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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Dissertation•
01 Jan 2003
TL;DR: The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani to allow for gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis.
Abstract: The aim of this study was to develop a genetic transformation system for the plant pathogenic fungus Rhizoctonia solani (teliomorph, Thanatephprus cucumeris Frank [Donk]). The availability of a transformation system would allow us to study gene exchange, epidemiology, and to use techniques such as gene disruption or gene silencing to investigate the role of fungal enzymes in pathogenesis.
The approach adopted was to use Agrobacterium tumefaciens to transform the fungus as reports in the literature suggested that this was the most efficient and easiest method to use. As a preliminary test, Fusarium oxysporum was transformed using a binary vector (pBINAN) containing a hygromycin resistance gene under control of an ascomycete promoter and terminator. Hygromycin resistant transformants were obtained after co-incubation of fungal conidia with the bacterium. The presence of the transgene was confirmed by analysis of DNA. The number of transformants depended on the genetic background of the A tumefaciens. Strains AGLO or AGRO gave higher numbers of transformants compared to LBA4404. No transformants were obtained when the hygromycin gene was under control of a basidiomycete (pBINHL1), or a plant (CaMV35S) promoter. Since the basidiomycete promoter used in pBINHL1 originates from Ustilago maydis, the vector was tested by transformation of Ustilago cynodontis. Stable transformants of U cynodontis were obtained with this vector.
A series of experiments were carried out on transformation of R. solani mycelium. Both the protoplast and the Agrobacterium transformation methods were tested. Parameters affecting protoplast production and regeneration were examined. Protoplast production varied with the age of the mycelium, with the osmotic stabilizer used, and with time of treatment with protoplasting enzymes. Regeneration of protoplasts was also affected by the osmotic stabilizer and the growth medium. Transformation of several isolates from different anastomosis groups (AG) was attempted by inducing protoplasts to take up DNA using polyethyleneglycol. Two plasmids were used; (1) pAN7-1 containing the resistance gene under control of an ascomycete promoter, and (2) pHL-1 in which the resistance gene is under control of a basidiomycete promoter. No transformants were obtained.
Attempts were then made to transform mycelium and protoplasts using A tumefaciens. The experiments used both mycelium and protoplasts as the recipient. A number of small resistant colonies were obtained using binary plasmid (pBINHL1) in which mycelium was transformed with the resistance gene was driven by the basidiomycete promoter. On transfer to fresh medium these colonies would grow to about 2cm diameter, and then stop growing. On a second transfer to fresh medium they failed to show any growth. No resistant colonies were obtained from A. tumefaciens transformation of protoplasts.
To improve transformation efficiency, a vector was constructed in which the hygromycin resistance gene was fused to an R solani laccase promoter sequence. No resistant colonies were obtained using this vector. Further experiments were carried out using a hygromycin resistance gene specially modified for expression in basidiomycetes by the insertion of artificial introns in the 5' and 3' untranslated regions, and a number of AT to CG conversions in the coding region. Most of the recipient isolates gave transformants with the unstable resistance phenotype. However, one AG 6 isolate gave transformants with a stable resistance phenotype. Of six transformants recovered from this isolate, five were shown by PCR and southern blotting to contain the transgene. In four of these transformants the resistance phenotype was stable in the absence of selection.
3 citations
01 Jan 1998
TL;DR: Bare patches due to natural and artificial infestation declined during successive croppings of bulbs, whereas bulb rot tended to increase, and temporal niche differentiation is one explanation for the decline phenomenon.
Abstract: Rhizoctonia disease causes severe losses during the production cycle of tulip. The complex nature of the disease requires a precise characterization of the causal pathogens. Typical bare patches are caused by R. solani AG 2-t. Bulb rot symptoms are, apart from AG 2-t isolates, caused by R. solani AG 5. AG 4 isolates seem of little importance in field-grown tulips. Anastomosis behaviour showed AG 2-t to be a homogeneous group, closely related to the heterogeneous group of AG 2-1 isolates. Pectic enzyme patterns discriminated tulip infecting AG 2-t isolates from AG 2 isolates not pathogenic to tulip. Geographically separated AG 2-t and AG 2-1 isolates, both pathogenic to tulip, differ in nucleotide number and sequence of ITS rDNA. Differential interaction between AG 2-t isolates and tulip cultivars was highly influenced by experimental conditions. According to geostatistical analysis field sampling intensity could be reduced down to 10% and still provided adequate disease severity maps. Bare patches due to natural and artificial infestation declined during successive croppings of bulbs, whereas bulb rot tended to increase. Temporal niche differentiation is one explanation for the decline phenomenon.
3 citations
Cites background from "Molecular markers in Australian iso..."
...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 the ribosomal DNA (rDNA) (Liu et al, 1992, 1993, 1995; Liu and Sinclair, 1993; Kanematsu and Naito, 1995), and pulsed field gel electrophoresis (Keijer et al, 1996)....
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01 Jan 1996
TL;DR: Progress in the development of identification methods based on the use of antibodies, or detection of DNA markers for identification of phytopathogenic fungi in general are reviewed, with special reference to R. solani.
Abstract: Techniques for identification of fungal pathogens are necessary for understanding the epidemiology and etiology of the organism and for assessing the effects of control strategies. Detection techniques also allow the level of the pathogen to be monitored so that disease outbreaks can be predicted and control strategies implemented at an early stage. In recent years considerable progress has been made in the development of identification methods based on the use of antibodies, or detection of DNA markers. Such tests are simple to use and have a high degree of specificity. This chapter reviews progress in the development of such tests for identification of phytopathogenic fungi in general, with special reference to R. solani.
2 citations
References
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Book•
15 Jan 2001
TL;DR: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years as mentioned in this paper and has been so popular, or so influential, that no other manual has been more widely used and influential.
Abstract: Molecular Cloning has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential. Molecular Cloning, Fourth Edition, by the celebrated founding author Joe Sambrook and new co-author, the distinguished HHMI investigator Michael Green, preserves the highly praised detail and clarity of previous editions and includes specific chapters and protocols commissioned for the book from expert practitioners at Yale, U Mass, Rockefeller University, Texas Tech, Cold Spring Harbor Laboratory, Washington University, and other leading institutions. The theoretical and historical underpinnings of techniques are prominent features of the presentation throughout, information that does much to help trouble-shoot experimental problems. For the fourth edition of this classic work, the content has been entirely recast to include nucleic-acid based methods selected as the most widely used and valuable in molecular and cellular biology laboratories. Core chapters from the third edition have been revised to feature current strategies and approaches to the preparation and cloning of nucleic acids, gene transfer, and expression analysis. They are augmented by 12 new chapters which show how DNA, RNA, and proteins should be prepared, evaluated, and manipulated, and how data generation and analysis can be handled. The new content includes methods for studying interactions between cellular components, such as microarrays, next-generation sequencing technologies, RNA interference, and epigenetic analysis using DNA methylation techniques and chromatin immunoprecipitation. To make sense of the wealth of data produced by these techniques, a bioinformatics chapter describes the use of analytical tools for comparing sequences of genes and proteins and identifying common expression patterns among sets of genes. Building on thirty years of trust, reliability, and authority, the fourth edition of Mol
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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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