rvYAlT!. INSTITUI'È 3r-ro"ß I,]BRÀRY

MYCOPHAGOUS AMOEBAE IN A SUPPRESSIVE

PASTURE SOIL IN RELATION TO THE

TAKE-ALL DISEASE OF

by

Sukunar Chakraborty B.Sc. (Ag. ) lbnours, M.Sc.

Departnent of Plant PathologY Waite Agricultural Research Institute The UniversitY of Adelaide South Australia

Thesis subnitted to The Unj-versity of Adelaide in fulfilrnent of the requirenents for the degree of Doctor of PhilosoPhY

March, 1983. TABLE OF CONTENTS

Page No.

SUMI/IARY 1)

STATfl/lENT uii

ACTAIOWLEDGEMENTS uiií

1 I I I NIRODUCTI ON

II. |VIATTRIALS AND lvlETt1ODS 9 PHYSICAL AND CHEMICAL PROPERTIES OF EXPERIMENTAL SOILS 9 ORIGIN AND MAINTENANCE OF FUNGI AND BACTERIA 9 TEST FOR SUPPRESSIVENESS OF SOILS 13 SUPPRESSIVENESS OF EXPERIMENTAL SOILS !4

III. STUDIES ON SOIL AIVIOEBAE 19 A. ISOLATION, MAINTENANCE AND CHARACTERIZATION OF AMOEBAE 19

Isolation of anoebae 20 (¿) Ihe soiL pLate method 20 (i¿) IsoLation from soiL suspension 2L ftii) So'LL er,tich¡nent method 22 (íu) Menbt'at¿.¿ ¡'iLter bw"LaL met\pd 22 Purification and Maintenance 25 Cultural characters of Amoebae 26 ft) Grouth Tesponses of ønoebae to diffez'ønt ternpet'atures 26 (¿i. ) IVprnaL death points of ønoebae 28 (í¿i) Effeet of antibíotics on the gz'oath of ønoebae 30 6u) Suítabilit¿ of uarioua brand.s of agar foz' the cultiuation of amoebae 32

B DESCRIPTTON AND IDENTIFICATION OF .NVÍOEBAE ISOLATED FROI{ THE IIIAITE PERIVANENT PASTURE 36

Methods 56 The anoebae studied 37 Aeanthøno eba p o Lgptøg a 37 Echinønoeba sp, 59 Gephyrønoeba sp. 4L Uni dentífi ed Leptomy æLd øno eba 44 Mayoz'eLLa sp. 47 -L1,-

Page No.

P'Laty øno eb a st enop o dia 49 PLatyønoeba sp. 51 Saceamoeba sp. 53 Thee øno eba quaÅtiLineat a 55 TVtecøno eba sphaeronuc Le o Lus 57 Theeønoeba granife.nø sub-species rrinor 58 Anidentifí ed vønpyv' eLLíd øno eba 62

C. IN VIIR? INTERACTION 0F SOIL AN'ÍOEBAE WITH G9Ú AND OTHER SOIL FUNGI 66

The feeding trials 67 Feeding activities of the a¡noebae ó8 Thp uwLdenti fi eC Leptornyrid ønoeba 70 Saeeønoeba sp. 79 Gephyrønoeba sp. 81 Theeøno eba granifera sub-species minor B3 MayoreLLa sp. 85 Feeding of nycophagous amoebae on other soil fungi 87 Discussion 89

IV. ROLE OF SOIL AÍIOEBAE IN SUPPRESSION OF Gfl 91 A. SOIL AIÍOEBAE AND SAPROPHYTIC SURVIVÃL oF Ggt 91 The nethod used 94 Amoebae and saprophytic survival of Ggt hyphae in suppressive and non-suppressive soil 95 Amoebae and saprophytic survival of Ggt hyphae in WPP, CW, MB and CS 101 Saprophytic survival of Ggt ín sterile soils amended with suspensions of nycophagous amoebae 106 Ability of mycophagous amoebae to invade infected wheat roots 109 Discussion 7L2

B POPULATIONS OF AMOEBAE IN SUPPRESSIVE AND NON- SUPPRESSIVE SOILS 174

Soils 115 Consi-deration of methods Lt6 Populations of amoebae in the soils L2T Populations of Ggt ín the survey soils 723 Effect of soil moisture on amoebal activity L25 Survival of amoebae under extremes of moisture stress L2B Page No.

Populations of amoebae in suppressive and non- suppressive soils r^Jith similar soil texture 129 Suitability of the survey soils as a nedium for anoebal gror^rth L32 Di scussion L32

c ASSOCIATION OF AI{OEBAE IN WHEAT RHIZOSPHERES IN SUPPRESSIVE AND NON_SUPPRESSIVE SOILS 139

Association of soil amoebae with wheat roots - a sulvey L47 Population dynanics of amoebae in suppressive and non-suppressive soí1s 143 Selection of soils and sanpling 143 Populations of mycophagous and other arnoebae in the PPltl, PeW and CIV soils 746 Anoebae in wheat rhizoplane in suppressive and non-suppressive soils 148 Correlation between soil moisture, population of Ggt and amoebae in the suppressive and non- suppressive soils 1s0

D REDUCTION OF TAKE-ALL BY MYCOPHAGOUS AN,{OEBAE IN POT BIOASSAYS 155

Effect of rnycophagous amoebae on the severity of take-al1 Is7 Reduction of take-al1 severity by soil cultures of nycoplìagous amoebae 158 Soil cultures of mycophagous amoebae 160 Effect of soil cultures of mycophagous anroebae on the severity of take-a11 160 Effect of initial population size of mycophagous amoebae on the severity of take-a11 764 The accompanying organisms of. Thecønoeba granifera sub-species m¿nor 764 Discussion 767

V, GEI'IERAL DISCUSSION 169 -LU-

Page No

VI . APPENDI CES APPENDIX I - Formulae for nedía and saline solutions 17s

APPENDIX IIA - Generic populatíons of amoebae in sanples of the Waite pernãnent pasture, Avon and Murray Bridge soil 179

APPENDTX IrB - _1- Population (no. tJ dry wt) of soil amoebae during twó wheat crop and inter-crop fallow in suppressive (PPW and PeW) ancl non-suppressive (CW) soil 180

APPENDIX IIC - lVheat rhizoplanc amoebae (percentage root plated) during two crops in suppressive (PPW and PeW) and non-suppïessive (CW) soil 181

APPENDIX III - Publications 183

VI I. BIBLIOGRAPHY 184 -ü-

SUMMARY

Á¡noebae were isolated, characterised and identified frorn soil of the Waite Institute perrnanent pasture plot which is suppressive in pot bioassays to the take-all disease of wheat. Nine species of amoebae belonging to eight geneïa were tested for their rnycophagy against three plant pathogenic fungi including GaeumannonrAees gr'ønínis tritíei.

Members of the geneïa, Gephyz,ønoeba, Ma¿oreLLa, Saccunoeba, TlPcønoeba and an unidentified species of the order Leptomyxida were nycophagous. Al1 nycophagous arnoebae, except the unidentified leptonyxid were able to feed on pigrnented (nelanised) fungal cells.

Populations of the various genera of soil amoebae were assessed fron samples of the naturally suppressive pasture soiI, a suppressive wheat-pasture rotation and three non-supplessive wheat-field soils-

The suppressive soils showed higher populations of both mycophagous and other amoebae and a higher frequency of occurrence of nycophagous genera. Soil texture and water holding capacity were not related with population 1eve1s of anoebae in these soils.

saprophytic survival of the take-al1 Ì.¡as studied by burying fungal hyphae in suppressive and non-suppressive soils. Ëtyphal density and survival of pigmented hyphae declined at a faster rate in the pernanent pasture soil than in thc non-suppressive soils. Hyphae recovered fron the suppressive soil showed a higher association of mycopl'ragous and other amoebae and scanning electron rnicroscopy of these

hyphae showed extensive erosion and discrete perforations in their lvalls The decline in survival of the fungus was related to the rate of decline in the density of pigrnented h1çhae in suporessive soil, irrespective of the soil type.

Studies on the population dynamics of anoebae showed a higher rhizosphere population and a higher rhizoplane association of nycophagous

and other amoebae in suppressive soi1s. Populations in the suppressive pasture-wheat soil did not correlate with soil moisture during two wheat crops and one inter-crop fal1ow.

Three urycophagous amoebae, Gephgramoeba, Saeeønoeba and Theeønoeba gnanifez'a sub-species mLnor" alone or nixed effectively reduced take-al1 severity in pot bioassays. The reduction in disease rating and the increase in height and dry weight of plants were comparable to that obtained with the suppressive pasture soil. Higher populations of

these amoebae in conbination were able to further reduce disease severity and improve plant grovrth.

Mycophagous amoebae are pïoposed as a component of the suppressive factors in the permanent pasture soil. The observations that nyco-

phagous amoebae associate themselves with the fungus in soi1s, can lyse

both pignented and hyaline hyphae during the pre-colonisation and parasitic phase of the fungus, and reduce take-all severity in pot bioassays substantiate this proposal. -o'?.1,-

STATø/IENT

This thesis contains no naterial which has been accepted for the award of any other degree or diploma,in any university and to the best of ny knowledge and belief contains no material previously pubtished or written by another person, except when due reference is nade in the text.

S. Chakraborty -Ð1-LL-

ACI

I wish to thank Dr. J.H. WarcuP, nY supervisor, for his interest, constructive criticisn and encouragenent and Prof. H.R. Wallace, chairman of the Departnent of Plant Pathology, llJaite Agricultural Research Institute, for the provision of facilities during this study.

Part of the research for this thesis was carried out in Canberra, at the C.S.I.R.O. Division of Forest Research and I wish to acknowledge Prof. H.R. Wallace and Dr. J.H. Warcup for their support of ny application for ] grant which financed my trips. I wish to thank Dr. K.Nl. Old of the Division of Forest Research, c.s.I.R.O., for his collaboration in studies on the in uitro interactions of Ggt with amoebae and also for the financial arrangements for one of ny trips to Canberra. The hospitality of Mr. J.M. Oros in Canberra is gratefully acknowledged.

I thank Mr. T. Hancock and Mr. R. Cook for advice on statistical analyses; Mr. B. Palk for preparing the photographs and Mrs. M. Brock for typing the thesis.

I gratefully acknor+ledge The University of Adelaide for the award of an University Research Grant Scholarship throughout this work and the award of rThe K.P. Barley Ptizer for outstanding post-graduate research in Agricultural Sciences toì tgge.

I wish to thank the staff and fellow students at the Departnent of Plant Pathology, I{aite Institute, for their assistance; ny palents and my wife Shukla, fot their encouïagement and understanding. 1 WAITÊ INSTITUTE I.IBRARY

I I NTRODUCTION

Leeuwenhoek (1677) was probably the first to record the occurrence of protozoa in soil and presented accounts of several protozoa includíng

Amoeba (Leeuwenhoek, 7702). It was alrnost thlo centuries later that

Russell and Hutchinson (1909) advanced their theory of rsoil sicknessr and proposed that the increase in soil fertility after partial sterili- sation was due mainly to the removal of bacteriophagous soil protozoa- Although current interpretations of partial sterilisation suggest that this explanation is incorrect, their hypothesis aroused great interest in the study of soil protozoa. Systernatic research on soil protozoa began in 1915 at Rothansted with the demonstration of active protozoa by Martin and Lewin. Cutler (1919, 1920) developed nethods of counting soil protozoa and protozoa were found to be ubiquitous; the sane species occurring in arctic, temperate and tropical soi1s. None of the soils exanined r4/ere completely devoid of protozoa. Sandon (1927) reviewed the geographical distribution of soil protozoa and suggested that temperature, noisture, soil reaction and texture are of litt1e importance in species distribution of rnajority of soil protozoa.

