- Email: [email protected]
Characteristics of some Rhizoctonia spp. from South Australian plant nurseries
Mycal. Res. 98 (1): 83-87 (1994)
83
Printed in Great Britain
Characteristics of some RhizDctonia spp. from South Australian plant nurseries
G. MASUHARA"', S. M. NEATE AND D. A. SCHISLERt CSIRO Division of Soils, Private Bag No.2, Glen Osmond, South Australia 5064, Australia
Forty-nine Rhizoctonia isolates were obtained from nursery plants, potting mix, vegetable seedlings and bedding plants in South Australia. Of these isolates, 75 % were binucleate Rhizoctonia and 25 % were multinucleate. Anastomosis tests showed that all of the multinucleate isolates were either Rhizoctonia solani AG-2-1 or AG-4 and that the binucleate Rhizoctonia belonged to either AG-F, AG-!, AG-K and CAG-5 or to four other groups of isolates which did not anastomose with the known isolates tested. Teleomorphs of 14 isolates were induced and were identified as Thanatephorus cucumeris, Cerafobasidium cornigerum, C. pseudocornigerum and two unknown species Ceratobasidium. Extracellular pectic enzyme patterns were not useful in determining likely anastomosis groups for the binucleate Rhizoctonia.
Though there are many reports of the identity of Rhizoctonia spp. from extensive agriculture, detailed research on Rhizocfonia isolated from plant nurseries throughout the world is limited (Cline et al., 1988; Stephens, Herr & Schmitthenner, 1982), and is absent for Australia. Rhizoctonia is divided into two major groups, multinucleate and binucleate. Thanatephorus cucumeris (Frank) Dank (anamorph; the multinucleate Rhizoctonia solani KUhn) is grouped by anastomosis between hyphae into AG-1 to AGIO and a bridging isolate AG-BI (Ogoshi, 1976; Kuninaga, Yokosawa & Ogoshi, 1978; Neate & Warcup, 1985; Carling & Kebler, 1987; Sneh, Burpee & Ogoshi, 1991). Binucleate isolates are also grouped by anastomosis into AG-A to AGS (Ogoshi et aI., 1979; Oniki et al., 1984, 1986; Sneh et aI., 1991). Independently, Burpee et ai. (1980) described the binucleate groups CAG-1 to CAG-7. AG-A, AG-D and AGF correspond to CAG-2, CAG-1 and CAG-4, respectively and AG-E with both CAG-3 and CAG-6. Induction of the teleomorphic stage of Rhizoctonia isolates is important for characterization of these fungi. Induction of teleomorphs of Rhizoctonia on agar media has been reported (Adams & Butler, 1983), but the method is not as successful as the soil-over-agar method of Stretton et al. (1964) and Warcup & Talbot (1965). Pectic enzyme patterns in pectic acrylamide gels have been found to be useful for determining likely anastomosis groups of isolates of Thanafephorus cucumeris and binucleate Rhizoctonia found in cereal fields in South Australia (Neate, Cruickshank & Rovira, 1988; Neate & Cruickshank, 1988). Sweetingham, Cruickshank & Wong (1986) suggested that these patterns may be used to characterize pathogenic groups.
Nuclear testing and anastomosis
Present addresses: • University of Tsukuba. Institute of Agriculture and Forestry, Tsukuba, Ibaraki 305, Japan. tUSDA-ARS. National Centre for Agricultural Utilization Research, Peoria, IL, 61604, U.S.A.
The nuclear condition of Rhizoctonia isolates was determined by a modified Giemsa method (Herr, 1979). Binucleate isolates were tested against the binucleate anastomosis groups AG-A,
This study examines the anastomosis group (AG), teleomorph and pectic enzyme patterns of Rhizoctonia isolated from bedding plants, nursery plants or soils from plant nurseries in South Australia. The pathogenicity of selected isolates is considered in detail elsewhere (Schisler, Neate & Masuhara, 1993).
MATERIALS AND METHODS Isolation of Rhizoctonia
Rhizoctonia was isolated from (a) diseased plants exhibiting Rhizoctonia-like symptoms: these are tan to brown sunken lesions on roots which in more severe cases in some host species lead to a rotting away of the cortex exposing the stele; eventually the stele too rots away leaving a pointed brown stub. (b) Apparently healthy plants. (c) New and used potting soils from 30 nurseries. The isolation methods are reported elsewhere (Schisler, Neate & Masuhara, 1993). In addition to the 39 isolates described by Schisler et al. (1993), 10 isolates from 5 additional sources were included (nurseries 12-16 in Table 1). Four of the sources were from root lesions on seedlings from vegetable growers and the fifth was obtained from a root lesion on an orchid from an orchid nursery. When Rhiwctonia - like hyphae (Parmeter, Sherwood & Platt, 1969) were found, they were transferred onto fresh 1/6 strength NDY agar (Czapek-Dox + yeast; Warcup, 1955) and were examined under a light microscope ( x 100) after growth for I month.
6-2
84
South Australian Rhizoctoria species Table 1. Origin, nuclear condition, anastomosis group (AG) and teleomorph of 49 Rhizoctonia isolates from nurseries in South Australia Isolate CIBI CIB2 CIB3 CIB4 CRESPI CRESP2 CRESP3 ENG4BI ENG4B2 ENG4B3 ENG4B4 GRI2PI BREIBI BREIB2 BRE1B3 BRE4BI BRE4B2 BRE4B3 JIBI JIB2 OISI R2BI R2B2 WOC3PI B1B3 CRESB1 CRESB2 CRESB3 ENG2B1 ENG2B2 MIBI M1B2 MIS2 MlS3 MIS4 ORCP1 ORCP2 BIBI B1B2 CAUPI CAUP2 CRESB4 CUCPI CUCP2 CUCP3 OIBI ONIIP3 ONIIP4 ONl2PI
Nursery I I I I 2 2 2 3 3 3 3
4 S S S S S S 6 6 7 8 8
9 10 2 2 2 3 3 11 11 11 11 11 12 12
10
10 13 13
2 14 14 14 I IS IS 16
Host·
Nuclei"
AG
Baiting Baiting Baiting Baiting
B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B M M M M M M M M M M M M
AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-K CAG-s CAG-S CAG-S Group A Group A Group B Group C Group 0 Group 0 Group E Untested Untested AG-2-I AG-2-I AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-2-I Untested AG-2-I
Pimelia Pimelia Pimelia
Baiting Baiting Baiting Baiting Cyclamen Baiting Baiting Baiting Baiting Baiting Baiting Baiting Baiting Soil Baiting Baiting Alfalfa Baiting Baiting Baiting Baiting Baiting Baiting Baiting Baiting Soil Soil Soil Thelymilra Thelymilra
Baiting Baiting Cauliflower Cauliflower Baiting Cucumber Cucumber Cucumber Baiting Onion Onion Onion
Teleomorph
Ceralobasidium comigerum
C. comigerum
C. romigenltn
C. pseudoromigerum
Unidentified Unidentified C. cornigerum C. comigerum C. romigerum
Thanalephorus cucumeris
T. cucumeris T. cucumeris
T. cucumeris
T. cucumeris
• Isolate made by baiting method described elsewhere (Schisler, Neate & Masuhara, 1993), b M, multinucleate; B, binucleate.
