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Perfect states of some rhizoctonias
[ 427 ] Trans. Br. mycol. Soc. 49 (3), 427-435 (1966) Printed in Great Britain
PERFECT STATES OF SOME RHIZOCTONIAS By]. H. WARCUP
AND
P. H. B. TALBOT
Waite Agricultural Research Institute, Adelaide, South Australia (With
2
Text-figures)
Perfect states of several Rhizoctonia isolates have been produced in culture. An isolate considered to be Rhizoctonia goodyerae-repentis Costantin and several other Rhizoctonia isolates are Ceratobasidium cornigerum (Bourd.) Rogers. R. globularis Saksena & Vaartaja is a Sebacina. A culture considered to be R. rubiginosa Sappa & Mosca is Ascophanus carneus (Pers, ex Fr.) Boud., and the perfect state of a Rhizoctonia from Orchis mascula (L.) L. is described as Thanatephorus orchidicola sp.nov.
Since De Candolle (1815) first described Rhiroctonia, a large number of sterile mycelia have been placed in this form-genus. In general these fungi are characterized by possessing relatively wide, coloured basal hyphae, with branch hyphae that arise at an acute angle when young but later are directed almost at right angles to the main axis. Typically a septum is formed in the branch hypha close to the point of branching, and the branch is slightly constricted at the junction or at the septum. Such mycelia may, or may not, also produce sclerotia. Many species of Rhiroctonia occur on plants of agricultural, silvicultural or horticultural importance and have been extensively studied, yet comparatively few attempts have been made to induce such sterile mycelia to fruit, thus enabling their perfect states to be identified. Some of the betterknown perfect states are the following: Buddin & Wakefield (1927, 1929) associated R. crocorum (Pers.) DC. ex Fr. with Helicobasidium purpureum (Tul.) Pat.; the perfect state of R. solani Kuhn is Thanatephorus cucumeris (Frank) Donk (Donk, 1956, 1958); recently Whitney & Parmeter (1964) reported that the perfect state of R. hiemalis Saksena & Vaartaja is an ascomycete, possibly Trichophaea bullata Kanouse. In our earlier studies on the ecology and identity of mycelia isolated from soil (Warcup & Talbot, 1962, 1963, 1965) it was noted that several of the fungi induced to fruit in culture had Rhizoctonia mycelial states. Moreover, a few of the isolates which fruited had been in culture for periods up to 11 years. This suggested that with the methods already available it might be possible to induce named cultures of Rhizoctonia, whether recently isolated or stored in culture collections, to fruit and thus allow them to be identified in the perfect state. This paper reports the results of such a study. It is important to note that because few morphological characters are available for characterizing species of Rhieoctonia there must always be an element of doubt in the determination of species in the mycelial state. On the other hand, the perfect states often afford a conclusive means of differentiating isolates which are extremely alike in the mycelial state.
428
Transactions British Mycological Society
We have found instances where the type culture of a particular Rhizoctonia species, and other isolates authentically determined as that species, have yielded quite different perfect states. In this paper we have connected certain Rhizoctonia isolates with their perfect states, but only when type cultures were used do we claim to relate Rhizoctonia species to their perfect states. METHODS
The main method used to induce fruiting of Rhizoctonia isolates was the soil-on-agar culture method (Stretton, McKenzie, Baker & Flentje, 1964; Warcup & Talbot, 1965). Urrbrae clay was the main 'casing soil' used, but in some experiments a variety of other soils or clays, both natural and sterilized, was used. In some cases a Rhizoctonia strain fruited on a range of casing soils, in other cases fruiting was more restricted. Details are given in the text. In a few cases, particularly with isolates suspected of being ascomycetes, the fungus was grown on sterilized wheat chaff, and after it had thoroughly permeated the chaff the whole block was covered with casing soil (Warcup & Talbot, 1962). Light was necessary for development of fructifications of some ascomycetes. The water softener 'Calgon' was found very effective in freeing soil-borne fructifications from clay and other particles. Fructifications were shaken lightly in a small volume of water containing a few drops of 10 % Calgon before being mounted. Treatment in Calgon did not impede further growth of fungi on agar media if it was desired to culture from fructifications. PERFECT STATES
(I)
(Bourd.) Rogers (1935, p. 5); Christiansen (1959, p. 48). Rhizoctonia goodyerae-repentis Costantini Hadley & Perombelon (1963), isolate Rgr. R. globularis Saksena & Vaartaja (1960, p. 940), isolate no. 2616 (authentic). Rhizoctonia sp.; Warcup (1957), isolate T 18. Warcup & Talbot (1965) have given a general description of this species, which has been isolated from South Australian soils 354 times over a period of ten years. Two strains, differing slightly in cultural appearance, were noted. The fructifications of these strains, however, proved almost identical except that the basal hyphae in the one had thicker, laminated walls. A further sixteen isolates were induced to fruit (Warcup & Talbot, 1965), were studied in detail (Table I) and were compared with reliably determined collections of C. cornigerum and C. obscurum Rogers on natural substrata. These isolates came from widely separated localities in South Australia, from Scotland and from Canada. In some isolates the spores were broad in relation to length, thus corresponding with those of C. obscurum, while in others they were relatively narrower and corresponded with those of C. cornigerum. The ratio of average length to average width of spores is 1'3-1'5 for C. obscurum, 1·6-2'0 CERATOBASIDlUM CORNIGERUM
Rhizoetonia. J. H. Warcup and P. H. B. Talbot
429
for C. cornigerum and 2,6 for C. pseudocornigerum Christiansen, these figures being calculated from measurements recorded in the literature and also made personally from herbarium material of the first two species. In South Australian, Scottish and Canadian isolates this ratio varies continuously between 1'5 and 2'1 (Table 1), and in this feature the local species corresponds with C. cornigerum. The actual shape and size of individual spores can be deceptive, Spores of C. obscurum are described Table
1. Comparison ofmicroscopic features infructifications of isolates referred to Ceratobasidium cornigerum
Spore measurements ~
Isolate number 84 79
Origin of isolates Gumeracha MtBurr
280g TI8 Rgr.