Soil protozoa have two najor ecological roles: influencing the structure of the nicrobial connunity and enhancing nutrient cycling; both activiti.es are related to their feeding on bacteria. Protozoa enhance mineralisation of nutrients inmobil.ised in the nicrobial bio- rnass by consuming bacteria and excreting excess nutrients thereby accelerating netabolic turnover in soils (Stout and Hea1, 1967; Stout, L1TS). Protozoa selectively lirnit the size of bacterial populations in soil (Singh, Lg4l, 1942; Danso and Alexander, 1975; Habte and and they are responsible Alexarrder , Ig77; Sardeshpande et aL. ' L977) -2-

for the decline of Rhizobiwn (Danso et aL., 1975) and plant pathogenic

Xanthomonas eønpestz"Ls (Panunel) Dowson in soil (Habte and Alexander, Ig75). However, protozoa ðo not elininate their bacterial prey (Danso arrd Alexander, 1975; Habte and Alexander, 1978a, 1978b).

The role of protozoa in the mineralisation of nutrients is well known. Azotobacteo spP. can fix nore nitrogen in the presence of protozoa (Cutler and Bal , L962) or amoebae (l'{asir, 7923) and nixed cultures of bacteria and plotozoa (Skinner, 1927; Meiklejohn, L932) produce nore amnonia than the bacteria alone. Recent studies in soil microcosms have shown greater nineralisation of carbon, nitrogen and phosphorus in soil containing bacteria and anoebae than in soil contain- ing bacteria without amoebae (Coleman et aL., 1977; Anderson et aL.' 1978; CoIe et aL., 1978; Colenan et aL., 1978; Elliott et aL., 1979). Recent reviews on the occurrence and activities of soil protozoa have been presented by Singh (1963), Pu-ssard (1967), Stout and Heal (1967) ' Viswanath and Pillai (1968), Nicoljuk and Geltzer (1972) and Darbyshire

(1s7s) .

Protozoa are represented in the soil mainly by rhizopods, which include the naked and , and ciliates. In tems of nunber per unit mass naked arnoebae constitute the dominant group. Their numbers vary from 2 x 103 to 1690 x 103 g-1 dry wt, whereas testate anoebae vary from 2 x 703 to 73 x 105 g-1 dry wt, flag- ellates fron 0.5 x 103 to 56 x 103 g-1 dty wt and ciliates approxinately 0.75 x tOs g-1 dry wt of soil (Darbyshire and Greaves, 1967; Hea1,

1971). As a component of the protozoan fauna, soil amoebae contribute to the decomposition of organic matter (Aristovskaya, 1963; Ten, 1967) and increase rhizosphere phosphate activity (Gould et aL., 1979). -3-

Soil anoebae are known to secrete heteroauxin (IAA) in lucelne rhizo- pheres (Nikoljuk and Tapilskaja, 1969), deconpose chitin in soil (Okafor, 1966), and to transport aninal viruses (Toorney et aL.,1948; Baron et aL., 1980). Of direct concern to mankind are the pathogenic amoebae which cause a variety of diseases, some fatal, collectiveLy known as amoebiasis (Ewers, 1981). The free-living soil amoebae which cause diseases including amoebic neningoencephalitis have recently been reviewed by Culbertson (1971) '

oehler (1916) demonstrated that the yeast SaecVmromgces eæiguus

Hansen was eaten by anoebae. In feeding trials with L2 species of soil.moulds and 4 species of yeasts, Severtzova (1928) found that amoebae nultiplied with 4 species of moulds and 2 yeasts and suggested that vegetative cells were preferred to spores. Castellani (1950), Negroni and Físcher (1941), Nero et aL. (1964) and Bunting et aL. (1979) showed that a¡noebae feed on yeasts but not on filamento'Js fungi. In 1963' Heal conducted feeding trials with 4 different amoebae using 16 fila- mentous fungi and 19 yeasts. Many yeasts were consumed, but only 2 fungi, Paecilorryces eLegans (Corda) Mason and Hughes ancl PoLyscyaLatum feeundissirm.tm Reiss. acted as food sources. Ingestion of fungal spores by anoebae and ciliates was lreported by Esser et aL. (1975). They foturd that conidia of ALtez,nar"La, Cuz'uuLaria, HeLmínthospot'ium and Fusarium were ingested by a Ihecønoeba sp. but only Fusarium spores were digested. Others have suggested an antagonistic action of amoebae towards plant pathogenic fungi (TapiIskaja, 1967; Mavtjanova and

Nasyrova, 1969) . aLbidø, develop in the rhizosphere of cotton plants, have a pronounced antagonistic effect on Vez'ticiLLiwn dahLiae

Kleb. and protect plants against rvilt (Tapilskaja, 1965; Nikoljuk and -4-

Tapilskaja, 1967). Treatment of cotton seeds with amoebae after arti- ficial contamination with V.dahLiae increased the germination rate and had a favourable effect on the overall developnent of the plant.

Studies on the survival of soil-borne fungi have shown that spores of pignented isolates of fungi survive longer in soil than those of hyaline isolates of the same fungus (01d and Robertson, L970; Clough, 1975). This resístance towards lysis is due to the presence of nelanin compounds in the walls of the pignented isolates (Kuo and Alexander , 7967; 8u11, 1970). However, direct observation of fungal spores, including pignented ones, after incubation in natural soils revealed that spores become perforated by holes varying fron 0.2 to 5.0 Um in diarneter (01d' 1969; 01d and Robertson, 1969; Clough and Patrick, 7972). This has been referred to as rperforation lysist in the review of 01d and Wong (f976) and autolysis, nechanical puncture by soil animals and enzymatic lysis by microorganisms were considered as possible causes of perforations.

Itt L977 01d showed that giant soil amoebae caused perforation lysis of spores of CoehlioboLus satiuus (Ito et Kuribay) Dreschl. ex. Dast. buried in natural soils (01d, L977a). The amoeba was later identified as irnpatiens Cienk. (01d and Darbyshire, 1980). The perforat- ing and lysing activities of this amoeba during attacks on fungal spores have been described by 01d (I977a) and 01d and Darbyshj-re (1978). 0f 50 fungal species tested, 2L were used as food" Conidia, chlamy- dospores, sporangia, teleutospores, basidiospores and hyphae of many fungi of a wide array of taxonomical groupings were suitable food for the anoeba. Anderson and Patrick (1978) also independently described nycophagous amoeboid organisms from soil that perforate spores of -5-

C1n1,ara eLegans Nag Raj and Kendrick l: IhieLatsiopsis basieola (Berk. and Br.) Ferr.) and C.satiuus and believed one to be a member of the VatrpyreLLa. These authors described two other nycophagous amoebae of the genera They,atromyæa and Vanrpgt,elLa which produced snaller perfora- tions of 1 un or less in diameter in conidia of C.satiuus (Anderson and Patrick, 1980). One of these genela, Ihez'atrornyæa is also known to be nenatophagous (lVeber et aL. , 1952¡ Zwillenberg, 1953; Sayre, L973).

Anong the 1

VønpyreLLa and Theratrontgæø belong to the farnily in the classification of the plotozoa proposed by Honigberg et aL. (1964). At least two other g"rr"t., Theeønoeba (Esser et aL., L97S; Alabouvette et aL., L979¡ Pussard et aL., 1979) and Cashia (Pussard et aL., 1980) have nycophagous species. Homrna et aL. (1979) isolated vanpyrellid amoebae frorn wheat field soil by burying pignented hyphae of GaewnannomAces grønLnis (Sacc.) Von Arx and 01ivier. The arnoebae caused perforations and annular depressions in the hyphae similar to those described by Old (I977a). Apart fron these reports information on the significance of amoebae in the ecology and control of soil firngi is generally lacking (01d and Patrick, 1979).

The take-all disease caused by Gaeumannomuces gr'øninis (Sacc.) Von Arx and Oli-vier var. trLticí Walker (C,gt) is considered as the nost inportant Ìoot rot of rvheat throughout the world (Butler, 1961) and is second in causing crop losses after stem (Garrett, 1942). Although take-all tr,as first recognised in 1852 in South Australia (Anon., 1868), it was Prillieux and Delacroix (1890) who reported that a fungus,

OphíoboLus gramLrrLs Sacc., was the causal agent. Since then, take-a1l has been the subject of much research and cornprehensive reviews on its various aspects appear ín Garrett (1942), Butler (1961), Nilsson (1969), Walker (1975), Asher and Shipton (1981). -6-

Despite consj.derable research, take-al1 is still a najor liniting factor of wheat production in South Australia (Stynes, 1975). The development of take-al1 in the field is influenced by cropping history (Louw, 7957 i- Glynne, 1965; Slope and Etheridge, L97L) and less take- all occurs in wheat following oats (McAlpine, 1904; Richardson, 1910; Itthite, 1947), peas (Zogg, 1969) and trefoil (Garrett, 1943) which makes crop rotation one of the rnain control measures against the disease (Butler,1961)

In continuous cereat cropping disease incidence and severity tends to decline (Broadfoot, I934a; Glynne, 1935; Garrett, 1943). However, White (1947) founil an increase in the severity of take-alI after 4 years of continuous wheat. A decline in the severity of take-all after a peak disease in cereal monoculture was reported by Slope and Cox (1964).

Take-all decline (TAD), as it is now known, occurs in nany countries including Netherlands (Gerlagh, 1968), France (Lemaire and Coppenet, 1968), Yugoslavia (Vojinovic, I973), Switzerland (Zogg, 1959), U.S.A. (Shipton et aL., 7973) and England (Slope, 1963; Cox, 1963; Shipton, 1969; Pope and Jackson, 1973; Pope and Hornby, 1975). TAD soils can suppress the saprophytic and parasitic growth of Ggt in pot bioassays (Lester and Shipton, L967; Shipton et aL.5 1973; Pope and Jackson,

I97 3; Wildermuth, 1977).

Suppression sinilar to TAD has been reported under variotrs soil, crop and environmental conditions and ltlalker (1975) distinguished six conditions of disease suppression of which three require the presence of both host and pathogen. Wildermuth (L977 ) found that suppressj.on can also be induced by infection of wheat by Gaewnar¿rpntAces graninis -7-

(Sacc. ) Von Arx and Olivier var. g?Øninis l¡lelker and Gibbez'eLLa, zeae (Schw.) Petch. His work also enphasized that some natural soils, without a history of wheat monoculture, can be suppressive to take-all. Palti (1981) cited more than 20 plant pathogenic fungi which are prevented by soil suppressiveness from attacking crops. However, as

Brown et aL. (1973) pointed out, the developnent of an antagonism to

Ggt in the absence of the host is not necessarily the same as TAD.

Interactions between rnembers of the soil nicroflora and the pathogen have been the frequent explanations of the nechanism of suppression (Gerlagh, 1968; Pope and Jackson, 1973; Vojinovic, I973;

Cook and Rovira, tSlO; Rovira and Wildermuth, 1981). Others have associated virus-1ike particles (Lapierre et aL., 1970), reduced virulence

(Cunninghan, 1975), changes in the NO'-N:NHO-N ratio of rhizosphere soil (Brown et aL., 7973), and hyperparasitisn (Zogg and Jaggi, 1974) with reduced infection by Ggt. Hornby (1979) reviewed the literature and concluded that no single hypothesis adequately explained TAD. The demonstration of nycophagous activities of amoebae in TAD soils (Honma et aL.r 1979) suggests that the role of soil anoebae in suppression of Ggt wa'r'rants further study.

