Trans. Br. mycol. Soc. 79 (1) 123-127(1982)

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Printed in Great Britain

RELATIONSHIP BETWEEN A RHIZOCTONIA SPECIES ANDGRASSLAND PLANTS

By R. CAMPBELL, E. 1. NEWMAN, R. A. LAWLEY* AND P. CHRISTIEtDepartment of Botany, The University, Bristol BS8 lUG

Rhizoctonia colonized the root surfaces of different species of grassland plants to differentextents, being more common on grasses than dicotyledons. No other fungi that were isolatedby plating out root segments showed a statistically significant difference between host species.The Rhizoctonia isolate used had little, if any, effect on the growth of Lolium perenne and wasnot pathogenic, either in normal soil with increased levels of Rhizoctonia or under gnotobioticconditions.

Papers on the fungal flora of rhizospheres or rootsurfaces often report the presence of sterilemycelium (Waid, 1957, 1974; Thornton, 1965;McIlwaine & Malone, 1976). This can be classifiedinto 'dark sterile' and 'hyaline sterile', and someof the 'dark sterile' mayor may not be distinguishedas Rhizoetonia spp, (Waid, 1957); however, conven-tional classification and naming is mostly preventedby lack of reproductive structures. This is no doubtthe principal reason why comparatively little hasbeen published about the distribution of these fungiand their relations to plants, subjects which deservestudy. Here we describe research on a sterile greyfungus, belonging to the genus Rhizoetonia,growing in or isolated from soil from a single field.The research had two aims : firstly to determinewhether the abundance of the fungus on rootsurfaces differed markedly between different speciesof grassland plants, and whether the difference wasreduced if the plants' root systems intermingled .This was investiga ted by examining plants from thefield, and by growing plants in pots with twospecies either separate or together. Secondly, weinvestigated whether the fungus is parasitic on theplants or purely saprophytic, as indicated by itsability to penetrate healthy root cells and to causereductions in growth. The fungus was inoculatedon to axenic plants, and also its abundance innon-sterile soil was altered by mixing in wheat seedon which it had been grown.

Sterile fungi have one experimental advantagewhich we have made use of in this study. When rootsegments are incubated on agar plates, growth of asterile fungus is good evidence that its hyphae werepreviously on or in the root, whereas a colony of a

* Present address: Public Analyst's Laboratory,220-222 Elgar Road, Reading RG2 oDG.

t Present address: BritishAntarctic Survey,Mading-ley Road, Cambridge CB3oET.

spore-forming fungal species could originate froma spore on the root.

METHODS

Site and soil

Plants and soil were collected from a field in AshtonPark, near Bristol, which has been under permanentgrassland for many years and is grazed by cattle.The soil is a well-structured clay-loam, pH 4'8 (2'5water: 1 soil), organic matter 6'2 % (by wetdigestion), phosphorus extractable by Truog'sreagent 2'4 #g/g. Seeds for the pot experimentswere collected from a nearby ungrazed field.

Estimating abundance of sterile grey fungi

Plant root systems were washed free of soil innon-sterile tap-water. Microscopic examinationconfirmed that the root surfaces were free of soil.The roots were spread out on a flat-bottomed dish,and individual root segments selected usingrandom points marked on the dish . The segmentswere rinsed in sterile distilled water, then a 4 mmlength was cut from each with sterile razor bladesand placed on an agar medium, 5 segments perplate. The medium was malt agar-l-rose bengal,except in Experiment 1 when it was Martin'speptone-dextrose agar + rose bengal. Incubationwas at 25°C in Expt 1, 15°C in the remainder. At7 and 14 days the numbers and identity, wherepossible, of all fungal colonies growing from theroot segments were recorded.

Preparation of inoculum

Fungicide-free wheat seed was autoclaved wet andplaced as a dense layer in covered Petri dishes. Itwas inoculated with an isolate of the sterile greyfungus derived from Lolium spp. growing inAshton Park soil and incubated for 14 days at 15°,

Statistical analysis

Because the numbers of fungal colonies perreplicate pot were usually small and sometimes 0 ,

non-parametric methods were used for assessmentof statistical significance ; these were the Fisherexact test , the Wilcoxon matched-pairs signed -ranktest or the Fri edman two-way analysis of variance,depending on the number of treatments andwhether or not the experiment involved blocks .Shoot weights and nutrient concentrations wereanalysed by conventional analysis of variance.