AG-Ba, AG-C, AG-D, AG-E, AG-F, AG-G, AG-I, AG-J, AGK, CAG-I, CAG-2, CAG-4, CAG-5, CAG-6, CAG-7 and multinucleate isolates against the multinucleate anastomosis groups AG-I, AG-2-I, AG-3, AG-4, AG-5, AG-6, AG-7 and AG-8, in both cases by the modified method of Sanders, Burpee & Cole (I978). Agar blocks of 2-3 mm 2, containing hyphae of the known AG group or the isolate to be tested, were opposed on cellophane overlying distilled water agar in a 2'5 em diam. plastic Petri plate and incubated for 24-48 h at 25°C. The area in which advancing hyphae overlapped was then scanned at 125 x magnification for anastomoses. An-
astomosis was only recorded where the 'killing reaction' occurred, Le. when one or more hyphal cells on either side of the anastomosis died and became vacuolate (Flentje & Stretton, I964). Where the unknown isolate was not able to be placed in a group, tests were repeated at least three times.
Inducing teleomorphs Isolates were grouped according to morphology, and induction of the teleomorph was attempted by the soil-over-agar method (Stretton ef al., 1964; Warcup & Talbot, I965).
85
G. Masuhara, S. M. Neate and D. A. Schisler Table 2. A comparison of the grouping of binucleate isolates of Rhiwetonia based on electrophoretic patterns with the grouping determined by anastomosis
Isolate CIBI CIB2 CIB3 R2Bl R2B2 CRESPI MIS3 CRESP2 CRESP3 ENG4Bl ENG4B2 ENG4B3 ENG4B4 GRI2Pl BREIBI BREIB2 BREIB3 BRE4Bl BRE4B2 BRE4B3 MIS4 JIBI JIB2 MIB2 01S1 WOOPI B1B3 CRESB1 CRESB2 CRESB3 ENG2Bl ENG2B2 M!S2
Electrophoresis group 1 1 1 1 1 2 2 3 4 S S 6 6 7 8 8 8 8 8 8 8 9 9 9
10 11
12 13 13 13
14 14 IS
Anastomosis group AG-F AG-F AG-F AG-I AG-I AG-F Group 0 AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-I AG-I AG-! AG-I AG-! AG-! Group E AG-I AG-I Group C AG-! AG-! AG-K CAG-S CAG-S CAG-s Group A Group A Group 0
Isolates were grown on NDY agar in individual 9 cm diam. Petri plates for 2 wk until the plate was densely covered by mycelium; the lid was then removed and the plates covered with a 1-2 cm layer of soil clods up to I' 5 cm diam. taken from the clay B horizon of Urrbrae Red-Brown Earth (calcic luvisol; Dudal, 1968), pH 7, 50-60% montomorillonite/illite clay, 20-25 % of both silt and fine sand, negligible coarse sand (Litchfield, 195 I). The soil was wetted to just short of saturation with deionized water, and this was maintained by watering every day. Plates were incubated at room temperature (15-25 0) in diffuse light with normal day/night cycles.
Extra-cellular perlic enzymes Isolates were grown in 2 ml of a weak broth culture medium containing pectin for 10 d (Sweetingham et al., 1986). Culture broth was used for electrophoresis using a modified method of Cruickshank (1983). Fifty IJI of culture broth was mixed with about 0'005 g of Sephadex G200 superfine (Pharmacia, Uppsala, Sweden), and 4-6 IJI of the resulting slurry was added to polyacrylamide gels containing 0'9% pectin. Electrophoresis was at a constant current of 10 rnA with a
starting voltage of 90 V, and was stopped when the 0'5 % bromophenol blue tracking dye had migrated 6'5 cm toward the anode. After electrophoresis the gels were incubated with shaking in 0'1 M DL-malic acid at 25° for 1'5 h, and then stained by shaking in a solution of 0'015 % ruthenium red overnight at 5°. RESULTS
Nuclear condition and anastomosis group of isolates The origin, nuclear condition, AG group and teleomorph of 49 isolates of Rhizocfonia from diseased plants, soil particles and by baiting soil with seedlings are shown in Table 1. Giemsa staining showed that 37 of the 49 isolates of Rhizocfonia were binucleate, 12 being multinucleate. Colony colours of 32 of the binucleate Rhizocotonia isolates were white to pale yellow; however, colonies of CRE5B1, CRE5B2, CRE5B3, ENG2B1 and ENG2B2 were a dark brownish colour on 1/6 strength NDY agar. Both multinucleate and binucleate isolates were recovered by baiting soil samples at nursery I, but in general only one type was recovered from an individual nursery. All the multinucleate isolates were either Rhizocfonia solani AG-2-1 or AG-4. Four of the 12 multinucleate isolates were AG-2-1 and seven isolates were AG-4, with one not tested. Binucleate Rhizocfonia belonged to either AG-F, AG-!, AG-K or CAG-5, or to four groups of isolates which do not anastomose with known isolates. Some 65 % of the binucleate isolates belonged to either AG-F or AG-!.
Teleomorphs Teleomorphs were obtained for 14 of the 30 isolates tested. Twelve of the isolates were identified as Thanatephorus cucumeris, Ceratobasidium comigerum (Bourdot) Rogers, or Ceratobasidium pseudocomigerum Christiansen on the basis of characteristics of hymenia, basidia and basidiospores as described by Talbot (1965) and Warcup & Talbot (1965, 1966). The C. pseudocomigerum was distinguished from C. comigerum by oblong to cylindrical spores with a ratio of length to width more than 2: 1. Normally, hymenia emerged 1-4 wk after covering the agar plate cultures with soil. An exception was D1B1 (Thanatephorus cucumeris), which formed a hymenium only after normal incubation for 4 wk, followed by complete drying of the colonized soil over 2 d and then a re-wetting period of 2 d. Fungal tissue from the hymenium was recultured on 1/6 strength NDY agar and confirmed as the isolate D1B1 by comparing morphology in culture. The teleomorphs of binucleate Rhizocfonia CRE5B3 and ENG2B2 were not consistent with currently described species. Basidia of CRE5B3 were holobasidiate, with metabasidia subglobose to broadly clavate, 12-15 IJm long and 7-8'5 IJm wide, with four sterigmata 6-9 IJffi long. The ratio of width of metabasidia to supporting hyphae was less than 2: 1. Basidia of ENG2B2 were holobasidiate, metabasidia were subglobose, 8'4-9'4 IJm long and 6'9-8'8 IJm wide, with four sterigmata 5'1-8'9 IJm long. The ratio of width of metabasidia to supporting hyphae was less than 2: 1.
