466 Notes and brief articles

DABOUSSI-BAREYRE, M.-J. & PARISOT, D . (1981). Nucleo­cytoplasmic interactions implicated in differentiationin Nectria haematococca. In Fusarium : Diseases,Biology, and Taxonomy (ed. P. E . Nelson, T . A. Tous­soun & R. J. Cook), pp . 30~317. University Park andLondon: Pennsylvania State University Press.

CASSE, F., BOUCHER, C., JUILLIOT, J. S., MICHEL, M . &DENARIS, J. (1979). Identification and characterisationof large plasmids in Rhizobium meliloti using agarosegel electrophoresis. Journal of General Microbiology113, 229-242.

COLLINS, R. A., STOHL, L. L., COLE, M. & LAMBOWITZ,A. M . (1981). Characterization of a novel plasmidDNA found in mitochondria of N. crassa. Cell 24,443-452.

GARBER, R. C., TURGEON, B. G . & YODER, O. C. (1984).A mitochondrial plasmid from the plant pathogenicfungus Cochliobolus heterostrophus. Molecular andGeneral Genetics 196,301-310.

GUARDIOLA, J., GRIMALDI, G., CONSTANTINO, P., MIC­HELI, G. & CERVONE, F. (1982) . Loss of nitrofuranresistance in Fusarium oxysporum is correlated with lossof a 46'7 kb circular DNA molecule.Journal of GeneralMicrobiology 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 fungusRhizoctonia solani.Journal ofGeneral Microbiology 130,2067-2°7°.

LLOYD, D . & POOLE, R. K. (1978). Subcellular fractiona­tion : isolation and characterization of organelles. InTechniques 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 SpringHarbor, N .Y. : Cold Spring Harbor Laboratory.

MARRIOTT, A. C., ARCHER, S. A. & BUCK, K. W. (1984).Mitochondrial DNA in Fusarium oxysporum is a 46'5kilobase pair circular molecule. Journal of GeneralMicrobiology 130, 3001-3°08.

MEYERS, J . A., SANCHEZ, D ., ELWELL, L. & FALKOW, S.(1976). Simple agarose gel electrophoretic method forthe 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). Thegenetics of Aspergillus nidulans. Advances in Genetics5,141- 238.

SIMON, E . W . (1957). The effect of digitonin on thecytochrome 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 afilamentous fungus, the ascomycete Podospora anserina.Molecular and General Genetics 162, 341-343.

STAHL, D., TUDZYNSKI, P., KUCK, D. & EsSER, K . (1982).Replication and expression ofa bacterial-mitochondrialhybrid pla smid in the fungus Podospora anserina.Proceedings of the National Academy of Sciences, USASo, 1058-1062.

WOOD, D . D . & LUCK, D. J . L. (1969). Hybridization ofmitochondrial RNA. Journal of Molecular Biology 41,211-2 24.

A SPECIES OF RHIZOCTONIA WITH UNINUCLEATE HYPHAE ISOLATEDFROM 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 formgenus 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 onagar of isolates producing or not producing microsclerotia was compared, but they did notdiffer substantially. The fungus was called Rhizoctonia Dz. It had narrow hyphae withuninucleate 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 100species of Rhizoctonia have been described, butthere 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.

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

Trans . Br. mycol. Soc. 87 (3), (1986) Printed in Great Britain

Notes and brief articlesinvestigators (Hollins, Iellis & Scott, 1983; Deacon& Scott, 1985).

During an investigation of the fungi associatedwith root senescence in winter wheat followingfour long-established crop rotations, 65-73 % offungi (depending on rotation type) isolated fromsurface-sterilized roots were sterile in culture.Sterile fungi were divided into seven types (Dl-4and Hl-3) on the basis of the morphologicalfeatures seen in agar culture and growth rate onthree artificial media. Type D2 had hyphaecharacteristic ofthe form-genus Rhizoctonia DC. Astudy was made to determine which species ofRhizoctonia it most closely resembled.

A total of 832 lengths of wheat root, each of1 em, were surface-sterilized with 1 % (vIv)NaOCI, washed three times with sterile distilledwater and plated on to 2 % water agar (WA:Difco-Bacto Agar, Difco Ltd, U.S.A.). They wereincubated for 28 d at 17°C and the fungi growingout from the root lengths were examined at x 100magnification. In all, 125 isolates of D2 wererecorded, of which 43 were selected for moredetailed study.

