A species of Rhizoctonia with uninucleate hyphae isolated from roots of winter wheat

Notes and brief articles

466

DABOUSSI-BAREYRE, M.-J. & PARISOT, D . (1981). Nucleocytoplasmic interactions implicated in differentiation in Nectria haematococca. In Fusarium : Diseases, Biology, and Ta xonomy (ed. P. E . Nelson, T . A . Toussoun & R . J. Cook), pp . 30~317 . University Park and London: Pennsylvania State University Press. CASSE, F., BOUCHER, C., JUILLIOT, J. S., MICHEL, M . & DENARIS, J. (1979 ). Identification and characterisation of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. Journal of General Microbiology 113, 229-242. COLLINS, R. A., STOHL, L. L., COLE, M. & LAMBOWITZ, A. M . (1981). Characterization of a novel plasmid DNA found in mitochondria of N. crassa. Cell 24, 443-45 2. GARBER, R. C., TURGEON, B. G . & YODER, O . C . (1984). A mitochondrial plasmid from the plant pathogenic fungus Cochliobolus heterostrophus. Molecular and General Genetics 196,301-310. GUARDIOLA, J., GRIMALDI, G., CONSTANTINO, P., MICHELI, G. & CERVONE, F. (1982) . Loss of nitrofuran resistance in Fusarium oxysporum is correlated with loss of a 46'7 kb circular DNA molecule. Journal of General Microbiology 128, 2235-2242. GUERRY, P., LEBLANC, D . J. & FALKOW, S. (1973). General method for the isolation of plasmid deoxyribonucleic acid . Journal of B acteriology 116 , 10641066. HASHIBA, T., HOMMA, Y., HYAKUMACHI, M . & MATSUDA, 1. ( 1984). Isolation of a DNA plasmid in the fungus Rhizoctonia solani.Journal of General Microbiology 130, 20 67-2°7° . LLOYD, D . & POOLE, R. K. (1978). Subcellular fractionation : isolation and characterization of organelles. In Techniques in M etabolic Research (ed . by T. R. Hes-

keth, H . L. Kornberg, J. C . Metcalf, D . H. Northcote, C. 1. Pogson & K . F. Tipton), pp. 1-27. New York : Elsevier/North-Holland. MANIATIS, T ., FRITSCH, E. F. & SAMBROOK, J . (1982). Molecular Cloning: a laboratory manual. Cold Spring Harbor, N .Y. : Cold Spring Harbor Laboratory. MARRIOTT, A. C ., ARCHER, S. A. & BUCK, K. W. (1984). Mitochondrial DNA in Fusarium oxysporum is a 46'5 kilobase pair circular molecule. Journal of General Microbiology 130, 3001-3°08. MEYERS, J . A., SANCHEZ, D ., ELWELL, L. & FALKOW, S. (1976). Simple agarose gel electrophoretic method for the isolation and characterization of plasmid deox yribonucleic acid . Journal of Ba cteriology 127, 15291537· PONTECORVO, G ., ROPER, J . A., HEMMONS, M. , MAcDONALD, K . D . & BUFTON, A. W. J . (1953). The genetics of Aspergillus nidulans. Advances in Genetics 5,14 1- 238. SIMON, E . W . (1957). The effect of digitonin on the cytochrome c oxidase activity of plant mitochondria. Biochemical Journal 69, 67-74. STAHL, D., LEMKE, P. A., TUOZVNSKI, P ., KUCK, D. & ESSER, K. (1978). Evidence for plasmid-like DNA in a filamentous fungus, the ascomycete Podospora anserina. Mol ecular and General Genetics 162, 341-343. STAHL, D., TUDZYNSKI, P., KUCK, D. & EsSER, K . (1982). Replication and expression ofa bacterial-mitochondrial hybrid pla smid in the fungus Podospora anserina. Proceedings of the National Academy of Sciences, USA So, 1058-1062. WOOD, D . D . & LUCK, D. J . L. (1969). Hybridization of mitochondrial RNA. Journal of Molecular Biology 41, 211-2 2 4.

