CSIRO PUBLISHING
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Australasian Plant Pathology, 2004, 33, 241–247
Characterisation of Rhizoctonia solani isolates causing root canker of lucerne in Australia J. R. AndersonA, S. BentleyB, J. A. G. IrwinA,D, J. M. MackieA, S. NeateC and J. A. PattemoreB A
Cooperative Research Centre for Tropical Plant Protection, John Hines Building, The University of Queensland, Qld 4072, Australia. B Cooperative Research Centre for Tropical Plant Protection, Molecular Diversity and Diagnostics Research Laboratory, Plant Pathology Building, 80 Meiers Road, Indooroopilly, Qld 4068, Australia. C Department of Plant Pathology, North Dakota State University, PO Box 5012, Fargo, ND 58105-5012, USA. D Corresponding author; email:
[email protected]
Abstract. The fungus causing Rhizoctonia root canker of lucerne in western Queensland has been characterised as a new subgroup within anastomosis group (AG 6) of Rhizoctonia solani. Isolates from two sites showed identical rDNA ITS sequence homology but could be differentiated based on DNA fingerprints. The lucerne isolates did not cause disease on wheat, indicating they are genetically different from the AG 6 subgroup that causes crater disease on wheat in South Africa. Root canker symptoms were produced on all commercial Australian cultivars of lucerne tested. AP04012 J.eCtRhal.rAantcderisaotni ofRhizoctoniasolani
Additional keywords: alfalfa, Medicago sativa.
Introduction A root canker disease of lucerne (Medicago sativa) caused by Rhizoctonia solani was first described by Smith (1943) in the USA. The disease, which is responsible for substantial economic losses of lucerne in the Imperial and Palo Verdi Valleys of California (Erwin 1956), and in south-western areas of Queensland, Australia (Irwin 1977), occurs only under high soil temperature conditions. It is characterised by the presence of elliptical, sunken, necrotic cankers that form at the junction of the tap and lateral roots. These cankers cincture the root, and the disease often results in plant death (Irwin 1977). R. solani also causes damping-off in lucerne, as well as crown bud rot, stem blight and crown necrosis (Stuteville and Erwin 1990). The first identification of the ‘praticola type’ of R. solani, later identified as AG 4, was from lucerne in 1929 (Kotila 1929), and other studies have shown AG 4 to be associated with damping-off and aerial diseases of lucerne (Anderson 1977). However, the identity of isolates causing root canker and the genetic relationships between the R. solani isolates causing these different diseases in lucerne have not been previously determined. R. solani refers to a species complex, comprising a number of genetically distinct and reproductively isolated groups (Anderson 1982; Ogoshi 1987), covering a wide range of variability in morphology, ecology and pathology. Consequently, R. solani has been subdivided into groups © Australasian Plant Pathology Society 2004
based on a range of criteria, the most commonly used being anastomosis group (AG) reflecting the capacity of different isolates to anastomose (Anderson 1982). When two isolates from the same AG are paired and subsequently anastomose, there are two possible reactions: perfect fusion (a C3 reaction) or imperfect fusion (a C2 reaction) (Carling et al. 1996). Isolates belonging to different AG groups show no fusion of cell walls. Since 1969, the number of AGs recognised has increased from four to 12 (Carling et al. 1994), and subgroups within some AGs based on anastomosis frequency, host range and other characteristics have also been reported (Carling et al. 1996). More recently, molecular tools such as DNA base sequence homology and DNA fingerprinting have been used to assess genetic relatedness within and among AGs (e.g. Kuninaga et al. 1997). This paper reports detailed biological characterisation of R. solani isolates that cause lucerne root canker (LRC) in Australia. We have investigated their AG allocation, host range and genetic and phylogenetic relationships using DNA analysis. Methods Isolates used in the studies R. solani cultures were obtained by isolation from root cankers on mature lucerne taproots collected from fields at Roma and Biloela, Qld. These sites are approximately 250 km apart. Additional Australian 10.1071/AP04012
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Table 1.
