Molecular markers in Australian isolates of Rhizoctonia solani
TL;DR: There was less variation in the RFLP patterns when random cloned fragments of DNA were used as probes and group specific patterns could be identified for all groups, but significant variation within some of the groups whereas other groups were more uniform.
About: This article is published in Fungal Biology.The article was published on 1994-06-01 and is currently open access. It has received 18 citations till now. The article focuses on the topics: Restriction fragment length polymorphism & Restriction enzyme.
Summary (2 min read)
Jump to: [Fungal strains] – [Growth and isolation of DNA] – [Southern blotting] – [RFLPs in ribosomal RNA genes] – [RFLPs with random cloned fragments] and [DISCUSSION]
Fungal strains
- The names and characteristics of the isolates used in this study are outlined in Table 1 .
- The isolates are divided into pectic zymograrn groups (ZG) on the basis of the pectic enzymes produced when grown on pectin (Sweetingharn.
- These pectic zymogram groups correspond to anastomosis groups.
- The pathogenicity of the isolates is: ZG I, cereals and legumes; ZG2, cereals and legumes; ZG3, legwnes; ZG4.
Growth and isolation of DNA
- For analysis of RFLPs mycelium was grown in Petri dishes WA, Western Australia; SA, South Australia; Tas, Tasmania; jap, Japan; NZ, New Zealand.
- Hind III digested pUC18 was treated with calf intestinal phosphatase (I unit of activity I-lg-1 DNA) at 37°for 30 min.
- Both DNA preparations were then extracted with phenol-chloroform, with chloroform, and ethanol precipitated.
- The pellet was washed with 70% ethanoL dried and dissolved in TE buffer.
- Transformants were selected on LB agar containing 50 I-lg ml-l ampicillin, and X-gal (Sambrook et ai., 1990) .
Southern blotting
- R. solani DNA was digested with restriction enzyme (5 units of activity I-lg-1 DNA) for 15 h under conditions specified by the supplier.
- The digest was fractionated by electrophoresis through a 0'8% agarose gel in TAE buffer at I Vern-I.
- After prehybridization, heat denatured probe was added to the bag and hybridization carried out overnight at 65°.
- The membrane was rinsed briefly in 2 x SSe, O' 1% SDS at room temperature and at 65°for 30 min in the same solution.
- The nick translation reagents were obtained from Amersham, and used in accordance with their instructions.
RFLPs in ribosomal RNA genes
- A Southern blot prepared with fcoR I digested R. solani DNA was probed with a wheat rRNA gene.
- All of the ZG3 isolates contain a 2'2 kb band which is unique to this group.
- Other bands observed in common among isolates from different groups included a 9'7, and a 0'6 kb band.
- On the basis of anastomosis behaviour this group is known to be highly polymorphic (Sneh et al., 1991) .
- Similarly, the ZG8 isolate AH-1 which can be differentiated from the other ZG8 isolates by its RFLP pattern (Fig. I , lane 22; Table 2 ), belongs to a different AG4 subgroup than the other AG4 isolates (Table I ).
RFLPs with random cloned fragments
- To generate additional probes for RFLP analysis, R. salani DNA was digested to completion with Hind III, and the digest cloned into the plasmid pUeI8 to generate a library.
- Size of fcoR I fragments homologous to pRGL2-IO were selected from this library at random, and screened to determine the insert size.
- Hind III and feaR V were also very useful whilst Pst I, and BamH I generated very few bands and thus were likely to be less useful in differentiating groups.
- Identical patterns were also obtained for these isolates with the pRGLl-AI probe.
- Probe pRGLZ-AI gave a more complex pattern with the ZG3 isolates than with isolates from other ZG.
DISCUSSION
- Ribosomal RNA genes are present in hundreds of copies per genome in fungi, and they contain sequences that are highly variable (Olsen et al., 1986) .
- They also showed that there was variation within each of the groups.
- This pattern of variability may reflect the degrees of divergence from the group.
- ZG4 and ZGS, appear to be quite different groups despite the fact that they belong to the same anastomosis group, AG2.