The morphology of the hyphae and micro­sclerotia was recorded by examining the isolates on2% WA with a light microscope at x 100 magnifi­cation. Isolates producing microsderotia wereplaced in subtype D 2S, others in subtype D zh,

Three agar media were used to examine in moredetail the production, or non-production ofmicrosclerotia by isolates of D 2S and D zh. Theywere 2 % WA, Czapek-Dox agar (CDA: OxoidLtd, U.K.) and soil extract agar (SEA), preparedaccording to a method of Nemec (1969), modifiedas follows. Soil from Cambridge University Fannwas bulked, mixed, air-dried, passed through a I insieve and 500 g were autoclaved with 1 I distilledwater for 30 min at 1210. Since filtration throughfilter paper was impracticable because of the highclay content, the soil and water were mixed beforeand after autoclaving and left to settle at roomtemperature overnight. The clear supernatant wasfiltered through 'Whatman no. i ' filter paper(Whatman Ltd, U.K.) and made up into agar. Inaddition, 11 isolates producing microsclerotia and32 isolates producing only hyphae were inoculatedon to 2 % WA and incubated at 15°. They wereobserved after 21 d and 42 d and changes inmorphology were recorded.

A study was made to determine whether thegrowth rates on agar of the two subtypes D2S andD2h were different. The colony diameter of threereplicates of nine isolates of subtype D2S and nineof D2h on Potato Dextrose Agar (PDA: OxoidLtd) at 25° was measured every 3 d over a 15 dperiod. This medium and temperature were chosen

so that results would be comparable with thosepublished by other workers, e.g, Hollins et al.(1983), Deacon & Scott (1985). All isolates grew soconsistently that only one colony diameter wasrecorded. For each isolate, a regression analysis ofcolony diameter against time (for linear effects) andagainst time" (for quadratic effects) was made .

Isolates of D2 were then compared with knownisolates of R. solani and R. cerealis. One isolate ofR. solani was obtained from the CAB InternationalMycological Institute (Kew, U.K.) and anotherfrom C. A. Gilligan (University of Cambridge,U .K .). One isolate of R . cerealiswas obtained fromthe Centraalbureau voor Schimmelcultures(Baarn , The Netherlands) and two more fromR. Hollins (Plant Breeding Institute, Cambridge,U.K.), who isolated them from sharp eyespotlesions on wheat stems, and labelled them R82/196(from a field at Bury St Edmunds, U .K.), andR83/260 from a field near Luton, U .K.).

The morphology of the colony and hyphae on2 % WA of all isolates of sterile type D2 wascompared with that of the known isolates byobservation with the light microscope at x 100magnification.

The number of nuclei and diameter of maturehyphal cells of 18 isolates ofD2 and the five namedisolates were determined by inoculating them on toPDA and placingpieces ofsterile PT300 cellophane,washed thoroughly to remove urea and glycerolplasticizers, around the periphery of the inoculum.Cultures were incubated at 25° for 9 d, then thecellophane was removed and the adherent fungaltissue fixed in a mixture of absolute ethanol andglacial acetic acid (3 : 1 vIv) for 10 min, beforestorage in 70 % ethanol. They were hydrolyzed in1 M-HCl (cold at room temperature for 5 min, thenhot at 60° for 7 min), stained in a solution ofGiemsa (BDH Ltd, U.K.) diluted 1: 5 withphosphate buffer (pH 6'9) and mounted on slidesin phosphate buffer (Hrushovetz, 1956). Thenumber of nuclei per cell and the hyphal diameterwere measured at x 1000 magnification.

To determine whether the isolates formedrecognizable structures in living roots, five ofsubtype D2S, five of D2h, and all the namedisolates were inoculated on to the roots of sterilewheat seedlings, prepared as described by Hall(1986). Gnotobiotic and uninfected control seed­lings were incubated at 25° for 14 d under a cycleof 18 h light/6 h darkness. Roots were severed,softened in a buffered (0'1 M acetic acid/sodiumacetate buffer, pH 4) solution of 15% pectinase(Sigma Ltd, U.K.) for 18 h at 25°, mounted onslides and examined at x 100 magnification.

All isolates of type D2 fungi growing out fromroots across 2 % WA had black septate hyphae

Trans. Br, mycol. Soc. 87 (3), (1986) Printed in Great Britain

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) Mainhypha showing septa, prominent inclusions and a lateral branch ; (B) formation of intercalary and terminalchlamydospores ( = ' monilioid cells ') with refractile inclusions in agar ; (C) aggregation of chlamydosporesto form a microsclerotium in agar ; (D) deposition of a dark pigment in a rnicrosclerotium in agar ; (E) shorthyphae with a few swollen regions and incipient chlamydospores in agar; (F) hypha stained with Giemsashowing 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 corticalcells, preparation softened by pectinase digest ion. Scale bars represent zo em unless otherwise indicated.