J. R. Anderson et al.
Sources of Rhizoctonia solani isolates studied in these investigations
Provider
Provider’s number
AG classification
Original host
Geographic origin
J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Mackie J. Dennis J.F. de Beer S. Neate ex H. Ogle S. Neate ex H. Ogle S. Neate ex H. Ogle
UQ2351 UQ3331B UQ3332B UQ3333B UQ3334B UQ3335B UQ3336B UQ3337B UQ3338B UQ3339B UQ3194 1342 1661 1664 1665
Australian isolates 6 subgroupA 6 subgroup 6 subgroup 6 subgroup 6 subgroup 6 subgroup 6 subgroup 6 subgroup 6 subgroup 6 subgroup 4 4 4 4 4
Lucerne Lucerne Lucerne Lucerne Lucerne Lucerne Lucerne Lucerne Lucerne Lucerne Medicago sp. Soil Soil debris Cotton Cotton
Roma, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD Biloela, QLD South Australia Moonta, SA Narrabri, NSW Gatton, QLD St George, QLD
A. Ogoshi R. Jones A. Ogoshi N. Anderson N. Anderson A. Ogoshi A. Ogoshi C. Windels R. Jones A. Ogoshi A. Ogoshi A. Ogoshi A. Ogoshi A. Ogoshi A. Ogoshi Y. Homma A. Ogoshi
Previously characterised tester isolates CS-2 (ATCC 66157) 1 IA Rice 1–2-013 1 IB Unknown F-2 (ATCC 66155) 1 IC Sugar beet 48 (ATCC 44658) 2-1 Soil K-15–76 2-2 Carrot C-96 2-2IIIB Unknown RI-64 2-2IV Sugar beet P-42 3 Potato Butler 283 4 Unknown AH-1 4 HGI Peanut, root RH165 4 HGI Sugar beet GM-10 5 Soybean NTA3–1 6 Soil NKN2–1 6 GV Non-cultivated soil OHT-1 6 HGI Non-cultivated soil HO-1556 7 Soil TE 2–4 BI Non-cultivated soil
Japan North Carolina, USA Japan Australia USA Japan Japan Grand Forks, USA Wisconsin Chiba, Japan Hokkaido, Japan Japan Hokkaido, Japan Nukanai, Japan Ohtakimura, Japan Hokkaido, Japan Hokkaido, Japan
A B
Determined in the present study. These isolates were obtained from different plants collected from the same field.
R. solani isolates from a range of other hosts are listed in Table 1. Hyphal tip cultures were made for each isolate before use. Additional AG tester isolates were imported and used under quarantine permit (Table 1). All isolates prefaced by UQ are deposited in BRIP and are available upon request to the BRIP curator, Dr R. Shivas, QDPI, Meiers Road, Indooroopilly, Qld. Cytology Hyphae from the growing edge of cultures grown on potato-dextrose agar (PDA) (Difco) were mounted on microscope slides, stained with alkaline Safranin O (Bandoni 1979), and observed at 400u magnification under interference contrast illumination. The mean number of nuclei per cell was calculated from two replicates (two slides), each replicate comprising ten observations per isolate. Hyphae from the periphery of actively growing 6-day-old PDA cultures were mounted on microscope slides and stained as above. Hyphal diameter was determined under interference contrast
illumination at 1000× magnification and the mean for each isolate was calculated from 40 observations. Anastomosis grouping Cultures were maintained on PDA at 24°C prior to testing. The procedure was based on that of Parmeter et al. (1969) as used by Carling et al. (1987). Sterile rectangles of cellophane (5 u 2 cm) were dipped in weak PDA (13 g/L) and placed on water-agar plates (WA; 20 g/L). Isolates were paired by excising a cube of agar containing mycelium from the growing margin of cultures and placing at opposite ends of a cellophane strip. Plates were incubated until hyphae from the two isolates started to interact, at which time the cellophane strip was removed to a microscope slide and stained with 0.05% trypan blue in lactophenol. The zone of contact was observed at 100× magnification to identify anastomosis events and reactions were graded according to the system proposed by Carling et al. (1988). Briefly, C0 and C1 reactions indicate the isolates belong to different AG groups with no cell-wall to cell-wall penetration; C2 and C3 reactions indicate they
Characterisation of Rhizoctonia solani
Australasian Plant Pathology
belong to the same AG group with cell-wall fusion occurring. Self anastomosis usually results in a C3 reaction.
