungal strains was performed using a microwave mini-prep extraction method (Goodwin and Lee, 1993). The precipitated DNA pellets were rinsed with 80 % ethanol, dried, dissolved in 100 μL TE 10:0.1 (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) and stored at − 20 • C for polymerase chain reaction (PCR) analyses. ...
... Nucleo Spin Plant II, a commercial kit, was used to perform the DNA extraction (Macherey-Nagel Germany), from the mycelium harvested by scraping the surface of the pure isolate culture in a Petri dish. The nucleic acids were isolated using the microwave mini-preparation method outlined by Goodwin & Lee (1993) by adding 100 μL of lysis buffer (50 mM Tris-HCl pH 7.5, 50 mM EDTA, 3% SDS and 1% 2-mercaptoethanol). The final DNA pellet was made in TE 100 μL buffer (10 mM Tris-HCl pH 8.0, 0.1 mM EDTA), and it was kept at -20°C until the use. ...
Fusarium crown rot (FCR), is one of the most serious wheat diseases. In this study one hundred and seventeen Fusarium symptomatic samples were collected in the Northeast of Algeria. The obtained isolates (34), were identified on the basis of the morphological and the molecular data using phylogenetics. Accession numbers MW366557 and MW448396, were assigned by NCBI GenBank to isolate FE6 and FE8, identified as Fusarium asiaticum and F. incarnatum, respectively. A pathogenicity test was conducted on seven bread wheat cultivars (cv) to test their ability to induce FCR by the disease index (DS) parameter. The impact on coleoptile length and weight was estimated by the reduction parameters (RCL and RCW%). The results showed that the strains were saprophytic rather than pathogenic with negligible DS. A statistically significant decrease in coleoptile weight was recorded by FE6 (P = 0.03 ˂ 0.05), and FE8 (P = 0.017 ˂ 0.05). The cv. Medracen was mostly affected by this reduction with RCW% (11.90 and 35.96%). To the best of our knowledge, for the first time this work confirmed the existence of F. asiaticum and F. incarnatum species in Algeria.
... From the mycelium collected by scraping the surface of the culture on a Petri dish of purified isolate. 100μl lysis buffer (50mM Tris-HCl pH 7.5, 50mM EDTA, 3% SDS and 1% 2mercaptoethanol) was added and the nucleic acids were isolated using the microwave minipreparation procedure described by Goodwin & Lee [22]. The last DNA pellet was completed in a TE 100μl buffer (10mM Tris-HCl pH 8.0, 0.1mM EDTA) and stored at -20 °C until use. ...
Description of the subject: Fusarium crown rot (FCR) is one of the serious wheat diseases. Objective: The isolation of Fusarium brachygibbosum, which has recently described in the Algerian territory especially for wheat, and evaluation of its pathogenicity and capacity to induce FCR on wheat. Methods : In this study, one hundred and seventeen Fusarium symptomatic samples were collected from the northeast of Algeria. The 34 isolates obtained were identified based on morphological data and confirmed by molecular identification. Isolate FE10, identified as Fusarium brachygibbosum, was assigned the accession number MW450596 by NCBI GenBank. Two pathogenicity tests were conducted on 9 cultivars (cv) of bread wheat. The In vitro test in the host with the parameters; percentage of germination inhibition (%GI), to determine the impact on the germination of sowing seeds, and the Area Under the Disease Progress Curve (AUDPC) to test the effect on the initial infection of germinated coleoptiles. In vivo test in the growth chamber to test the F. brachygibbosum ability to induce FCR by the disease severity (DS) parameter. Results : The results showed that FE10 induced FCR at all tested cultivars with a significant decrease in germination rate and coleoptile emergence with GI% and AUDPC values reaching up to 17.36, and 38.86%, respectively for cv. Nif Encer. In addition F. brachygibbosum negatively affected the vegetative system length and fresh weight (RCL% and RCW%) up to 34.49 and 48.43%, respectively with cv. Arz. Conclusion : The moderate pathogenicity of F. brachygibbosum was observed in this study. Description du sujet : La pourriture fusariene du collet (FCR) est l'une des maladies graves du blé. Objectifs : L'isolement de Fusarium brachygibbosum, qui a été décrit récemment dans le territoire algérien, en particulier pour le blé, et l'évaluation de son pouvoir pathogène et de sa capacité à induire la FCR sur le blé. Méthodes : Dans cette étude, cent dix-sept échantillons symptomatiques de Fusarium ont été collectés dans le nord-est de l'Algérie. Les 34 isolats obtenus ont été identifiés sur la base de données morphologiques et confirmés par identification moléculaire. L'isolat FE10, identifié comme Fusarium brachygibbosum, le numéro d'accession MW450596 lui attribuer par NCBI GenBank. Deux tests de pathogénicité ont été réalisés sur 9 cultivars (cv) de blé panifiable. Le test in vitro sur l'hôte avec les paramètres : pourcentage d'inhibition de la germination (%GI), pour déterminer l'impact sur la germination des graines de semis, et l'Area Under the Disease Progress Curve (AUDPC) pour tester l'effet sur l'infection initiale de coléoptiles germées. Test in vivo dans la chambre de culture pour tester la capacité de F. brachygibbosum à induire la FCR par le paramètre de sévérité de la maladie (DS). Résultats : Les résultats ont montré que FE10 a induit la FCR chez tous les cultivars testés avec une diminution significative du taux de germination et de l'émergence des coléoptiles avec des valeurs GI% et AUDPC atteignant jusqu'à 17,36, et 38,86%, respectivement pour cv. Nif Encer. En outre, F. brachygibbosum a affecté négativement la longueur du système végétatif et son poids frais (RCL% et RCW%) jusqu'à 34,49 et 48,43%, respectivement avec le cv. Arz. Conclusion : La pathogénicité modérée de F. brachygibbosum a été constatée dans ce travail.
... Some researchers used dry ice for breaking the cells while some use enzymatic digestions and homogenization for DNA extractions (Griffin et al., 2002). The microwave method was also used for lysing the cell for extracting DNA (Goodwin and Lee, 1993). Izumitsu et al. (2012) used microwave method in combination with cooling to isolate DNA in 16 minutes. ...
Black root rot (BRR) caused by a soilborne Berkeleyomyces rouxiae is a pandemic disease on cotton seedlings in Australia. BRR of cotton was reported for the first time in northern New South Wales (NSW), Australia in 1990. Now, the disease is widespread across cotton growing regions in NSW. Much research has focused exclusively on control management; however, relatively little work has been conducted to understand the BRR pathogen population for their temporal and spatial distributions. A total of 294 B. rouxiae isolates that were freshly collected across NSW over five cropping seasons (2017–2022) were assessed for their genetic diversity based on sequences of the nuclear ribosomal DNA internal transcribed spacer, mini-chromosome maintenance complex component 7, translation elongation factor 1-alpha and RNA polymerase II second largest subunit. Additionally, these isolates were subjected to a specific duplex PCR assay for mating type determinations. Multiple sequence alignments revealed that the prevailing cotton-B. rouxiae was 100% identical; however, the population can be divided into two subgroups based on the presence of mating idiomorphs. The MAT1-1 type was predominant and accounted for 62.2% of the population. A total of 25/77 fields were confirmed to harbour both MAT1-1 and MAT1-2 isolates. However, we failed observe sexual structures in crossing experiments. Based on the sequence uniformity of the cotton-B. rouxiae population, we suggest that the pathogen has spread from one field to another. Therefore, stricter farm hygiene practices should be enforced to minimise a further spreading risk.
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