Trans. Br, mycol. Soc. 87 (3), (1986) Printed in Great Britain

Notes and brief articles

Table 1. Morphologicalfeatures of two subtypes oj a species of Rhizoctonia on 2 % WA at 15°C after 21 d,then 42 d incubation

Trans-No. of Micro- formationisolates sclerotia Swollen Neither MS to hyaline

Subtype examined (M S) regions (SR) nor SR form

D2S 11 1,4 4,6 6,1 1, 1D2h 32 4,16 15, 12 13,4 7,7

24

O+-----y----,,---...,-----r---,o 3 6 9 12 15

Time (d)

Fig. 2. Growth of two subtypes of a species ofRhizaaonia, ( e ) D2S and (0) D2h, on PDA at 25 °C.Results are three replicates of nine isolates of eachsubtype, The S.E. of the x and x2 coefficients, and theV-intercepts for a comparison are± 1'201, 0·602 and0"225 respectively.

containing prominent inclusions. Mature hyphaewere 4 pm diam and produced branches at somedistance behind their apices and at right angles tothe main hypha, just below a septum. Brancheswere 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 theswelling of a hyphal tip, usually on a hyphal branchwhich delimited usually less than ten swollenregions behind it as it extended. These regions werecut off by cross-walls, forming discrete cells inwhich refractile inclusions developed (Fig . 1B). Adark pigment was deposited in the cells, and thissequence was repeated by other neighbouringbranch hyphae until a small cluster of cells wasformed (Fig. 1 C-D), Microsclerotia usually ma­tured within 21 d. Isolates producing only hyphae(subtype D2h) sometimes produced short brancheswith swollen regions (Fig. 1E). On all agars tested,D2h formed a ring of aerial mycelium in aperipheral zone 1-2 em wide within two weeks ofincubation, but this feature was not recorded inD2S until after three to four weeks incubation.

Subcultures of D2S isolates on 2% WA, SEAand CDA sometimes failed to produce micro­sclerotia, whereas Dzh isolates produced swollenregions . 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 samemorphological features when subcultured on WA(T able 1). For this reason, all isolates weremaintained on root agar (RA), prepared by placingtwice-autoclaved pieces of wheat root around acentral inoculum disk on WA.

When colony growth rates were examined, ananalysis of the variance in the linear and quadraticcoefficients and the Y-intercepts between D2S andDzh isolates showed that the Y-intercepts weredifferent at P < 0'05, and the quadratic coefficients

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21

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Trans . Br . mycol. Soc. 87 (3), (1986) Printed in Great Br itain

47° Notes and brief articles

Table 2. Cytological f eatures offiv e named Rhizoctonia isolates

Averagenumber ofnuclei perhyphal cell± S,E,M.

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

Range ofnumber ofnuclei

Rhizoetonia solani4'7 ± 0 '29 (30) 1- 10S'7 ± 0 '34 (30) 2--9

Averagecell width(pm)± S,E.M.

9'S ±O'S4 (30)8'S ±O'33 (30)

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

Rhizoctonia cerealis2':Z ±O '12 (30) 1-3 3'9±0'24 (30)1'9±0'18 (30) 1-3 4'3 ±0'32 (30)1'9 ±0'1O (30) 1-3 S'9±0'22 (30)

were different at P < 0'01 , but the linear coefficientswere not different (Fig. 2). There was nosignificant difference between the growth rates ofthe two subgroups from day three to day twelve,when the growth rate of isolates in subgroup D2Sbegan to decelerate,

On these pieces of evidence it is most likely thatsubtypes 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 namedisolates produced large pseudosclerotia, but isolatesof D2 did not. The hyphal morphology of thenamed fungi was identical to that of isolates of D2 ,but the hyphae of all D2 isolates were much moreheavily pigmented than those of the namedisolates.

Nuclear counts and hyphaI diameters of the fivenamed isolates (Table 2) are consistent with thosepublished by Hollins et at. (1983) and Boerema &Van Hoeven (1975) for these two species. In noisolate of D2 could nuclei be seen in older regionsof the hyphae, which were heavily pigmented,vacuolate, and contained many inclusions. Nucleicould only be seen in young hyphae which wererelatively hyaline, and it was established that eachof the hyphal cells examined invariably containeda single, large, central nucleus (Fig. 1 F). Thewidths of mature hyphae were uniformly 4 pm.

Isolates of D2 produced micro sclerotia in rootcortical cells (Fig. 1G), those of R . solani producedcoarsely granular pseudoparenchyma (F ig. 1H),but those of R . cerealis produced only hyalinehyphae. None of these features was seen in roots ofuninfected control seedlings.