A SPECIES OF RHIZOCTONIA WITH UNINUCLEATE HYPHAE ISOLATED FROM ROOTS OF WINTER WHEAT BY GEOFFREY

HALL*

Department of Applied Biology, University of Cambridge, Pembroke Street, Cambridge CB2 3DX, U.K.

A dark sterile fungus isolated from roots of winter wheat had hyphae characteristic of the form genus Rhizoctonia. Some isolates produced microsclerotia in agar culture, but others did not. The production of microsclerotia on three agar media was examined, and the growth rate on agar of isolates producing or not producing microsclerotia was compared, but they did not differ substantially. The fungus was called Rhizoctonia Dz. It had narrow hyphae with uninucleate cells and formed micro sclerotia in the roots of sterile wheat seedlings, distinguishing it from R. solani and R . cerealis. Parmeter & Whitney (1970) state that over 100 species of Rhizoctonia have been described, but there is no published work comparing all these . The most common species on wheat are R . solani

* Current address : Health and Safety Executive, Occupational Medicine and Hygiene Laboratories, 403-405 Edgware Road, London NW2 6LN, D.K. Trans . Br. mycol. S oc. 87 (3), (1986)

(Frank) Donk, which inhabits roots, and R . cerealis Boerema (sharp eyespot) which inhabits stems, but which so far has not been isolated from roots. Boerema & van Hoeven (1977) distinguished R. cerealis from R. solani by the former's possession of regularly binucleate hyphal cells and a much slower growth rate in culture. Since then these species have regularly been compared by other

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Notes and brief articles investigators (Hollins, Iellis & Scott, 1983; Deacon & Scott, 1985). During an investigation of the fungi associated with root senescence in winter wheat following four long-established crop rotations, 65-73 % of fungi (depending on rotation type) isolated from surface-sterilized roots were sterile in culture. Sterile fungi were divided into seven types (Dl-4 and Hl-3) on the basis of the morphological features seen in agar culture and growth rate on three artificial media. Type D2 had hyphae characteristic ofthe form-genus Rhizoctonia DC. A study was made to determine which species of Rhizoctonia it most closely resembled. A total of 832 lengths of wheat root, each of 1 em, were surface-sterilized with 1 % (vIv) NaOCI, washed three times with sterile distilled water and plated on to 2 % water agar (WA: Difco-Bacto Agar, Difco Ltd, U.S.A.). They were incubated for 28 d at 17°C and the fungi growing out from the root lengths were examined at x 100 magnification. In all, 125 isolates of D2 were recorded, of which 43 were selected for more detailed study. The morphology of the hyphae and microsclerotia was recorded by examining the isolates on 2 % WA with a light microscope at x 100 magnification. Isolates producing microsderotia were placed in subtype D 2S, others in subtype D zh, Three agar media were used to examine in more detail the production, or non-production of microsclerotia by isolates of D 2S and D zh. They were 2 % WA, Czapek-Dox agar (CDA: Oxoid Ltd, U.K.) and soil extract agar (SEA), prepared according to a method of Nemec (1969), modified as follows. Soil from Cambridge University Fann was bulked, mixed, air-dried, passed through a I in sieve and 500 g were autoclaved with 1 I distilled water for 30 min at 1210. Since filtration through filter paper was impracticable because of the high clay content, the soil and water were mixed before and after autoclaving and left to settle at room temperature overnight. The clear supernatant was filtered through 'Whatman no. i ' filter paper (Whatman Ltd, U.K.) and made up into agar. In addition, 11 isolates producing microsclerotia and 32 isolates producing only hyphae were inoculated on to 2 % WA and incubated at 15°. They were observed after 21 d and 42 d and changes in morphology were recorded. A study was made to determine whether the growth rates on agar of the two subtypes D2S and D2h were different. The colony diameter of three replicates of nine isolates of subtype D2S and nine of D2h on Potato Dextrose Agar (PDA: Oxoid Ltd) at 25° was measured every 3 d over a 15 d period. This medium and temperature were chosen Trans. Br, mycol. Soc. 87 (3), (1986)