- Unlike ZG4 isolates which are very homogenous in their RFLP patterns, ZGS isolates are very heterogeneous except perhaps with probe pRGLl-IO.
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TL;DR: RAPD-PCR was used to characterize isolates of Rhizoctonia solani from bare patches in cereal and pasture crops at two locations in Western Australia, Newdegate and Esperance, and there was no difference in RAPD- PCR pattern between highly virulent and weakly virulent isolates at Newdegates.
Abstract: RAPD-PCR was used to characterize isolates of Rhizoctonia solani from bare patches in cereal and pasture crops at two locations in Western Australia, Newdegate and Esperance. All of the isolates belonged to anastomosis group 8, pectic zymogram group 1-1 or 2. The pectic zymogram assignment could be confirmed by RAPD-PCR. There was no difference in RAPD-PCR pattern between highly virulent and weakly virulent isolates at Newdegate, or between isolates from different patches at Newdegate. The Newdegate isolates were identical to isolates from Esperance, and to isolates from various locations in South Australia.
21 citations
TL;DR: The genetic diversity and population structure among 72 rice-infecting isolates of Rhizoctonia solani AG-1 IA, collected from 12 counties (subpopulations) of Guangdong, Guangxi and Hainan provinces in south China, were investigated using nine inter-simple sequence repeat (ISSR) markers.
Abstract: The genetic diversity and population structure among 72 rice-infecting isolates of Rhizoctonia solani AG-1 IA, collected from 12 counties (subpopulations) of Guangdong, Guangxi and Hainan provinces (populations) in south China, were investigated using nine inter-simple sequence repeat (ISSR) markers. A total of 116 bands were amplified, with a majority of amplified fragments ranging from 500 bp to 2500 bp in size, of which 110 (94.8%) were polymorphic. Seventy-two isolates were grouped into six major clusters at 73% genetic similarity coefficient by the unweighted pair group method with arithmetic mean (UPGMA) with Dice’s distance matrices. The genetic diversity was high [percentage of polymorphic bands (P %) = 94.83%; Shannon’s diversity index (I) = 0.3175; Nei’s diversity (h) = 0.2034] at the population level, but low within populations [P % = 53.38%; Shannon’s diversity index (I) = 0.2734; Nei’s diversity (h) = 0.1811]. The mean coefficient of gene differentiation (Gst) was 0.165, indicating th...
15 citations
Cites methods from "Molecular markers in Australian iso..."
...These genetic markers included isoenzymes (Liu & Nickrent 1990; Neeraja et al. 2003), DNA base sequence complementary analysis (Cubeta & Vilgalys 1997; Ceresini et al. 2002), restriction fragment length polymorphisms (RFLP) (O’Brien 1994; Rosewich et al. 1999; Ceresini et al. 2002), random amplified polymorphic DNA (RAPD) (Duncan et al. 1993; Zhou et al. 2002; Dubey et al. 2012; Mirmajlessi et al. 2012; Wang et al. 2013), amplified fragment length polymorphism (AFLP) (Taheri et al. 2007), simple sequence repeats (SSR) (Bernardes-de-Assis et al. 2009) and inter-simple sequence repeats (ISSR) (Sharma et al. 2005; Guleria et al. 2007; Stodart et al. 2007; Khodayari et al. 2009; Dubey et al. 2012; Mirmajlessi et al. 2012)....
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...…2003), DNA base sequence complementary analysis (Cubeta & Vilgalys 1997; Ceresini et al. 2002), restriction fragment length polymorphisms (RFLP) (O’Brien 1994; Rosewich et al. 1999; Ceresini et al. 2002), random amplified polymorphic DNA (RAPD) (Duncan et al. 1993; Zhou et al. 2002; Dubey et…...
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TL;DR: The results suggest that MoSDR1 functions as a key metabolic enzyme in the regulation of development and pathogenicity in M. oryzae.