86
South Australian Rhizoctoria species (a)
... 1
(b)
,,'
~_
'i"
~
~
".~
..4~':~~~~~:·~ : c
•
-
~
,
(c)
Fig. 1. Pectic zymograms of selected isolates of binucleate Rhizocfonia. (Fig. Ia) similarity of patterns of four isolates from the same nursery. (Fig. I b) range of variation of patterns of four isolates identified as AG~F (Fig. 1 c) range of variation of patterns of four isolates identified as AG-L
Extracellular pectic enzymes All multinucleate isolates produced characteristic banding patterns on pectin - acrylamide gels and were able to be placed in either AG-2-1 or AG-4 based on their patterns. All binucleate isolates except C1B4, M1BI, ORCP1 and ORCP2 also produced characteristic banding patterns. Binucleate isolates were placed in IS groups according to similarity of pattern (Table 2) and compared with patterns of known anastomosis groups. The II AG-F isolates fell into seven electrophoretic pattern groups, the 12 AG-I isolates fell into five groups and the three CAG-5 isolates (all from the same nursery) fell into one group. Pectic zymogram patterns of selected binucleate isolates are shown in Figure 1. Isolates from the same anastomosis group and the same nursery showed similar patterns, but isolates from within AG-F or AG-I from different nurseries showed a range of patterns. When the likely anastomosis group could not be identified by the authors from the pectic enzyme banding patterns obtained, the patterns were assessed independently. Assessors were only rarely able to match the pattern of an isolate with the pattern of a known anastomosis group. Where many isolates in a single anastomosis group were tested, there was significant variation among the patterns of the isolates. Furthermore, when isolates in AG-A were compared with CAG-2, AG-D with CAG-I, AG-E with CAG-3 and CAG-6, and AG-F with CAG-4, i.e. pairs that should be similar since they are recognized to be from the same anastomosis group, there was also significant variation between zymogram patterns.
DISCUSSION Forty-nine Rhizoctonia isolates, including both multinucleate and binucleate types, were obtained from nursery plants and soils in South Australia. The multinucleate Rhizoctonia identified in the present investigation have been isolated previously in South Australia; AG-2-1 from wheat roots and AG-4 from soiL wheat roots, and Sagina procumbens (pearlwort) (Neate & Warcup, 1985). However, the present investigation showed that in South Australia AG-2-1 also colonizes onion seedlings and nursery soils, and AG-4, cauliflower and cucumber seedlings. These species are within the previously described host ranges for the fungus and it is expected that with more work other hosts for these fungi will be found in South Australia. Pathogenicity tests of these isolates were reported by Schisler, Neate & Masuhara (1993). Anastomosis tests of binucleate Rhizoctonia confirmed the existence of AGF and AG-K in Australia, and showed for the first time the presence of AG-I and CAG-5. The groups AG-F and AG-K had previously been associated with root rot of subterranean clover or bare-patch disease of cereals (Wong & Sivasithamparam, 1985; Roberts & Sivasithamparam, 1986). The isolate of AG-K also anastomosed with the AG-A tester isolate, but at a much lower frequency than occurred with the AG-K tester isolate. This suggests that AG-A and AG-K are closely related, and may even be two subgroups of the same anastomosis group. The association between these two groups deserves further study. The inability to identify the teleomorphs of CRE5B3 and ENG2B2 is consistent with the findings of Parmeter, Whitney & Platt (1967), who described difficulties of identification of teleomorphs of binucleate Rhizoctonia; Ogoshi et al. (1983) apparently had similar problems, as they induced teleomorphs of 1 I of IS binucleate anastomosis groups, but did not identify the species. Most problems have been due to large variations in size and shape of basidia and spores. CRE5BI, CRE5B2 and ENG2B1 which did not form teleomorphs and CRE5B3 and ENG2B2 which formed unidentified teleomorphs all have dark brown colonies. As CRESS1 was identified as belonging to CAG-5 it confirms the previous record of that group having brown colonies (Burpee et al., 1980). Although Wong & Sivasithamparam (1985) found brown-coloured binucleate isolates associated with root rot of subterranean clover in Western Australia, this is the first record of brown-coloured binucleate Rhizoctonia in South Australia. Neate (1985) found that colony colour was a useful means of separating cultures of Rhizoctonia from South Australian wheat fields into likely species. However, the usefulness of this character in separating isolates in nursery soils is limited by the existence of isolates of Ceratobasidium showing brown coloration in culture. This is the first time teleomorphs have been produced for isolates in AG-I, and it suggests that there may be two species in the group, C. cornigerum and C. pseudocornigerum. More isolates need to be studied to confirm this. This study confirms the finding of Ogoshi et al. (1983) that AG-F has a Ceratobasidium sp. teleomorph. The two AG-F isolates which produced a teleomorph were both identified as C. cornigerum. Although the multinucleate isolates showed some degree
G. Masuhara, S. M. Neate and D. A. Schisler of variability, pectic enzyme patterns could be used to predict likely anastomosis groupings with 100 % success. For the binucleate isolates, pectic enzyme patterns were useful for determining the degree of relatedness of morphologically similar strains. For example, CRE5Bl, CRE5B2 and CRE5B3 isolates from the same nursery showed similar enzyme patterns to each other, and isolates JIB1 and JIB2, both from another nursery, also showed similar patterns to each other. However, overall the usefulness of the technique within binucleate anastomosis groups was limited by variability in patterns obtained from isolates later shown to be of the same anastomosis group, and in comparison with those of the anastomosis group tester isolates. As these results differed from those of Neate & Cruickshank (1988) the patterns were independently assessed with the same result. Currently, there are 19 binucleate tester isolates recognized in the system of Ogoshi ef al. (Ogoshi ef a/., 1979; Oniki et a/., 1984, 1986; Sneh ef al., 1991) and seven in the system of Burpee et