Direct PCR detection, cloning, and characterization of fungi communities in soils and compost
| Autor: | Luis Jimenez, Joy Bochis, Tina Choe, Matthew Gardner, Victorya Ramos, Mahtab Tazehabadi, Theranda Jashari, Stephanie Zapata, Jenifer Vasquez, Victoria Ellman |
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| Rok vydání: | 2019 |
| Předmět: |
Plasmid preparation
biology Ascomycota Microbial DNA Phylum General Engineering Basidiomycota 010501 environmental sciences Ribosomal RNA biology.organism_classification complex mixtures 01 natural sciences food.food 03 medical and health sciences 0302 clinical medicine food Stellaria media Botany 030212 general & internal medicine 0105 earth and related environmental sciences Zygomycota |
| Zdroj: | BIOS. 90:87 |
| ISSN: | 0005-3155 |
| Popis: | A procedure was developed to detect and identify fungi in garden soil, forest soil, and compost samples. Microbial DNA was extracted using the Zymo Microbe DNA MiniPrep protocol. Fungi ribosomal internal transcribed space sequences (ITS) were amplified by PCR using primers ITS1 and ITS4. DNA fragments of approximately 640 base pair were detected in all positive samples. Clone libraries were constructed with amplified DNA by ligating the detected fragments with vector pCR®4-TOPO. Transformations were performed using competent Mix and Go Escherichia coli cells. Plasmids were isolated from each clone using the Zyppy plasmid miniprep protocol and inserts were screened by PCR using M13 DNA primers. Most of the identified fungal species were aligned to the phyla Ascomycota and Basidiomycota. BLAST analysis of clone libraries showed that sequences from compost samples were only comprised of the Ascomycota species Thermomyces lanuginosus. Clones from garden soils were mostly unidentified species closely aligned to Ascomycota and Zygomycota while the most abundant sequences in forest soils were related to the Basidiomycota species Cortinarius flexipes. In garden soil more than 50% of ITS sequences belonged to the Kingdom Plantae with species such as Cardamine hirsute, Stellaria media, and Cerastium dinaricum. However, there were no plant ITS sequences in compost and forest soil. Fungal species belonging to the phylum Ascomycota were widely distributed in the 3 environments studied with the forest soil showing the highest fungal diversity. |
| Databáze: | OpenAIRE |
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