In conclusion, the hyphal morphology of isolatesof 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

sterile wheat seedlings, show that they cannot beconsidered to be either R. solani or R. cerealis.Fungi in this type are therefore referred to asRhizoctonia D2. The structures each form in theroots of sterile wheat seedlings can be used todistinguish between these three fungi.

Comparison of the growth rate of RhizoctoniaD2 with reported rates for R . solani and R . cerealiswas difficult because there has been little consensusin the use of the term ' radial growth rate': someauthors report it as the increase in radius (inrom h- 1 or rom d"), others as the increase incolony diameter, Hollins et al. (1983) reportedradial growth rates on PDA at 25° of 15 to16 rom d- 1 for R , solani isolated from potato scabs,and 4 to 8 mm d-1 for R . cerealis isolated fromwheat stems . Flentje (1956) reported growth ratesof 12 to 15 rom d-1 for R . solani on PDA, but hisisolates 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. solanifrom roots of wheat suffering from crater disease inSouth Africa . These are all faster than thel '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 therange 0'7-17'0 mm d-1 • A study of publishedresults led Sherwood (1970) to conclude thatgrowth rate could not be used to distinguish R.solani from most other species. In addition, in thisstudy PDA was used in prepared form. Otherworkers have preferred to prepare it from freshingredients, which may be an additional source ofvariation in reported growth rates . Since isolates ofR . solani are variable in culture, standardization ofmethods and terminology is required before com­parison between the growth rates of isolates fromdifferent sources, and between different species, can

Tram. Br . mycol. Soc. 87 (3), (1986) Printed in Great Br itain

Notes and brief articles 471

usefully be made. Therefore, growth rate was notused to compare the species of Rhizoctonia investi­gated in this study.

R. solani amd R. cerealis have received muchattention because of their importance as plantpathogens and have often been compared. Otherspecies of Rhizoctonia have been poorly studied.Campbell et al. (1982) isolated a species ofRhizoctonia from the roots of grassland plantswhich they described briefly, but they provided noillustration of it. It was more common on the rootsof grasses than dicotyledons, but had little effect onthe growth of Lolium perenne L. Deacon & Scott(1985) isolated an atypical Rhizoctonia solani whichformed bead-like swellings on wheat roots andabundant monilioid cells on PDA, but which didnot anastomose with other R. solani isolates andwhich 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 inrelatively large numbers (Waid, 1974). These fungiare usually discarded by investigators. A moredetailed study of sterile fungi would add much toknowledge about the behaviour and ecology offungi in roots, accounts of which usually understatetheir role because of lack of information. A studyof fungi, including sterile fungi, in the roots ofwinter wheat in East Anglia will soon be reported.

I wish to thank Dr H. T. Tribe for usefulcomments and Mr L. Guarino for assistance withcomputing. This work was done while the authorwas in receipt of a MAFF studentship.

REFERENCES

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

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 aRhizoctonia species and grassland plants. Transactionsof the British Mycological Society 79, 123-127.

DEACON, J. W. & SCOTT, D. B. (1985). Rhizoctonia solaniassociated with crater disease (stunting) of wheat inSouth Africa. Transactions of the British MycologicalSociety 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 andevidence 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 Rhizoctoniasolani 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 fungiisolated from root-rot diseased strawberry plants.Phytopathology 59, 1552-1553.

PAPAVIZAS, G. C. (1965). Comparative studies of single­basidiospore isolates of Pellicularia filamentosa andPellicularia praticola. Mycologia 57, 91-103.

PARMETER, J. R. Jr & WHITNEY, H. S. (1970). Taxonomyand nomenclature of the imperfect state. In Rhizoctoniasolani: Biology and Pathology (ed. J. R. Parameter, jr),pp. 7-19. Berkeley, U.S.A.: University of CaliforniaPress.

SHERWOOD, R T. (1970). Physiology of Rhizoctoniasolani. 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 TheBiology of Plant Litter Decomposition, 1 (ed.e. H. Dickinson & G. J. F. Pugh), pp. 175-210.London, U.K.: Academic Press.

of nematodes is exceedingly sparse due to theirdelicate structure. The same holds true for fungi,but one Palaeozoic fungus, presumably an ascomy­cete, has been dated to the Permian era, about 240

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, appearedto have been parasitized by fungi showing a striking resemblance to present-day nemato­phagous species.

Nematodes are an ancient group of animals,probably originating in the early Palaeozoic orpossibly Precambrian era (Poinar, 1983). This viewis based on indirect evidence, since the fossil record

Trans. Br. mycol. Soc. 87 (3), (1986) Printed in Great Britain