Table 2. Reaction of lucerne cultivars to Rhizoctonia solani isolate UQ2351
Molecular analysis
Cultivar
Deoxyribonucleic acid amplification fingerprinting. Isolates from lucerne root cankers and cultures representing each AG were cultured in duplicate on PDA. A cube of agar from the growing margin of the plate was excised and used to inoculate 200 mL of potato-dextrose broth (PDB) in 250 mL Erlenmeyer flasks. The cultures were incubated for 7 days at room temperature and shaken daily. The mycelial mat was harvested by filtration through Miracloth (Calbiochem Inc.) and stored no longer than 5 days at –20°C prior to DNA extraction. Deoxyribonucleic acid was extracted according to Bentley et al. (1994) and quantified spectrophotometrically using a GeneQuant RNA/DNA Calculator (Pharmacia LKB Biochrom Ltd). Genetic variation was determined by DNA amplification fingerprinting (DAF) according to Bentley and Bassam (1996). Three DAF primers [IMBR (5c GAA ACG CC 3c), DINQ (5c CTG GCC CA 3c), DJDH (5c ACC AGC CA 3c)] were used in separate, duplicated polymerase chain reaction (PCR) runs, and amplification products were separated on polyacrylamide gels (10% total monomer, 2% cross-linker) and visualised by silver staining. Deoxyribonucleic acid bands were manually scored. Ribosomal DNA ITS1 and ITS2 sequence analysis. Deoxyribonucleic acid was extracted from two replicates of each isolate. The ITS region was amplified by PCR using primers ITS1 and ITS4 according to White et al. (1990) including a no template control reaction. Amplification of PCR products was confirmed on 1% agarose gel stained with ethidium bromide using an appropriate size marker. ITS PCR products were purified using Wizard PCR Preps DNA Purification System (Promega Corp., Australia) according to manufacturer’s instructions. Both strands of the ITS1, 5.8S and ITS2 regions inclusive were direct sequenced using primers ITS1, ITS2, ITS3 and ITS4 (White et al. 1990). The sequencing reaction was conducted using ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, California) as per manufacturer’s instructions. Sequencing was carried out by the Australian Genome Research Facility, University of Queensland, St Lucia. A consensus sequence for each isolate was compiled from sequence fragments using Sequencher v3.0 computer software. Gene sequence data analysis. Two datasets of alignments were generated for phylogenetic analysis. Sequence data generated in this study from the ITS region of LRC isolates and from GenBank sequences representing each AG of R. solani (Gonzalez et al. 2001) were included in the initial dataset. A further dataset was generated including the LRC sequences and additional GenBank sequences from isolates in AG 6, using AG 1-IA as an outgroup. ITS sequences were aligned using ClustalX (Thompson et al. 1997) and checked visually. Phylogenetic analyses were performed and trees constructed from distance matrix values generated by the neighbour-joining method (Saitou and Nei 1987) using the computer software package MEGA v2.0 (Kumar et al. 2001). Distances were determined using Kimura’s 2-parameter model (Kimura 1980), omitting all sites with gaps. A bootstrap analysis of the sequence data was carried out with 1000 replications.
Sequel HR Hallmark WL516 Sceptre PL5929 Aquarius Eureka Hunter River Trifecta Sequel
Plant inoculations Lucerne cultivar reaction. A range of Australian-grown lucerne cultivars was inoculated with UQ2351 (Table 2). Fifteen replications, each with five plants per pot, were planted into 15-cm-diameter pots filled with UC mix (Baker 1957). These were grown for 10 weeks under natural illumination at 25°C. Inoculum was 10-day-old cultures grown on autoclaved wheat grains. The infested grains were poured onto the surface of the pot, at 20 g/pot then incorporated into the upper 2–3 cm
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% Resistant 33.8A 27.3A 27.0A 25.4A 24.4A 23.0A 20.8A 18.1A 15.9A 13.0A
No significant differences (P d 0.05).