Wildermuth (L977) found reduced numbers of infective hyphae of Ggt in the rhizosphere of wheat in the presence of several suppressive soils including soil from a permanent pasture plot at the Waite Agricultural

Research Institrrte, South Australia. The amoebae of the suppressive pernanent pasture soil and their role in the suppression of take-aI1 have been investigated. lr{ycophagous activities of isolated arnoebae were studied in detail and species which fed on Ggt hyphae ín uitro were -8-

tested for their ability to reduce the severity of take-all in pot bioassays. Saprophytic survival of the fungus in relation to arnoebal populations in suppressive and non-suppressive soils was studied. Population dynanics of mycophagous and other amoebae in wheat rhizospheresin suppressive and non-suppressive soils was also studied to determine the associati-on of these anoebae with the pathogen in its parasitic phase. -9-

II. Í'IATERIALS AND ÍVIETHODS

PHYSICAL AND CHEMICAL PROPERTIES OF EXPERIMENTAL SOILS

The location, classification, particle size distribution, pH and cropping history of the three soils used are listed in Table 1. particle size distribution was determined fron three sanples of each soil using the hydrometer nethod (Piper, lgs0) for nechanical analysis. Three sanples of each soil, passed through a 2 mm sieve were noistened to -0.98 kPa, placed on noist filter papeï inside sintered glass funnels and suctions of -0.98 to -19.6 kpa (10 to 200 cn of water) were applied for two weeks before estination of soil moisture contents. Suctions of -100, -500 and -1500 kPa were obtained using pïessure chanbers. Plastic conduits (2.5 cm 1ong, 3.5 cn internal diameter) with the noist soils were placed on top of a high pressure ceramic plate in the chamber and pressure applied from a compressed air cylinder for two weeks. The moisture characteristic curve of each soil is shown in Fig.l.

ORIGIN AND MAINTENANCE OF FTJNGI AND BACTERIA

Table 2 sltows the origin of the different fungi and bacteria used in this work. The naint"rrtrr." nedia for these organisrns are as follows (composition of the nedia in Appendix I).

cochLiobolus satiuus czapek-Dox agar (in: Raper and Fenne1l, 1965) Gaeumannomyees gratninis tr"iticí NDy/6 (Warcup, 1gS5) PfuiaLophora. sp. NDy/6 Phytophthora e'innønowL Rands. vB juice agar (Miller, 195s) Saccharomyees ceaeÐisíae 29o malt agar Meyen a-nd Hansen KlebsieLLq sp. Trypto'e-Glucose-yeast agar (TGY, Cavender and Raper, 1965) - 10-

TABLE 1: Physical and chenical properties and cropping history of the experirnental soi1s.

(%) Particle size distribution pH Cropping Locality Classification clay silt sand (1: 5 Ilistorya (.2um) (2-20vtt) (>20ym) wat EI )

Avon Gc 1.11 solonised-** 16.9 8.2 74.8 8.6 WP brown soil

Murray Gc 1. 12* Bridge solonised-** 9.5 0.6 89.8 7.7 WPPBPPIV brovrn soil

Waite ' * Institute Dr 2.23 red-brown 24.5 17.6 57.8 6.4 PPPPPPP permanent * pasture earth*

:k Factual key (Northcote, 1979) ** Great soil group (Stace et aL., 1968) a Itl = lVheat P = Pasture B = Barley - 11-

Fig.l: Moisture characteristic curves of experimental soils.

Waite perrnanent pasture.

Avon. Murray Bridge. MOTSTURE CONTENT (o/o)

19 (¡t Þ o o o o o

(, o o t- o o o GI o U' .co -{ à õ z

! ¡

(,r

o

o, -12-

TABLE 2: Origin of the fungi and bacteria used.

Organisn Isolate No. Origin Supplier

CoehLioboLus CS-2 Pine roots, A.C.T. K'M.01d satíuus

GaewnannomAees G.1 Soil, Waite S. Chakraborty grønLnis tritiei pernanent Pasture \4aize, PR-II P .T.W.ltlong PhiaLophorq, sp. (DAR 32oe8) Cambridge, U.K.

Phytophthoz'a Banksía sp., lV.A. K.M.01d cinnønomí ^-2t R?rizoctonia solani xihn F91 Wheat, S.A. J. H.Warcup

WARH/6 Soil, W.A. J. H.Warcup

Saecløromyces J . Iì. Warcup cereuisiae 729

KLebsíeLLa sp. J. H.Warcup -13-

TEST FOR SUPPRESSIVENESS OF SOILS

The pot bioassay (shipton et aL., ]-gTs) was used with nodifica- tions to test suppressiveness of experimental soils. A prelirninary experinent was conducted using a steríle coarse sand (cs) instead of fi:nigated soil. washed and dried sand was thinly spread in flat metal trays, covered with tAlfoilt and autoclaved under dry cycre at tzf} for I h for 3 consecutive days.

ïnoculum of Ggt was prepared by grinding oat grains colonised by the fungus. Oat grains soaked in distilred water overnight were autoclaved at rzl?c for t h on each of three successive days. These were inoculated with agar cultures of the fungus and incubated in darkness at 25oc for B-10 rveeks until the fungus had extensively colonised the oats. The grain was shaken every two to three days to prevent it fron natting together with mycelíun. Colonised oat grains were dried in a laminar flow, ground and sieved through a 2 mt sieve.

GrounÀoat inoculurn was added at a rate of 0.2% (w/w) to 1750 g cs and nixed thoroughly by shaking these in an inflated plastic bag. Soil of the pernanent pasture plot (l\tPP) in the rotation experiment at the lVaite Agricultural Research Institute, South Australia, was usecl as the suppressive soil (wildermuth, rg77). The wpp soil was added at rates of r% and 70% (w/w) and the rnixing repeated. I'hree hundred and fifty g of the mixture was aclded to each 300 cm3 plastic pot and 5 wheat seeds of cultivar Halberd were spread over the surface. rn control pots, au-uoclaved ground oat inoculrrn (0.2% w/w) of Ggt was added. Seeds were coverecl lvith 50 g cs (without Ggt o'r lvPp soil) in -74-

each pot and 50 nl distilled water added. This resulted in 15% moisture in CS (oven dry wt basis). Five replicates were used for each treatnent and pots were randonised. All plant growth experiments were conducted in growth rooms with a L2 h day-length, naintained at

15t1oc and provided a light intensity of 10760 1ux at the height of the plants. Eveqy second day, pots were $iatered to the original wt and height of plants neasured.

Plants were harvested four weeks after seeding, roots washed free of adhering CS and rated for take-all (Cook and Rovira in Wildermuth, 1977). Details of the scale of disease rating is as follows:

Disease ratlng Description of typical plants

0 No visible sign of disease 1 Lesions on < three serninal roots; no lesions on stem 2 Lesions on > three seminal roots; no lesions on stem 3 Lesions on all Toots and discrete lesions on the base of stem 4 Lesions on all roots and lesions coalesced around base of stern 5 As 4 but necrosis around stem base more severe 6 Seedling so severely diseased that only the first leaf produced; leaf still green 7 Seedling conpletely necrotic

The growth rate of plants in the different treatments is shown in

Fíg.2. The results (Table 3) show that CS can be used as a growth nediurn to test suppressi-veness of soils.

SUPPRESSIVENESS OF EXPERIMENTAL SOILS

Suppressiveness of the Âvon, Murray Bridge OfB), and WPP, along with soils from tlrree other permanent rotation plots at the Itraite - 15-

Iig. ¿t Mean height of wheat plants during growth in sterilized coarse sand, inoculated with dead or live G. graminis trit¿eL, or live Ggt pLus two levels of the suppressive Waite pernanent pasture soil.

o-.-{ Control (autoclaved Ggt). H Live Ggt plus Waite permanent pasture soil 10%. EH Live Ggt plus Waite pernanent pasture soil 1%. o----o Li-ve Ggt. 30

20 E I J¿ t- ñ-t It O-u ul T o-o t-z 10 J o- I

812 16 20 2/. 28 DAYS AFTER SOWING -16-

TABLE 3: Height and disease rating of wheat gror¡In in sterilized coarse sand, inoculated with dead or Li-ve Ggt or live Ggt plus suppressive soiL.

Treatment P1ant height Disease rating (cn)

Ißgt + ItrPP soil (70v") 2t.t 2.0

LGgt + WPP soil (l%) 16.8 3.2

LGgt 14. 5 4.7

DGgt 24.0 0.0

Least significant difference P = 0.01 2.33 0.62

* L = Live D = Dead -17 -

Institute was determined using the nodified pot bioassay described earlier. The length of rotation in the Waite Institute plots was

27 years in WPP, 55 years in the continuous-wheat (CW), 53 years in the pea-wheat (PeW) and 27 years in the Pasture-pasture-wheat (PPW). The test soils were used at a rate of J'% (w/w) in all cases.

Results (Table 4) show that the WPP, PeW and PPW are suppressive, and

Avon, MB and CW are non-suppressive to take-all. -18-

TABLE 4: Disease rating and plant heights obtained with Ggt and different soils added at a rate of I% to sterilized coarse sand.

Treatnent Disease rating Plant height (cm)

* LGgt + Avon soíI 4.6 15.0

+ MB soil 4.4 L3.4

+ I.IPP soil 2.9 16 .6

+ PPIV soil 2.0 16. 5

+ Peltl soil 3.3 15. 1

+ CW soil 4.L 14.0

LGgt 5.4 12.9

DGgt 0.0 18.8

Least significant difference P = 0.01 r.42 2.0

* L = Live D = Dead -19-

III. STUD I ES ON SO I L AI4OEBAE

A. ISOLATION, NÍAINTENANCE AND CHARACTERIZATION OF AMOEBAE

The ¡nethods of examination and isolation of soil amoebae can be broadly placed into three groups - (i) direct examination; (ii) ex- traction fron soil; and (iii) culture (Heal, I97l). Koffmann (1932) used direct microscopic exanination for counting numbers of soil amoebae. Although this seens to be the logical nethod to overcone selective culture techniques (Bunt and Tchan, 1955), its application is linited nostly to freshwa,ter rhizopods (Leidy, 1879; Page, 1976) as generally microscopical observation of soils does not reveal more than shells of testaceous rhizopods and occasional ciliates (Sandon , L927). Martin and Lewin (1915) extracted trophic amoebae using picric acid and by bubbling a steam of air through soil suspensions. Howevet, due to the ease of culturing and identifying anoebae on agar plates, soil plating (Sandon, L927; Page, 1976) and soil dilution cultures (Cutler, L920;

Singh, 1946a) have been widely used.

For isolation of mycophagous amoebae, 01d. (I977b) deposited spoles of CochLiobolus satiuus on mernbr:ane filters, enclosed thern in filter packets and buried thcse in soil. Arnoebae from the recovered rnembranes were cultured with fresh spores of the fungus in water. Anderson and Patrick (1978) baited soil suspensions with conidia of the sane fungus and isolated, by pipetting, digestive cysts of rnycophagous anoebae that surrounded partially lysed spores. Pussard et aL. (1979) userl neomycin -1 -1 (10-100 ug ml ^) and rifamycine (L00 ug ml ') for selective isolation of the strictly mycophagous amoeba Thecønoeba gz,anifera (Greeff, 1866) sub- species mínor Pussarcl et aL., 1979. -20-

Water agar (Cutler, 1920), nutrient agar and dilute hay infusion

agaT (Sandon, 1927; Bodenheiner and Reich, 1933), soil extract agar (Dixon, Lg37), NaCl-agar (singh, 1946a), silica gel (singh, 1946b) and several other nedia (Page, I976), enriched with a food bacteriun, have been used successfully in culturing soil anoebae. Axenic cultures are difficult to obtain and although pure culture of anoebae has been

known since 1898 (Tsujitani, 1898) few anoebae have been grown in axenic culture (Neff, 1957; Griffin, L973; De Jonckeere, 1977; Surek and Melkonian, 1980). Most soil amoebae are naintained in ¡nono-axenic cultures using bacteria, algae or other protozoa as food (Page, 1976).