Canada Adelaide Scotland
2616
Canada
40 62 9S 85 94 83 136
Adelaide Mt Burr Keyneton Echunga Cungena Gumeracha Caroline
Source Lupin Pinus radiata Soil Goodyera repens Pinus banksiana Soil Pinus pinaster Oats Pinus log Wheat Lupin Pinus radiata
Laminated Maximum hyphae Ratio length of present (+) av, length! metabasor av. breadth idium absent( -)
Length (/l)
Width (/l)
6'5-8'0 5"3-9'S
4'5-5'5 3'7-6'4
1'5 1'5
17'5 13'0
5"1-8'2 8,8-g'2 6'0-8'5
4'0-5'0 4'7-5"9 4'0-5"3
1'5 1'5 1,6
15'0 17'0 14'0
7'o-g'2
4,6-5"8
1,6
10'0
+
6'5-7'8 6'4-9'o
3,8-5"2 4'3-5"2
1,6 1,6
13'5 13'0
+
8'0-9'4 6,8--8,6 6,8-g'S 6'5- 10'0 7'(}-9'4
4,6-6'1 4'0-5'1 4'0-5'6 4'0-5'5 4'(}-S'2
1,6 1'7 1'7 1'7 1,8
17'0 14'0 18'0
1,8 5"S-g'o 3'5-4'6 13'0 + Pinus 7,6-10'0 4'1-5"0 14'5 is radiata Bundaleer Pinus 2'1 67 6'5-9'7 3'3-4'6 13'0 radiata 6g Bundaleer Pinus 2'1 16'5 7"5- 10'0 4'0-4'4 + radiata Bundaleer Pinus 2'1 7'2-g'5 3,6-4'4 15"0 71 + radiata • With the exception of the Canadian and Scottish isolates, the place-names refer to places in South Australia, 2788 82
Canada Tarpeena
as 'broadly ellipsoid, 7'5-8 x 6 fl' while those of C. cornigerum are' broadly fusiform in one aspect, asymmetrical, obliquely attenuated in the other, (6'5-)7'5-9'5 x 4-4'5-6 fl' (Rogers, 1935). In size, the measurements for spores of C, obscurum are overlapped by those for C. cornigerum. The spores of both species are much the same shape when they commence development, but those of C. cornigerum later elongate more in proportion to their width than do those of C. obscurum. Thus, depending upon the stage at which individual spores are shed, it is possible for spores of both species to have approximately the same size and shape, The differences are best brought out by comparing the ratio oflength to breadth in a large number of spores in several isolates. C. obscurum was originally differentiated also by its possession of short-
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celled basal hyphae with thick, laminated walls. Lamination of the hyphal walls was noted in two Canadian and three South Australian isolates but could not be correlated with any other taxonomic features suggesting C. obscurum. Such laminated hyphae were narrower (7'5 ft wide) than the corresponding hyphae in C. obscurum (up to 14 ft wide) and were not shortcelled. The metabasidia of C. obscurum are described as 'ovoid-clavate, 19-24 x 9- t t u", and those of C. cornigerum as 'ovate or pyriform, 12-24 x 7'5-9-II ft' (Rogers, 1935). In ten of our isolates the metabasidia were 15ft or less in length and in the others did not exceed 18ft in length; again the isolates correspond more closely with C. cornigerum. A feature of interest was the high proportion of sterigmata which became secondarily septate or branched; this was also found with secondary sterigmata formed during spore repetition. It was observed that an overwatered soil-on-agar culture of Isolate 2616 gave rise to an exceptionally large number of basidia with distorted sterigmata, some forming two or three branches tipped by spicules; this suggests that the presence of free water on the basidia, or perhaps high humidity, may be a cause of this phenomenon. Some basidia are delimited by a basal septum at the point where the basidial body narrows abruptly into the basidium-bearing hypha; in other basidia of the same preparation the basal septum may be set back further in the hypha so that the basidium appears pedicellate. This feature is therefore of little taxonomic value and cannot be used, for example, to differentiate the parasitic web-blights of coffee (Pellicularia koleroga sensu Rogers, 1943; Koleroga noxia Donk, 1958) from the genus Ceratobasidium. In soil-on-agar cultures of all our isolates, groups of branched, monilioid hyphae were produced and sometimes became aggregated into semistromatic masses, possibly the beginnings of sclerotia. In all isolates these hyphae were essentially similar, originating as branches of relatively narrow hyphae and being composed of subcylindrical to barrel-shaped cells, 21-32(-37) x 10-17 ft, constricted at the transverse septa. The cells are formed in acropetal succession; the terminal cell of a chain, after swelling, develops an apical prolongation which then swells and becomes cut off by a septum. In their manner of formation these cells are blastosporic, not arthrosporic or chlamydosporic; they have dense contents, thin walls, and appear to be binucleate, Dolipores are usually clearly visible in the septa. Isolate Rgr, (Table 1) was isolated by Dr G. Hadley, Botany Department, University of Aberdeen, from the roots of the orchid Goodyera repens (L.) R.Br. growing in a pinewood at Dinnet, Aberdeenshire and represents , a typical endophyte from Goodyera repens as studied by Downie (1940) ,. The isolates of Ceratobasidium studied differed markedly in their ability to fruit under the conditions used, Some fruited abundantly and regularly on a wide range of casing soils; others fruited infrequently on a restricted range of soils. C. cornigerum is widely distributed and has also been isolated from a number of widely separated plant genera and from soil.