so that results would be comparable with those published by other workers, e.g, Hollins et al. (1983), Deacon & Scott (1985). All isolates grew so consistently that only one colony diameter was recorded. For each isolate, a regression analysis of colony diameter against time (for linear effects) and against time" (for quadratic effects) was made . Isolates of D2 were then compared with known isolates of R. solani and R. cerealis. One isolate of R. solani was obtained from the CAB International Mycological Institute (Kew, U.K.) and another from C. A. Gilligan (University of Cambridge, U .K .). One isolate of R . cerealis was obtained from the Centraalbureau voor Schimmelcultures (Baarn , The Netherlands) and two more from R. Hollins (Plant Breeding Institute, Cambridge, U.K.), who isolated them from sharp eyespot lesions on wheat stems, and labelled them R82/196 (from a field at Bury St Edmunds, U .K.), and R83/260 from a field near Luton, U .K.). The morphology of the colony and hyphae on 2 % WA of all isolates of sterile type D2 was compared with that of the known isolates by observation with the light microscope at x 100 magnification. The number of nuclei and diameter of mature hyphal cells of 18 isolates of D2 and the five named isolates were determined by inoculating them on to PDA and placing pieces ofsterile PT300 cellophane, washed thoroughly to remove urea and glycerol plasticizers, around the periphery of the inoculum. Cultures were incubated at 25° for 9 d, then the cellophane was removed and the adherent fungal tissue fixed in a mixture of absolute ethanol and glacial acetic acid (3 : 1 vIv) for 10 min, before storage in 70 % ethanol. They were hydrolyzed in 1 M-HCl (cold at room temperature for 5 min, then hot at 60° for 7 min), stained in a solution of Giemsa (BD H Ltd, U.K.) diluted 1: 5 with phosphate buffer (pH 6'9) and mounted on slides in phosphate buffer (Hrushovetz, 1956). The number of nuclei per cell and the hyphal diameter were measured at x 1000 magnification. To determine whether the isolates formed recognizable structures in living roots, five of subtype D2S, five of D2h, and all the named isolates were inoculated on to the roots of sterile wheat seedlings, prepared as described by Hall (1986). Gnotobiotic and uninfected control seedlings were incubated at 25° for 14 d under a cycle of 18 h light/6 h darkness. Roots were severed, softened in a buffered (0'1 M acetic acid/sodium acetate buffer, pH 4) solution of 15 % pectinase (Sigma Ltd, U.K.) for 18 h at 25°, mounted on slides and examined at x 100 magnification. All isolates of type D2 fungi growing out from roots across 2 % WA had black septate hyphae

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Notes and brief articles lA

'/ 1B

IG

IO,um I

Fig. 1. (A-G) Morphological features of a species of Rhizoctonia from the roots of winter wheat. (A) Main hypha showing septa, prominent inclusions and a lateral branch ; (B) formation of int ercalary and terminal chlamydospores ( = ' monilioid cells ') with refractile inclusions in agar ; (C) aggregation of chlamydospores to form a microsclerotium in agar ; (D) deposition of a dark pigment in a rnicrosclerotium in agar ; (E) short hyphae with a few swollen regions and incipient chlamydospores in agar; (F) hypha stained with Giemsa showing large, single nuclei; (G) microsclerotia composed of clusters of dark-pigmented chlamydospores, root softened by pectinase digestion. (H) pseudoparenchyma produced by R. so/ani in wheat root cortical cells, preparation softened by pectinase digest ion. Scale bars represent zo em unless otherwise indicated.

Trans. Br, mycol. S oc. 87 (3), (1986)

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"'

Notes and brief articles Table

1.