Experiments 4 and 5 : altering the abundance of thesterile grey fungus on soil-grown plants

Experiment 4 was started in Nov . 1977. Into eachlitre offreshly collected Ashton Park soil was mixed20 ml of wheat seeds inocu lated with the sterile greyfungus as described above. Each litre of soil wasthen placed in a 13 cm pot. There were two controltreatments, (a) an equal quantity of sterile wheatseed was added, (b) no add ition . Four L. perenneseedlings were planted in each pot. There wereeight replicate pots per treatment. After nine weeksthe plants were harvested, and their shoot weightsand nutrient contents determined. Abundance ofthe sterile grey fungus was determined by platingfifteen 4 mm root segments per replicate.

Experiment 5 used ground up wheat seed asinoculum, and the 'wheat control' pots receivedsterile ground wheat seed. It was started on 1 June1979 and harvested nine weeks later. There wereagain eight pots per treatment, but 20 rootsegments were plated per replicate. In otherrespects it was the same as Expt 4.

124 Rhizoctonia on grassland plants

by which time there was profuse hypha I prolifera- examined undertion . The 'whole-seed' inoculum was used for colonization.Expts 3 and 4. For Expt 5 the wheat seed wasground up before inoculation, but otherwise thetechnique was the same.

Individual experiments

Abundance in the field

Plants of H olcus lanatus L. (Yorkshire fog), Loliumperenne L. (perennial ryegrass ), Plantago lanceolataL. (ribwort plantain) and Trifolium repens L. (whiteclover) were collected from the field site on 31 Aug.to 4 Sept. 1977. On each of the 5 days , two plantsof each species were collected, giving ten plants perspecies in all. Abundance of the sterile grey funguswas determined as described above .

Experiments I and 2: abundance in pors

In these experiments L. perenne and P. lanceolatawere grown in pots of Ashton Park soil, unsterilizedand without added fertilizer, in a glasshouse. Each13 ern pot contained either four plants of onespecies or two plants of each. At the end, abundanceof the sterile grey fungus and other fungal speciesor groups was determined as described above . Theplant shoots were dried, ground up , digested insulphuric acid, and the ir nitrogen and phosphorusconcentrations determined on a Technicon Auto-analyzer. Experiment 1 started on 9 Nov. 1973 andthe plants were harvested after 16 weeks. Therewere 10 replicate pots and 15 root segments perreplicate, making a total of 150 segment s per treat-ment. Experiment 2 started on 23 July 1976 andthe plants were harvested after 11 weeks. Therewere 14 replicate pots and 25 root segments perrepl icate, making a total of 350 segments pertreatment.

a microscope for fungal

Exp erim ent 3 : gnotobiotic culture

Seeds of L. perenne were sterilized with silvernitrate (Christie, 1976) and germinated on maltagar . Fourteen large glass boiling tubes 4 x 25 emcontaining moist sterile sand and closed by a cottonwool bung, were autoclaved, and separately-autoclaved Hoagland's solution was then added.Young seedlings which showed no contaminationwere aseptically planted into the sand. Into seventubes a single wheat seed on which the sterile greyfungus had been grown (see above) was placed inthe sand close to the seedling. The remaining seventubes received a sterile wheat seed instead .

The tubes were placed under a light bank(approx. 500 foot candles ) in a room at about 22°.

After 17 days the plants were harvested, the shootsdried and weighed, the roots stained, some inphenyl acetic an iline blue , some in tr ypan blue, and

RE SUL TS

Identification of fungi

Isolates from Expt 1 which produced spores wereidentified, using standard keys, to genus or species .Of the eleven macroscopically distinct types of

Table 1. Abundance of Rhizoctonia on roors offourspecies collected f rom fi eld, expressed as percentage of4 mm segments infected

LoJium perenne 11 ' 3 c·Holcus lanatus 6 '0 bPlantago lanceolata 3"3 abTrifolium repens 0 a

., Overall statistical sign ificance P < O'Ot. Any twovalues not followed by the same letter are significan tlydifferent at P >:: 0'05.