al. (1980). Our results and those of others have shown that many isolates do not fit into any of the currently recognized groups, suggesting there are many groups yet to be described. This variation in pectic enzyme pattern within binucleate anastomosis groups and the potential numbers of groups in existence limits the use of pectic iso-enzyme identification of binucleate Rhizoctonia to ecological systems where more is known about the range of groups of isolates present. The authors thank Ann Benger for her competent technical assistance. One of us, D.A.S., thanks Incitec Ltd, Australian for a portion of his salary and research support. REFERENCES Adams, G. C. jr & Butler, E. E. (1983). Influence of nutrition on the formation of basidiospores in Thanatephorus cucumeris. Phytopathology 73, 147-151. Burpee, L. L., Sanders, P. L., Cole, H. Jr. & ShelWood, R. T. (1980). Anastomosis groups among isolates of Ceratobasidium cornigerum and related fungi. Mycologia 72, 689--701. Carling, D. E. & Kebler, K. M. (1987). Characterization of a new anastomosis group (AG-9) of Rhizoclonia solani. Phytopathology 77, 1609--1612. Cline, M. N.. Chastagner, G. A., Aragaki, M., Baker, R., Daughtrey, M. L., Lawson, R. H., Macdonald, j. D., Tammem, j. F. & Worf, G. L. (1988). Current and future research directions of ornamental pathology. Plant Disease 72, 926--934. Cruickshank, R. H. (1983). Distinction between Sclerotinia species by their pectic zymograms. Transaclions of the British Mycological Society 80, 117-119. Dudal. R. (1968). Definition of soil units for the soil map of the world. World Soil Resources Report No. 33, FAO: Rome. Flentje, N. T. & Stretton, H. M. (1964). Mechanisms of variation in Thanatephorus cucumeris and T. praticolus. Australian journal of Biological Science 17, 686--704. Herr, L. J. (1979). Practical nuclear staining procedures for Rhizoctonia-like fungi. Phytopathology 69, 958-961. Kuninaga, S., Yokosawa, R. & Ogoshi, A. (1978). Anastomosis grouping of Rhizoclonia solani Kuhn isolated from non-cultivated soils. Phytopathological Society of japan 49, 184-190. Litchfield, W. H. (1951). Soil survey of the Waite Agricultural Research Institute, Glen Osmond S.A. CSIRO Australia, Division of Soils, Divisional Report No. 2/51. (Accepted 16 june 1993)
87 Neate, S. M. (1985). Rhizoctonia in South Australian wheatfields. In Ecology and Management of Soil-Borne Plant Pathogens (ed. C. A. Parker, A. D. Rovira, K. j. Moore, P. T. W. Wong and j. F. Kollmorgen), pp. 54-56. American Phytopathological Society, St Paul. MN. Neate, S. M. & Cruickshank R. H. (1988). Pectic enzyme patterns of Ceratobasidium and Rhizoclonia spp. associated with sharp eyespot-like lesions on cereals in South Australia. Transaclions of the British Mycological Society 91, 267-272. Neate, S. M., Cruickshank, R. H. & Rovira, A. D. (1988). Pectic enzyme patterns of Rhizoclonia solani isolates from agricultural soil in South Australia. Transaclions of the British Mycological Society 90, 37-42. Neate, S. M. & Warcup, j. H. (1985). Anastomosis grouping of some isolates of Thanatephorus cucumeris from agricultural soils in South Australia. Transactions of the British Mycological Society 85, 615-620. Ogoshi, A. (1976). Studies on the grouping of Rhizoclonia solani Kuhn with hyphal anastomosis and on the perfect stages of groups. Bulletin of the National Institutes of Agricultural Science, Tokyo, Series C 30, 1-63. Ogoshi, A., Oniki, M., Araki, T. & Ui, T. (1983). Anastomosis groups of binucleate Rhizoclonia in japan and North America and their perfect states. Transaclions of the Mycological Society of japan 24, 79--87. Ogoshi, A., Oniki, M., Sakai, R. & Ui, T. (1979). Anastomosis grouping among isolates of binucleate Rhizoclonia. Transaclions of the Mycological Society of japan 20, 33-39. Oniki, M., Araki, T., Ogoshi, A., Kasai, K., Hamaya, E. & Ando, Y. (1984). Some properties and taxonomic assessment of the tea black rot fungus. Study of Tea 6, 7-14. Oniki, M., Kobayashi, K., Araki, T. & Ogoshi, A. (1986). A new disease of turf-grass caused by binucleate Rhizoclonia AG-Q. Annals of the Phytopathological Society of japan 52, 850--853. Parmeter, j. R. jr, ShelWood, R. T. & Platt, W. D. (1969). Anastomosis grouping among isolates of Thanatephorus cucumeris. Phytopathology 59, 1270--1278. Parmeter, j. R. jr, Whitney, H. S. & Platt, W. D. (1967). Affinities of some Rhizoclonia species that resemble mycelium of Thanatephorus cucumeris. Phytopathology 57, 218-223. Roberts, F. A. & Sivasithamparam, K. (1986). Identity and pathogenicity of Rhizoclonia spp. associated with bare patch disease of cereals at a field site in Western Australia. Netherlands journal of Planf Pathology 92, 185-195. Sanders, P. L., Burpee, L. L. & Cole, H. jr (1978). Preliminary studies on binucleate turfgrass pathogens that resemble Rhizoclonia solani. Phytopathology 68, 145-148. Schisler, D. A., Neate, S. M. & Masuhara, G. (1993). The occurrence and pathogenicity of Rhizoclonia fungi in South Australian plant nurseries. Mycological Research 97, 77-82. Sneh, B., Burpee, L. & Ogoshi, A. (1991). Identification of Rhizoclonia Species. APS Press: St Paul. Minnesota. Stephens, C. T., Herr, L. j. & Schmitthenner, A. F. (1982). Characterisation of Rhizoclonia isolates associated with damping-off of bedding plants. Plant Disease 66, 700--703. Stretton, H. M., McKenzie, A. R., Butler, K. F. & Flente, N. T. (1964). Formation of the basidial stage of some isolates of Rhiwclonia. Phytopathology 54, 1093-1095. Sweetingham, M. W., Cruickshank, R. H. & Wong, D. H. (1986). Pectic zymograms and taxonomy and pathogenicity of the Ceratobasidiaceae. Transactions of the British Mycological Society 86, 305-311. Talbot, P. H. B. (1965). Studies of 'Pellicularia' and associated genera of Hymenomycetes. Persoonia 3, 371-406. Warcup, j. H. (1955). Isolation of fungi from hyphae present in soil. Nature, London 175, 953. Warcup, j. H. & Talbot, P. H. B. (1965). Ecology and identity of mycelia from soil. III. Transaclions of the British Mycological Society 48, 249--259. Warcup, j. H. & Talbot, P. H. B. (1966). Perfect states of some Rhizoctonias. Transactions of the British Mycological Society 49, 427-435. Wong, D. H. & Sivasithamparam, K. (1985). Rhizoclonia spp. associated with root rots of subterranean clover in Western Australia. Transactions of the British Mycological Society 85, 21-27.