Morphologicalfeatures of two subtypes oj a species of Rhizoctonia on 2 % WA at 15°C after then 42 d incubation

Subtype

No. of isolates examined

Microsclerotia (M S)

Swollen regions (SR)

11

1,4 4,16

4,6 15, 12

D2S D2h

32

24

21

18

~

c

15

(;).

I

e ,5 ,3 12

....~

.,...... !'::

"0

",'

>.

I

l:::

.9

II

8

~

9

6

3

O+-----y----,,---...,-----r---,

o

3

6

9

12

15

Time (d) Fig. 2. Growth of two subtypes of a species of Rhizaaonia, ( e ) D2S and (0) D2h, on PDA at 25 °C. Results are three replicates of nine isolates of each subtype, The S.E. of the x and x 2 coefficients, and the V-intercepts for a comparison are± 1'201, 0·602 and 0"225 respectively.

Trans . Br . mycol. Soc. 87 (3), (1986)

Neither MS nor SR 6,1 13,4

21

d,

Transformation to hyaline form 1, 1 7,7

containing prominent inclusions. Mature hyphae were 4 pm diam and produced branches at some distance behind their apices and at right angles to the main hypha, just below a septum. Branches were constricted at their point of origin (Fig. 1A). After 14 d incubation, hyphae sometimes developed clusters of darkly pigmented chlamydospores, 12-120 pm diam, best considered as microsclerotia (subtype D2S). Microsclerotia were initiated by the swelling of a hyphal tip, usually on a hyphal branch which delimited usually less than ten swollen regions behind it as it extended. These regions were cut off by cross-walls, forming discrete cells in which refractile inclusions developed (Fig . 1B). A dark pigment was deposited in the cells, and this sequence was repeated by other neighbouring branch hyphae until a small cluster of cells was formed (Fig. 1 C-D), Microsclerotia usually matured within 21 d. Isolates producing only hyphae (subtype D2h) sometimes produced short branches with swollen regions (Fig. 1E). On all agars tested, D2h formed a ring of aerial mycelium in a peripheral zone 1-2 em wide within two weeks of incubation, but this feature was not recorded in D2S until after three to four weeks incubation. Subcultures of D2S isolates on 2 % WA, SEA and CDA sometimes failed to produce microsclerotia, whereas Dzh isolates produced swollen regions . This effect was most pronounced on 2 % WA, on which one isolate also produced thick, septate hyaline hyphae with prominent inclusions. Neither subtype consistently produced the same morphological features when subcultured on WA (T able 1). For this reason, all isolates were maintained on root agar (RA), prepared by placing twice-autoclaved pieces of wheat root around a central inoculum disk on WA. When colony growth rates were examined, an analysis of the variance in the linear and quadratic coefficients and the Y-intercepts between D2S and Dzh isolates showed that the Y-intercepts were different at P < 0'05, and the quadratic coefficients

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Notes and brief articles

47°

Table 2. Cytological f eatures offiv e named Rhizoctonia isolates Average number of nuclei per hyphal cell ± S,E,M .

Range of number of nuclei

Average cell width (pm) ± S,E.M .

British, ex C.A .B.I.M,I. Briti sh, ex C, Gilligan

Rhizoetonia solani 4'7 ± 0 '29 (30) 1- 10 9'S ±O'S4 (30) S'7 ± 0 '34 (30) 2--9 8'S ±O'33 (30)

Dutch, ex c.B.S. British, R82/ 196 British, R83/260

2':Z ±O '12 (30) 1'9±0'18 (30) 1'9 ±0'1O (30)