R. Campbell, E. I. Newman, R. A. Lawley and P. Christie 125

Table 2. Abundance of Rhizoctonia in two glasshouse experiments, expressed as percentage of 4 mm rootsegments infected

Expt 1 Expt 2

Alone Mixed Alone Mixed

Lolium perennePlantago lanceolaraStatistical significance :

Lolium v. Plantagoalone v. mixed

8'0 11'32'0 6'0

P < 0'05

Not significant

22'4 21'6

4'0 4'4

P < 0'001

Not significant

Table 3, Total amount offungal mycelium ofall typeson root surface (mmjmm2) in Expt I, determined bydirect microscopic observation

Alone MixedLatium perenne 8'4 a" 11 '3 aPlantago lanceolata 8,8 a 1S'S b

* Values not followed by the same letter are significantlydifferent at P ~ o-oj.

sterile grey fungus isolated, one predominated.This produced brown or black carbonaceoussclerotia in culture. The hyphae, both in cultureand on the roots, characteristically branched atright angles, with a septum near the base of thebranch. Such isolates were identified as belongingto the genus Rhizoctonia, and the assessments ofabundance and the inoculation experiments arebased on isolates of this type only.

The second most common sterile grey typeproduced no sclerotia or characteristic branchingand was present in only 2 % of all root segments inExpt 1, The other nine isolates occurred only rarely(most of them only once out of 600 root segmentsinExpt 1); they were distinguishable on macroscopiccharacters such as colour, growth rate and lack ofsclerotia. These ten less common types were notconsidered further,

Abundance of Rhizoctonia

Tables 1 and 2 show the abundance of Rhizoctoniaon root surfaces. There were significant differencesbetween the four plant species collected from thepermanent pasture (Table 1), the grasses havingmuch higher abundance of the fungus than the twodicotyledons . In both the glasshouse experiments(Table 2) L. perennehad a much higher abundanceof the fungus than P, lanceolata whether the speciesgrew in separate pots or together, In Expt 1 thefungus was on average more abundant on plants in

the mixture pots than in monoculture, but this wasnot statistically significant and failed to recur in thesecond experiment. Table 3 shows the total lengthof hyphae of all fungal species per unit area of rootin Expt 1. The data were previously published byChristie et al. (1978), where details of the methodwere given. Comparison with Table 2 indicates thatthe apparent greater abundance of Rhizoctonia inmixture pots may (if not merely due to samplingerror) reflect simply the greater abundance offungiin general, whereas the greater abundance ofRhizoctonia on L. perenne than P. lanceolata is adifference in the proportion of this fungal species.

None of the other species or higher taxa of fungiwhich were counted showed a significant differencein abundance between L. perenneand P. lanceolataor between mixture and monoculture, in eitherexperiment. The most abundant groups werePenicillium spp. (61 % of root segments in Expt 1and 22 % in Expt 2) and Zygomycotina (5 % and18%). Some of these may have grown from sporeson the root surface, Species identified in Expt 1 arelisted by Christie (1976),

Growth in gnotobiotic conditions

When L. perenne plants were grown in sand culturewith no microorganisms except the Rhizoctonia sp.,there was after 17 days a profuse mat of hyphae onthe root surfaces, but careful examination of stainedroots under a microscope failed to detect any hyphaepenetrating into root tissue. This experiment waskcpr short (17 days) so that little root tissue woulddie from old age, and hence allow penetration of asaprophyte: studies on wheat (Holden, 1975)indicate that many cortical cells may die in the firstfew weeks of root life, while the root as a wholeremains apparently healthy.

