Stable transformation of Rhizoctonia solani with a modifiedhygromycin resistance gene

Jiang WuA,B and Philip A. O’BrienA,C

ASchool of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia.BPresent address: Agritechnology, Orange, NSW 2800, Australia.CCorresponding author. Email: P.Obrien@murdoch.edu.au

Abstract. A vector for Agrobacterium tumefaciens-mediated transformation of basidiomycetes was constructed byinsertion of a modified hygromycin resistance gene into the plant binary vector pBin19. The hygromycin coding region isflanked by a basidiomycete promoter and terminator. Isolates from different anastomosis groupings (AG) of thephytopathogenic 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 sixtransformants isolated,five showed enhanced growth on agar containing either 25 or 50mg/mLhygromycin.However, as thehygromycin concentration increased, the difference between the transformants and the control reduced, until at 100mg/mLthere was no difference. The resistance phenotype was retained through repeated subcultures on non-selective media. Thepresence of the gene in the transformants was confirmed by PCR analysis and Southern hybridisation.

Introduction

Rhizoctonia solani Kuhn [teliomorph Thanatephorus cucumeris(Frank) Donk] is a serious pathogen of many crops worldwideunder many different types of environmental conditions(Parmeter and Whitney 1970). Studies of the genetics,pathology and ecology of this pathogen have been hindered bya lack of a transformation system.A transformation systemwouldallow the introduction of marker genes, which could be used tostudy gene exchange (both vegetative and sexual), and enable theuse of techniques such as gene disruption or gene silencing(Meyer 2008) to investigate mechanisms of virulence.

There has been only one reported attempt at developing atransformation system forR. solani. Robinson andDeacon (2001)transformed protoplasts with a vector containing a hygromycinresistance gene. Although they obtained resistant colonies, thesestopped growing after a short interval and failed to show anyfurther growth even when transferred to fresh media. Southernhybridisation analysis showed that the transgene had notintegrated into the endogenome. In recent years there havebeen several reports showing that fungi can be transformed byAgrobacterium tumefaciens (deGroot et al. 1998; Mullins et al.2001). Transformants can be isolated on selective media after aperiod of co-cultivating conidia or protoplasts withA. tumefaciens cells containing a suitable binary vector. Thetechnique is easier and more efficient compared with traditionalmethods of protoplast transformation by polyethyleneglycol orelectroporation.

Studies of transgene expression in the basidiomycetesSchizophyllum commune and Agaricus bisporus have revealedthat, in general in basidiomycetes, cDNA transgenes areexpressed less efficiently than genomic clones which contain

introns (Lugones et al. 1999). Scholtmeijer et al. (2001)demonstrated that the insertion of artificial introns at the 50

and 30 untranslated regions of the E. coli hph (hygromycinphosphotransferase) gene greatly increased the stability of thehph transcript in transformed cells of S. commune andconsequently increased the transformation efficiency. Theyalso reported that conversion of an AT rich sequence at the50 end of the hph coding sequence to a GC rich sequencefurther increased expression and transformation efficiency.In this study, we report the A. tumefaciens-mediated stabletransformation of R. solani with a hygromycin resistance genemodified for enhanced expression in basidiomycetes.

MethodsIsolates

TheR. solani isolates used are described in Table 1. Isolates weremaintained on potato dextrose agar (PDA) at 4�Cand subculturedat intervals onto fresh PDA agar and grown at 26�C.

Construction of a binary vector

AnAgrobacterium binary vectorwas constructedwith amodifiedhph gene (Fig. 1). This modified gene contains introns in the50 and 30 untranslated regions, several A–C conversions in the50 part of the coding region and is flanked by the Pgpd and Tsc3

of the basidiomycete S. commune (Scholtmeijer et al. 2001).The gene was transferred from pHYM2.2 (kindly provided byK. Scholtmeijer) to the plant binary vector pBinl9 (Bevan 1984)as a HindIII/EcoR1 fragment to create pJW2.2hyg-15. ThepJW2.2hyg-15 plasmid was transferred to A. tumefaciensAGLO (Lazo et al. 1991) by conjugation (Hooykaas 1988).