KLebsieLLa pneunoníae (Schroeter) Trevisan (=Aetobacter aerogenes (Kruse) Beijerinck) supports the growth of a wide number of amoebae (Singh, 1946a), Bacteria ancl sna11 protozoa, which are associated with anoebae in soil and which usually grow in nedia used for culturing arnoebae can also serve as food organisms (Page, L976); although some accompanying bacteria can have antagonistic effects on amoebae (Groscop and Morgan,

1e64) .

Anoebae ale most abundant in the top 0-10 cm of the soil profile (Crump, 1920; Stout, 1968) and both population and diversity rapidly

decline wj th increasing depth (Sandotr, 1927).

Isolatiorr of amoebae

Soil was collected from the top 0-5 cm layer of the l{PP plot and kept in sealed plastic bags. Four nethods of isolation were used:

G) The soiL pLate method The nethod consists of incubating snall anounts (approx. 0.5 g) of soil on a suitable aga'r rnediun enrj.ched with a food bacterium and -2L-

exanining for amoebae. Initially 1.0% Bacto agar' in distilled water spread with a suspension of a KLebsieLla sp. was used. Plates were incubated at 25oC for 7-74 d,ays and examined using a phase contrast microscope. 9"ty Acanthønoeba sp. was present 9 ro b abtY on these plates. This was(due to the osmotic shock in water agar (Page, 1976) and therefore the agar nedium was amended with a salt solution. The modified medium was 1.0% Bacto agar in Prescott and Jamest solution (Prescott and Janes, 1955; PJA, Appendix I).

Petri, plates poured with this nedium tlere similarly enriched with bacterial suspension and inoculated with a soí1 crurnb (about 0.2 Ð. On incubation several arnoebae, including Aeanthamoeba sp., PLatyamoeba sp., Thecønoeba sp., and unidentified leptonyxid and varnpyrellid amoebae, were observed on these plates. Although srnaller species rlke AcantLnmoeba, PLatgamoeba etc' could be seen on 1 ,.ì the clear agar meclium after about 1 week, the larger lepto- nyxid. and vanpyrellicl anoebae took up to 2 weeks to appear. Sinrilar results \¡/ere reported by Page (L976), tvho recornmends an incubation of at least 2 rveeks and the same was followed throughout this work.

(¿¿) Isolation from soiL suspension

TWo grams of soil were suspended in 20 nl sterile rnodified

Neff's amoeba saline (Page, I967a; AS, Appendix I), shaken and allowed to settle for a few min (5-5 min). Snall poTtions (about 50 ul) of the suspension l\tere plated on PJA with a KlebsieLLa sp. Plates were incubated as previously and when -22-

exanined showed Aeanthømoeba spp.' Echinønoeba spp.' Gephyz'ønoeba sp.¡ PLatyønoeba spp., Iheeamoeba spp., and the unidentified leptomyxid and varnpyrellid anoebae.

(í,i¿) SoiL ew"Lehment method soil noistened to -8 kPa was dispensed into 15 cn Petri plates to a depth of 7-8 nm. Mycelial mats of Ggt, obtained from

one week old cultures grown in 0. 4eo malt extract broth at 25oC

were washed twjce in sterile distilled water (SDW), placed in auto- claved silk cloth bags (5x5 cm) and buried in the noist soil. After 3 weóks at 25oC, nycelial mats were ïecovered and honogenised to forn a suspension, portions of which were streaked on PJA- Acant?n¡noeba spp,, Echinamoeba spp., Gephyz'amoeba sp. ' PLatyamoeba spp., Mayot'eLLa sPP., Ihacamoeba sPP., and several unidentified

arnoebae besides the unidentifieci leptomyxid and vampyrellid anoebae which migrated on to PJA medirrm were j'solated'

6p) Membnane f'LLter bt'n"taL method Five fungi and one bacteriurn, KLebsieLLa sp., ü¡e1.e buried

in the IVPP and MB soils using the membrane filter burial technique of old (Lg77b). Myceiial suspensions of. Ggt' Phialophora sp-

and Rhizoctonía soLani (F91) rvere obtained by homogenisíng 0.4% nalt extract broth gror\tn cultures of the fungi after washing in SDW' Conidia of CoehLioboLus satí¿uus were harvested from Czapek-Dox cultures by flooding plates with SDI{ and lightly rubbing each colony with a sterile 1oop. Suspensions of conidia were filtered through sterile cheese cloth and washed twice in SDItt' Suspensions

of Saachal,ornAces eeTeuisiae and KLebsieLLa sp. were obtained by -23-

washing 2eo na1-t extract agar and TGY cultures respectively with

SDW. One nl of suspension of each organism was drawn up with a sterile syringe and deposited on a L2 mm diameter nillipore filter (pore size 0.45 un) held in a rswinexf membrane holder (Millipore Corpn., Bedford, Massachusetts). The nillipore filter'was sandwiched between two 25 nm diarneter nuclepore filters, pore size 5.0 pm (Nuclepore Corpn., Pleasanton, California), the edges sealed with vacuum grease and buried in the test soils.

WPP and MB soils were sieved through a 2 mm sieve, noistened to -B kPa and -2 kPa respectively and dispensed into 9 cn dianeter

Petri plates before membrane burial. Petri plates were incubated at 25oC for two weeks. SDW was used as a control.

At recovery, membranes were trinmed to exclude the peripheral areas and each was cut into 4 approx. 1 mm squares using a sterile sca1pe1. These squares were plated on PJA, incubated at 25oC for two rveeks and examined for amoebae. Several amoebae were iso- lated frorn these soils: genera ITke Aeartthamoeba, PLatyamoeba and the unidentified vampyrellid amoeba were isolated using almost all the test organisms, whereas, Echiramoeba, Ihecamoeba and the lepto- nyxids were isolated only using fungi (Table 5).

The previously described nycophagous genera, At'achnuLa,

Theratxontyæa and (01d, L977a; 01d and Darbyshire, 1978;

Anderson and Patrick, L978, f9B0) were not isolated from these soils using these techniques. 01d (1977a) recovered Anacht'tuLa from a garden soil using the rnembrane filter burial technique and naintained cultures of the amoeba in SDIV rvith suspensions of C.eatiuus conidia. Menbranes recovered fron I{PP and MB soils were TABLE 5: percent recovery of different genera of soil arnoebae fron two soils using different organisms as baits

Acanth- EehLn- Leptonyxid MayoreLLa PLaty- Theeamoeba T.granífena Unidentified Vanpyrellid 0rganisn amoeba ønoeba amoeba ønoeba ssp. mínoz' amoeba anoeba spp. spp. spp. spp. spP.

Waite permanent p asture soil': uvvn^+ 20 5 0 5 40 0 20 40 5 C. satiuus 20 15 10 5 40 0 60 70 0 20 5 4S 90 30 I PVrLaLophora sp, 75 30 15 0 Ì!À I RVrLzoctonia 10 0 5 0 30 60 45 solani (F91) 55 5 S. cet,euisiae 30 10 0 0 30 5 10 60 30 KLebsieL1.a sp. 55 0 0 0 15 0 0 35 15 Control 20 0 0 0 10 0 0 0 20 Murray Bridge soil:

Ggt 55 0 0 5 55 0 6s 100 90 C. satiuus 75 ^ 0 0 20 5 25 70 70 PhiaLopho?a. sp. 65 45 0 0 70 0 80 95 75 Rhizocotonia 0 70 5 75 85 50 soLarri (F91) 70 20 0 S. eet'euisiae 70 0 0 5 45 10 0 65 80 KLebsíeLLc¿ sp. 45 0 0 0 20 0 0 55 35 Control 10 0 0 0 10 0 0 15 0 -25-

therefore placed in SDW and exanined with an inverted nicroscope using phase contrast. Both Az'achnula and Thez'atz'ornyæa wete observed in the suspensions recovered fron these soils. So far, atteilpts to grow these amoebae on agar nedia have been unsuccess- ful (K.M.01d, personal conmunication) and this nay be the reason for not obtaining these genera on agar. plates from the WPP and

MB soil using these techniques.

Purification and Maintenance

Single trophozoites of arnoebae fron nixed cultures on isolation plates were mark"¿ un¿ transferred to fresh PJA + KLebsieLLa plates by cutting out small blocks of agar. Single arnoeba cultures were establ- ished by repeated transfers of single trophozoites and gradually freed fron accompanying soil bacteriaby using Neff's (1958) technique as modi- fied by Culbertson (1971). Four to five pieces of filter paper, each soaked with approx. 20 uI of neomycin solution (concentration 3.5 units _1 U1-') were placed on the periphery of a PJA plate inoculated in the centre with an anoebal culture. funoebae which nigrated to the bacteria-free zone around the filter papeT were subcultured on PJA +

KLebsieLLa plates. This nethod is suitable for removal of surface-borne bacteria but does not elininate endosymbionts (Griffin, 1973), therefore, these cultures can not be referred to as monoaxenic cultuTes. The mycophagous Thecatnoeba granífena sub-species nrLnot'was associated witl'r a

FusarLum sp. rvhen isolated and was rnaintained rvith this fungus without KLebsieLla sp. -26-

Cultural characters of Amoebae

ft) Gtouth responses of ønoebae to diffeTent tenrpeTatunes In rnany pTotozoa increasing temperatute up to about 30oC favours netabolism and reproduction (Reich, 1933) ' Higher temperatures a]:e adverse with a death point in the range of

35-40oC in some protozoa (Maguire, 1960), but cysts can survive

nuch higher temperatures (Stout, 1955). The effect of tenperatuÏe on the nultiplication rate is different for different amoebae or

even strains of the same amoeba (Hawkins and Danie1li, 1965)' however, nost of the work on temperature sensitivity of arnoebae

has been done with pathogenic species. TernpeTature tolerance is often used to distinguish between pathogenic and non-pathogenic

amoebae (singh, 1975). Most soil arnoebae grow within a range of

18 to 25oC ancl pathogenic strains need 37oC (Page, 1976), although not all amoebae which grow at 37oC are pathogenic (Griffin, 1972) and sone potential pathogens do not grow at this tenpeÏatuTe. Non-cystic pathogenic arnoebae in tissue specimens are destroyed by ordinary freezing at -zOoC (Ensminger and Culbertson, 1966) whereas cysts can survive storage in liquid nitrogen for up to

seven yeal-s (Neal et aL., Lg74). Sinple relationships between temperature and amoebal activity in soil have not been shown.

Nine different amoebae were tested for their growth

responses to tenperatures from soC to 35oC. Fíve mm diameter discs from 2-5 weeks old cultules vleTe inoculated to PJA +

KLebsieLLa plates. Three replicates of each of the test arnoebae were incubated at each test tenperature for 10 days ' Results on the colony diarnters (Table 6) show that rrtost of the amoebae grel -27-

TABLE 6: Growth lesponse of anoebae to different tempelatules.

Mean colony diameters (cm) at various tenPeratures (oC) Anoeba 51 01520 25 30 5s

AcantVømo eba p olypVng a 0.6 8.2 8.5 8.5 8.5 8.5 0.7

Gephyz,amoeba sp. 0 0 3.5 5.1 4.2 5.4 0

Unidentified lePtomYxid 0 1.8 8.5 8.5 8.5 0 0

PLaty amo eba st enoP o &La 0 2.7 5. t 8.5 8.5 8.5 0

Saccamoeba sp. 0 5.6 7.5 8.5 8.5 0 0

The cøno efu quaårí Lineat a 0 8.1 8.5 6.0 5.6 0 0

T, grarrLf er'q. ssp .