were different at P < 0'01 , but the linear coefficients were not different (Fig. 2). There was no significant difference between the growth rates of the two subgroups from day three to day twelve, when the growth rate of isolates in subgroup D2S began to decelerate, On these pieces of evidence it is most likely that subtypes D2S and D2h represent the same fungus, which exhibits a range of morphological features. Therefore, all isolates were placed in one type, i.e. D2. After 21 d on PDA at 25°, all of the named isolates produced large pseudosclerotia, but isolates of D2 did not. The hyphal morphology of the named fungi was identical to that of isolates of D2 , but the hyphae of all D2 isolates were much more heavily pigmented than those of the named isolates. Nuclear counts and hypha I diameters of the five named isolates (Table 2) are consistent with those published by Hollins et at. (1983) and Boerema & Van Hoeven (1975) for these two species. In no isolate of D2 could nuclei be seen in older regions of the hyphae, which were heavily pigmented, vacuolate, and contained many inclusions. Nuclei could only be seen in young hyphae which were relatively hyaline, and it was established that each of the hyphal cells examined invariably contained a single, large, central nucleus (Fig. 1 F). The widths of mature hyphae were uniformly 4 pm. Isolates of D2 produced micro sclerotia in root cortical cells (Fig. 1G), those of R . solani produced coarsely granular pseudoparenchyma (F ig. 1H ), but those of R . cerealis produced only hyaline hyphae. None of these features was seen in roots of uninfected control seedlings. In conclusion, the hyphal morphology of isolates of D2 resembled that of a species of Rhizoctonia, but studies of the number of nuclei per hyphal cell, and the structures they formed in the roots of Tram. Br . mycol. Soc. 87 (3), (1986)

Rhizoctonia cerealis 1-3 3'9±0'24 (30) 1-3 4'3 ±0'32 (30) 1-3 S'9±0'22 (30)

sterile wheat seedlings, show that they cannot be considered to be either R. solani or R. cerealis. Fungi in this type are therefore referred to as Rhizoctonia D2. The structures each form in the roots of sterile wheat seedlings can be used to distinguish between these three fungi. Comparison of the growth rate of Rhizoctonia D2 with reported rates for R . solani and R . cerealis was difficult because there has been little consensus in the use of the term ' radial growth rate': some authors report it as the increase in radius (in rom h- 1 or rom d"), others as the increase in colony diameter, Hollins et al. (1983) reported radial growth rates on PDA at 25° of 15 to 16 rom d- 1 for R , solani isolated from potato scabs, and 4 to 8 mm d- 1 for R . cerealis isolated from wheat stems . Flentje (1956) reported growth rates of 12 to 15 rom d- 1 for R . solani on PDA, but his isolates included some from wheat stems . Deacon & SCOtt (1985) reported radial growth rates of 4'0, 5'4 and 5'9 rom d- 1 for three isolates of R. solani from roots of wheat suffering from crater disease in South Africa . These are all faster than the l '8mmd- 1 average (range l 'l-3 '3mmd- 1 ) increase reported here for the 18 isolates of D2 between days 3 and 12. However, Papavizas (1965) reported growth rates on PDA for 60 single basidiospore isolates from one isolate of R . solani in the range 0'7-17'0 mm d- 1 • A study of published results led Sherwood (1970) to conclude that growth rate could not be used to distinguish R. solani from most other spec ies. In addition, in this study PDA was used in prepared form. Other workers have preferred to prepare it from fresh ingredients, which may be an additional source of variation in reported growth rates . Since isolates of R . solani are variable in culture, standardization of methods and terminology is required before comparison between the growth rates of isolates from different sources, and between different species, can

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Notes and brief articles usefully be made. Therefore, growth rate was not used to compare the species of Rhizoctonia investigated in this study. R. solani amd R. cerealis have received much attention because of their importance as plant pathogens and have often been compared. Other species of Rhizoctonia have been poorly studied. Campbell et al. (1982) isolated a species of Rhizoctonia from the roots of grassland plants which they described briefly, but they provided no illustration of it. It was more common on the roots of grasses than dicotyledons, but had little effect on the growth of Lolium perenne L. Deacon & Scott (1985) isolated an atypical Rhizoctonia solani which formed bead-like swellings on wheat roots and abundant monilioid cells on PDA, but which did not anastomose with other R. solani isolates and which has yet to produce basidiospores in culture. Most other species of Rhizoctonia have, no doubt, been assigned to the group 'sterile dark fungi', which has often been isolated from plant roots in relatively large numbers (Waid, 1974). These fungi are usually discarded by investigators. A more detailed study of sterile fungi would add much to knowledge about the behaviour and ecology of fungi in roots, accounts of which usually understate their role because of lack of information. A study of fungi, including sterile fungi, in the roots of winter wheat in East Anglia will soon be reported. I wish to thank Dr H. T. Tribe for useful comments and Mr L. Guarino for assistance with computing. This work was done while the author was in receipt of a MAFF studentship.