Rhizoetonia. J. H. Warcup and P. H. B. Talbot ( 2 ) SEBA CINA
431
sp. (Fig. I)
Rhizoctonia globularis Saksena & Vaartaja (1960, p. 939), isolate no. 2287 (type). Subcultures of the type culture of R. globularis were induced to form small fructifications in soil-on-agar plates, using Urrbrae red clay as the soil. The fructifications were whitish, subgelatinous to waxy. This perfect state proved to be a species of Sebacina with ovoid, cruciately-septate probasidia, 9-11 x 6'5-8 p: The spores were amygdaliform to subcylindrical with an attenuated base, repetitive, 6'5-10 x 3-4 p, hyaline, smooth. The hyphae were hyaline, tortuous, 2-4 p wide, without clamp connexions, Fruiting was poor, however, and insufficient information could be gleaned to enable the species to be determined with certainty.
DGoOOO~J) 10p
(b)
20" Fig.
I.
(e) Sebacina sp., the perfect stat e of Rhiroctonia globularis (type culture). a, Spores; b, basidia; c, monilioid hyphae.
10"
Saksena & Vaartaja (1960) reported that basidia with tiny sterigmata and basidiospores were observed twice in cornmeal agar cultures of Isolate 2287, but they did not have enough material to allow identification.
(3)
A SCOPHANUS CARNE US
(Pers, ex Fr.) Boud.
Rhiroctonia rubiginosa Sappa & Mosca; Saksena & Vaartaja (1960, p. 633), isolate no. 2747. This isolate of Saksena & Vaartaja, which they considered on cultural, mycelial and chlamydospore characters to represent R. rubiginosa, formed small pink apothecia on soil-on-agar cultures in the light. Fructifications were formed both on Urrbrae red clay and on a potting soil (U .C. mix: Baker, 1957). The apothecia proved to be those of Ascophanus carneus.
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Transactions British Mycological Society
Apothecia of this species had previously been obtained from an isolate derived from hyphae from wheat-field soil (Warcup & Talbot, 1963). Two further isolates have also been obtained from other soils in South Australia. It was noted that the conidial state of A. carneus, Oedocephalum glomerulosum (Bull.) Sacc., was sparsely produced by all cultures grown on cornmeal agar, but that conidial heads were generally more abundant on soil agar. As reported by Gamundi & Ranalli (1964), cultures of A. carneus will sometimes produce apothecia on a medium of 0'03 % yeast extract, filter paper and 2'5 % agar kept in continuous light.
(4)
sp.; Whitney & Parmeter (1964). Rhiroctonia hiemalis Saksena & Vaartaja (1961, p. 940), isolate no. 2785 TRICHOPHAEA
(type). Subcultures of the type culture of R. hiemalis were induced to fruit by the soil-on-agar method in the light, and gave rise to apothecia as described by Whitney & Parmeter (1964). The apothecia were cupulate, 2-6 mm. diam., with a greyish-white hymenium and cream to pale brown excipulum bearing conspicuous hyaline to dark brown hairs. Asci cylindrical, up to 156 x 12-14'5 fl. Ascospores broad-ellipsoid, thin-walled, smooth, hyaline, not amyloid, 11'5-12'5 x 7'5 fl. Paraphyses hyaline, smooth, branched in fascicles near the base, cylindrical, septate, somewhat constricted at the septa, with rounded apex, not capitate, up to 160 x 2'4-3'6 fl. Hairs hyaline or brown with a paler rounded or truncate apex, subfusoid to subcylindrical and tapering to the apex, up to 450 x 14'5 fl, septate, firm-walled, arising from spherical hyaline or pale brown excipular cells up to 28 fl in diam. Whitney & Parameter (1964) considered this perfect state to be very close to or a variant of Trichophaea bullata Kanouse, agreeing well with this species except in having smaller ascospores whose size might have been affected by cultural conditions. In our work we noted marked differences in the size of apothecia (2-3 mm.) formed on clay-on-agar culture compared with apothecia (4-6 mm.) formed on potting compost covering the mycelium growing on wheat chaff; however, the ascospore size was the same on both media and somewhat smaller than the ascospores recorded by Whitney & Parmeter (13-16'5 x 8-9'5 fl, avo I4 x 8'5 fl)·
(5) Thanatephorus orchidicola sp.nov. (Fig. 2) Fungus resupinatus, albidus vel eervinus, ex hyphis brunneis basalibus ad 17 P latis erasse tunieatis et ex hyphis superioribus: angustioribus ereetis hyalinis haud nodososeptatis, Rami subhymenii subeymosi, 8'5- lOp diam, Basidia (13-) 16-21 X 9- IIp, cylindraeea vel subcylindracea, Sterigmata 4, recta vel curvata divers a, ad 9-12 p longa, 3 I" crassa, Basidiosporae eervinae, 9- 12 X 7---9'5 u, leves, haud amyloideae, obpyriformiae vel obovatae, interdum uno latere compressae, Typus-Herb. W.A.R,I. no. 15844.
Culture sparse and slow-growing; hyphae dark brown. Basal hyphae of fructification repent, brown even when young, up to 17 fl in diameter, with slightly thickened walls. Hymenium formed in discontinuous tufts composed of subcymose branches giving rise to basidia; sub-basidial branches
Rhizoetonia. J. H. Warcup and P. H. B. Talbot
433
l..-..-J 10Jl
(d) I I
'0·'·.." ·,···,,,········.·
I
t
::
,
I I
:
:
~
, I
I . •.•..•.•.
·:n .··.·.·. ·.·.·...···:·.·.·,·.·····
l ::.·..
Fig.
2.
i
\,~:::::::::::::..:::::==='::::::::::::::===::=::::~·~ :::=: ··· · ·;:,:.:.; · ·· · .. ···· .. .. :
:
:.:'.
Thanatephorus orchidicola. a, Spores; b, hymenium; c, basidia; d, basal hyphae (type).