¡nLnot' 0 r.4 8.5 8.5 8.5 8.5 0

Unidentified amoeba (Un.1) 0 8.5 8.5 8.5 8.5 0 0

Unidentified vanPYrel lid 0 0 0.7 2.9 2.0 1.0 0 -28-

well at temperatures between 15oC and 25oC' and only Aeqnthamoeba poLyphaga (Puschkarew, 1913) was able to grow at temperatures of soc and sSoC. The results are in agreement with those obtained with other soil arnoebae (Page, 1976). The najority of the amoebae grew wel I at 25oC and this has been used as the incubation temperature throughout this work.

(¿i) ThermaL death points of ønoebae Trophozoite and cyst suspensions of individual amoebae were prepared by washing the surface of 2 weeks old PJA cultures with 5 ¡nl AS. ,Two nl of these suspensions $IeTe placed in snall test tubes and partially inrnersed in a lvater bath equipped with a TotaTy shaker (Orbital shaking vlater bath, Model OltI 1412, Paton Industries Ltd., S.A. 5069) which was pre-heated to a constant temperature. Control tubes had 2 nl of AS with a thermometer in then. Treatment time of 10 nin was recorded fron the tine when the AS in the contïol tubes reached the water bath tenperature. About 0.1 ml of cooled (roon tenp.) suspension was plated on PJA + KLebsielLa plates. No bacteria were added for T.granifena sub- species minoy. Plates were examined for antoebal glowth after two

weeks at 25oC.

Acanthqmoeba poLyphaga and Echinamoeba sp. snrvived tenpera- tures of up to B4oC (Table 7). Gephyr'ønoeba sp. rvas nost sensi- tive and did not survive beyond 40oC. The najority of the

amoebae were killed at or below 70oC. Of the cyst-forming arnoebae,

AeantLn¡noeba and EcLrLnamoeba survived very high temperatures,

whereas Gephyratnoeba and the unidentified leptonyxid and the

vampyrellid anoebae hrer.e killed at relatively lower temperatuTes' -29-

TABLE 7: Thermal death points of anoebae.

Amoeba Temperature (oc)

Ac anthØho eba p o Lyphng a 84

Eehi,nønoeba sp. B4

Gephytønoeba sp. 40

Platy øno eba st enop odia 70

Saecønoefu, sp. 65

Thee ønoeba quadr"L Lineat a ó5

T.g,anifena ssp. minor 65 Unidentified leptonYxid 60

Unidentified vanpyrel 1id 55 -30-

Although cysts are generaLLy regarded as ¡nore tolerant to high tenperatuïes than vegetative amoebae (Singh, I946a; Stout, 1955), cysts of all amoebae do not have sinilar temperature tolerances (Bodenheiner and Reich, L933; Sopina, 1968).

ftä) Effeet of antibioties on the grouth of amoebae A considerable amount of infonnation is available on the effect of various antibiotics on free-living soil amoebae (Hawkins, Ig73). These include both antibacterial and antifungal antibiotics (Loefer, 1951; Loefer and Matney, I9S2; Blumberg and Loefer, L952; l(alinina, 1969). Actinomycin D and othel anti- biotics produced by the Actinonrycetales nake these bactería un- suitable as food for anoebae and also suppress amoebal growth in soil (Geltzer, 1963).

The effects of four concentrations of each of two antibacterial and one antifungal antibiotic on the grolth of ten different

amoebae were studiecl usì-ng the poison food technique (Batenan' 1933). Stock solutions of each of the three antíbiotics, streptomycin, neonycin and actidione tvere rnixeci with 100 ml luke

warm PJA to obtain final concentrations of 10, 50, 100 and 200 Ug _1 m1-I and plates were poured rvith approx. 20 nl mediun. All plates except those to be used for T.gz'anífera sub-species minot', were enriched witin KLebsieLLa sp. Plates were inoculated with a 5 nun dianeter disc from 2 weeks o1d arnoebal cultures. Colony dianeters after two rveeks of incubation at 25oC show that the responses vary

with the antibiotic and the anoebal species (Table 8) ' TABLE B: Mean colony diarneters (cn) of amoebae on PJA incorporated with different levels of antibiotics.

Str 1n Neomyc Actidi u ) u 1- Amoeba Control 50 100 20 50 0 10 s0

Acanthamceba 8.5 8.5 7 .9 6.8 2.6 8.5 8.5 8.5 8.5 8.5 8. s 3.0 + poLyphnga +Jr + + + + + + + +++ Gephyramoeba sp. 2.9 3.0 7.4 0.2 7.7 7.2 1.9 L.4 + + + + + + + +++ + 7.4 8.5 6.1 4.7 0.1 7.3 6.t 3.5 PLatyamoeba I stenopodia + + + + + + + + + + + + (^ I Saecønoeba sp. 8.5 8.s 8"5 7.0 8.5 8.5 8.5 8.5 8.5 8.5 8.s 8.s 8.s + + + + + + + + + +

Thecamoeba 8.5 1.5 0.4 3.7 2.6 8.5 0. 3 4 .2 0.6 sphaeronucleolus + + + + + + + + + + + + Thecamoeba 8.5 6.9 4.9 2.8 1.s 8.s 8.s 8.5 7.9 quadxiLinea'ba + + + + + + + + + + + + I.gz,anifera ssp. 8.5 8.5 8.5 8.5 1.1 8.5 8. s I .5 8.5 ninaz, + + + + + + + + + + + Unidentified 8.5 8.1 8.4 7.4 5.5 8.s 8.1 8.5 8.5 8 .3 8.5 8.5 8.5 amoeba (Un.1) + + + + + + + + + + + + Unidentified 7.5 8. 1 2.0 I .5 0.7 s.8 7.4 7.8 7.7 leptonyxid + + + + + + + + + + + Unident ified 3.7 1.1 0.6 1.6 1.0 1.s 0.8 vanpyrellid + + + + + + + + + + + *Growth of the food organisns i K\ebsieLLa sp. for all arnoeba, except for ?. granifera ssp. minor" in whi.ch case it was Fusaz'iwn sp. -32-

The growth of the food bacterium was effectively inhibited at

200 ¡rg n1-1 of streptonycín and that of tine Eusariwn sp.,used to -1 maintain T.granifez"a sub-species minoz', at 100 ug ml of actidione. Saceunoeba and an unidentified anoeba (Un-l) were not affected by any of the antibiotics at the concentrations used, P\atyanoeba stenopodia (Page, 1969), Thecønoeba sphaet'o- nucLeoLus (Greeff, 1891) and AcantVtamoeba poLypVnga were affected at higher concentrations of some or all the antibiotics. One significant observation $¡as the effect of actidione on the growth of Theea¡noeba qu.aáriLineata (Carter, 1856) , I.granifera sub-species nrinor, GepVtyramoeba and the unidentified leptornyxid and vampyrellid amoebae, which failed to gTow even at the lowest concentration of _1 10 pg rnl-' of this antibiotic. $¡ith actidione treatments, although the food organisms glew at lower concentrations, 5 out of

10 amoebae failed to gïovl at any concentration of this antibiotic. Thus, it appears that the effect of actidione at aLI concentrations and streptonycin and neonycin at higher concentlations had direct effects on the growth of the amoebae. The effectivity of acti- dione treated soil in liniting protozoan activity is known (llabte and Alexander, 1975).

(iu) SuitabLlity of uarLous brands of agat for the cuLtiuation of ønoebae Two concentrations of each of three different brands of agar, Difco bacto, Davis, Oxoid No.3, and agarose were tested for their suitability to support the growth of seven soil anoebae. Gel strength of 1.0e" Difco bacto hlater agar was used as the standard and concentrations of other agars were varied to natch their ge1 strength with that of the standard. Gel strength was subjectively -35-

tested by cutting uniform thicknesses of each agar with a scalpel after they had solidified. WateI agar plates v/ere inoculated in the centre with a 5 nm diameter disc taken from a stock culture of one of the test anoebae after a uniforrn suspension of KlebsieLLd sp. had been spread on each plate. Colony diameters were measured after an incubation of 2 weeks at 25oC, Difco bacto and 0xoid no.S proved to be better than Davis agar (Table 9).

Agarose was the least effective and GephyrØnoebq did not gIo14, on either concentration.

The difficulties in obtaining axenic cultures of free-living arnoebae is rnainly due to the lack of knowledge on their nutrition- al requirements (Griffin, 1973). Reich (1948) found that the respiratory rate of fed MayoreLLa paLestinensis Reich, 1933 is greater than that of starved ce11s and nuch greater than that of the cysts. The respiratoTy Tate was also slightly higher in a nutrient solution than in a balanced salt solution and the amoeba could metabolise added peptone. Nutrient toxicity, however, is

mor.e conmon and amoebae generally do not grow well on high nutrient nedia which support the growth of many inedible and toxin producing bacteria (Singh, 1948). Ionic strength of a nediun has a profound effect on the growth and reproduction of anoebae (Griffin, I973).

NaegLerLa gruber"L (Schardinger, 1899) rnoves four tines faster in

20 nM ICl than in deionised water and non-electrolytes do not affect loconotion (King et aL., 1979). Elevated osmotic pressure,

Cr2*, Mg2* and a decrease i. carbon sotlrce on the other hand,

causes encystation in HattmanneLLa rhgsodes Singh, 1952 (Band, 1963). A weak saline solution favours the gro$tth of nany soil

amoebae (Singh, 1955; Page, 7976). Of the four brancls of agar -34-

TABLE 9: Growth of amoebae on various water agars.

Colony diameter (cm) Anoeba Di acto AV ES no 0.5 0.7, 0 1.0

Gephynamoeba sp, 0 0 4.0 3.0 2.5 3.1 5.3 2.9

PLatyatnoeba 4.7 stenopodia 2.6 s.2 4.0 4.6 5.3 4.L 2.7

Theeamoeba 6.1 quadz,iLineata 1.2 0.9 6.8 6.0 4.2 4.8 3.9

T.granifeTa ssp. 3 5 8. s 8.5 m1,nor. ' 4.2 2.6 6.3 6.7 8.2 * Unidentified 8.s amoeba (Un.1) 4.9 3.9 8.5 8.s 8.5 8.s Unidentified 7 7 .7 leptomyxid 4.L 4.4 7 .L 8.5 6.0 6. 0 .2

Unidenti fied 0.7 varnpyrellid L.7 0"8 2.0 1.3 0.6 0.6 2.4

* Not tested -35-

tested, Difco bacto, which proved better in supporting the growth of the amoebae tested, had a higher concentration of Na and K than the other brands tested (J.H.Warcup, unpublished data). -36-

B DESCRIPTION AND IDENTIFICATION OF AMOEBAE ISOI.A,TED FROM THE IVAITE PERMANENT PASTURE

Dangeard (1900) considered that nothing ís more difficult than to identify an amoeba. Similar opinions have been voiced before and since (Page, L967a). The difficulty was attríbuted to the nature of the organisns (Gruber, 1885) and inadequate descriptions (Arndt, 1924;

Jepps, 1956). Both Penard (1902) and Schaeffer (1926) held that arnoebae do have characteristics naking identification possible. Bovee and Jahn (1973) listed rnore than 40 characters, including biochemical and physio- logical ones, used by various workers to distinguish beÛveen anoebal groups. They cónsidered that no genus based solely on norphology is adequate. Horvever, most work on amoebal identifícation, including the nore recent (Bovee, L972; Page, 1972,L976,1977; Pussard and Pons, L977; Page and Elakey, 1979), is based mainly on the norphological characters.

Two distinct trends have occurred rvith respect to the criteria used: structure of pseudopods and method of locomotion, and patterns of nuclear division (Singh, 1952; Bovee, 1953; Page, 1967a; Pussard, I973).