A
of the potting mix. Two control pots of each cultivar were inoculated with autoclaved wheat grain only. Pots were moved to 33°C under natural illumination and were assessed for symptom development 3 weeks later. Reaction was assessed on the percentage of the root surface covered by cankers, with any plant having < 25% of the root surface covered by cankers being classified as partially resistant. The number of partially resistant plants was expressed as a percentage for each cultivar. Data were subject to ANOVA. Test for pathogenicity to wheat. To test if LRC isolates were pathogenic to wheat, three isolates (UQ2351, UQ3333 and UQ3335) were used to inoculate white, spring wheat cv. Hartog and a positive control, lucerne cv. Hunter River. Five replicates of 15 seeds of each were planted into UC mix in 15-cm-diameter pots and allowed to grow under natural light conditions at 25°C for 7 weeks. They were inoculated with Rhizoctonia-infested wheat grains as described above and then grown at 33°C for 2 weeks; controls (two replicates) were inoculated with sterile, steamed grain only. Plants were then assessed for development of disease in the roots.
Results Cytology All LRC isolates showed the morphological characteristics of Rhizoctonia and were multinucleate, confirming that they all belong to the taxon R. solani (Parmeter et al. 1969). Number of nuclei per cell ranged from 3 to 13, with an average of 5.1 nuclei per cell. Average hyphal diameter of LRC isolates was 7.1 Pm with a range of 4.8–10 Pm. Both nuclear number per cell and average hyphal diameter for the LRC isolates were within the expected range for R. solani (Butler and Bracker 1970). Anastomosis groupings When paired with self, complete fusion of walls and membranes (C3 reaction) occurred with each of the LRC isolates. Pairings between all combinations of LRC isolates from Biloela (UQ3331 – UQ3339) yielded C3 reactions. Pairings between these LRC isolates from Biloela and the isolate from Roma (UQ2351) yielded C2 reactions (wall contact and pore plus death of anastomosing and adjacent cells). The highest level of relationship in the anastomosis reaction of LRC isolates when paired with the known tester
negative
UQ3339
UQ3338
UQ3337
UQ3336
J. R. Anderson et al.
UQ3336
UQ3335
UQ3334
UQ3333
UQ3333
UQ2351
UQ2351
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Marker
244
Lucerne cultivar reaction to LRC isolate UQ2351
500
All control plants had healthy white roots and showed substantial new growth. No significant differences for disease reaction were observed between any of the cultivars tested. Percentage of plants partially resistant (cankers covering < 25% of the root surface) ranged from 13.8% for Sequel to 33.9% for Sequel HR (Table 2). Of the 750 plants inoculated across the ten cultivars, all except 28 plants (3.7%) showed disease symptoms. Subsequent re-inoculation using the above methods showed these 28 plants to be disease escapes.
400
Test for pathogenicity to wheat of three LRC isolates
300
All lucerne and wheat control plants that were not inoculated had healthy white roots. Symptom development in lucerne inoculated with LRC isolates ranged from necrotic lesions at the junction of tap and lateral roots, or cinctured tap root, through to plant death. All inoculated wheat plants had healthy white root systems, indicating an incompatible reaction between wheat and the LRC isolates.