REFERENCES

BOEREMA,G. H. & VAN HOEVEN,A. A. (1977). Check-list for the scientific names of common parasitic fungi.

47 1

Series zb. Fungi on field crops: cereals and grasses. Netherlands Journal of Plant Pathology 83, 165-2°4. CAMPBELL, R, NEWMAN, E. 1., LAWLEY, R. A. & CHRISTIE, P. (1982). The relationship between a Rhizoctonia species and grassland plants. Transactions of the British Mycological Society 79, 123-127. DEACON, J. W. & SCOTT, D. B. (1985). Rhizoctonia solani associated with crater disease (stunting) of wheat in South Africa. Transactions of the British Mycological Society 85, 319-327. FLENTJE, N. T. (1956). Studies in Pelliculariafilamentosa (Pat.) Rogers. 1. Formation of the perfect state. Transactions of the British Mycological Society 39, 343-356. HALL, G. (1986). Demonstration of chlamydospores and evidence for microsclerotia in Periconia macrospinosa. Transactions of the British Mycological Society 86, 347-349· HOLLINS, T. W., JELLIS, G. J. & SCOTT, P. R. (1983). Infection of potato and wheat by isolates of Rhizoctonia solani and Rhizoctonia cerealis. Plant Pathology 32, 303-310. HRUSHOVETZ, S. B. (1956). Cytological studies of Helminthosporium sativum. Canadian Journal of Botany 34, 321-3 27. NEMEC, S. (1969). Sporulation and identification of fungi isolated from root-rot diseased strawberry plants. Phytopathology 59, 1552-1553. PAPAVIZAS, G. C. (1965). Comparative studies of singlebasidiospore isolates of Pellicularia filamentosa and Pellicularia praticola. Mycologia 57, 91-103. PARMETER, J. R. Jr & WHITNEY, H. S. (1970). Taxonomy and nomenclature of the imperfect state. In Rhizoctonia solani: Biology and Pathology (ed. J. R. Parameter, jr), pp. 7-19. Berkeley, U.S.A.: University of California Press. SHERWOOD, R T. (1970). Physiology of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology (ed. J. R Parmeter, Jr), pp. 69-70 & 91. Berkeley, U.S.A.: University of California Press. WAID, J. S. (1974). The decomposition of roots. In The Biology of Plant Litter Decomposition, 1 (ed. e. H. Dickinson & G. J. F. Pugh), pp. 175-210. London, U.K.: Academic Press.

SOME POSSIBLE FOSSIL NEMATOPHAGOUS FUNGI BY HANS-BORJE JANSSON

Department of Microbial Ecology, University of Lund, S-223 62 Lund, Sweden AND GEORGE O. POINAR, JR

Department of Entomological Sciences, University of California, Berkeley, California 94720, U.S.A.

Fossil nematodes in pieces of Mexican amber, approximately 25 million years old, appeared to have been parasitized by fungi showing a striking resemblance to present-day nematophagous species. Nematodes are an ancient group of animals, of nematodes is exceedingly sparse due to their probably originating in the early Palaeozoic or delicate structure. The same holds true for fungi, possibly Precambrian era (Poinar, 1983). This view but one Palaeozoic fungus, presumably an ascomyis based on indirect evidence, since the fossil record cete, has been dated to the Permian era, about 240 Trans. Br. mycol. Soc. 87 (3), (1986)

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