CSIRO PUBLISHING

www.publish.csiro.au/journals/app Australasian Plant Pathology, 2009, 38, 79–84

� Australasian Plant Pathology Society 2009 10.1071/AP08081 0815-3191/09/010079

Transformation procedure

For transformation an inoculum block (5mm3) was transferredfrom a stock agar plate of the fungal isolate to a 9-cm Petri dishcontaining 20mL potato dextrose broth and incubated at 26�Cfor 3 days. The mycelium was harvested, resuspended in anequal volume of fresh growth medium and blended for 1min toproduce a suspension ofmycelial fragments. This suspensionwasincubated at 26�C for 24 h without shaking. The myceliumwas harvested, resuspended in induction medium (Hooykaas1988) and blended to produce a suspension. The transformationprocedure was essentially as described by deGroot et al. (1998)except that the bacterial suspension was diluted 100-fold withinduction medium before mixing with the mycelium, andmycelium bacterial mixes were spread onto induction mediumplates without filters. At the end of the co-cultivation periodthe mycelium was scraped off the plates and made into a slurryby blending. Aliquots of the slurry were plated onto selectivePDA plates containing hygromycin 50mg/mL (Boehringer-Mannheim, Mannheim), cefotaxim 200mg/mL (Sigma,St Louis, MO), and timentin 150mg/mL (SmithKline Beacham,London), which were incubated at 26�C until the resistantcolonies grew. Mycelium growing on the plates wassubcultured on fresh selective agar for further testing.

Stability of the transgene

The stability of the transformed phenotype was determined bytransferring inoculum from a selective PDA plate to the edge of anon-selective PDA plate and incubating at 26�C. When themycelium was close to the opposite edge of the plate,inoculum was taken and transferred to a fresh, non-selectivePDA plate, and also to a selective PDAplate to test for resistance.

PCR analysis

The presence of the transgene was detected by PCR analysis withhph primers (hph-l, 50-CTGAACTCACCGCGACG-30; hph-2,50-ACGGACGCACTGACGTG-30), which amplify a 510-bpregion (241–751) within the hph gene. DNA for PCR analysiswas extracted from mycelium as described previously (O’Brien1994). The presence of contaminating A. tumefaciens cells waschecked by use of the vir primers (Sawada et al. 1995).Amplifications were carried out in 10-mL reactions containingl unit of Tth Plus DNA polymerase (Fisher Biotech, Perth, WA),1mM MgC12, 25 pmol of each primer, 40mM dNTPs, and5–50 ng of template DNA in a buffer supplied with theenzyme. The temperature cycling conditions for the hphprimers were: 95�C for 5min, followed by 30 cycles of 95�Cfor 30 s, 55�Cfor 30 s, 72�Cfor 30 s, and1 cycleof 72�Cfor 7min.The vir primers were used as described previously (Sawada et al.1995).

Southern hybridisation

The transformants were grown in Petri dishes containing 20mLV8 liquid medium (Miller 1955) containing 15mg/mLhygromycin at 26�C for 4 days. Harvesting of the mycelium,extraction ofDNA, digestion of theDNA (5mg)withHindIII, andSouthern hybridisation were carried out as described previously(O’Brien 1994) with 5mg DNA per well. The probe sequencewas derived from the hygromycin gene by amplification withthe hph primers. The PCR product was labelled with 32P usingthe Megaprime labelling kit from Amersham-Pharmacia (CastleHill, NSW) as described by the manufacturer.

Results

Isolates from different anastomosis groups (AG) of R. solani(Table 1) were tested because of observed high variation intransformation competency in other organisms (Gelvin 2003).Mycelial growth during the 48-h co-cultivation period withA. tumefaciens resulted in a mycelial mat across the surface ofthe co-cultivation plate. In order to select transformants, themycelial mat was recovered by scraping the mycelium off thesurface of the plate, added to 1mL sterile saline (containingcefotaxim and timentin) and homogenised to produce a fine

Table 1. Isolates of Rhizoctonia solani used in this study

Isolate AGA ZGB Origin Source

ST-11-6 3 7 Japan A. OgoshiRH-165 4-HGII 8 Japan A. Ogoshi1342 4 8 South Australia S. NeateSCR122 6 Not

determinedSouth Australia J. Harris

AAG, anastomosis grouping (Carling 1996).BZG, pectic zymogram grouping (Cruickshank 1990).