1500 1000 700
200
Fig. 1. Genetic variation between LRC isolates of Rhizoctonia solani from Roma (UQ2351) and Biloela (UQ3333–3339 inclusive). Deoxyribonucleic acid fingerprints were generated using primer IMBR. Accession numbers are indicated at the top of the figure and molecular size (bp) markers are indicated to the left.
isolates (Table 1) occurred with AG 6. All LRC isolates produced C2 reactions when paired with AG 6 GV and AG 6 HGI isolates. None of the LRC isolates anastomosed with any of the remaining tester isolates representing AG-1,2,3,4,5,7 or AG-BI. All produced C0 interactions indicating the absence of anastomosis relationships between these AGs and LRC isolates of R. solani. Deoxyribonucleic acid amplification fingerprinting LRC isolates UQ3333–3339 inclusive produced identical DNA fingerprints to each other using all three primers (Fig. 1). Isolate UQ2351 shared 67% of bands with UQ3333–3339 isolates, when the total number of bands generated using all three primers were pooled. Ribosomal DNA ITS1 and ITS2 sequence analysis The nucleotide sequence of the ITS region of LRC isolates UQ2351 and UQ3333 were identical. When compared with ITS nucleotide sequences from all known AGs (Gonzalez et al. 2001), LRC sequences were most similar to AG 6 GV (96.2%) and AG 6 HG (95.4%). Phylogeny of LRC isolates and representatives of all known AGs was inferred using the neighbour-joining method and was consistent with recent studies (Gonzalez et al. 2001). LRC isolates formed a clade and clustered with AG 6 isolates (Fig. 2). Additional isolates from AG 6 from GenBank were included and the analysis repeated to further resolve LRC isolates and AG 6 isolates. AG1-IA was used as an outgroup. LRC isolates clustered separately and basal to the known AG 6 isolates (Fig. 3).
Discussion We conclude that Rhizoctonia root canker of lucerne in western areas of Queensland is caused by a hitherto undescribed AG subgroup of R. solani. The LRC isolates produced C2 AG reactions when paired with other AG 6 subgroups and C0 reactions when paired with all other tester isolates. This is the first report that isolates of R. solani causing lucerne root canker belong to a subgroup of AG 6. Crater disease of wheat in the hot humid Springbok Flats of South Africa was reported as caused by a R. solani strain in AG 6 (Carling et al. 1996; Meyer et al. 1998), with the crater disease pathogen showing a close affinity to tester isolate AG 6 GV. In our study, LRC isolate ITS sequences were also slightly more similar to the AG 6 GV subgroup (96.2%) than the AG 6 HG subgroup (95.4%). Phylogenetically, LRC isolates clustered basal to the two previously described AG 6 subgroups with fewer steps separating LRC isolates from AG 6 GV isolates. We have conclusively demonstrated that the lucerne LRC isolates are not pathogenic to wheat, indicating the LRC isolates are different from the wheat pathogen reported by Meyer et al. (1998). The relationship between the organism causing LRC in Australia and that in the U.S.A. remains to be determined. A bridging (C1 reaction) relationship between AG 6 isolates from South Africa and Tanzania causing crater disease and AG-1 IA was reported by Carling et al. (1996). This relationship was also shown to exist for AG-4 isolates and the crater disease isolates (Meyer et al. 1998). Our studies revealed no interactions (C0 reactions) between LRC isolates and isolates from groups other than AG 6. Genetic differences also exist between various AG 6 subgroups which have so far been identified from wheat, soil and
Characterisation of Rhizoctonia solani
Australasian Plant Pathology
UQ2351
Anastomosis group LRC
UQ3333
LRC
75RS
AG6 GV
HAM1 1
AG6 HG
SHIBA 1
AG1 1B
CS GI
AG1 1A
PS 1
AG1 1C
91ST8057 2
AG7
45RS
AG4 HIII
PF 10
AG4 HI
RH 131
AG4 HII
ZG1 2 SA50
AG8
K31
AG5
89
ROTH 16
AG11
59
P2
AG2-1
65RS
AG9
W45B3
AG10
AI1 4
AG B1
15RS
AG2-2 IIIB
BC 10
AG2-2 IV
100 80
93
55
99 74
55
99
55
100
50
245
Fig. 2. Consensus neighbour-joining tree illustrating genetic relationships in terms of distance derived from sequences of ITS-5.8S rDNA regions of LRC isolates and known AGs of Rhizoctonia solani. Distances were determined using Kimura’s two-parameter model. Support for each clade is represented by bootstrap values on each branch.