83
Printed in Great Britain
Characteristics of some RhizDctonia spp. from South Australian plant nurseries
G. MASUHARA"', S. M. NEATE AND D. A. SCHISLERt CSIRO Division of Soils, Private Bag No.2, Glen Osmond, South Australia 5064, Australia
Forty-nine Rhizoctonia isolates were obtained from nursery plants, potting mix, vegetable seedlings and bedding plants in South Australia. Of these isolates, 75 % were binucleate Rhizoctonia and 25 % were multinucleate. Anastomosis tests showed that all of the multinucleate isolates were either Rhizoctonia solani AG-2-1 or AG-4 and that the binucleate Rhizoctonia belonged to either AG-F, AG-!, AG-K and CAG-5 or to four other groups of isolates which did not anastomose with the known isolates tested. Teleomorphs of 14 isolates were induced and were identified as Thanatephorus cucumeris, Cerafobasidium cornigerum, C. pseudocornigerum and two unknown species Ceratobasidium. Extracellular pectic enzyme patterns were not useful in determining likely anastomosis groups for the binucleate Rhizoctonia.
Though there are many reports of the identity of Rhizoctonia spp. from extensive agriculture, detailed research on Rhizocfonia isolated from plant nurseries throughout the world is limited (Cline et al., 1988; Stephens, Herr & Schmitthenner, 1982), and is absent for Australia. Rhizoctonia is divided into two major groups, multinucleate and binucleate. Thanatephorus cucumeris (Frank) Dank (anamorph; the multinucleate Rhizoctonia solani KUhn) is grouped by anastomosis between hyphae into AG-1 to AGIO and a bridging isolate AG-BI (Ogoshi, 1976; Kuninaga, Yokosawa & Ogoshi, 1978; Neate & Warcup, 1985; Carling & Kebler, 1987; Sneh, Burpee & Ogoshi, 1991). Binucleate isolates are also grouped by anastomosis into AG-A to AGS (Ogoshi et aI., 1979; Oniki et al., 1984, 1986; Sneh et aI., 1991). Independently, Burpee et ai. (1980) described the binucleate groups CAG-1 to CAG-7. AG-A, AG-D and AGF correspond to CAG-2, CAG-1 and CAG-4, respectively and AG-E with both CAG-3 and CAG-6. Induction of the teleomorphic stage of Rhizoctonia isolates is important for characterization of these fungi. Induction of teleomorphs of Rhizoctonia on agar media has been reported (Adams & Butler, 1983), but the method is not as successful as the soil-over-agar method of Stretton et al. (1964) and Warcup & Talbot (1965). Pectic enzyme patterns in pectic acrylamide gels have been found to be useful for determining likely anastomosis groups of isolates of Thanafephorus cucumeris and binucleate Rhizoctonia found in cereal fields in South Australia (Neate, Cruickshank & Rovira, 1988; Neate & Cruickshank, 1988). Sweetingham, Cruickshank & Wong (1986) suggested that these patterns may be used to characterize pathogenic groups.
Nuclear testing and anastomosis
Present addresses: • University of Tsukuba. Institute of Agriculture and Forestry, Tsukuba, Ibaraki 305, Japan. tUSDA-ARS. National Centre for Agricultural Utilization Research, Peoria, IL, 61604, U.S.A.
The nuclear condition of Rhizoctonia isolates was determined by a modified Giemsa method (Herr, 1979). Binucleate isolates were tested against the binucleate anastomosis groups AG-A,
This study examines the anastomosis group (AG), teleomorph and pectic enzyme patterns of Rhizoctonia isolated from bedding plants, nursery plants or soils from plant nurseries in South Australia. The pathogenicity of selected isolates is considered in detail elsewhere (Schisler, Neate & Masuhara, 1993).
MATERIALS AND METHODS Isolation of Rhizoctonia
Rhizoctonia was isolated from (a) diseased plants exhibiting Rhizoctonia-like symptoms: these are tan to brown sunken lesions on roots which in more severe cases in some host species lead to a rotting away of the cortex exposing the stele; eventually the stele too rots away leaving a pointed brown stub. (b) Apparently healthy plants. (c) New and used potting soils from 30 nurseries. The isolation methods are reported elsewhere (Schisler, Neate & Masuhara, 1993). In addition to the 39 isolates described by Schisler et al. (1993), 10 isolates from 5 additional sources were included (nurseries 12-16 in Table 1). Four of the sources were from root lesions on seedlings from vegetable growers and the fifth was obtained from a root lesion on an orchid from an orchid nursery. When Rhiwctonia - like hyphae (Parmeter, Sherwood & Platt, 1969) were found, they were transferred onto fresh 1/6 strength NDY agar (Czapek-Dox + yeast; Warcup, 1955) and were examined under a light microscope ( x 100) after growth for I month.
6-2
84
South Australian Rhizoctoria species Table 1. Origin, nuclear condition, anastomosis group (AG) and teleomorph of 49 Rhizoctonia isolates from nurseries in South Australia Isolate CIBI CIB2 CIB3 CIB4 CRESPI CRESP2 CRESP3 ENG4BI ENG4B2 ENG4B3 ENG4B4 GRI2PI BREIBI BREIB2 BRE1B3 BRE4BI BRE4B2 BRE4B3 JIBI JIB2 OISI R2BI R2B2 WOC3PI B1B3 CRESB1 CRESB2 CRESB3 ENG2B1 ENG2B2 MIBI M1B2 MIS2 MlS3 MIS4 ORCP1 ORCP2 BIBI B1B2 CAUPI CAUP2 CRESB4 CUCPI CUCP2 CUCP3 OIBI ONIIP3 ONIIP4 ONl2PI
Nursery I I I I 2 2 2 3 3 3 3
4 S S S S S S 6 6 7 8 8
9 10 2 2 2 3 3 11 11 11 11 11 12 12
10
10 13 13
2 14 14 14 I IS IS 16
Host·
Nuclei"
AG
Baiting Baiting Baiting Baiting
B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B B M M M M M M M M M M M M
AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-I AG-K CAG-s CAG-S CAG-S Group A Group A Group B Group C Group 0 Group 0 Group E Untested Untested AG-2-I AG-2-I AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-4 AG-2-I Untested AG-2-I
Pimelia Pimelia Pimelia
Baiting Baiting Baiting Baiting Cyclamen Baiting Baiting Baiting Baiting Baiting Baiting Baiting Baiting Soil Baiting Baiting Alfalfa Baiting Baiting Baiting Baiting Baiting Baiting Baiting Baiting Soil Soil Soil Thelymilra Thelymilra
Baiting Baiting Cauliflower Cauliflower Baiting Cucumber Cucumber Cucumber Baiting Onion Onion Onion
Teleomorph
Ceralobasidium comigerum
C. comigerum
C. romigenltn
C. pseudoromigerum
Unidentified Unidentified C. cornigerum C. comigerum C. romigerum
Thanalephorus cucumeris
T. cucumeris T. cucumeris
T. cucumeris
T. cucumeris
• Isolate made by baiting method described elsewhere (Schisler, Neate & Masuhara, 1993), b M, multinucleate; B, binucleate.