Gpd

LB RBIn1KmplantIn2

HindIIIEcoR1

SC3 terminatorhph

A A AT TA A

CCCGGCG

5′

Fig. 1. Structure of the T-DNA region of the plasmid pJW2.2hyg. The region is defined by the left (LB) and right(RB) border sequences of the Agrobacterium tumefaciens T-DNA. Gpd represents the promoter sequence used todrive expressionof the hygromycin (hph) gene. The introns are indicated by In1 and In2.Conversion of the sequenceAAATTAA at the 50 end of the hph gene to CCCGGCG increases expression (Scholtmeijer et al. 2001). The vectorpBin19 contains theM13 Lacmultipurpose cloning cassette and a kanamycin resistance gene with plant expressionsignals (Bevan 1984). The lac sequences are indicated by white boxes adjacent to the EcoR1 andHindIII sites. Themodified hph gene was inserted as an EcoR1/HindIII insert.

80 Australasian Plant Pathology J. Wu and P. A. O’Brien

suspension of small mycelial fragments. An aliquot of this wasspread on the surface of the selective plate. Transformants wereconsidered to be independent only if they came from separate co-cultivation mixes. After 7–10 days of incubation, fresh growth inthe form of small mycelial colonies was observed on selectiveplates with all isolates. However, when subcultured onto freshselective agar only the subcultures from isolate SCR 122 wereable to grow. Small colonies observed on selective plates spreadwith the untransformed SCR 122 also failed to grow whensubcultured to fresh medium.

The transformants grew faster than the control on hygromycinagar (Fig. 2); however, growth was considerably slower than

on non-selective agar. Growth of the untransformed controlwas not completely inhibited by the concentration ofhygromycin used in the selective medium, but continued ata slow rate. We tested the degree of resistance of thetransformants by measuring radial growth in differentconcentrations of hygromycin. The transformants grew betterthan the control at both 25 and 50mg/mL hygromycin but not at75 or 100mg/mL (Fig. 3). All of the transformants wereprogressively inhibited as the concentration of hygromycinincreased from 25 to 100mg/mL. None had a high level ofresistance. Transformant 6 did not exhibit a resistant phenotypeat any concentration of hygromycin.

Six independent transformants of SCR 122 were analysed forthe presence of the transgene by amplification of a sequence fromthe hph gene. Of the six transformants analysed, onlytransformant 6 failed to amplify with the hph primer pair(Fig. 4). All DNA extracts gave a product with PCR primersfor fungal rRNA genes (data not shown) demonstrating that thenegative result obtained with transformant 6 was not due toinhibition of PCR. The possibility that the PCR products weredue to contaminating A. tumefaciens cells was checked by PCRanalysis using primers to the vir region of the Ti plasmid. Sincethis region is not transferred to the recipient fungus, its detectionwould indicate the presence of contaminating A. tumefacienscells. None of the transformants gave a product with theseprimers, although the bacterial culture did (Fig. 4).

Integration of the T-DNA into the genome of thetransformants was confirmed by Southern blotting with aprobe from within the hph gene. Bands greater in size than15 kbp were observed in all five transformants (Fig. 5). As thereis only a single HindIII site within the T-DNA, these bandsextend from the HindIII site within the T-DNA to a HindIII sitewithin the adjoininghostDNA.Thepresence of these bands thusconfirmed integration as non-integrated T-DNA would give aband of 6.2 kbp. Two bands of smaller size were observed in

4.5

3.5CON

TR6

TR5

TR4

TR3

TR2

TR12.5

2

1.5

Rad

ial g

row

th (

cm)

d incubation

0.5

–0.51 2 3 4 5 7 9 11 13

0

1

4

3

Fig. 2. Growth curves of transformants on hygromycin agar. Isolates weregrown on potato dextrose agar plates with 50mg/mL hygromycin at26�C. Three plates were set up for each isolate, and four measurementswere made from each plate. The bars indicate the standard error.

7

6

5

4

3

2

1

0H25 H50

Hygromycin (mg/L)

Col

ony

diam

eter

(cm

)

H75 H100

Fig. 3. Growth of transformants (Tr) at different concentrations of hygromycin. The growthconditions were as described in the legend to Fig. 2. In each group the measurements are:non-transformed, Tr6, Tr5, Tr4, Tr3, Tr2, Tr1. Bars indicate the standard error.

Transformation of Rhizoctonia solani Australasian Plant Pathology 81

transformant 4, possibly representing integration of more thanone copy.