I have attenpted identification of isolates using trophozoite and cyst nrorphology, locomotive behaviour and cytological structures. Nuclear division and anoebo- transformation were not studied.

Methods

The hanging drop technique, Cruickshank culture chanbers (Sterilin Ltd., Teddington, Middlesex, England) and agar cultures vJere used to examine live trophozc¡ites by phase contrast nicroscopy. Occasionally agar plates were also exarnined by bright field illtrmination by adjusting the condenser. -37 -

For preparing hanging drops, PJA cultures were flooded with AS and suspensions of trophozoites were drawn into sterile pasteur pipettes. Drops of suspensions were placed on cavity slides and vacuum grease applied to seal the coverslip placed on the cavity. Slides were left inverted for up to 6 h before exanination. Measurements, except in most cases those of nuclei, were made on live trophozoites.

For studies of the nuclei, the orcein staining nethod for nematodes (Triantaphyllou, 1975) was used. Trophozoite suspensions deposited on glass slides were incubated in Petri dish noist chanbers overnight at

25oC to a1low better adhesion and production of well spread forns of trophozoites. Plates were flooded with 2.5% glutaraldehyde in AS and the anoebae fixed at zSoC for 6 h to overnight. Amoebae were stained with 1% aceto-orcein for 25-30 min, destained with 45% acetic acid for

3-5 sec, mounted in 45% acetic acid using a coveTslip and sealed with nail polish.

The anoebae studied

Acanthamoefu polypløga (Puschkarew, 1913) Trophozoites are more oï less elongated to triangular in shape with a broad hyaline ectoplasnic zone at the advancing nargin (Fig.5a).

Numerous slender and tapering , often v¡ith a pointed tip arise from the ectoplasmic zone (Fig.5a). Acanthopodia, as these are conmonly called, are also present in the postelior part of the body. Often bright field observation provides a better outline of the amoeba with the acanthopodia. One to many contractile vacoules are prorninent within the dense cytoplasm (Fig.5b). Trophozoites measure 10-25 pn in length and 6-15 pm in breadth. Rate of loconotion is of the order of -38-

Fig. 3: Aeantln¡noeba poLyphaga .

a A live trophozoite showing the hyaline ectoplasn (Ec) and acanthopodia (arrows).

b Glutaraldehyde-fixed trophozoite after orcein staining showing a contractile vacuole (Cv) and the nucleus (N).

c Cysts showing endocyst (End) and ectocyst (Ect).

(Scale bar = 10 pn for all Figs.)

-39-

0.33 - 0.9 un ,"r-1 and is usually unidirectional. Trophozoites are uninucleate and nuclei can be observed in phase contrast. Stained nuclei (Fig.3b) are spherical to elongate, measure between 2-6 ym in diameter and contain one darkly stained nucleolus.

Cysts are rectangular to polygonal in shape with two distinctly visible walls (Fig.3c). The thick endocyst often appears as 4-6 radiating rays. The ectocyst is thin wal1ed, often wrinkled and loosely applied and usually does not confom to the shape of the endocyst

(Fig.3c). Cyst diameters range fron 11 - 18 un.

The genus AeantVwnoeba Yolkonsky, 1951 is well characterised, specially by the cyst r4rhich forns the basis for species differentiation (Page, 1975,1976; Pussard and Pons, 1977). According to the tropho- zite and cyst rnorphology, this anoeba resembles Aean'bhamoeba polyphaga (Page, I976).

Eehinønoeba sp.

Active trophozoites are sornewhat triangular or elongate with a distinct anterior hyaline ectoplasmic zone. Several fine long and slender pseudopodia, often with a pointed tip, and commonly known as filopodia are produced fron the ectoplasn (Fig.4a), The filopodia are generally longer and möre slender than the acanthopodia typical of the genus Acanthcmoeba, The flattened nature of the trophozoites is evident in bright fielcl (Fig.ab). Their dimensions, including filopods range fron l0 - 45 um in length and 5-35 um in breadth. Generally 1-5 contractile vacuoles are seen in the finely granular endoplasn. Tropho- zoites are uninucleate and the nucleus measures 2-4.5 pn in diameter with a darkly stained nucleolus 0.6 - 1.5 Um in diameter. Cysts are -40-

Fig..j_t Echinamoeba sp.

a Live trophozoite showing the ectoplasn (Ec) and filoPodia (F).

b Live trophozoite in bright-fie1d.

c Cyst showing the single nucleus (N).

' d. Cysts showing a range of commonly observed shapes and sizes.

(Scale bar = 5 prn in all Figs.) Ç*' 't -41-

generally oval, elongate or obpyriform (Fig.4d) and measuTe between

10-30 Uur in diameter/length. They have a thick wall apparently con- sisting of three layers (Fig.4c). The prorninent single cyst nucleus can be easily seen in wet mounts (Fig.4c).

In 1975, Page created a new family rEchinanoebidaer to accornmo- date snal1 solitary non-parasitic amoebae with fine, non-anastomosing pseudopodia. He included two of his newly described genera Echinønoeba and Staeltyamoeba along wítln FíLamoeba Page, 1967 in this fanily; differentiating then nainly on the basis of their filopod and tropho- zoite dinensions. While the cysts of. Stachgamoeba are clistinct from those of the other two genera, Ech:inamoeba and FiLamoeba have sone over- lapping cystic and trophozoite characters. Trophozoites of my isolate uratch Filatnoefu. noLaná.i Page, 1967 (Page, L967b) in their size, however filopodial and cystic characters are nore like those of Eehirnrrtoeba and I have tentatively placed it in the latter genus. Two species of

Eehírwnoeba, E.erudnns Page, 1975 (HartmanneLLa eæudans, Page 1967) and E.siLuestris Page, 1975, are known (Page, 1975) but ny isolate does not entirely fit the description of either.

Gephyramoeba sp.

The trophozoites are more or less filamentous and branched and vary widely in their size due to anastomosis between individuals. In liquid nedium trophozoites may extend over a wide area, up to 700 sq. Uil., and branch profusely, with the branches generally of uniforn rvidth to give a ribbon-like appealance (Fig.5a). On agar, branching is linrited and tends to be dichotomous (Fig.5b). A limax forn has not been observed for this anoeba. The size of a trophozoite varies depending on the -42-

Fig.5: GepVtynanoeba sp.

a Trophozoite in liquid mediu¡n showing its ribbon-like appearance.

b Dichotomously branched trophozoite on agar.

c Part of a trophozoite showing an orcein- stained nucleus (N).

d Trophozoites withdrarving pseudopodia to round-up as a result of linited oxygen supply.

Cysts.

(Scale bar = 10 um in all Figs. )

-43-

nurrber of anastomoses it has undergone. A range of 80 - 750 pm in length, and 50 - 130 pn in breadth has been observed in liquid medium.

In phase contrast, the cytoplasn of live amoebae appears to be devoid of any prominent organelles. The distinction between the hyaline ectoplasm and the finely granular endoplasm is only visible at high nagnification. Pulsating vacuoles, 1 - 30 or more per trophozoite, with a diameter of approx. 1.5 - 17 pm, are the only cytoplasnic structures visible in live trophozoites. Nuclei can only be observed in stained specimens (Fig.5c). Young trophozoites formed by the excyst- ment of uninucleate cysts are uninucleate but soon develop into multi- nucleated individuals by anastomosis. The number of nuclei in a trophozoite varied from 1 - 25 or more. The nucleus is spherical to oval in shape, with a diameter of 1.6 - 4.1 um and contains one darkly stained nucleolus which varies fron 0.8 - 2.5 ym in dianeter (Fig.5c). The tips of pseudopodial branches often have a cluster of lobose pseudo- podia (Fig.Sc). The anoeba has a very slow rate of movernent. Tropho- zoites in and around the centre of Cruickshank chambers often cease locomotion, rvithdraw pseudopodia, gradually round up (Fig.5d) and finally die, presumably due to the lack of oxygen. A sinilar pathologi- cal process has been observed ín GepVtynamoeba deLícatuLa Goodey, 1915

(Pussard and Pons, 1976.c).

The anoeba forrns thin-wa11ed spherical, oval or almost oblong cysts

(Fig.5e), L2.8 - 37 pn in dianeter, rvhich resemble those fonned by many other amoebae. Cysts are generally uninucleate. 0f 100 cysts examined, only 6 had nore than 1 nucleus (up to 4 in nunrber). The cyst nucleus is specially prominent before germination. -44-

Based on trophozoite and cyst morphology, this amoeba appears to belong to the family Gephyramoebidae (Pussard and Pons, I976a), and it has been tentatively placed in the genus Gephynatnoeba Goodey, 1915.

The arnoeba is sinilar to G.deLieatula Goodey,1915 in appearance as described by Pussard and Pons (1976c) but differs in being multinucleate and anastomosing.

Unidentífíed Leptornyæid amoeba

The amoebal trophozoite exists in two distinct forms, a branched flattened form (Figs.6a,b,c) ranging fron B0 un to 150 pn in length and

28 ym to 95 pn in'breadth; and a lirnax forrn (Fig.6b). The change between the two forms can be readily observed (Fig.6d). The linax form is conrnon in aqueous nedia which are disturbed and can be found at the interface between mediun and air. Amoebae in non-disturbed cultures tend to settle to the botton of the dish and produce the branched forn.

The latter forn is also conmon on agar. Lirnax forrns are narrower at the posterior end than at the anterior one and neasure from 50 pm to 180 pm in length and 20 pm to 40 pn in width. Both forns are uninucleate (Figs.6a,c,d), the nucleus rneasures 6 pm - 10 un in dianeter and contains an elongated nucleolus (3 Un - 5 Urn). The protoplast is distinctly divided into a hyaline ectoplasm and a granular, compact endoplasm. The branched form contains one to Tnany vacuoles up to 16 pn in díaneter.

The linax form normally has a single large vacuole at the posterior end of the cell.

Fron the advancing nargin of the ce11 srna1l eruptive pseudopodia of the type lobosa are forned by the ectoplasm into which the granular endoplasrn flows. As the trophozoite advances the posterior rnargin forms -45-

Fig.6: The unidentified leptonyxid amoeba.

a Large flattened form of a trophozoite showing the anterior lobopodia (L), contractile vacuole (Cv) and single nucleus (N).

b Tubular limax forn.

c Trophozoite showing anterier lobo- podia (L), posterior filopodia (F), nucleus (N) and the typical branched norphology.

d Forn internediate between branched and linax showing the single nucleus (N), hyaline ectoplasn (Ec) and granular endoplasm (En).

e Cysts and endocyst (arrow).

(Scale bar = 10 prn in all Figs.)

t

I

I

I

I

,

I I

-46-

a variable number of fine filopods (Fig.6c). The rate of locomotion can be as rapid as 5 pn r".-1 in branched forms. Pseudopods can be fo::ned in a nunber of directions sinultaneously, but ultinately flow in one direetion. Sinilar filopods appear when the lobose pseudopod is retracted. True polyaxial locomotion has not been observed.

Anastomosis between trophozoites has not been observed so far even though crowding is common on agar. Durì-ng cell division the tropho- zoite extends linearly with pseudopods occurring at both ends. Two nuclei can be clearly seen at this stage. The central portion of the protoplast becomes constricted and finally two daughter ce1ls separate

The generation tine on PJA + KLebsiella sp. as food varies from 3-5 h.

The amoeba forms cysts which are usually spherical in shape

(Fig.6e), but can vary if individuals are crowded or cysts forn rvithin the confines of lysed fungal ce1ls. Cysts are smooth rvalled and measure 15-30 un in dj-aneter. The single nucleus can be seen very clearly just prior to gernination. Rarely up to three nuclei have been seen at this stage. A single trophozoite invariably energes from the cyst.