Abstract: The phytopathogenic fungus Magnaporthe oryzae is a major limiting factor in rice production. To understand the genetic basis of M. oryzae pathogenic development, we previously analyzed a library of T-DNA insertional mutants of M. oryzae, and identified ATMT0879A1 as one of the pathogenicity-defective mutants. Molecular analyses and database searches revealed that a single TDNA insertion in ATMT0879A1 resulted in functional interference with an annotated gene, MGG00056, which encodes a short-chain dehydrogenase/reductase (SDR). The mutant and annotated gene were designated as MoSDR1T-DNA and MoSDR1, respectively. Like other SDR family members, MoSDR1 possesses both a cofactor- binding motif and a catalytic site. The expression pattern of MoSDR1 suggests that the gene is associated with pathogenicity and plays an important role in M. oryzae development. To understand the roles of MoSDR1, the deletion mutant ΔMosdr1 for the gene was obtained via homology-dependent gene replacement. As expected, ΔMosdr1 was nonpathogenic; moreover, the mutant displayed pleiotropic defects in conidiation, conidial germination, appressorium formation, penetration, and growth inside host tissues. These results suggest that MoSDR1 functions as a key metabolic enzyme in the regulation of development and pathogenicity in M. oryzae.
12 citations
TL;DR: Taxonomic position of 46 Korean isolates of Rhizoctoniasolani which were classified into nine intraspecific groups by anastomosis and cultural characteristics was ana-lyzed by randomly amplified polymorphic DNA (RAPD) and sequence analyses of the internal transcribed spacer(ITS) regions of ribosomal DNA) showed that sequence analysis with ITS regions could be a rapid anduseful method for identification of intrapecific group of R. solani.
Abstract: National Academy of Agricultural Science, RDA, Suwon 441-707, Korea(Received on June 24, 2009; Accepted on January 13, 2010)Taxonomic position of 46 Korean isolates of Rhizoctoniasolani which were classified into nine intraspecific groupsby anastomosis and cultural characteristics was ana-lyzed by randomly amplified polymorphic DNA (RAPD)and sequence analyses of the internal transcribed spacer(ITS) regions of ribosomal DNA. All the isolates withineach group showed highly similar band patterns inRAPD. The ITS regions of the isolates within the samegroups showed a high level of sequence similarity above96.0% whereas similarities among different groups werebelow 94.4%. When compared with several referencestrains of R. solani from foreign countries, all theKorean isolates were clustered with the foreign isolatesbelonging to the same groups in the phylogenetic tree.All six Korean strains of AG-4 were identified as HG-1out of 3 subgroup of AG-4. We discussed taxonomicposition of Korean isolates of R. solani and showed thatsequence analysis with ITS regions could be a rapid anduseful method for identification of intraspecific group ofR. solani.Keywords : anastomosis, ITS, randomly amplified poly-morphic DNA, Rhizoctonia solaniRhizoctonia solani Kuhn [teleomorph: Thanatephoruscucumeris (Frank) Donk], the most recognized specieswithin genus Rhizoctonia, is potent pathogen of economi-cally important plant species and shows considerable diver-sity in morphology, geographic location, host specificityand pathogenicity (Ogoshi, 1987). Kim et al. (1994; 1995)reported that R. solani had caused 65 diseases to wide rangeof plants including rice, potato, pepper, vegetables, orna-mentals, turf grasses, pine tree etc. in Korea. The concept ofanastomosis group (AG) is a widely accepted principle foridentifying intraspecific groups in the R. solani complex(Carling, 1996). Fourteen AGs, AG-1 through AG-13 andAG-BI (bridging isolates), had been described for R. solaniby this concept (Carling, 1996; Ogoshi, 1987). However,AG concept is not an ideal method for classification of R.solani as misidentification is caused from the varied fre-quency of hyphal fusion in some AG (Liu and Sinclair,1992). For the biological bases of AG, vitamin requirementanalysis (Ogoshi and Ui, 1979), serological studies (Adamsand Butler, 1979), fatty acid analyses (Stevens Johnk andJones, 1993; Stevens Johnk et al., 1993), isozyme analy-ses (Reynolds et al., 1983), total soluble protein pattern(Liu and Ge, 1988), GC content analysis and