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Journal ArticleDOI

Molecular markers in Australian isolates of Rhizoctonia solani

01 Jun 1994-Fungal Biology (Elsevier)-Vol. 98, Iss: 6, pp 665-671
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)

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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Citations
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Journal ArticleDOI
TL;DR: Results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.
Abstract: Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to evaluate the genetic diversity of 45 isolates within and between anastomosis groups (AGs) in Rhizoctonia solani. The 5.8s rDNA sequence was completely conserved across all the AGs examined, whereas the ITS rDNA sequence was found to be highly variable among isolates. The sequence homology in the ITS regions was above 96% for isolates of the same subgroup, 66-100% for isolates of different subgroups within an AG, and 55-96% for isolates of different AGs. In neighbor-joining trees based on distances derived from ITS-5.8s rDNA sequences, subgroups IA, IB and IC within AG-1 and subgroups HG-I and HG-II within AG-4 were placed on statistically significant branches as assessed by bootstrap analysis. These results suggest that sequence analysis of ITS rDNA regions of R. solani may be a valuable tool for identifying AG subgroups of biological significance.

212 citations

Journal ArticleDOI
TL;DR: This listing covers the period May 1, 1997 through to June 30, 1997, which roughly corresponds with the British Mycological Society's Special Interest Committees.

211 citations

Journal ArticleDOI
TL;DR: Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields and it is proposed to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1.
Abstract: Methods based on internal transcribed spacers (ITS) ribosomal DNA (rDNA) polymorphism and pectic zymograms (ZG) were compared for their use in routine identification of Rhizoctonia solani isolates occurring in flower bulb fields. Thirty three AG 2-t isolates, pathogenic to tulips, could be distinguished from AG 1-IC, AG 2-2IIIB and AG 2-2IV, AG 3 and AG 5 by means of ITS rDNA fragment length and after digestion with EcoR I from AG 4 and AG 5. AG 2-t isolates and two Japanese isolates, pathogenic to crucifers and tulips, had an estimated fragment size of 710 bp, whereas Dutch AG 2-1 isolates, non-pathogenic to tulips, showed an estimated fragment size of 705 bp on agarose gel. Digestion of AG 2-t and AG 2-1 isolates with EcoR I, Sau3A I, Hae III and Hinc II revealed four and five distinct ITS rDNA digestion patterns, respectively. In AG 2 isolates 2tR114, 21R14 and 21R61 a double digestion pattern, indicating different ITS sequences within an isolate, was found. The observed ITS fragment length polymorphism between isolates pathogenic and non-pathogenic to tulips were considered too small to be used in routine screening of field isolates. Sequencing of AG 2 isolates 21R01, 21R06, 2tR002 and 2tR144 showed a total ITS rDNA fragment length of 715, 713, 714, and 728 bp. As an alternative to ITS rDNA fragment length polymorphism, pectic enzyme patterns were studied using a commercially available vertical gel-electrophoresis system and non-denaturing polyacrylamide gels amended with pectin. Anastomosis tester isolates AG 1 to AG 11 revealed different ZG. Fifty AG 2-t isolates and five AG 2-1 isolates belonged to a homogeneous pectic zymogram group. We propose to assign AG 2 isolates pathogenic to crucifers and tulip to ZG5-1. AG 2-1 isolates, non-pathogenic to tulip, formed a heterogeneous group with 4 distinct ZG. Pectic zymography provides an easy, quick and unambiguous method for routine identification of large numbers of field isolates. Such a technique is needed for research on the dynamics of Rhizoctonia populations to develop environmentally friendly control measures of rhizoctonia disease in field-grown flower bulbs.

48 citations


Cites background from "Molecular markers in Australian iso..."

  • ...…of isolates of R. solani include genomic restriction fragment length polymorphism (Vilgalys and Gonzalez, 1990; Jabaji-Hare et al., 1990; O’Brien, 1994), random amplified polymorphic DNA (Duncan et al.,1993; Yang et al., 1995), polymorphism of the internal transcribed spacer (ITS) of…...

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Journal ArticleDOI
TL;DR: The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied, and PCR-ribotyping with AluI and MspI is sufficient for analysis of distance between populations.
Abstract: Forty seven strains of the black yeasts, Aureobasidium pullulans and Hormonema dematioides, and the type strain of Hormonema macrosporum were examined using PCR-ribotyping and universally primed PCR with subsequent hybridization. Four groups (populations) were distinguished within A. pullulans with PCR-ribotyping, which largely coincided with UP-PCR/hybridization groups. The UP-PCR technique revealed a greater degree of heterogeneity between the groups studied. Five strains identified as Hormonena dematioides on the basis of physiological and morphological data formed a group recognizable with PCR-ribotyping and UP-PCR/hybridization, which also included H. macrosporum. Aureobasidium pullulans is characterized by the absence of RsaI restriction sites in rDNA amplified with primers 5.8S-R and LR7, while Hormonema species possessed several bands after RsaI digestion. For analysis of distance between populations, PCR-ribotyping with AluI and MspI is sufficient. Strains of A. pullulans produce exopolysaccharides in liquid media with different nitrogen sources, while the strains of Hormonema synthesize minor amounts of polysaccharides in media with peptone. Populations of A. pullulans differ slightly from each other in their optimal, medium-dependent production of polysaccharides.