AG-Ba, AG-C, AG-D, AG-E, AG-F, AG-G, AG-I, AG-J, AGK, CAG-I, CAG-2, CAG-4, CAG-5, CAG-6, CAG-7 and multinucleate isolates against the multinucleate anastomosis groups AG-I, AG-2-I, AG-3, AG-4, AG-5, AG-6, AG-7 and AG-8, in both cases by the modified method of Sanders, Burpee & Cole (I978). Agar blocks of 2-3 mm 2, containing hyphae of the known AG group or the isolate to be tested, were opposed on cellophane overlying distilled water agar in a 2'5 em diam. plastic Petri plate and incubated for 24-48 h at 25°C. The area in which advancing hyphae overlapped was then scanned at 125 x magnification for anastomoses. An-
astomosis was only recorded where the 'killing reaction' occurred, Le. when one or more hyphal cells on either side of the anastomosis died and became vacuolate (Flentje & Stretton, I964). Where the unknown isolate was not able to be placed in a group, tests were repeated at least three times.
Inducing teleomorphs Isolates were grouped according to morphology, and induction of the teleomorph was attempted by the soil-over-agar method (Stretton ef al., 1964; Warcup & Talbot, I965).
85
G. Masuhara, S. M. Neate and D. A. Schisler Table 2. A comparison of the grouping of binucleate isolates of Rhiwetonia based on electrophoretic patterns with the grouping determined by anastomosis
Isolate CIBI CIB2 CIB3 R2Bl R2B2 CRESPI MIS3 CRESP2 CRESP3 ENG4Bl ENG4B2 ENG4B3 ENG4B4 GRI2Pl BREIBI BREIB2 BREIB3 BRE4Bl BRE4B2 BRE4B3 MIS4 JIBI JIB2 MIB2 01S1 WOOPI B1B3 CRESB1 CRESB2 CRESB3 ENG2Bl ENG2B2 M!S2
Electrophoresis group 1 1 1 1 1 2 2 3 4 S S 6 6 7 8 8 8 8 8 8 8 9 9 9
10 11
12 13 13 13
14 14 IS
Anastomosis group AG-F AG-F AG-F AG-I AG-I AG-F Group 0 AG-F AG-F AG-F AG-F AG-F AG-F AG-F AG-I AG-I AG-! AG-I AG-! AG-! Group E AG-I AG-I Group C AG-! AG-! AG-K CAG-S CAG-S CAG-s Group A Group A Group 0
Isolates were grown on NDY agar in individual 9 cm diam. Petri plates for 2 wk until the plate was densely covered by mycelium; the lid was then removed and the plates covered with a 1-2 cm layer of soil clods up to I' 5 cm diam. taken from the clay B horizon of Urrbrae Red-Brown Earth (calcic luvisol; Dudal, 1968), pH 7, 50-60% montomorillonite/illite clay, 20-25 % of both silt and fine sand, negligible coarse sand (Litchfield, 195 I). The soil was wetted to just short of saturation with deionized water, and this was maintained by watering every day. Plates were incubated at room temperature (15-25 0) in diffuse light with normal day/night cycles.
Extra-cellular perlic enzymes Isolates were grown in 2 ml of a weak broth culture medium containing pectin for 10 d (Sweetingham et al., 1986). Culture broth was used for electrophoresis using a modified method of Cruickshank (1983). Fifty IJI of culture broth was mixed with about 0'005 g of Sephadex G200 superfine (Pharmacia, Uppsala, Sweden), and 4-6 IJI of the resulting slurry was added to polyacrylamide gels containing 0'9% pectin. Electrophoresis was at a constant current of 10 rnA with a
starting voltage of 90 V, and was stopped when the 0'5 % bromophenol blue tracking dye had migrated 6'5 cm toward the anode. After electrophoresis the gels were incubated with shaking in 0'1 M DL-malic acid at 25° for 1'5 h, and then stained by shaking in a solution of 0'015 % ruthenium red overnight at 5°. RESULTS
Nuclear condition and anastomosis group of isolates The origin, nuclear condition, AG group and teleomorph of 49 isolates of Rhizocfonia from diseased plants, soil particles and by baiting soil with seedlings are shown in Table 1. Giemsa staining showed that 37 of the 49 isolates of Rhizocfonia were binucleate, 12 being multinucleate. Colony colours of 32 of the binucleate Rhizocotonia isolates were white to pale yellow; however, colonies of CRE5B1, CRE5B2, CRE5B3, ENG2B1 and ENG2B2 were a dark brownish colour on 1/6 strength NDY agar. Both multinucleate and binucleate isolates were recovered by baiting soil samples at nursery I, but in general only one type was recovered from an individual nursery. All the multinucleate isolates were either Rhizocfonia solani AG-2-1 or AG-4. Four of the 12 multinucleate isolates were AG-2-1 and seven isolates were AG-4, with one not tested. Binucleate Rhizocfonia belonged to either AG-F, AG-!, AG-K or CAG-5, or to four groups of isolates which do not anastomose with known isolates. Some 65 % of the binucleate isolates belonged to either AG-F or AG-!.
Teleomorphs Teleomorphs were obtained for 14 of the 30 isolates tested. Twelve of the isolates were identified as Thanatephorus cucumeris, Ceratobasidium comigerum (Bourdot) Rogers, or Ceratobasidium pseudocomigerum Christiansen on the basis of characteristics of hymenia, basidia and basidiospores as described by Talbot (1965) and Warcup & Talbot (1965, 1966). The C. pseudocomigerum was distinguished from C. comigerum by oblong to cylindrical spores with a ratio of length to width more than 2: 1. Normally, hymenia emerged 1-4 wk after covering the agar plate cultures with soil. An exception was D1B1 (Thanatephorus cucumeris), which formed a hymenium only after normal incubation for 4 wk, followed by complete drying of the colonized soil over 2 d and then a re-wetting period of 2 d. Fungal tissue from the hymenium was recultured on 1/6 strength NDY agar and confirmed as the isolate D1B1 by comparing morphology in culture. The teleomorphs of binucleate Rhizocfonia CRE5B3 and ENG2B2 were not consistent with currently described species. Basidia of CRE5B3 were holobasidiate, with metabasidia subglobose to broadly clavate, 12-15 IJm long and 7-8'5 IJm wide, with four sterigmata 6-9 IJffi long. The ratio of width of metabasidia to supporting hyphae was less than 2: 1. Basidia of ENG2B2 were holobasidiate, metabasidia were subglobose, 8'4-9'4 IJm long and 6'9-8'8 IJm wide, with four sterigmata 5'1-8'9 IJm long. The ratio of width of metabasidia to supporting hyphae was less than 2: 1.