Excystrnent is stimulated by placing the cysts into AS or on PJA + KLebsieLLa rnedium. In older cultures, a second type of cyst which is apparently snaller in size and formed inside an existing cyst wa11, has been observed (Fig.6e). Sinilar rendocystsr have been observed in other lcptomyxid amoebae before (Pussard and Pons , I976a, 7976b).

The order Leptomyxida includes arnoebae whj ch form thin spreading reticulate trophozoites, have polyaxial locomotion and produce lobose pseudopodia (Pussard and Pons , I976a). I'he order contains two families, Leptomyxidae and Gephyramoebidae, distinguisherl on the basis of nulti- nucleate trophozoites in the former and uninucleate trophozoites in the -47 -

latter. Leptornyæa fLabeLLata Goodey, 1915 was retained r4rithin the same genus as L.reticulatø Goodey, 1915, the type species (Pussard and Pons, 1976b), despite significant differences in trophozoite morphology. In L.fLabeLLata, trophozoites are branched rather than reticulate and loco- ¡notion is not polyaxial. Another significant characteristic is the production of linax forrns when growing in a water fi1n. rn all these characters this amoeba closely resembles L. fl-abeLLata. The principal difference appears to be in the number of nuclei. L.fLabeLLata is reported to have 1-48 nuclei per trophozoite. Up to 3 nuclei were seen in cysts of this arnoeba. As nuclear nurnber is the basis for separating the fanilies Leptomyxidae and Gephyranoebidae the amceba can not be attributed to tr. fLabeLLata and it has been placed under the order Leptomyxida as an unidentified amoeba of the order.

MayorelLa sp.

Trophozoites are more or less elonga.ted and flattened. In live trophozoites the singte nucleus, 4-8 ¡rm in diarneter appears as the rnost proninent structure and is more or less spherical in shape with a rounded nucleolus neasuring 2.5 - 5.5 pm in diameter (Fig.7a). The nunber of large contractile vacuoles, 2 - rr pm in diameter may vary from l - 20 or nore in one trophozoite and their abundance often gives a reticulate appearance to the trophozoite. Coarsely granular endoplasm covers the major part of a trophozoite and the hyalíne ectoplasrnic region is present in the pseudopoclial projections and also appears as a fine filnr around the cell. From the hyaline ectoplas¡nic zone appear a nunber of pseudopodia in almost all directi.ons. Pseudopodia are usually conical nanilliform but can be digitiform and sometirnes quite extended with rounded end (Fig.7a). Sometimes trophozoites constantly change their -48-

Fig.7: MagoreLLa sp.

a. Trophozoite with its proninent nucleus (N), digitiforn pseudopodia (Ps) and contractile vacuol-es (Cv). b. Trophozoite showing the irregularly bulbous uroid (arrow). c. Trophozoite showing paral1el streaks (arrows) along its length.

(Scale bar = 10 pm in all Figs.)

-4.9-

direction of novenent, while at other tines a more or less unidirectional novement ¡nay continue for a longer period. Rapid unidirectional loco- motion is cornmon in rnany limax arnoebae and although this amoeba does not have a limax form, the trophozoites move as fast as 0.5 - 1.8 u* r".-1. Under continuous locomotion, the posterior end (uroid) appears irre- gularly bulbous (Fig.7b). Often the ridges forned on rnamilliforrn pseduo- podia extend beyond the pseudopodial length and can be seen as nore or less paral1el streaks along the entire length of the trophozoite (Fig.7c). Trophozoites rneasure from 50 - 130 pm in length and 20 - 100 pm in breadth. No cysts have been observed in culture.

Trophozoites of this anoeba resenbLe MayoreLLa penardi Page, L972 (Page, L976) except for the morphology of floating forns. Floating forms of M.penardi ale irregularly rounded but those observed here of this amoeba have radiating slencler pseudopodia.

PLatyønoeba stenopodia Page, 1969

Trophozoites are generally elongated rvith a truncate anterior end (Figs.8a,b,d). The anterior portion covering half or more of the trophozoitets length is hyaline andappears flat whereas, the posterior half is granular and somewhat thicker (Fig.8d), Usually trophozoites are broader at the anterior half than at the posterior (Fíg.8a).

Floating forms commonly have blunt radiating pseudopodia rvhich may be larger than the diarneter of the central body and alrnost alrvays have rounded ends (Fig.Bc). The single nucleus and generally one contractile vacuole are the prominent features of the coa::sely granular endoplasn.

Itlhen stainecl, the nucleus appears ova1, elongate or spherical in shape and rneasures between 1.4 - 2.5 um in diameter rvith a nucleolus 0.9 to -50-

Fig.8: PLatyatnoeba stenopodia.

a Trophozoite showing the broad anterior ectoplasm (Ec) and a contractile vacuole (Cv).

b Trophozoites. Note the granular endo- plasrn (En) and the truncate anterj.or (arrow).

c Floating form.

d A typical elongated trophozoite ín loco- motion. Note the ratio of ecto- (Ec) and endoplasn (En) and the tTuncate anterior (arrow). e. Orcein-stained nucleu-s (N).

f Uninucleate cysts. Note the dense spherical body (S) besides the nucleus (N).

(Scale bar = 10 pn in all Figs. ) a b c

\ \l Cv a I l E /t Ç

t Ec \

e d

ú

1 N

En t

Õ

I Ec -51-

1.6 um in dianeter (Fig.Be). Trophozoites under continuous locornotion are elongated with the length breadth ratio often reaching 3:1 and have 2-3 folds approxinately paral1e1 to the longitudinal axis (Fig.8d). The rate of locomotion is between 0.2 - 0.4 pn sec-l. Trophozoites range from 6 - 30 ¡rn in length and 5 - 20 pm in breadth.

The amoeba forrns spherical to oval cysts, 4-72 pm in diameter. Cysts are uninucleate and contain a dense spherical body apart from the nucleus (Fig.8f). Sinilar dense bodies have been reported in cysts of

PLatyønoeba tpp. (Page, 1976).

Trophozoite, and cyst morphology of this amoeba matches the description of PLatyønoeba stenopodia Page, 1969, a freshwater and leaf litter species described by Page (I969).

PLatycvnoeba sp. Locomotive forms are ovoid, ellipsoid, flabellate or triangular in shape rr'ith a rounded or flat anterior end (Fígs.9a,b). Movement is more gliding than pseudopodial, although occasional short pseudopodia nay be noticed. Rate of locomotion varies between 0.3 - 0.8 p^ sec-l.

This is a snall amoeba ancl cytological details are only visible at higher magnifications, although the distinction between the ecto- and endoplasm can be readily observed'at loler rnagnifications. The hyaline ectoplasn extends as a broad fan shaped area at the anterior and often appears wrinkled or wavy (Fig.9b). The nucleus and usually one contractile vacuole (1.5 - 10 um in dianeter) are the prominent features of the finely granular endoplasm. Stained nuclei (Fig.9c) measure between

2.0 - 5.0 un in dianeter and nucleoli fron 1.2 - 5.0 um in diameter. Trophozoite dinensions: 15-90 pm in length and 8-55 pm in breadth. -s2-

Li&_9_: PLatyønoebo tp.

a Trophozoite in loco¡notion shorving ectoplasm (Ec), granular endoplasrn (En) and contractile vacuole (Cv).

b Trophozoite with wavy anterior end (arrows ) .

c Nucleus (N) in a stained arnoeba.

d The spherical and elongated cysts. Note the dense spherical body (S) apart fron the single nucleus (N).

(Scale bar = 10 un in all Figs.)

-53-

Cysts of two distinctly different shapes have been consistently observed in single trophozoite cultures under sirnilar conditions. More nulnerous are the spherical to oval cysts (Fig.9d) which measure betleen

7.5 and 20 pn in dianeter. The other type is considerably larger and elongated in shape (Fig.9d) and has a maximurn diameter around the niddle and tapers towards the snooth and rounded ends. Their dimensions vary from 18-35 ¡un in length and 9-15 ¡-rrn in breadth. Both types are uni- nucleate, have a smooth thin wa11 and contain a dense spherical structure apart fron the nucleus (Fig.9d).

Trophozoites of this anoeba closely resemble those of the genus PLatyamoeba Page, 1969 (Page, 1969). They also have resemblances with Schaefferts Rugipes biLzi Schaeffer, 1926 as redefined by Sarq¡er (1975) as CLydoneLLa uíua.æ (Schaeffer, 7926) Sawyer, L975. However, C.uiuan is a much srnaller amoeba, known only fron rnarine habitats and does not form cysts. 0n the other hand, my isolate does not resemble any known

PLatyønoeba ín cyst morphology and hence has been placed in this genus as an unidentified species.

Saccamoeba sp.

Trophozoites of this anoeba are 1ong, slender and broader at the anterior than at the posterior end (Figs.10a,d). Occasionally it pro- duces lobose pseudopodia both in liquid and agar cultures (Figs.10b,c). 0n agar however, trophozoites conrnonly appear as a rounded nass varying in shape and containing a number of refringent granules (Fig.10c); they either form small lobose pseudopodia or assume a limax form to continue locomotion. Trophozoites often penetrate into the agar nediun.

In a newly formed pseudopod (Fig.l0c) and at the advancing nargin of a -54-

Fig.10: Saceamoeba sp.

a Trophozoite with the ectoplasrnic cap (C).

b Trophozoites in liquid culture with short pseudopodia (Ps) .

c Trophozoite on agar appearing as a rounded nass containing refringent granules. Note the pseudopodia (Ps).

d A typical limax amoeba in locomotion, showing the ectoplasnic crescent (Cr) and the crystals (arrows).

e Orcein-stained nucleus (N) in a rounded-up trophozoite.

(Scale bar = 10 pn in all Figs.)

-55-

linax amoeba (Fig.10a), a hyaline ectoplasmic crescent is usually present. However, the ectoplasnic cap may be absent in limax amoebae under continuous locomotion. A characteristic feature of this amoeba is the occurïence of 10 - 28 rectangular to cuboidal crystals in its cytoplasrn (Figs.10b,c,d). Crystals vary from 0.75 pm to 1.5 pn in length. Trophozoite dinensions vary ftom 24 - 45 pn in length and 8 - 33 pn in breadth. The single nucleus and its dense nucleolus can be seen in live trophozoites, but the abundance of cyto- plasmic granules may obscure this. Stained nuclei (Fig.10e) measure between 3.2 - 8.8 um in diarneter and nucleoli fron L.6 - 7.2 vm. No cysts have been óbserved in cultures.

Trophozoites of this anoeba match the generic description of

Saecamoeba FrenzeL, 1892; ernend. Bovee, L972 (Bovee, 7972; Page, 7976). Nunber of crystals, trophozoite size and norphology of the villous-bulb uroid are used to distinguish between the non-cystic species of

Saccqnoefu (Page, 1976). The posterior end of the amoeba described here is snooth and round, and does not have villous bulb or rigid villi in the urodium. The size of the trophozoites is also small.er than the non-cystic species of Saeca¡noeba described by Bovee (I972) or Page (1976) The anoeba therefore has been placed in this genus as an unidentified species.

Ihecamoeba quadriLineata (Carter, 1856)

Arnoebae in locomotion are flattened, oblong or elongately elliptical in outline with broadest point usually at or near the niddle.