8·5-1o,u wide. Metabasidia (13-) 16-21 X 9-1t u, cylindrical or somewhat wider at the middle. Sterigmata 4 per basidium, 9-12,u long, up to 3 ,u wide at the base, straight or slightly curved, divergent. Basidiospores fawn coloured, pale brownish in the microscope, 9-12 x 7-9'5,u, smooth,
434
Transactions British Mycological Society
repetitive, not amyloid, obpyriform to obovate, sometimes with one side flattened. Isolated from Orchis mascula (L.)L. as Rhizoctonia sp., Widgham Wood, Cambs., England, by Miss S. E. Harley, 1963. This species differs from Thanatephorus cucumeris (Frank) Donk particularly in the colour, shape and size of the spores. The spores of T. cucumeris are never coloured and in shape are oblong-ellipsoid with one side flattened; in size they range from 6-14 x 4-8 It but average about 7-9 x 4-6'5 It. The hyphae of T. orchidicola are, on" the average, wider than those of T. cucumeris. In T. cucumeris the basal hyphae may sometimes reach 17 It in diameter though about 10 It diam, is more usual, and the sub-basidial hyphae are usually about 6-8 It wide. In T. orchidicola the sub-basidial hyphae are 8'5- 10 It wide and the basal hyphae frequently 15- 17 It wide. The Rhizoctonia culture isolated by Miss S. E. Harley fruited on an alkaline soil (Cambridge Botanic Garden soil) and a calcareous clay but not in several neutral or acid soils in the same series of experiments. DISCUSSION
Although it may sometimes be necessary to propose new species of Rhiroctonia, and it is legitimate to do so, the above results show that many sterile mycelia can be induced to fruit in culture. The cultural conditions employed in fruiting experiments are sometimes conducive to the expression of variation in the fructifications obtained and it may therefore be difficult in some instances to determine the species; nevertheless, we are satisfied that the effort needed to produce fructifications is well worth while. We wish to thank the following for kindly providing cultures of Rhizoctonia: Dr O. Vaartaja, Forest Pathology Laboratory, Maple, Ontario, for Canadian and some South Australian isolates; Miss S. E. Harley, Botany School, Cambridge, for various orchid rhizoctonias, and Mr R. Dodman, Waite Institute for isolates 94 and 95 of Ceratobasidium cornigerum. This work was done mainly at the Waite Institute, Adelaide, but some experiments on orchid endophytes were carried out when one of us (J.H.W.) was visiting the Botany School, Cambridge. REFERENCES
BAKER, K. F. (1957). The V.C. system for producing healthy container-grown plants. Calif. Agr. Expt. Stn Manual 23. BUDDIN, W. & WAKEFIELD, E. M. (1927). Studies on Rhizoctonia crocorum (Pers.) DC. and Helicobasidium purpureum (Tul.) Pat. Trans. Br. mycol. Soc. 12, 116-14°. BUDDIN, W. & WAKEFIELD, E. M. (1929). Further notes on the connexion between Rhizoctonia crocorum and Helicobasidium purpureum. Trans. Br. mycol. Soc. 14, 97-99. CHRISTIANSEN, M. P. (1959). Danish resupinate fungi. Part I. Ascomycetes and Heterobasidiomycetes. Dansk bot. Ark. 19, I-55. DE CANDOLLE, A. P. (18 I5). Memoire sur les rhizoctones, nouveau genre de champignons qui attaque les racines des plantes et en particular celle de la luzerne cultivee, Mem. Mus. natn. Hist. nat., Paris 2,209-216. DONK, M. A. (1956). Notes on resupinate Hymenomycetes. I I. The tulasnelloid fungi. Reinwardtia 3, 363-379.
Rhizoetonia.J. H. Warcup and P. H. B. Talbot
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DONK, M. A. (1958). Notes on resupinate Hymenomycetes. V. Fungus 28, 16-36. DOWNIE, D. G. (1940). On the germination and growth of Goodyera repens. Trans. Proc. bot. Soc. Edinb. 33, 36-51. GAMUNDI, I. J. & RANALLI, M. E. (1964). Estudio sistematico y biol6gico de las Ascobolaceas de Argentina. I. Nova Hedwigia 7,517-533. HADLEY, G. & PEROMBELON, M. (1963). Production of pectic enzymes by Rhizoctonia solani and orchid endophytes. Nature, Lond. 200, 1337. ROGERS, D. P. (1935). Notes on the lower Basidiomycetes. Stud. nat. Hist. Iowa Univ. 17, 1-43· ROGERS, D. P. (1943). The genus Pellicularia (Thelephoraceae). Farlowia I, 95-118. SAKSENA, H. K. & VAARTAJA, O. (1960). Descriptions of new species of Rhizoctonia. Can. ]. Bot. 38, 931-943. SAKSENA, H. K. & VAARTAJA, O. (1961). Taxonomy, morphology, and pathogenicity of Rhizoctonia species from forest nurseries. Can. ]. Bot. 39, 627-647. STRETTON, H. M., McKENZIE, A. R., BAKER, K. F. & FLENTJE, N. T. (1964). Formation of the basidial stage of some isolates of Rhizoctonia. Phytopathology 54, 1093-1095. WARCUP, J. H. (1957). Studies on the occurrence and activity of fungi in a wheat-field soil. Trans. Br. mycol. Soc. 40, 237-262. WARCUP, J. H. & TALBOT, P. H. B. (1962). Ecology and identity of mycelia isolated from soil. Trans. Br. mycol. Soc. 45, 495-518. WARCUP, J. H. & TALBOT, P. H. B. (1963). Ecology and identity of mycelia isolated from soil. II. Trans. Br. Mycol. Soc. 46, 465-472. WARCUP, J. H. & TALBOT, P. H. B. (1965). Ecology and identity of mycelia isolated from soil. III. Trans. Br. mycol. Soc. 48, 249-259. WHITNEY, H. S. & PARMETER, J. R. Jm. (1964). The perfect stage of Rhizoctonia hiemalis. Mycologia 56, 114-118.