86
South Australian Rhizoctoria species (a)
... 1
(b)
,,'
~_
'i"
~
~
".~
..4~':~~~~~:·~ : c
•
-
~
,
(c)
Fig. 1. Pectic zymograms of selected isolates of binucleate Rhizocfonia. (Fig. Ia) similarity of patterns of four isolates from the same nursery. (Fig. I b) range of variation of patterns of four isolates identified as AG~F (Fig. 1 c) range of variation of patterns of four isolates identified as AG-L
Extracellular pectic enzymes All multinucleate isolates produced characteristic banding patterns on pectin - acrylamide gels and were able to be placed in either AG-2-1 or AG-4 based on their patterns. All binucleate isolates except C1B4, M1BI, ORCP1 and ORCP2 also produced characteristic banding patterns. Binucleate isolates were placed in IS groups according to similarity of pattern (Table 2) and compared with patterns of known anastomosis groups. The II AG-F isolates fell into seven electrophoretic pattern groups, the 12 AG-I isolates fell into five groups and the three CAG-5 isolates (all from the same nursery) fell into one group. Pectic zymogram patterns of selected binucleate isolates are shown in Figure 1. Isolates from the same anastomosis group and the same nursery showed similar patterns, but isolates from within AG-F or AG-I from different nurseries showed a range of patterns. When the likely anastomosis group could not be identified by the authors from the pectic enzyme banding patterns obtained, the patterns were assessed independently. Assessors were only rarely able to match the pattern of an isolate with the pattern of a known anastomosis group. Where many isolates in a single anastomosis group were tested, there was significant variation among the patterns of the isolates. Furthermore, when isolates in AG-A were compared with CAG-2, AG-D with CAG-I, AG-E with CAG-3 and CAG-6, and AG-F with CAG-4, i.e. pairs that should be similar since they are recognized to be from the same anastomosis group, there was also significant variation between zymogram patterns.
DISCUSSION Forty-nine Rhizoctonia isolates, including both multinucleate and binucleate types, were obtained from nursery plants and soils in South Australia. The multinucleate Rhizoctonia identified in the present investigation have been isolated previously in South Australia; AG-2-1 from wheat roots and AG-4 from soiL wheat roots, and Sagina procumbens (pearlwort) (Neate & Warcup, 1985). However, the present investigation showed that in South Australia AG-2-1 also colonizes onion seedlings and nursery soils, and AG-4, cauliflower and cucumber seedlings. These species are within the previously described host ranges for the fungus and it is expected that with more work other hosts for these fungi will be found in South Australia. Pathogenicity tests of these isolates were reported by Schisler, Neate & Masuhara (1993). Anastomosis tests of binucleate Rhizoctonia confirmed the existence of AGF and AG-K in Australia, and showed for the first time the presence of AG-I and CAG-5. The groups AG-F and AG-K had previously been associated with root rot of subterranean clover or bare-patch disease of cereals (Wong & Sivasithamparam, 1985; Roberts & Sivasithamparam, 1986). The isolate of AG-K also anastomosed with the AG-A tester isolate, but at a much lower frequency than occurred with the AG-K tester isolate. This suggests that AG-A and AG-K are closely related, and may even be two subgroups of the same anastomosis group. The association between these two groups deserves further study. The inability to identify the teleomorphs of CRE5B3 and ENG2B2 is consistent with the findings of Parmeter, Whitney & Platt (1967), who described difficulties of identification of teleomorphs of binucleate Rhizoctonia; Ogoshi et al. (1983) apparently had similar problems, as they induced teleomorphs of 1 I of IS binucleate anastomosis groups, but did not identify the species. Most problems have been due to large variations in size and shape of basidia and spores. CRE5BI, CRE5B2 and ENG2B1 which did not form teleomorphs and CRE5B3 and ENG2B2 which formed unidentified teleomorphs all have dark brown colonies. As CRESS1 was identified as belonging to CAG-5 it confirms the previous record of that group having brown colonies (Burpee et al., 1980). Although Wong & Sivasithamparam (1985) found brown-coloured binucleate isolates associated with root rot of subterranean clover in Western Australia, this is the first record of brown-coloured binucleate Rhizoctonia in South Australia. Neate (1985) found that colony colour was a useful means of separating cultures of Rhizoctonia from South Australian wheat fields into likely species. However, the usefulness of this character in separating isolates in nursery soils is limited by the existence of isolates of Ceratobasidium showing brown coloration in culture. This is the first time teleomorphs have been produced for isolates in AG-I, and it suggests that there may be two species in the group, C. cornigerum and C. pseudocornigerum. More isolates need to be studied to confirm this. This study confirms the finding of Ogoshi et al. (1983) that AG-F has a Ceratobasidium sp. teleomorph. The two AG-F isolates which produced a teleomorph were both identified as C. cornigerum. Although the multinucleate isolates showed some degree
G. Masuhara, S. M. Neate and D. A. Schisler of variability, pectic enzyme patterns could be used to predict likely anastomosis groupings with 100 % success. For the binucleate isolates, pectic enzyme patterns were useful for determining the degree of relatedness of morphologically similar strains. For example, CRE5Bl, CRE5B2 and CRE5B3 isolates from the same nursery showed similar enzyme patterns to each other, and isolates JIB1 and JIB2, both from another nursery, also showed similar patterns to each other. However, overall the usefulness of the technique within binucleate anastomosis groups was limited by variability in patterns obtained from isolates later shown to be of the same anastomosis group, and in comparison with those of the anastomosis group tester isolates. As these results differed from those of Neate & Cruickshank (1988) the patterns were independently assessed with the same result. Currently, there are 19 binucleate tester isolates recognized in the system of Ogoshi ef al. (Ogoshi ef a/., 1979; Oniki et a/., 1984, 1986; Sneh ef al., 1991) and seven in the system of Burpee et