The stability of the transformed phenotype was tested bygrowing the transformants on non-selective media. Four of thetransformants showed a stable hygromycin resistant phenotypeand were capable of growth on selective media after eighttransfers on non-selective media. Although transformant 3 lostthe resistant phenotype after a single round of selection on non-selective medium.

Discussion

In this studywe obtained stable transformants ofR. solani using ahygromycin resistance gene especiallymodified for expression inbasidiomycetes. The transformants grew better than the non-transformed parent on selection agar, and the presence of thetransgene was confirmed by DNA analysis. This contrasts withthe failure of previous attempts to transform R. solani with anunmodified hph gene (Robinson and Deacon 2001).

In these experiments the transformed strains were clearlydifferentiated from the non-transformed strains by theirincreased growth rate on selection agar. However, the

non-transformed strain was not completely inhibited forgrowth, but continued to show some residual growth even atthe highest concentration of hygromycin tested. We have foundthat Rhizoctonia shows a high degree of resistance to antibioticsthat are commonly used for selection of transformed strains(P. A. O’Brien, unpubl. data). This may be related to theproduction of laccases. These enzymes are involved in thedegradation of lignin in the host cell walls enabling the fungustomore effectively colonise the plant (Breen and Singleton 1999;Claus 2004; Bora et al. 2005). Laccases are also involved in thesynthesis of pigments (Litvintseva and Henson 2002) and arecapable of detoxifying a wide range of compounds (Mayer andStaples 2002; Couto and Herrera 2006).

Traditionally, it has proved more difficult to obtain stabletransformants of basidiomycetes compared with ascomycetes(Ullrich et al. 1985). This has been ascribed to difficultiesin finding an efficiently expressed selectable marker.Complementation of nutritional deficiencies has been used forCoprinus cinereus (Mellon et al. 1987). However, this requiresisolation of the mutant strains, and the basidiomycete version ofthe complementing gene. Although hygromycin resistance wasused successfully with Ustilago maydis (Wang et al. 1988), andU. violaceae (Bej and Perlin 1989), attempts to use it with otherspecies of basidiomycetes were less successful. Lugones et al.(1999) reported that the presence of introns within the 50 and 30

untranslated regions of the hph gene were necessary for stabletransformation of the basidiomycete S. commune. Their resultsshow that the intron sequences increase the stability of themRNA. Further increases in transformation efficiency wereachieved by changing some of the A/T bp at the 50 end of thehph coding region to C/T bp (Scholtmeijer et al. 2001). In theseexperiments we observed that the non-stably transformedcolonies obtained on the primary selective agar plates with allisolates of R. solani were larger than those obtained using anunmodifiedhygromycingene (datanot shown).This is ascribed tothe higher level of expression of the modified gene in R. solani.

The presence of the transgene in transformants 1–5 wasconfirmed by PCR and Southern hybridisation. Whiletransformants 1, 2, 3, and 5 appear to contain single copies ofthe transgene, the presence of two bands in transformant 4suggests that it contains more than one copy. That thetransgene was not due to the presence of residualAgrobacterium cells adhering to and growing along with thefungal hyphae was demonstrated by the lack of a PCR productwith primers that amplify a region outside of the T-DNA on thevector (Sawada et al. 1995). The sequence amplified by theseprimers would not be transferred to Rhizoctonia.

Transformant 6 appears to be an escape. Although it appearsnot to contain the transgene, itwas originally identifiedbecause ofgrowth on the primary selection plate. This may have been due tothe presence of a clump of mycelium that masked the inhibitoryeffect of the antibiotic. However, given the fact that theuntransformed parent is also capable of some growth onhygromycin agar, it is not surprising that such escapes shouldoccur. Perhaps a better method for selection of transformantswould be to overlay the primary selection platewith a layer of softagar containing hygromycin. Transformants that grow throughthe top agar would be selected. It has also been reported that suchescapes are commonly observed when hygromycin is used for

1(a)

(b)

0.56

0.56

2 3 4 5 6 7 8 9

Fig. 4. PCR analysis of transformants (Tr). (a) Analysis with the hphprimers. lanes: 1, DNA markers; 2, pJW2.2hyg-15 DNA; 3, non-transformed Rhizoctonia solani; 4–9, R. solani Tr 1–6, respectively.(b) Analysis with the vir primers: lanes: 1, DNA markers; 2–4,Agrobacterium tumefaciens LBA4404, AGLO and AGL1, respectively;5–9, Tr 1–5, respectively. Marker sizes are in kbp.