(Accepted for publication 28 September 1965)
MYC·49
PERFECT STATES OF SOME RHIZOCTONIAS By]. H. WARCUP
AND
P. H. B. TALBOT
Waite Agricultural Research Institute, Adelaide, South Australia (With
2
Text-figures)
Perfect states of several Rhizoctonia isolates have been produced in culture. An isolate considered to be Rhizoctonia goodyerae-repentis Costantin and several other Rhizoctonia isolates are Ceratobasidium cornigerum (Bourd.) Rogers. R. globularis Saksena & Vaartaja is a Sebacina. A culture considered to be R. rubiginosa Sappa & Mosca is Ascophanus carneus (Pers, ex Fr.) Boud., and the perfect state of a Rhizoctonia from Orchis mascula (L.) L. is described as Thanatephorus orchidicola sp.nov.
Since De Candolle (1815) first described Rhiroctonia, a large number of sterile mycelia have been placed in this form-genus. In general these fungi are characterized by possessing relatively wide, coloured basal hyphae, with branch hyphae that arise at an acute angle when young but later are directed almost at right angles to the main axis. Typically a septum is formed in the branch hypha close to the point of branching, and the branch is slightly constricted at the junction or at the septum. Such mycelia may, or may not, also produce sclerotia. Many species of Rhiroctonia occur on plants of agricultural, silvicultural or horticultural importance and have been extensively studied, yet comparatively few attempts have been made to induce such sterile mycelia to fruit, thus enabling their perfect states to be identified. Some of the betterknown perfect states are the following: Buddin & Wakefield (1927, 1929) associated R. crocorum (Pers.) DC. ex Fr. with Helicobasidium purpureum (Tul.) Pat.; the perfect state of R. solani Kuhn is Thanatephorus cucumeris (Frank) Donk (Donk, 1956, 1958); recently Whitney & Parmeter (1964) reported that the perfect state of R. hiemalis Saksena & Vaartaja is an ascomycete, possibly Trichophaea bullata Kanouse. In our earlier studies on the ecology and identity of mycelia isolated from soil (Warcup & Talbot, 1962, 1963, 1965) it was noted that several of the fungi induced to fruit in culture had Rhizoctonia mycelial states. Moreover, a few of the isolates which fruited had been in culture for periods up to 11 years. This suggested that with the methods already available it might be possible to induce named cultures of Rhizoctonia, whether recently isolated or stored in culture collections, to fruit and thus allow them to be identified in the perfect state. This paper reports the results of such a study. It is important to note that because few morphological characters are available for characterizing species of Rhieoctonia there must always be an element of doubt in the determination of species in the mycelial state. On the other hand, the perfect states often afford a conclusive means of differentiating isolates which are extremely alike in the mycelial state.
428
Transactions British Mycological Society
We have found instances where the type culture of a particular Rhizoctonia species, and other isolates authentically determined as that species, have yielded quite different perfect states. In this paper we have connected certain Rhizoctonia isolates with their perfect states, but only when type cultures were used do we claim to relate Rhizoctonia species to their perfect states. METHODS
The main method used to induce fruiting of Rhizoctonia isolates was the soil-on-agar culture method (Stretton, McKenzie, Baker & Flentje, 1964; Warcup & Talbot, 1965). Urrbrae clay was the main 'casing soil' used, but in some experiments a variety of other soils or clays, both natural and sterilized, was used. In some cases a Rhizoctonia strain fruited on a range of casing soils, in other cases fruiting was more restricted. Details are given in the text. In a few cases, particularly with isolates suspected of being ascomycetes, the fungus was grown on sterilized wheat chaff, and after it had thoroughly permeated the chaff the whole block was covered with casing soil (Warcup & Talbot, 1962). Light was necessary for development of fructifications of some ascomycetes. The water softener 'Calgon' was found very effective in freeing soil-borne fructifications from clay and other particles. Fructifications were shaken lightly in a small volume of water containing a few drops of 10 % Calgon before being mounted. Treatment in Calgon did not impede further growth of fungi on agar media if it was desired to culture from fructifications. PERFECT STATES
(I)
(Bourd.) Rogers (1935, p. 5); Christiansen (1959, p. 48). Rhizoctonia goodyerae-repentis Costantini Hadley & Perombelon (1963), isolate Rgr. R. globularis Saksena & Vaartaja (1960, p. 940), isolate no. 2616 (authentic). Rhizoctonia sp.; Warcup (1957), isolate T 18. Warcup & Talbot (1965) have given a general description of this species, which has been isolated from South Australian soils 354 times over a period of ten years. Two strains, differing slightly in cultural appearance, were noted. The fructifications of these strains, however, proved almost identical except that the basal hyphae in the one had thicker, laminated walls. A further sixteen isolates were induced to fruit (Warcup & Talbot, 1965), were studied in detail (Table I) and were compared with reliably determined collections of C. cornigerum and C. obscurum Rogers on natural substrata. These isolates came from widely separated localities in South Australia, from Scotland and from Canada. In some isolates the spores were broad in relation to length, thus corresponding with those of C. obscurum, while in others they were relatively narrower and corresponded with those of C. cornigerum. The ratio of average length to average width of spores is 1'3-1'5 for C. obscurum, 1·6-2'0 CERATOBASIDlUM CORNIGERUM
Rhizoetonia. J. H. Warcup and P. H. B. Talbot
429
for C. cornigerum and 2,6 for C. pseudocornigerum Christiansen, these figures being calculated from measurements recorded in the literature and also made personally from herbarium material of the first two species. In South Australian, Scottish and Canadian isolates this ratio varies continuously between 1'5 and 2'1 (Table 1), and in this feature the local species corresponds with C. cornigerum. The actual shape and size of individual spores can be deceptive, Spores of C. obscurum are described Table
1. Comparison ofmicroscopic features infructifications of isolates referred to Ceratobasidium cornigerum
Spore measurements ~
Isolate number 84 79
Origin of isolates Gumeracha MtBurr
280g TI8 Rgr.