al. (1980). Our results and those of others have shown that many isolates do not fit into any of the currently recognized groups, suggesting there are many groups yet to be described. This variation in pectic enzyme pattern within binucleate anastomosis groups and the potential numbers of groups in existence limits the use of pectic iso-enzyme identification of binucleate Rhizoctonia to ecological systems where more is known about the range of groups of isolates present. The authors thank Ann Benger for her competent technical assistance. One of us, D.A.S., thanks Incitec Ltd, Australian for a portion of his salary and research support. REFERENCES Adams, G. C. jr & Butler, E. E. (1983). Influence of nutrition on the formation of basidiospores in Thanatephorus cucumeris. Phytopathology 73, 147-151. Burpee, L. L., Sanders, P. L., Cole, H. Jr. & ShelWood, R. T. (1980). Anastomosis groups among isolates of Ceratobasidium cornigerum and related fungi. Mycologia 72, 689--701. Carling, D. E. & Kebler, K. M. (1987). Characterization of a new anastomosis group (AG-9) of Rhizoclonia solani. Phytopathology 77, 1609--1612. Cline, M. N.. Chastagner, G. A., Aragaki, M., Baker, R., Daughtrey, M. L., Lawson, R. H., Macdonald, j. D., Tammem, j. F. & Worf, G. L. (1988). Current and future research directions of ornamental pathology. Plant Disease 72, 926--934. Cruickshank, R. H. (1983). Distinction between Sclerotinia species by their pectic zymograms. Transaclions of the British Mycological Society 80, 117-119. Dudal. R. (1968). Definition of soil units for the soil map of the world. World Soil Resources Report No. 33, FAO: Rome. Flentje, N. T. & Stretton, H. M. (1964). Mechanisms of variation in Thanatephorus cucumeris and T. praticolus. Australian journal of Biological Science 17, 686--704. Herr, L. J. (1979). Practical nuclear staining procedures for Rhizoctonia-like fungi. Phytopathology 69, 958-961. Kuninaga, S., Yokosawa, R. & Ogoshi, A. (1978). Anastomosis grouping of Rhizoclonia solani Kuhn isolated from non-cultivated soils. Phytopathological Society of japan 49, 184-190. Litchfield, W. H. (1951). Soil survey of the Waite Agricultural Research Institute, Glen Osmond S.A. CSIRO Australia, Division of Soils, Divisional Report No. 2/51. (Accepted 16 june 1993)
87 Neate, S. M. (1985). Rhizoctonia in South Australian wheatfields. In Ecology and Management of Soil-Borne Plant Pathogens (ed. C. A. Parker, A. D. Rovira, K. j. Moore, P. T. W. Wong and j. F. Kollmorgen), pp. 54-56. American Phytopathological Society, St Paul. MN. Neate, S. M. & Cruickshank R. H. (1988). Pectic enzyme patterns of Ceratobasidium and Rhizoclonia spp. associated with sharp eyespot-like lesions on cereals in South Australia. Transaclions of the British Mycological Society 91, 267-272. Neate, S. M., Cruickshank, R. H. & Rovira, A. D. (1988). Pectic enzyme patterns of Rhizoclonia solani isolates from agricultural soil in South Australia. Transaclions of the British Mycological Society 90, 37-42. Neate, S. M. & Warcup, j. H. (1985). Anastomosis grouping of some isolates of Thanatephorus cucumeris from agricultural soils in South Australia. Transactions of the British Mycological Society 85, 615-620. Ogoshi, A. (1976). Studies on the grouping of Rhizoclonia solani Kuhn with hyphal anastomosis and on the perfect stages of groups. Bulletin of the National Institutes of Agricultural Science, Tokyo, Series C 30, 1-63. Ogoshi, A., Oniki, M., Araki, T. & Ui, T. (1983). Anastomosis groups of binucleate Rhizoclonia in japan and North America and their perfect states. Transaclions of the Mycological Society of japan 24, 79--87. Ogoshi, A., Oniki, M., Sakai, R. & Ui, T. (1979). Anastomosis grouping among isolates of binucleate Rhizoclonia. Transaclions of the Mycological Society of japan 20, 33-39. Oniki, M., Araki, T., Ogoshi, A., Kasai, K., Hamaya, E. & Ando, Y. (1984). Some properties and taxonomic assessment of the tea black rot fungus. Study of Tea 6, 7-14. Oniki, M., Kobayashi, K., Araki, T. & Ogoshi, A. (1986). A new disease of turf-grass caused by binucleate Rhizoclonia AG-Q. Annals of the Phytopathological Society of japan 52, 850--853. Parmeter, j. R. jr, ShelWood, R. T. & Platt, W. D. (1969). Anastomosis grouping among isolates of Thanatephorus cucumeris. Phytopathology 59, 1270--1278. Parmeter, j. R. jr, Whitney, H. S. & Platt, W. D. (1967). Affinities of some Rhizoclonia species that resemble mycelium of Thanatephorus cucumeris. Phytopathology 57, 218-223. Roberts, F. A. & Sivasithamparam, K. (1986). Identity and pathogenicity of Rhizoclonia spp. associated with bare patch disease of cereals at a field site in Western Australia. Netherlands journal of Planf Pathology 92, 185-195. Sanders, P. L., Burpee, L. L. & Cole, H. jr (1978). Preliminary studies on binucleate turfgrass pathogens that resemble Rhizoclonia solani. Phytopathology 68, 145-148. Schisler, D. A., Neate, S. M. & Masuhara, G. (1993). The occurrence and pathogenicity of Rhizoclonia fungi in South Australian plant nurseries. Mycological Research 97, 77-82. Sneh, B., Burpee, L. & Ogoshi, A. (1991). Identification of Rhizoclonia Species. APS Press: St Paul. Minnesota. Stephens, C. T., Herr, L. j. & Schmitthenner, A. F. (1982). Characterisation of Rhizoclonia isolates associated with damping-off of bedding plants. Plant Disease 66, 700--703. Stretton, H. M., McKenzie, A. R., Butler, K. F. & Flente, N. T. (1964). Formation of the basidial stage of some isolates of Rhiwclonia. Phytopathology 54, 1093-1095. Sweetingham, M. W., Cruickshank, R. H. & Wong, D. H. (1986). Pectic zymograms and taxonomy and pathogenicity of the Ceratobasidiaceae. Transactions of the British Mycological Society 86, 305-311. Talbot, P. H. B. (1965). Studies of 'Pellicularia' and associated genera of Hymenomycetes. Persoonia 3, 371-406. Warcup, j. H. (1955). Isolation of fungi from hyphae present in soil. Nature, London 175, 953. Warcup, j. H. & Talbot, P. H. B. (1965). Ecology and identity of mycelia from soil. III. Transaclions of the British Mycological Society 48, 249--259. Warcup, j. H. & Talbot, P. H. B. (1966). Perfect states of some Rhizoctonias. Transactions of the British Mycological Society 49, 427-435. Wong, D. H. & Sivasithamparam, K. (1985). Rhizoclonia spp. associated with root rots of subterranean clover in Western Australia. Transactions of the British Mycological Society 85, 21-27.