Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue

Liu, L; Wang, C L; Peng, Wayne; Yang, Jie; Lan, Mu; Zhang, B; Li, J B; Zhu, Youyong; Li, C Y

doi:10.4238/2015.december.28.1

Cited by 16 publications

(10 citation statements)

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“…Using Chelex for DNA stabilization is cheap and easy (Liu et al 2015). It allowed the amplification of fragments of both genes of interest (HIS and TEF) for the Harris Uni-Core samples; however, the amplification of the HIS gene fragment failed for two samples, while the TEF primers delivered an amplification.…”

Section: Pcr As the Basis For The Identification Of Fungimentioning

confidence: 99%

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

et al. 2020

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Apple replant disease (ARD) negatively affects growth and yield of apple plants worldwide. Fungi belonging to the Nectriaceae have often been isolated from roots grown in replant soils and thus are proposed among others as one biotic cause of the disease complex. Microscopic analyses of ARD-affected roots revealed characteristic symptoms associated with fungal infection sites. Here, two extraction methods of such tissue sites were applied to directly identify an unknown fungus that forms typical cauliflower-like structures in diseased root cortex cells. Punching small tissue samples of about 0.5 mm3 volume with the Harris Uni-Core is a quick and easy method to harvest symptomatic material. Secondly, a laser microdissection (LMD) protocol for apple roots was established. This technique allows the extraction of defined cell or tissue fractions from thin cryo-sections. Tissue harvesting was followed by the identification of fungi via PCR amplification of two gene fragments and Sanger sequencing. For Harris samples, Chelex was used for DNA stabilization, while LMD samples were directly submitted to PCR. In Harris samples, mainly the Nectriaceae species Dactylonectria torresensis, Ilyonectria robusta and Rugonectria rugulosa were identified. In addition to these, in LMD samples Cylindrocladiella sp. and Ilyonectria europaea were detected. Thus, the intracellular CF structures contained different species of Nectriaceae in the ARD-affected cortex cells. These results contribute considerably to the etiology of the ARD. Both protocols offer the possibility to identify fungi from selected symptomatic small root sections by molecular tools avoiding isolation and subsequent axenic pure cultures of single fungal isolates.

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Section: Pcr As the Basis For The Identification Of Fungimentioning

confidence: 99%

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

et al. 2020

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“…For pure fungal cultures, hyphae were collected and ground in liquid Benihoppe leaves from each of the three plants were randomly harvested and pooled as one replicate; three replicates were conducted for each time point. After being ground into a powder in liquid nitrogen, genomic DNA was extracted using the modified cetyltrimethylammonium bromide (CTAB) method (Liu et al, 2015). DNA purity was then measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, USA) using the A 260/280 and A 260/230 ratios.…”

Section: Dna Extraction From Pure Fungal Cultures and Inoculated Stra...mentioning

confidence: 99%

Method to detect and quantify colonization of anthracnose causal agent Colletotrichum gloeosporioides species complex in strawberry by real‐time PCR

Yang

et al. 2022

Journal of Phytopathology

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Anthracnose, a destructive disease in strawberry worldwide, is caused by the pathogenic fungus Colletotrichum ssp.. To date, numerous studies focusing on its identification, pathogenicity and biological characteristics have been published. However, research on quantifying the colonization of this pathogen is still lacking, which is crucial to the study of anthracnose resistance. In this study, we developed a sensitive primer pair targeting the cutinase gene that is capable of distinguishing Colletotrichum gloeosporioides (C. gloeosporioides) species complex, the main species causing strawberry anthracnose in China, including C. fructicola, C. gloeosporioides, C. aenigma and C. siamense, from other Colletotrichum species and typical fungal pathogens. On this basis, we established a quantitative real‐time PCR (qRT‐PCR) assay to accurately quantify pathogen amounts from both pure cultures and infected strawberries, with detection limits of 105–101 copies. Using this method, we characterized quantitative traits of C. fructicola colonization in strawberry leaves and discovered differences in pathogen populations at different positions. Moreover, monitoring of pathogen growth during infection showed a dynamic change in the amount of C. fructicola. Finally, a comparison of the absolute and relative quantification results further proved this method's validity. Therefore, we propose that this specific PCR method enables rapid detection and quantification of C. gloeosporioides species complex colonization in strawberry and will be a useful tool for anthracnose management and strawberry resistance studies.

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“…Fungal mycelia were grown at 25°C for 5 days, collected and grounded into powder with liquid nitrogen. Genomic DNA was extracted with the cetyltrimethylammonium bromide (CTAB) method [52]. The harvested DNA was detected by agarose gel electrophoresis and quantified by Nanodrop.…”

Section: Fungal Culture and Genomic Dna Extractionmentioning

confidence: 99%

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites

et al. 2020

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Background: Secondary metabolites as natural products from endophytic fungi are important sources of pharmaceuticals. However, there is currently little understanding of endophytic fungi at the omics levels about their potential in secondary metabolites. Calcarisporium arbuscula, an endophytic fungus from the fruit bodies of Russulaceae, produces a variety of secondary metabolites with anti-cancer, anti-nematode and antibiotic activities. A comprehensive survey of the genome and transcriptome of this endophytic fungus will help to understand its capacity to biosynthesize secondary metabolites and will lay the foundation for the development of this precious resource. Results: In this study, we reported the high-quality genome sequence of C. arbuscula NRRL 3705 based on Single Molecule Real-Time sequencing technology. The genome of this fungus is over 45 Mb in size, larger than other typical filamentous fungi, and comprises 10,001 predicted genes, encoding at least 762 secretory-proteins, 386 carbohydrate-active enzymes and 177 P450 enzymes. 398 virulence factors and 228 genes related to pathogen-host interactions were also predicted in this fungus. Moreover, 65 secondary metabolite biosynthetic gene clusters were revealed, including the gene cluster for the mycotoxin aurovertins. In addition, several gene clusters were predicted to produce mycotoxins, including aflatoxin, alternariol, destruxin, citrinin and isoflavipucine. Notably, two independent gene clusters were shown that are potentially involved in the biosynthesis of alternariol. Furthermore, RNA-Seq assays showed that only expression of the aurovertin gene cluster is much stronger than expression of the housekeeping genes under laboratory conditions, consistent with the observation that aurovertins are the predominant metabolites. Gene expression of the remaining 64 gene clusters for compound backbone biosynthesis was all lower than expression of the housekeeping genes, which partially explained poor production of other secondary metabolites in this fungus. Conclusions: Our omics data, along with bioinformatics analysis, indicated that C. arbuscula NRRL 3705 contains a large number of biosynthetic gene clusters and has a huge potential to produce a profound number of secondary metabolites. This work also provides the basis for development of endophytic fungi as a new resource of natural products with promising biological activities.

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Direct DNA extraction method of an obligate parasitic fungus from infected plant tissue

Cited by 16 publications

References 0 publications

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

Molecular identification of Nectriaceae in infections of apple replant disease affected roots collected by Harris Uni-Core punching or laser microdissection

Method to detect and quantify colonization of anthracnose causal agent Colletotrichum gloeosporioides species complex in strawberry by real‐time PCR

Genomic and transcriptomic survey of an endophytic fungus Calcarisporium arbuscula NRRL 3705 and potential overview of its secondary metabolites

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