Canada Adelaide Scotland
2616
Canada
40 62 9S 85 94 83 136
Adelaide Mt Burr Keyneton Echunga Cungena Gumeracha Caroline
Source Lupin Pinus radiata Soil Goodyera repens Pinus banksiana Soil Pinus pinaster Oats Pinus log Wheat Lupin Pinus radiata
Laminated Maximum hyphae Ratio length of present (+) av, length! metabasor av. breadth idium absent( -)
Length (/l)
Width (/l)
6'5-8'0 5"3-9'S
4'5-5'5 3'7-6'4
1'5 1'5
17'5 13'0
5"1-8'2 8,8-g'2 6'0-8'5
4'0-5'0 4'7-5"9 4'0-5"3
1'5 1'5 1,6
15'0 17'0 14'0
7'o-g'2
4,6-5"8
1,6
10'0
+
6'5-7'8 6'4-9'o
3,8-5"2 4'3-5"2
1,6 1,6
13'5 13'0
+
8'0-9'4 6,8--8,6 6,8-g'S 6'5- 10'0 7'(}-9'4
4,6-6'1 4'0-5'1 4'0-5'6 4'0-5'5 4'(}-S'2
1,6 1'7 1'7 1'7 1,8
17'0 14'0 18'0
1,8 5"S-g'o 3'5-4'6 13'0 + Pinus 7,6-10'0 4'1-5"0 14'5 is radiata Bundaleer Pinus 2'1 67 6'5-9'7 3'3-4'6 13'0 radiata 6g Bundaleer Pinus 2'1 16'5 7"5- 10'0 4'0-4'4 + radiata Bundaleer Pinus 2'1 7'2-g'5 3,6-4'4 15"0 71 + radiata • With the exception of the Canadian and Scottish isolates, the place-names refer to places in South Australia, 2788 82
Canada Tarpeena
as 'broadly ellipsoid, 7'5-8 x 6 fl' while those of C. cornigerum are' broadly fusiform in one aspect, asymmetrical, obliquely attenuated in the other, (6'5-)7'5-9'5 x 4-4'5-6 fl' (Rogers, 1935). In size, the measurements for spores of C, obscurum are overlapped by those for C. cornigerum. The spores of both species are much the same shape when they commence development, but those of C. cornigerum later elongate more in proportion to their width than do those of C. obscurum. Thus, depending upon the stage at which individual spores are shed, it is possible for spores of both species to have approximately the same size and shape, The differences are best brought out by comparing the ratio oflength to breadth in a large number of spores in several isolates. C. obscurum was originally differentiated also by its possession of short-
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celled basal hyphae with thick, laminated walls. Lamination of the hyphal walls was noted in two Canadian and three South Australian isolates but could not be correlated with any other taxonomic features suggesting C. obscurum. Such laminated hyphae were narrower (7'5 ft wide) than the corresponding hyphae in C. obscurum (up to 14 ft wide) and were not shortcelled. The metabasidia of C. obscurum are described as 'ovoid-clavate, 19-24 x 9- t t u", and those of C. cornigerum as 'ovate or pyriform, 12-24 x 7'5-9-II ft' (Rogers, 1935). In ten of our isolates the metabasidia were 15ft or less in length and in the others did not exceed 18ft in length; again the isolates correspond more closely with C. cornigerum. A feature of interest was the high proportion of sterigmata which became secondarily septate or branched; this was also found with secondary sterigmata formed during spore repetition. It was observed that an overwatered soil-on-agar culture of Isolate 2616 gave rise to an exceptionally large number of basidia with distorted sterigmata, some forming two or three branches tipped by spicules; this suggests that the presence of free water on the basidia, or perhaps high humidity, may be a cause of this phenomenon. Some basidia are delimited by a basal septum at the point where the basidial body narrows abruptly into the basidium-bearing hypha; in other basidia of the same preparation the basal septum may be set back further in the hypha so that the basidium appears pedicellate. This feature is therefore of little taxonomic value and cannot be used, for example, to differentiate the parasitic web-blights of coffee (Pellicularia koleroga sensu Rogers, 1943; Koleroga noxia Donk, 1958) from the genus Ceratobasidium. In soil-on-agar cultures of all our isolates, groups of branched, monilioid hyphae were produced and sometimes became aggregated into semistromatic masses, possibly the beginnings of sclerotia. In all isolates these hyphae were essentially similar, originating as branches of relatively narrow hyphae and being composed of subcylindrical to barrel-shaped cells, 21-32(-37) x 10-17 ft, constricted at the transverse septa. The cells are formed in acropetal succession; the terminal cell of a chain, after swelling, develops an apical prolongation which then swells and becomes cut off by a septum. In their manner of formation these cells are blastosporic, not arthrosporic or chlamydosporic; they have dense contents, thin walls, and appear to be binucleate, Dolipores are usually clearly visible in the septa. Isolate Rgr, (Table 1) was isolated by Dr G. Hadley, Botany Department, University of Aberdeen, from the roots of the orchid Goodyera repens (L.) R.Br. growing in a pinewood at Dinnet, Aberdeenshire and represents , a typical endophyte from Goodyera repens as studied by Downie (1940) ,. The isolates of Ceratobasidium studied differed markedly in their ability to fruit under the conditions used, Some fruited abundantly and regularly on a wide range of casing soils; others fruited infrequently on a restricted range of soils. C. cornigerum is widely distributed and has also been isolated from a number of widely separated plant genera and from soil.