1

9.3

6.4

2 3 4 5 6 7

Fig. 5. Southern blot analysis of the transformants (Tr). HindIII-digestedDNA was electrophoresed, blotted and hybridised with a labelled sequencefrom within the hygromycin resistance gene. Lanes: 1, DNA markers; 2–6,Tr 1–5; 7, non-transformed control.

82 Australasian Plant Pathology J. Wu and P. A. O’Brien

selection of transformants of S. commune (Mooibroek et al.1990).

For transformants 1–5, the resistance phenotype was stablymaintained through repeated cycles of growth on non-selectiveagar. In contrast, transformant 3 rapidly lost its resistance on non-selective agar. This may be due to excision of the transgene, or topost-transcriptional gene silencing. Transgene excision has beenobserved in transformants of Cochliobolus heterostrophus(Keller et al. 1991), and Penicillium crysogenum (Renno et al.1992). In both studies it appears to be associated with tandemlyrepeated copies of the transgene. Post-transcriptional genesilencing is triggered by the formation of double-strandedRNA as a result of transcription of inverted copies of thetransgene (Muskens et al. 2000; Cogoni 2001). This effectivelyshuts down expression of all copies of the transgene irrespectiveof their location. It is not known whether the loss of resistancein transformant 3 is due to excision of the transgene or post-transcriptional gene silencing.

R. solani is a complex species consisting of several AGs (Snehet al. 1991). These groups are often considered to be separatespecies because they are genetically isolated from each other(Carling 1996). Although originally erected on the basis ofanastomosis behaviour the AG can also be differentiated onthe basis of pectic zymograms (Sweetingham et al. 1986),rDNA sequence (Sharon et al. 2008), and random amplifiedpolymorphic DNA-PCR (Duncan et al. 1993; MacNish andO’Brien 2005). In this study, we obtained stabletransformation of a single isolate from AG6. We did notrecover transformants from AG3 or AG4 isolates that wereincluded in the study. Variation in the formation of stabletransformants is a common observation in plant transformation(Gelvin 2000). Accessions or varieties with a plant species showconsiderable variation in their ability to be transformed.Usually itis found that where one, or a few, varieties can be transformed,many cannot. When developing a transformation system for aplant species, multiple varieties have to be evaluated for theirability to be transformed. In view of these observationswith plantspecies, it is, therefore, not surprising to find similar variation inR. solani. The main obstacle appears to be related to theintegration of the transforming DNA into the endogenome.With the plant species Arabidopsis thaliana there is an~10 000-fold difference in uptake of DNA between ecotypesas measured by transient expression, and integration of the DNAas measured by stable transformation (Gelvin 2000). Piers et al.(1996) also reported a large difference between uptake andintegration of DNA in the yeast Saccharomyces cerevisiae. Inthis study, we observed small colonies on the primary selectionplates with all of the isolates, but when sections from the edge ofthese colonies were transferred to fresh plates of selection agarthey did not grow. This is consistent with uptake of the DNAwithout integration. Expression of the non-integrated transgeneresults in resistance, but because the transgene is not integratedand, therefore, not replicated, the degree of resistance rapidlyreduces as the colonygrows. Similar conclusionswere reachedbyRobinson andDeacon (2001) in their studies on transformation ofR. solani.

This is thefirst report of stable transformation inR. solani. Theresults suggest that there is variation between isolates of differentAGs in the ability to be stably transformed. As with plants, and

with other fungal species, the main obstacle appears to beintegration of the transforming DNA into the endogenome.The possibility of increasing the integration by inserting afragment of R. solani DNA into the vector to provide a regionof homology with the endogenome should be explored. This hasbeen found to improve transformation in yeast (Bundock andHooykas 1996).

Acknowledgement

This project was supported by the Grains Research Committee of WesternAustralia.