DNA-DNAhybridization (Kuninaga and Yokozawa, 1980) had beenstudied. Recently, molecular biological techniques havebeen used in combination with morphological and physio-logical markers for the analysis of R. solani population(Guleria et al., 2007). The restriction fragment lengthpolymorphism (RFLP) studies on ribosomal DNA regionswere performed to differentiate AGs and their subgroups(O'Brien, 1994; Vilgalys and Gonzales, 1990). Randomlyamplified polymorphic DNA PCR (RAPD-PCR) analysiswas performed to detect genetic variability among theisolates of R. solani (Sharma et al., 2005). Sequenceanalyses of the internal transcribed spacer (ITS) regions ofribosomal DNA have been used to study the geneticrelationships between AGs of R. solani by many authors(Boysen et al., 1996; Gonzales et al., 2001; Kuninaga etal., 1997; 2000; Salazar et al., 1999). Several studies were performed with Korean isolates ofthe species. Kim et al. (1994; 1995) reported culturalcharacteristics of R. solani isolates collected from variouscrop plants in Korea and Hong et al. (1998) performedPCR-RFLP analysis of their partial 18S-ITS-5.8S regionof ribosomal DNA. However, there has been no report onthe taxonomic position of Korean isolates using sequenceanalysis of ribosomal ITS region. In this study, we ex-amined genetic diversity of 46 Korean isolates of R. solani,representing 9 groups (6 anastomosis group and 5 culturaltypes) using randomly amplified polymorphic DNA (RAPD)and DNA sequences of ribosomal ITS region. The objec-tives of this study were to: (1) determine the completeDNA sequence of the ribosomal ITS regions of Koreanisolates, (2) confirm the relatedness between molecular
10 citations
Cites methods from "Molecular markers in Australian iso..."
...The restriction fragment length polymorphism (RFLP) studies on ribosomal DNA regions were performed to differentiate AGs and their subgroups (O'Brien, 1994; Vilgalys and Gonzales, 1990)....
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TL;DR: A vector for Agrobacterium tumefaciens-mediated transformation of basidiomycetes was constructed by insertion of a modified hygromycin resistance gene into the plant binary vector pBin19, which confirmed the presence of the gene in the transformants by PCR analysis and Southern hybridisation.
Abstract: A vector for Agrobacterium tumefaciens-mediated transformation of basidiomycetes was constructed by insertion of a modified hygromycin resistance gene into the plant binary vector pBin19. The hygromycin coding region is flanked by a basidiomycete promoter and terminator. Isolates from different anastomosis groupings (AG) of the phytopathogenic fungus Rhizoctonia solani (Kuhn) were transformed with this vector using A. tumefaciens. Hygromycin-resistant transformants were isolated from a single AG6 isolate, but not from AG3 or AG4 isolates. Of six transformants isolated, five showed enhanced growth on agar containing either 25 or 50 ⧎g/mL hygromycin. However, as the hygromycin concentration increased, the difference between the transformants and the control reduced, until at 100 ⧎ g/mL there was no difference. The resistance phenotype was retained through repeated subcultures on non-selective media. The presence of the gene in the transformants was confirmed by PCR analysis and Southern hybridisation.
9 citations
Cites methods from "Molecular markers in Australian iso..."
...Harvesting of the mycelium, extraction ofDNA, digestion of theDNA (5mg)withHindIII, and Southern hybridisation were carried out as described previously (O’Brien 1994) with 5mg DNA per well....
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...DNA for PCR analysis was extracted from mycelium as described previously (O’Brien 1994)....
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References
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"Molecular markers in Australian iso..." refers background in this paper
...Ribosomal RNA genes are present in hundreds of copies per genome in fungi, and they contain sequences that are highly variable (Olsen et al., 1986)....
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...This may be a consequence of the fact that the rRNA genes can undergo significant variation without affecting biological function (Olsen et al., 1986)....
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