Rhizoetonia. J. H. Warcup and P. H. B. Talbot ( 2 ) SEBA CINA
431
sp. (Fig. I)
Rhizoctonia globularis Saksena & Vaartaja (1960, p. 939), isolate no. 2287 (type). Subcultures of the type culture of R. globularis were induced to form small fructifications in soil-on-agar plates, using Urrbrae red clay as the soil. The fructifications were whitish, subgelatinous to waxy. This perfect state proved to be a species of Sebacina with ovoid, cruciately-septate probasidia, 9-11 x 6'5-8 p: The spores were amygdaliform to subcylindrical with an attenuated base, repetitive, 6'5-10 x 3-4 p, hyaline, smooth. The hyphae were hyaline, tortuous, 2-4 p wide, without clamp connexions, Fruiting was poor, however, and insufficient information could be gleaned to enable the species to be determined with certainty.
DGoOOO~J) 10p
(b)
20" Fig.
I.
(e) Sebacina sp., the perfect stat e of Rhiroctonia globularis (type culture). a, Spores; b, basidia; c, monilioid hyphae.
10"
Saksena & Vaartaja (1960) reported that basidia with tiny sterigmata and basidiospores were observed twice in cornmeal agar cultures of Isolate 2287, but they did not have enough material to allow identification.
(3)
A SCOPHANUS CARNE US
(Pers, ex Fr.) Boud.
Rhiroctonia rubiginosa Sappa & Mosca; Saksena & Vaartaja (1960, p. 633), isolate no. 2747. This isolate of Saksena & Vaartaja, which they considered on cultural, mycelial and chlamydospore characters to represent R. rubiginosa, formed small pink apothecia on soil-on-agar cultures in the light. Fructifications were formed both on Urrbrae red clay and on a potting soil (U .C. mix: Baker, 1957). The apothecia proved to be those of Ascophanus carneus.
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Transactions British Mycological Society
Apothecia of this species had previously been obtained from an isolate derived from hyphae from wheat-field soil (Warcup & Talbot, 1963). Two further isolates have also been obtained from other soils in South Australia. It was noted that the conidial state of A. carneus, Oedocephalum glomerulosum (Bull.) Sacc., was sparsely produced by all cultures grown on cornmeal agar, but that conidial heads were generally more abundant on soil agar. As reported by Gamundi & Ranalli (1964), cultures of A. carneus will sometimes produce apothecia on a medium of 0'03 % yeast extract, filter paper and 2'5 % agar kept in continuous light.
(4)
sp.; Whitney & Parmeter (1964). Rhiroctonia hiemalis Saksena & Vaartaja (1961, p. 940), isolate no. 2785 TRICHOPHAEA
(type). Subcultures of the type culture of R. hiemalis were induced to fruit by the soil-on-agar method in the light, and gave rise to apothecia as described by Whitney & Parmeter (1964). The apothecia were cupulate, 2-6 mm. diam., with a greyish-white hymenium and cream to pale brown excipulum bearing conspicuous hyaline to dark brown hairs. Asci cylindrical, up to 156 x 12-14'5 fl. Ascospores broad-ellipsoid, thin-walled, smooth, hyaline, not amyloid, 11'5-12'5 x 7'5 fl. Paraphyses hyaline, smooth, branched in fascicles near the base, cylindrical, septate, somewhat constricted at the septa, with rounded apex, not capitate, up to 160 x 2'4-3'6 fl. Hairs hyaline or brown with a paler rounded or truncate apex, subfusoid to subcylindrical and tapering to the apex, up to 450 x 14'5 fl, septate, firm-walled, arising from spherical hyaline or pale brown excipular cells up to 28 fl in diam. Whitney & Parameter (1964) considered this perfect state to be very close to or a variant of Trichophaea bullata Kanouse, agreeing well with this species except in having smaller ascospores whose size might have been affected by cultural conditions. In our work we noted marked differences in the size of apothecia (2-3 mm.) formed on clay-on-agar culture compared with apothecia (4-6 mm.) formed on potting compost covering the mycelium growing on wheat chaff; however, the ascospore size was the same on both media and somewhat smaller than the ascospores recorded by Whitney & Parmeter (13-16'5 x 8-9'5 fl, avo I4 x 8'5 fl)·
(5) Thanatephorus orchidicola sp.nov. (Fig. 2) Fungus resupinatus, albidus vel eervinus, ex hyphis brunneis basalibus ad 17 P latis erasse tunieatis et ex hyphis superioribus: angustioribus ereetis hyalinis haud nodososeptatis, Rami subhymenii subeymosi, 8'5- lOp diam, Basidia (13-) 16-21 X 9- IIp, cylindraeea vel subcylindracea, Sterigmata 4, recta vel curvata divers a, ad 9-12 p longa, 3 I" crassa, Basidiosporae eervinae, 9- 12 X 7---9'5 u, leves, haud amyloideae, obpyriformiae vel obovatae, interdum uno latere compressae, Typus-Herb. W.A.R,I. no. 15844.
Culture sparse and slow-growing; hyphae dark brown. Basal hyphae of fructification repent, brown even when young, up to 17 fl in diameter, with slightly thickened walls. Hymenium formed in discontinuous tufts composed of subcymose branches giving rise to basidia; sub-basidial branches
Rhizoetonia. J. H. Warcup and P. H. B. Talbot
433
l..-..-J 10Jl
(d) I I
'0·'·.." ·,···,,,········.·
I
t
::
,
I I
:
:
~
, I
I . •.•..•.•.
·:n .··.·.·. ·.·.·...···:·.·.·,·.·····
l ::.·..
Fig.
2.
i
\,~:::::::::::::..:::::==='::::::::::::::===::=::::~·~ :::=: ··· · ·;:,:.:.; · ·· · .. ···· .. .. :
:
:.:'.
Thanatephorus orchidicola. a, Spores; b, hymenium; c, basidia; d, basal hyphae (type).