References

Bej AK, Perlin MH (1989) A high efficiency transformation system for thebasidiomyceteUstilagoviolaceae.Gene80, 171–176.doi: 10.1016/0378-1119(89)90263-1

Bevan MW (1984) Binary Agrobacterium vectors for plant transformation.Nucleic Acids Research 12, 8711–8721. doi: 10.1093/nar/12.22.8711

Bora P, Hardy GEStJ, O’Brien PA (2005) Laccase is required for macerationof lupin radicle tissue by Rhizoctonia solani. Australasian PlantPathology 34, 591–594. doi: 10.1071/AP05077

Breen A, Singleton F (1999) Fungi in lignocellulose breakdown andbiopulping. Current Opinion in Biotechnology 10, 252–258.doi: 10.1016/S0958-1669(99)80044-5

Bundock P, Hooykas PJJ (1996) Integration of Agrobacterium tumefaciensT-DNA in the Saccharomyces cerevisiae genome by illegitimaterecombination. Proceedings of the National Academy of Science of theUnited States of America 93, 15272–15275. doi: 10.1073/pnas.93.26.15272

CarlingDE (1996) Grouping inRhizoctonia solani by hyphal anastomosis. In‘Rhizoctonia species: taxonomy, molecular biology, pathology, anddisease control’. (Eds B Sneh, S Jabaji-Hare, SN Dijst) pp. 35–48.(Kluwer Academic Publishers: Dordrecht, the Netherlands)

Claus H (2004) Laccases: structure, reactions, distribution. Micron (Oxford,England) 35, 93–96.

Cogoni C (2001) Homology-dependent gene silencing mechanisms in fungi.Annual Review of Microbiology 55, 381–406. doi: 10.1146/annurev.micro.55.1.381

CoutoSR,HerreraLT (2006) Inhibitors of laccases: a review.CurrentEnzymeInhibition 2, 343–352. doi: 10.2174/157340806778699262

Cruickshank RH (1990) Pectic zymograms as criteria in taxonomy ofRhizoctonia. Mycological Research 94, 938–946.

deGroot MJA, Bundock P, Hooykaas PJJ, Beijersbergen AGM (1998)Agrobacterium tumefaciens-mediated transformation of filamentousfungi. Nature Biotechnology 16, 839–842. doi: 10.1038/nbt0998-839

Duncan S, Barton JE, O’Brien PA (1993) Analysis of variation in isolates ofRhizoctonia solani by random amplified polymorphic DNA assay.Mycological Research 97, 1075–1082.

GelvinSB (2000)Agrobacterium andplant genes involved inT-DNAtransferand integration. Annual Review of Plant Physiology and Plant MolecularBiology 51, 223–256. doi: 10.1146/annurev.arplant.51.1.223

Gelvin SB (2003)Agrobacterium-mediated plant transformation: the biologybehind the ‘gene-jockeying’ tool. Microbiology and Molecular BiologyReviews 67, 16–37. doi: 10.1128/MMBR.67.1.16-37.2003

Hooykaas PJJ (1988) Agrobacteriummolecular genetics. In ‘Plant molecularbiology manual’. (Eds SB Gelvin, RA Schilperoort, DPS Verma)pp.A4 : 1–13. (KluwerAcademicPublishers:Dordrecht, theNetherlands)

KellerN,BergstromG,YoderO (1991)Mitotic stability of transformingDNAis determined by its chromosomal configuration in the fungusCochliobolus heterostrophus. Current Genetics 19, 227–233.doi: 10.1007/BF00336491

Transformation of Rhizoctonia solani Australasian Plant Pathology 83

Lazo G, Stein P, Ludwig R (1991) A DNA transformation-competentArabidopsis genomic library in Agrobacterium. Biotechnology 9,963–967. doi: 10.1038/nbt1091-963

Litvintseva A, Henson J (2002) Characterization of two laccase genes ofGaeumannomyces graminis var. graminis and their differentialtranscription in melanin mutants and wild type. Mycological Research106, 808–814. doi: 10.1017/S0953756202005841

Lugones K, Schioltmeijer K, Klootwijk R, Wessels J (1999) Introns arenecessary for mRNA accumulation in Schizophyllum commune.Molecular Microbiology 32, 681–689. doi: 10.1046/j.1365-2958.1999.01373.x

MacNishGC,O’BrienPA (2005)RAPD-PCRused to confirm that four pecticisozyme (zymogram) groupswithin theAustralianRhizoctonia solaniAG8 population are true intraspecific groups. Australasian Plant Pathology34, 245–250. doi: 10.1071/AP05004

Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme.Phytochemistry 60, 551–565. doi: 10.1016/S0031-9422(02)00171-1

Mellon FM, Little PFR, Casselton LA (1987) Gene cloning andtransformation in the basidiomycete fungus Coprinus cinereus:isolation and expression of the isocitrate lyase gene (acu-7). Molecular& General Genetics 210, 352–357. doi: 10.1007/BF00325705

Meyer V (2008) Genetic engineering of filamentous fungi – progress,obstacles and future trends. Biotechnology Advances 26, 177–185.doi: 10.1016/j.biotechadv.2007.12.001

Miller P (1955) V-8 juice as a general purposemedium for fungi and bacteria.Phytopathology 45, 461–462.