21 citations

References
More filters
Journal ArticleDOI
TL;DR: It is demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups.
Abstract: The poor definition of pathotype variation in the rice blast fungus has historically handicapped strategies for reducing blast disease damage to the world's rice crop. We have employed a probe for a dispersed repeated DNA sequence called MGR [Hamer et al. (1989). Proc. Natl. Acad. Sci. USA 86, 9981-9985] to construct genotype-specific, EcoRl restriction fragment length profiles (MGR-DNA fingerprints) from United States field isolates of this fungus. By using a blind-test design, we demonstrated that MGR-DNA fingerprints distinguished the major pathotypes in the United States, accurately identified the pathotypes of isolates collected over a 30-year period, and defined the organization of clonal lineages within and among pathotype groups. These results resolved a lingering controversy regarding rice blast pathotype stability and illustrated new opportunities for tracking the population dynamics and evolution of this important crop pathogen.

245 citations

Journal ArticleDOI
TL;DR: It is proposed that the pattern of RFLP variation observed is most likely the result of genetic divergence within and among intraspecific groups.
Abstract: (...) Isolates from anastomosis groups 3, 4, 7, 8, and BI all possess a single, invariant RFLP unique to each intraspecific group. A relatively low level of RFLP variation also was characteristic of isolates within AG-1-IB, AG-1-IC, AG-2-2, and AG-9. In contrast, a relatively high level of RFLP diversity was observed among isolates within AG-1-IA, AG-2-1, AG-5, and AG-6. We did not observe any obvious relationship between levels of RFLP within intraspecific groups and variation of other biological factors (host range, morphological variation, etc). Instead, we propose that the pattern of RFLP variation observed is most likely the result of genetic divergence within and among intraspecific groups

141 citations

Journal ArticleDOI
TL;DR: The pectic enzymes of 140 isolates of Rhizoctonia-1ike fungi from the Western Australian grainbelt were examined by electrophoresis and found to fall into 11 distinct zymogram groups (ZG).
Abstract: The pectic enzymes of 140 isolates of Rhizoctonia-1ike fungi from the Western Australian grainbelt were examined by electrophoresis and found to fall into 11 distinct zymogram groups (ZG). Isolates within a ZG had a similar cultural and morphological appearance and were either all multinucleate or all binucleate. Some isolates from most ZGs sporulated when transferred from potato-dextrose-marmite agar to water agar. Isolates from within a ZG had the same teleomorph. Pathogenicity of the isolates was tested against wheat and lupins. All isolates from within a ZG were consistent in the type of lesions they produced and in their virulence towards these hosts. Rhizoctonia patch disease of cereals and lupins appears to be caused by isolates from ZG 1 and ZG 2. Severe reddish-brown and brown hypocotyl rots of lupins were caused by ZG 3 and ZG 4 isolates respectively. Five Ceratobasidium groups (CZG), one Waitea group (WZG) and ZG 5 had weak to nil pathogenicity towards wheat and lupins.

116 citations

Journal ArticleDOI
TL;DR: It is shown that, although the amount of bound DNA is indeed higher with a short alkaline transfer, transfer in ammonium acetate buffer (NH4AC) (2) and binding of DNA on membrane by conventional baking gives upon subsequent hybridization a five to ten fold better sensitivity, the probe hybridizes about ten times better with NH4Ac than NaOH transferred DNA leading to a drastic increase in sensitivity.
Abstract: Recently a protocol for rapid DNA transfer (Southern blotting) to nylon membranes using sodium hydroxyde as the transfer solvent, has been described (l). We show here that, although the amount of bound DNA is indeed higher with a short alkaline transfer, transfer in ammonium acetate buffer (NH4AC) (2) and binding of DNA on membrane by conventional baking gives upon subsequent hybridization a five to ten fold better sensitivity. Two DNA preparations, one labelled, one unlabelled, to estimate respectively the binding and the hybridization efficiency were electrophoresed side by side on 1% agarose gels. All gels were then soaked in 0,25 N HC1 2x15 min. before transfer of the DNA to Zeta probe nylon membrane (Bio-Rad). For NaOH transfer the DNA was directly transferred in 0,4 N NaOH as described (1). The other gels were equilibrated twice in 0,5 N NaOH, 1,5 M NaCl for 20 minutes, twice in 1 M NHAAc, 0,02 N NaOH for 30 minutes, before transfer of the DNA in 1 M NH4Ac, 0,02 N NaOH. After 24 hours of prehybridization, the DNA from the six membranes were incubated with the same amount (10^ cpm/ml, 2x108 cpm/ng) of nick translated p86 DNA for 24 hours in 50% formamide as described (3). The membranes were then washed three times 30 minutes with the last wash in 0.1 x SSC, 0.1% SDS at 65°C and autoradiographied. The comparison of these autoradiographies leads to three conclusions : 1) baking has no effect on the signal obtained (binding and hybridization) to DNA transferred in NaOH, but increase about two fold thr retention and several fold the hybridization of the DNA transferred in NH4Ac. 2) vith a short (4 hours) transfer there is more DNA bound to the membranes with alkaline transfer as judged by the intensity of the signal given by the labeled DNA. 3) after longer (16 hours) transfers, the amount of DNA on the membrane after tne two types of transfer tend to become equal. However, the probe hybridizes about ten times better with NH4Ac than NaOH transferred DNA leading to a drastic increase in sensitivity. The same results were obtained with transfer of genomic DNA and visualization of a single copy gene (not shown). REFERENCES : 1. Reed K.C. and Mann D.A. (1985) Nucl. Acids Res. L5 : 7207-7221. 2. Smith G.E. and Summers M.D. (1980) Anal. Biochem. JJ39 : 123-129. 3. Maniatis T., Fritsch E.F. and Sambrook J. (1982) Molecular Cloning : A laboratory Manual. Cold Spring Harbor Laboratory. Cold Spring Harbor, N.Y.

80 citations


"Molecular markers in Australian iso..." refers methods in this paper

  • ...The DNA was then denaturated and blotted onto Zeta Probe (BioRad) for 16 h using I M ammonium acetate 20 mM NaOH as the transfer buffer (Rigaud et al., 1987)....

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