8·5-1o,u wide. Metabasidia (13-) 16-21 X 9-1t u, cylindrical or somewhat wider at the middle. Sterigmata 4 per basidium, 9-12,u long, up to 3 ,u wide at the base, straight or slightly curved, divergent. Basidiospores fawn coloured, pale brownish in the microscope, 9-12 x 7-9'5,u, smooth,
434
Transactions British Mycological Society
repetitive, not amyloid, obpyriform to obovate, sometimes with one side flattened. Isolated from Orchis mascula (L.)L. as Rhizoctonia sp., Widgham Wood, Cambs., England, by Miss S. E. Harley, 1963. This species differs from Thanatephorus cucumeris (Frank) Donk particularly in the colour, shape and size of the spores. The spores of T. cucumeris are never coloured and in shape are oblong-ellipsoid with one side flattened; in size they range from 6-14 x 4-8 It but average about 7-9 x 4-6'5 It. The hyphae of T. orchidicola are, on" the average, wider than those of T. cucumeris. In T. cucumeris the basal hyphae may sometimes reach 17 It in diameter though about 10 It diam, is more usual, and the sub-basidial hyphae are usually about 6-8 It wide. In T. orchidicola the sub-basidial hyphae are 8'5- 10 It wide and the basal hyphae frequently 15- 17 It wide. The Rhizoctonia culture isolated by Miss S. E. Harley fruited on an alkaline soil (Cambridge Botanic Garden soil) and a calcareous clay but not in several neutral or acid soils in the same series of experiments. DISCUSSION
Although it may sometimes be necessary to propose new species of Rhiroctonia, and it is legitimate to do so, the above results show that many sterile mycelia can be induced to fruit in culture. The cultural conditions employed in fruiting experiments are sometimes conducive to the expression of variation in the fructifications obtained and it may therefore be difficult in some instances to determine the species; nevertheless, we are satisfied that the effort needed to produce fructifications is well worth while. We wish to thank the following for kindly providing cultures of Rhizoctonia: Dr O. Vaartaja, Forest Pathology Laboratory, Maple, Ontario, for Canadian and some South Australian isolates; Miss S. E. Harley, Botany School, Cambridge, for various orchid rhizoctonias, and Mr R. Dodman, Waite Institute for isolates 94 and 95 of Ceratobasidium cornigerum. This work was done mainly at the Waite Institute, Adelaide, but some experiments on orchid endophytes were carried out when one of us (J.H.W.) was visiting the Botany School, Cambridge. REFERENCES
BAKER, K. F. (1957). The V.C. system for producing healthy container-grown plants. Calif. Agr. Expt. Stn Manual 23. BUDDIN, W. & WAKEFIELD, E. M. (1927). Studies on Rhizoctonia crocorum (Pers.) DC. and Helicobasidium purpureum (Tul.) Pat. Trans. Br. mycol. Soc. 12, 116-14°. BUDDIN, W. & WAKEFIELD, E. M. (1929). Further notes on the connexion between Rhizoctonia crocorum and Helicobasidium purpureum. Trans. Br. mycol. Soc. 14, 97-99. CHRISTIANSEN, M. P. (1959). Danish resupinate fungi. Part I. Ascomycetes and Heterobasidiomycetes. Dansk bot. Ark. 19, I-55. DE CANDOLLE, A. P. (18 I5). Memoire sur les rhizoctones, nouveau genre de champignons qui attaque les racines des plantes et en particular celle de la luzerne cultivee, Mem. Mus. natn. Hist. nat., Paris 2,209-216. DONK, M. A. (1956). Notes on resupinate Hymenomycetes. I I. The tulasnelloid fungi. Reinwardtia 3, 363-379.
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DONK, M. A. (1958). Notes on resupinate Hymenomycetes. V. Fungus 28, 16-36. DOWNIE, D. G. (1940). On the germination and growth of Goodyera repens. Trans. Proc. bot. Soc. Edinb. 33, 36-51. GAMUNDI, I. J. & RANALLI, M. E. (1964). Estudio sistematico y biol6gico de las Ascobolaceas de Argentina. I. Nova Hedwigia 7,517-533. HADLEY, G. & PEROMBELON, M. (1963). Production of pectic enzymes by Rhizoctonia solani and orchid endophytes. Nature, Lond. 200, 1337. ROGERS, D. P. (1935). Notes on the lower Basidiomycetes. Stud. nat. Hist. Iowa Univ. 17, 1-43· ROGERS, D. P. (1943). The genus Pellicularia (Thelephoraceae). Farlowia I, 95-118. SAKSENA, H. K. & VAARTAJA, O. (1960). Descriptions of new species of Rhizoctonia. Can. ]. Bot. 38, 931-943. SAKSENA, H. K. & VAARTAJA, O. (1961). Taxonomy, morphology, and pathogenicity of Rhizoctonia species from forest nurseries. Can. ]. Bot. 39, 627-647. STRETTON, H. M., McKENZIE, A. R., BAKER, K. F. & FLENTJE, N. T. (1964). Formation of the basidial stage of some isolates of Rhizoctonia. Phytopathology 54, 1093-1095. WARCUP, J. H. (1957). Studies on the occurrence and activity of fungi in a wheat-field soil. Trans. Br. mycol. Soc. 40, 237-262. WARCUP, J. H. & TALBOT, P. H. B. (1962). Ecology and identity of mycelia isolated from soil. Trans. Br. mycol. Soc. 45, 495-518. WARCUP, J. H. & TALBOT, P. H. B. (1963). Ecology and identity of mycelia isolated from soil. II. Trans. Br. Mycol. Soc. 46, 465-472. WARCUP, J. H. & TALBOT, P. H. B. (1965). Ecology and identity of mycelia isolated from soil. III. Trans. Br. mycol. Soc. 48, 249-259. WHITNEY, H. S. & PARMETER, J. R. Jm. (1964). The perfect stage of Rhizoctonia hiemalis. Mycologia 56, 114-118.
(Accepted for publication 28 September 1965)
MYC·49