Mooibroek H, Juipers A, Sietsma J, Punt P, Wessels J (1990) Introduction ofhygromycin B resistance into Schizophyllum commune. Molecular &General Genetics 222, 41–48.

Mullins ED, Chen X, Romaine P, Raina D, Geiser DM, Kang S (2001)Agrobacterium-mediated transformation of Fusarium oxysporum: anefficient tool for insertional mutagenesis and gene transfer.Phytopathology 91, 173–180. doi: 10.1094/PHYTO.2001.91.2.173

MuskensMWM,Vissers APA,Mol JNM,Kooter JM (2000) Role of invertedDNA repeats in transcriptional and post-transcriptional gene silencing.Plant Molecular Biology 43, 243–260. doi: 10.1023/A:1006491613768

O’Brien PA (1994) Molecular markers in Australian isolates of Rhizoctoniasolani. Mycological Research 98, 665–671.

Parmeter JR, Whitney HS (1970) Taxonomy and nomenclature of theimperfect state. In ‘Rhizoctonia solani, biology and pathology’.(Ed. JR Parmeter) pp. 7–19. (University of California Press:Berkeley, CA)

Piers KL,Heath JD, LiangX, StevensKM,Nester EW (1996)Agrobacteriumtumefaciens-mediated transformation of yeast. Proceedings of theNational Academy of Science of the United States of America 93,1613–1618. doi: 10.1073/pnas.93.4.1613

Renno D, Saunders G, Smith P, Bull A, Holt G (1992) Mitotic instability ofintegratedplasmids inPenicilliumchrysogenum transformants.JournalofBiotechnology 24, 291–298. doi: 10.1016/0168-1656(92)90038-B

Robinson H, Deacon J (2001) Protoplast preparation and transienttransformation of Rhizoctonia solani. Mycological Research 105,1295–1303. doi: 10.1017/S0953756201005159

SawadaH, IekiH,Matsuda I (1995) PCRdetectionof Ti andRi plasmids fromphytopathogenic Agrobacterium strains. Applied and EnvironmentalMicrobiology 61, 828–831.

Scholtmeijer K,Wosten H, Springer J, Wessels J (2001) Effect of introns andAT-rich sequences on expressionof the bacterial hygromycinB resistancegene in the basidiomycete Schizophyllum commune. Applied andEnvironmental Microbiology 67, 481–483. doi: 10.1128/AEM.67.1.481-483.2001

SharonM,KuninagaS,HyakumachiM,NaitoS,SnehB (2008)Classificationof Rhizoctonia spp. using rDNA-ITS sequence analysis supports thegenetic basis of the classical anastomosis grouping. Mycoscience 49,93–114. doi: 10.1007/s10267-007-0394-0

Sneh B, Burpee LL, Ogoshi A (1991) ‘Identification of Rhizoctonia species.’(APS Press: St Paul, MN)

Sweetingham MW, Cruickshank RH, Wong DH (1986) Pectic zymogramsand taxonomy and pathogenicity of theCeratobasidiaceae. Transactionsof the British Mycological Society 86, 305–311.

Ullrich R, Novotny C, Specht C, Froelinger E, Miunoz-Rivas A (1985)Transforming basidiomycetes. In ‘Molecular genetics of filamentousfungi’. (Ed. W Timberlake) pp. 39–57. (AR Liss Inc.: New York)

Wang J, Holden DW, Leong SA (1988) Gene transfer system for thephytopathogenic fungus Ustilago maydis. Proceedings of the NationalAcademy of Science of the United States of America 85, 865–869.doi: 10.1073/pnas.85.3.865

Manuscript received 29 July 2008, accepted 30 September 2008

84 Australasian Plant Pathology J. Wu and P. A. O’Brien

http://www.publish.csiro.au/journals/app