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Spore isolation. A. Filtered liquid containing spores and the club roots left over after Miracloth filtering. B. A pellet with two layers: spores (brown) and starch (white). C. Spore layer after washing steps. D. Spores from Step C7 on top of two-step Ficoll gradient. E. Three layers after Ficoll gradient centrifugation (Step C9): upper layer with bacteria, middle layer mainly spores and bottom layers with starch and debris. F. Five layers (L1 to L5) after Ficoll gradient (Step C16), upper layer (L1) with bacteria, middle layers mainly spores (L2 and L4) and bottom
Source publication
Isolation of DNA from obligate biotrophic soil-borne plant pathogens is challenging. This is because of their strict requirement of living plant tissue for their growth and propagation. A soil habitat further imposes risk of contamination from other microorganisms living in close vicinity of the plant roots. Here we present a protocol on how to pre...
Contexts in source publication
Context 1
... over-heating of the material by running the mixer for 30 sec followed by a 30 sec break. Repeat until a colloidal suspension has formed ( Figure 3A). ...
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... Filter the homogenized tissue through 4 layers of Miracloth to remove debris. Depending on the club size and amount of debris this step may require a new round of filtering ( Figure 3A). ...
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... A pellet with two layers will form: a brown upper layer containing spores and a white layer with starch ( Figure 3B). Carefully remove the supernatant with a pipet using a 1 ml tip. ...
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... Wash the spores by re-suspending them in about 40 ml H2O and repeat centrifugation ( Figure 3C). Repeat washing and centrifugation if a white layer of starch is visible. ...
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... and add the re-suspended spores from Step C7 on top (3v 32% Ficoll:2v 16% Ficoll:1v spores). Make sure the interphase between 3 layers is undisturbed ( Figure 3D). 15. ...
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... Collect the spores, layers 2 and 4 ( Figure 3F) and wash them with 10 ml H2O and centrifuge. Dissolve the pellet in 2 ml TE buffer containing 20 μl of DNase I and incubate for 2 h at 37 °C. ...
Context 7
... over-heating of the material by running the mixer for 30 sec followed by a 30 sec break. Repeat until a colloidal suspension has formed ( Figure 3A). ...
Context 8
... Filter the homogenized tissue through 4 layers of Miracloth to remove debris. Depending on the club size and amount of debris this step may require a new round of filtering ( Figure 3A). ...
Context 9
... A pellet with two layers will form: a brown upper layer containing spores and a white layer with starch ( Figure 3B). Carefully remove the supernatant with a pipet using a 1 ml tip. ...
Context 10
... Wash the spores by re-suspending them in about 40 ml H2O and repeat centrifugation ( Figure 3C). Repeat washing and centrifugation if a white layer of starch is visible. ...
Context 11
... and add the re-suspended spores from Step C7 on top (3v 32% Ficoll:2v 16% Ficoll:1v spores). Make sure the interphase between 3 layers is undisturbed ( Figure 3D). 15. ...
Citations
... The homogenate was filtered through two layers of 220 mm filter paper (Universal Hygia, The Netherlands) and the filtrate was sedimented by centrifugation. Spores were further purified using a two-step ficoll gradient (32 and 16 %, Ficoll 400 (w/v), Carl Roth GmbH & Co. KG, Germany) centrifugation (Mehrabi et al., 2018) to separate the resting spores from bacteria and soil particles. Spores were collected from the interphase of the 16-32 % ficoll layer, and sedimented by centrifugation (11,800 rpm for 1 min) in a standard microcentrifuge (Eppendorf 5417, Germany). ...
... strain e3 infested soils were isolated and used for DNA extraction 55 . High-quality DNA was sent to SciLifeLab, Uppsala, Sweden for PacBio RSII sequencing according to the manufacturer's protocol. ...
Plasmodiophora brassicae is a soil-borne pathogen that attacks roots of cruciferous plants causing clubroot disease. The pathogen belongs to the Plasmodiophorida order in Phytomyxea. Here we used long-read SMRT technology to clarify the P. brassicae e3 genomic constituents along with comparative and phylogenetic analyses. Twenty contigs representing the nuclear genome and one mitochondrial (mt) contig were generated, together comprising 25.1 Mbp. Thirteen of the 20 nuclear contigs represented chromosomes from telomere to telomere characterized by [TTTTAGGG] sequences. Seven active gene candidates encoding synaptonemal complex-associated and meiotic-related protein homologs were identified, a finding that argues for possible genetic recombination events. The circular mt genome is large (114,663 bp), gene dense and intron rich. It shares high synteny with the mt genome of Spongospora subterranea, except in a unique 12 kb region delimited by shifts in GC content and containing tandem minisatellite- and microsatellite repeats with partially palindromic sequences. De novo annotation identified 32 protein-coding genes, 28 structural RNA genes and 19 ORFs. ORFs predicted in the repeat-rich region showed similarities to diverse organisms suggesting possible evolutionary connections. The data generated here form a refined platform for the next step involving functional analysis, all to clarify the complex biology of P. brassicae.
The characteristics of 41 native Trichoderma isolates collected from diverse microclimatic domains in Nepal and two Ohio isolates and their efficacy in biocontrol of plant diseases were assessed. Species assignment was based on a maximum likelihood phylogenetic analysis constructed on a concatenated dataset using the internal transcribed spacer region ( ITS), translocation elongation factor ( tef1), and RNA polymerase II subunit ( rpb2). Most of the Nepal isolates ( n = 37) were assigned to the Viride clade and identified as T. asperellum or T. asperelloides, whereas a minority ( n = 4) were assigned to the Harzianum clade and identified as T. lixii or T. rugulosum. One of the Ohio isolates belonged to the Hamatum clade and was closely related to the T. hamatum reference strain, whereas the other Ohio strain was closely related to T. ghanense (Longibrachiatum clade). Confrontation assays conducted to evaluate mycelial growth reduction of Rhizoctonia solani indicated that mechanisms of action included competition, mycoparasitism, and antibiosis. Trichoderma asperellum isolates NT22, NT8, NT24, and NT25 and T. asperelloides isolates NT1 and NT30 reduced Rhizoctonia root rot severity in greenhouse assays by more than 50% compared with the nontreated controls. Trichoderma asperellum isolates NT4, NT25, NT28, and NT17 and T. asperelloides NT8 suppressed clubroot severity in greenhouse assays by more than 75% compared with the nontreated controls. A significant negative correlation was observed between the percentage of mycelial growth reduction in vitro (confrontation assay) of R. solani and root rot severity. This study provides a basis for additional investigations of Trichoderma diversity and biocontrol potential in Nepal.
[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