The mean shoot dry weight of inoculated plantswas 7'3 mg and of controls 5'3 mg and thedifference was not statistically significant. Evidentlythe fungus was not harmful to the plants during thisstage of growth,

126 Rhizoctonia on grassland plants

Table 4. Resultsfrom Expts 4 and 5, in which Lolium perenne was grown in soil with or without inoculationby Rhizoctonia on wheat seed

Cantrall(soilonly)

Control :z(soil+wheat) Inoculated

Statisticalsignificance (P)*

Percentageof root segments infectedExpt 4

By Rhizoctonia 13'3 b* 1'7 a 20'0 b < 0'001

By Trichoderma 3'3 a 27.5 b 11'7 a < 0'002Expt 5

By Rhizoctonia n.d. 8 '1 a 33'8 b < 0'001

By Zygorhynchus n.d. 55'0 b 10'6 a < 0'001Shoot dry weight (gjplant)

Expt 4 0'39 a 1 '05 c 0'79 b < 0'001

Expt 5 0'13 a 0'60 b 0'63 b < 0'001

* Within any row any two valuesnot followed by the same letter are significantly different(P < 0-05). n.d. = notdetermined,

Influence of Rhizoctonia in soil

Table 4 shows results from the two experiments inwhich the Rhizotonia sp. was inoculated into soil inpots. The technique successfully produced markeddifferences in infection between the soil+wheatcontrol and the inoculated plants, but this was atleast partly because the sterile wheat reduced theinfection below that in untreated soil. The wheatacted asa bait for other fungi, especially Trichodermaspp . in Expt 4 and Zygorhynchus spp. in Expt 5, andthese may have competed against or antagonizedthe Rhizoctonia sp. in colonization of the rootsurface. The sterile wheat seed greatly stimulatedplant growth. The mechanism for this is not clear:plant concentrations of nitrogen and phosphorus,elements known to be limiting to L. perennegrowthon this soil, suggested that supply ofthese elementsby wheat seed was not the main reason for growthstimulation.

For assessing the effect of the Rhizoctonia sp. ongrowth of L. perenne, comparison of the inoculatedtreatment with Control 2 (soil +wheat) is mostrelevant. The results from the two experimentsdiffer: in Expt 4 inoculated plants were 25 'Yosmaller than the controls, but in Expt 5 there wasno difference in weight . After Expt 4 we thoughtthat the difference in size might be because somestimulatory material in the wheat seed had beenreleased by the fungus during its growth periodbefore it was mixed into the soil. Therefore inExpt 5 the wheat seed was finely ground to allowrelease of soluble substances from both inoculatedand control seed. In this experiment the differencein size between inoculated and control plantsdisappeared, which supports this explanation. Inneither experiment did the inoculated plants appearunhealthy or show any signs of pathogen attack.

DISCUSSION

Experiment 3 showed that the Rhizoetonia sp. is agenuine saprophyte which does not penetratehealthy root cells under the conditions used. Thisis in agreement with Waid (1957, 1974) whoconcluded, on the basis of limited evidence, thatRhizoetonia on Lolium spp , and Dactylis spp . wassaprophytic, colonizing the root surface oflive rootsbut penetrating the outer cortex only when it wasdead. Other sterile dark hyphae could penetratedeeply into the 'inner cortex'. It is thereforeinteresting that we found such marked differencesin abundance between different plant species, bothin the field and in pots . Waid (1974) collectedLolium perenne and Trifolium repens from pastureand found Rhizoctonia on 17% of L. perenne rootsegments but absent from T. repens roots. Thisagrees with our results (Table 1) . Waid found littledifference between the two hosts in abundance ofother dark sterile fungi, The occurrence ofRhizoctonia may be affected by the soil type orother unknown factors, for Thornton (1965)showed that both L. perenne and T. repens, fromseveral soil types in New Zealand, frequently hadnon-pathogenic sterile dark mycelium on theirroots (up to 44 % of root segments), but Rhizoctoniaspp. were isolated only rarely. This latter pointconflicts with Waid's (1974) findings and our own.

We have investigated the responses of plants toa single isolate, though our estimates of fieldabundance will have assessed many differentstrains . Some races of Rhizoctonia solani (= Thana-tephorus cucumeris Dank) have been shown to bepathogenic to Lolium spp, in North America(Sprague, 1950) but these diseases are uncommonin Britain even though R. solani is widespread(Sampson & Walker, 1954). Rhizoctonia is a large

R. Campbell, E. I . Newman, R. A. Lawley and P. Christie 127genus and different species and races mayor maynot be pathogenic on different hosts.