lucerne. AG 6 is a genetically variable group composed of subgroups which demonstrate partial phylogenetic relatedness (Kuninaga and Yokosawa 1984). Our studies have identified further genetic complexity within AG 6. Deoxyribonucleic acid amplification fingerprinting and rDNA ITS sequence analysis supported the anastomosis groupings found in this study. All LRC isolates obtained from different plants collected in the same field at Biloela had identical DNA banding patterns, indicating their clonality. These isolates were also genetically similar to the LRC isolate from Roma sharing identical rDNA ITS sequences, and generally similar but not identical DNA banding patterns. Other studies have used DNA/DNA hybridisation (Carling et al. 1988; Kuninaga and Yokosawa 1984), random amplified polymorphic DNA (RAPD) (Duncan et al. 1993), restriction fragment length polymorphism (RFLP) (Vilgalys
and Gonzalez 1990) and ITS sequence analysis (Carling et al. 2002; Gonzalez et al. 2001; Kuninaga et al. 1997) to characterise and differentiate anastomosis sub-groups of R. solani. In this study, ITS sequence analysis did not differentiate LRC isolates from Biloela or Roma. Here we demonstrate the utility of DNA amplification fingerprinting in further resolving genetic variation between closely related isolates of R. solani. The failure to find resistance to LRC isolates among a small collection of Australian lucerne cultivars is consistent with several other studies with diseases in other crops incited by R. solani where it has not been possible to identify heritable sources of disease resistance (Bains et al. 2002; Bradley et al. 2001). However, lucerne cultivars have been bred with increased resistance to seedling damping-off (Anderson 1982; Barnes et al. 1980). The importance of using individual AGs of R. solani in germplasm screening
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UBU 1 A 99 53
HAM1 1
AG 6 HG
70RS
95
NKN2 1 75RS 99
UQ2351 99
AG 6 GV
HN1 1 LRC isolates
UQ3333 CS GI
AG 1-1A
Fig. 3. Neighbour-joining tree illustrating genetic variation in terms of distance of LRC isolates and AG 6 isolates of Rhizoctonia solani. See Fig. 2 for details.
and breeding to improve the chances of successfully identifying useful resistance was highlighted by Ogoshi (1987). A wider range of lucerne genetic material should be screened using the AG subgroups identified in this work. The work reported in this paper has placed the LRC-causing isolates of R. solani within AG 6. Considering the results from molecular analysis, AG testing and pathogenicity testing, it is proposed the LRC pathogen represents a distinct intraspecific subgroup of AG 6. Acknowledgements Professor A. Ogoshi provided standard tester isolates for determination of AG group. Funding for this research was provided by the Grains Research and Development Corporation. References Anderson NA (1977) Evaluation of the Rhizoctonia complex in relation to seedling blight of flax. Plant Disease Reporter 61, 140–142. Anderson NA (1982) The genetics and pathology of Rhizoctonia solani. Annual Review of Phytopathology 20, 329–347. doi:10.1146/ ANNUREV.PY.20.090182.001553 Bains PS, Bennypaul HS, Lynch DR, Kawchuk LM, Schaupmeyer CA (2002) Rhizoctonia disease of potatoes (Rhizoctonia solani): fungicidal efficacy and cultivar susceptibility. American Journal of Potato Research 79, 99–106. Baker KF (1957) The UC system for producing healthy container-grown plants. Californian Agricultural Experiment Station Manual 23. Bandoni RJ (1979) Safranin O as a rapid nuclear stain for fungi. Mycologia 71, 873–874. Barnes DK, Sarojak DJ, Frosheiser FI, Anderson NA (1980) Registration of alfalfa germplasm with seedling resistance to Rhizoctonia solani (Reg. Nos. GP 111 to GP113). Crop Science 20, 675. Bentley S, Bassam BJ (1996) A robust DNA amplification fingerprinting system applied to analysis of genetic variation within Fusarium oxysporum f. sp. cubense. Journal of Phytopathology 144, 207–213.
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Received 20 September 2003, accepted 19 November 2003
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