When two plant species are grown together inpots the abundance of fungal mycelium on theirroot surfaces, assessed by direct observation, isoften intermediate between the abundance on thetwo species growing separately (Christie, Newman& Campbell, 1978; Lawley, Newman & Campbell,1981), though L. perenne and P. lanceolata show adifferent interaction (Table 3). One possibleexplanation is that hyphae can grow from the rootof one plant to the root of another, using substratefrom both. If the difference in abundance of theRhizoctonia between host species is related todifferences in exudatioan of suitable substrates itmight be expected to show such a response, i.e. inExpts 1 and 2 the difference in abundance betweenL. perenne and P. lanceolata should have beenreduced when they were in the same pot . In fact thisdid not happen, indicating that whatever stimulatesthe fungus on L. perenne roots or inhibits it on P.lanceolata roots is confined closely to the rootsurface.

The aim of Expts 4 and 5 was to investigate theeffects of the Rhizoctonia sp . on L. perenne undernormal soil conditions, rather than gnotobioticconditions where the fungus might have unusual orenhanced effects. It is known, for example, thatpathogenic fungi which are normally restricted tograsses and cereals may attack dicotyledons undergnotobiotic conditions (quoted in Walker, 1975).The aim was not fully achieved, since the wheatseed itself evidently had a marked effect on both thegrowth of the plant and the colonization of the rootby other fungi. Taking Expts 3, 4 and 5 togetherit appears that the Rhizoctonia sp. either has noeffect on L. perenne growth or it reduces it slightly.It is not clear whether the 25 % reduction in Expt4 is a genuine effect of the fungus which onlyappears under certain conditions, or is produced bysomething else, e.g. the Trichoderma sp.

Our conclusion is that if the Rhizoctonia sp. does

have an effect on L. perenne growth it is a small one.The isolate studied was a saprophyte with noparasitic ability but yet a strong specificity amonggrassland plants.

This research was supported by a grant from theNatural Environment Research Council.

REFERENCES

CHRISTIE, P. (1976). Interactions between root micro-organismsand grasslandplant speciesin mixtures andmonocultures. Ph.D. Thesis, University of Bristol.

CHRISTIE, P., NEWMAN, E. I. & CAMPBELL, R. (1978). Theinfluence of neighbouring grassland plants on eachothers' endomycorrhizas and root surface micro-organisms. Soil Biology & Biochemistry 10,521-527.

HOLDEN, J. (1975). Use of nuclear staining to assessratesof cell death in corticesof cereal roots. Soil Biology &Biochemistry 7, 333-334·

LAWLEY, R. A., NEWMAN, E. I. & CAMPBELL, R. (1981).Abundance of endomycorrhizas and root surfacemicro-organisms on three grassesgrownseparately andinmixtures. Soil Biology & Biochemistry (In the Press).

McILWAINE, R. S. & MALONE, J. P. (1976) . Effects ofchloropicrinsoiltreatmenton the microflora of soilandryegrassroots and on ryegrassyield. Transactions of theBritish Mycological Society. 67, 113-120.

SAMPSON, K. & WESTERN, J. H. (1954). Diseasesof BritishGrasses and Herbage Legumes. znd Edit. CambridgeUniversity Press.

SPRAGUE, R. (1950). Diseases of Cereals and Gra.'5es inNorth America. Ronald Press, New York.

THORNTON, R. H. (1965). Studies of fungi in pasturesoils. 1. Fungi associated with live roots. New ZealandJournal of Agricultral Research 8, 417-419.

WAID, J. S. (1957). Distribution of fungi within thedecomposing tissues of ryegrassroots. Transactions ofthe British Mycological Society, 40, 391-406.

WAID, J. S. (1974) . Decompostionof roots. In Biology ofPlant Litter Decomposition (ed. C. H. Dickinson &G. F. Pugh) pp. 175-211. Academic Press, London

WALKER, J. (1975) . Take-all diseases of Gramineae: areview of recent work. Review of Plant Pathology 54,113-144.

(Received for publication 17 September I98I)