The advancing nargin is usually rounded (Figs.1la,c), but can be wavy (Fig.11b) or truncate (Fig.1ld). fn locornotive forms 2-4 áorsa1 folds which form parallel along the length of the arnoeba can be observed -56-

Fig.11: The cøno eba qøãrùLineata'

showing a Trophozoite in locornotion the large contractile vacuole (Cv) ãr,ã trr""ttterior ectoPlasnic zone [Ec)'

b Tronhozoite t^Iith the wavy anterior r""!itt (arrows)' dorsal c Live trophozoite showing two folds (arrows) ' with d Glutaraldehyde-fixed trophozoite i*o .t""t dorsal folds (arrows) and the truncate anterior' granular endo- e Live trophozoite showing ;i;;''- (;;j ánd wavY advancing nargin (arrows). f Orcein-stained nucleus with a single elongated nucleolus (arrow) '

(Scale bar = 10 um in all Figs')

-57 -

(Figs.11c,d). Trophozoites usually move by gliding in one direction,

however, occasional lateral shifts do occur on agar rnedia whereby trophozoites aggregate together hrith their posterior ends oriented

towards the centre. Lateral shifts are relatively faster and rnore conmon in young cultures. The rate of gliding varies fron 0.2 to 0.9 un sec-1 ^.

Cel1 cytoplasn is distinctly divisible into a granular endoplasrn whichcontainsrefractile bodies, about 1.5 un in dianeter (Fig.11e), and the hyaline ectoplasn. The ectoplasm is broader at the anterior end. Usually one contTactile vacuole, up to 20 un in diameter is present at the posterior end of each trophozoite (Figs.11a,b,d,e). Anoebae are uninucleate and nuclei can be observed in live trophozoites; when stained, they appear elongated to oval in shape with an elongated

nucleolus (Fig.11f). In live amoebae, reniform, triangular or

spherical nuclei have also been seen.

The trophozoites natch the generic description of Thecønoeba Fromentel, I874 (Page, L977). Two species of Theeønoeba, T.strLata (Penard, 1890) and T.quadrLlíneata (Carter, 1856), have prominent dorsal

folds in loconotive arnoebae and nuclear stTuctrlres have been used to

distinguish them. In T.strLata the nucleolus is nade up of two or more distinct pieces, whereas , T.quaã.r"Llineata has a single nucleolus (Page,

7977). The isolate described here has beerr identified as Thecamoeba quadrLLi,neata.

Theccnnoeba splu.eronucLeoLus (Greeff, 1S91)

The arnoeba in hanging drop preparations often sink to the bottom during examjnation and in general poorJ-y adhere to glass surfaces. -58-

Floating forms appear highfy wrinkled. Trophic forns, however, are smooth and flat (Figs .I2a,b), and nore or less oblong, oval or sometimes broad fan-shaped with an expanded anterior region. There appears to be a sharp line of denarcation betr,Ieen the ecto- and endoplasm irr loco- notive forms. As the trophozoite glides avtay, a series of wavy patterns which originate in the endoplasn and travel to the anterior extrernity can be observed (Fig. Lzb). Rate of loconotion is 0.4 - 1,5 u* ,".-1. In general, there is only one contTactile vacuole, 6-L6 pm in diarneter, located at the posterior of the cell (Fig.72a), but occasionally several smaller vacuoles can be seen as craters in the endoplasm (Fig. 12b). Trophozoite dimensions are 35 - 80 pm in length and 25 - 65 pn in breadth, Nuclei in live amoebae appear spherical, oval or elongate with centrally located 1obed, spherical or elongated nuc1eo1i. When stained the nucleolus, however, almost alrvays appeared fragrnented in to 2-3 lobes (Fig.L2c). In live trophozoites nuclear diameter varied from

8-12 um and that of the nucleolus from 5.5 - 9 un.

Except for their size, trophozoítes resembLe Theeønoeba sphaero- nueLeoLus (Greeff , 1891; Page, I977). Trophozoites of T.sphaerorn,tcl-eo- Lus range from 65 - 140 um (nean 98 un) in length, whereas those of this isolate measure from 55 - 80 pm (mean 55 um). The nucleus is also larger in size and contains a single or fragmented but centrally located nucleolus (Page, L977). The nain difference, therefore, appears to be in the overall dinensions of the anoebal cel1 and its nucleus.

The isolate rnay be a sub-species of T.spLnercnucleoLus.

L'Vteconoeba gnan'ífera (Greeff, 1S66) sub-speci es m'Lnov' Pussard et aL., 1979

The trophozoite of this amoeba is more or less e11iptical, oval or elongated in shape (Figs.I3a,b,c,d), measuring between 24.4 - 40.8 un -59-

Fig.I2: Thecanoeba sphaez,onueLeolus.

a A typical fan-shaped trophozoite on agar. Note the sharp line of de- marcation (arrows) between the ecto- and endoplasm.

b Trophozoite in locomotion showing wavy pattern in the anterior ectoplasn (Ec) and crater-like endoplasm due to the abundance of contractile vacuoles (Cv).

c Orcein-stained nucleus with two nucleolar fragnìents.

(Scale bat = 10.pn in all Figs.)

-60-

Fig. 13: Theeønoeba granifera sub-species minor.

a Trophozoite showing anterior ecto- plasm (Ec) and the highly granular endoplasn (En).

b The ectoplasm appearing as a deep anterior crescent (arrow) in a trophic a¡noeba.

c Trophozoites showing the variation in the number of granules in them. A young trophozoite (1) has less granules than a fu1l-grown one (2). d. Trophozoite showing the anterior ecto- plasm crescent and its extension as a narror.{ border (arrow) towards the posterior region. e. Orcein-stained nucleus (N).

(Scale bar = 10 um in all Figs. )

I a v

En Ec t ¡1.-- e t - - a ,

b c t a'Dl I a r¡ , 1 t

h \l 2

e

o d- I

o

Å

- -61-

in length and 15 - 24.5 Um in breadth. The endoplasn is character- istically granular with refringent granules, L-3 pn in diameter, which appeal yellow or brown in colour, the intensity of colour varying con- siderably between individuals. A sma1l trophozoite nay have few or no granules (Fig.13c) and as it grows the granules increase in number. The hyaline ectoplasm is broader at the anterior end and appears as a deep anterior crescent (Figs.L3b,d) which extends posteriorLy as a narrow border (Fig.15d) when in contirruous locomotion. There is usually one pulsating vacuole, 1 - 6 Un in diameter, in the posterior part of the body. Locomotion is rapid and can be 1.5 Um r""-1. In a live trophozoite the nucleus is rendered inconspicuous by the abundance of granules. ltlhen stained, nuclei appear elongate to oval in shape (Fig.13e) and measure between 8 - 72 7n in diameter. Cysts have not

been observed.

The amoeba appeaïs closely related to Thecamoeba granifez'a Gteeff,

1866 as redescribed by Page (1976, L977). The trophozoites of

T.gz,anife.nø however, are larger in size and almost always contain more than one nucleolus in their nucleus. Pussatd et aL. (1979) described a snall cyst-forming rnycophagous amoeba which closely resenbled T.granife.nø except for its size and they named it T-granifeta sub- species minor. They reported one to three nucleoli in their isolates.

The anoeba described here also frequently associates itself with hyphae

of Fusarium and other fungi on agar plates. The amoeba has been main- tained in monoaxenic culture with an associated Fusatiwrt sp. and the sinilarity in sizes of the amoebal cysts with that of the fungal

chlamydospores probably explains the reason for the failure to distinguish cysts in cultures. The isolate has been identified as Î.granifera sub- species minor. -62-

Unidentífi ed vanrpy t' eLLíd atno eba

In this amoeba the concept of a trophozoite is almost non-existent due to the extensive anastomosis which occurs between individuals '

Anastornosis between individuals leads to the fornation of a giant reti- culum, 70-12 rrn in size, with an advancing front (Fig.14a) which is the most active part of the organism. Advancing fronts have fine filopodia which occur singly or in groups. Snaller individuals, several ¡'rm in length can be seen in liquid cultures (Fig.14b), but they soon anasto- mose and form the giant reticulun. The cytoplasn is finely granular and contains rnany snall pulsating vacuol es 2.2 - I pm in dianeter' No distinction can be made between ecto- and endoplasm. Loconotion is slow and in short term undetectable. If positions of filopods in an advancing nargin are marked on the occular micrometer graticule, and observed aftet 2 - 4 h, they can be seen to have changed. More direct evidence of locomotion can be obtained by increasing the illumination.

Then the reticulate forn will round up and forrn cysts. Active movements however, occur in the terninal filopodia which are slowly and regularly forned and rvithdra!/n.

On agar plates, this arnoeba nay be cont'used with the aphanoplasm- odiun of a nyxonycete (Alexopoulos, 1969, Fig.2, PaBe 222)' Its proteo-

nyxan nature is evident by the absence of a regular cytoplasrnic streaning, fornation of cysts and continuous formation and bursting of vacuoles in the cytoplasm. The amoeba is multinucleate (Fig'14d) and the number of nuclei varies with the size of an individual. Nuclei

neasure betr.reen 2.4 - 7 um in dianeter with a darkly stained nucleolus

1.6 - 4 u¡n in dianeter. -63-

Fig.14: The unidentified vanpyrellid a¡noeba.

a Advancing front of a giant reticulum formed on agar. Note the fine filopodia (arrows). b. A snaller individual in liquid mediun.

c The giant reticulun of the amoeba has concentrated at several points (anows) prior to cyst fornation. Note the thin-walled (1) and thick-wal1ed (2) cysts.

d Stained nuclei (arrows) in a giant reticulum of the arnoeba.

e Thick-walled cysts showing the range of shapes and sizes and three distinctly visible layers of the cyst-wa11 (arrows).

(Scale bar = 25 pm in all Figs.)

i; ì

-64-

The amoeba forms two tyPes of cysts. During cyst formation, the whole mass of protoplasm concentrates at several points (Fig.14c) which gradually becorne detached fron each other. These areas appear dense and finally round up to fonn single thin-walled cysts (Fig.14c). These cysts germinate easily. The other type of cyst has thick walls composed of three distinctly visible layers (Fig.14c) and is formed from the thin-wa11ed cysts by further contraction of the protoplasm. Often the thin wall of the original cyst is visible as an outline outside the thick-wal1ed cyst. Germination of these thick-wa11ed cysts has not been observed. Cysts can be of any shape or size (Figs.l4c,e). In the thick-walted cysts, a lange of 10 - 569 pm in dianreter or length, de- pending on their shapq has been observed. Cysts are conmonly elongate to oval in shape.

Many earlier workers who observed freshwater and soil amoebae described them as nel species and often erected ner^J genera on the basis of differences in their specinens. Zwillenberg (1953) in describing a new species of Theratrornyra considered 19 genera as possible synonyms. Other genera rvith filopodia which received detailed modern treatment are

ArachnuLa (01d and Darbyshire, 1980), Vatnpyz'elLa (Anderson and Patrick,

1978, 1980) , VønpgreLlidiwn (Surek and Melkonian, 1980) and NucLearia (Pernin, I976; l,fignot ànd Savoie, 1979). Two other genera, Biomyæa

and Groniø also produce filose pseudopodia (Leidy, 1879; Hedley, 1962), of which Gronria is a testate anoeba and Biomyæa has a central body from which radiating filopodia are produced.

The farnily Varnpyrellidae Zopf, 1885 includes organisms with hyaline

filiform pseudopodia, often branching, sometimes anastomosing which cío not produc.e spores and do not have a flagellate stage in their life cycle -65-

(Loeblich and Tappan, 1961). The amoeba described here does not match any species description I have noted, however, its characters generally are sinilar to those of the members of the fanily Vanpyrellidae and I have tentatively placed it in this fanily as an unidentified spec'ies. -66-

C. rN VITRO INTERACTION 0F SOIL AMOEBAE I{ITH Ggt AND OTHER SOIL FUNGI

There is a trvo way interaction between soil anoebae and fungi; firstly, several fungi, mostly Phycomycetes of the order Zoopagales, are predaceous and parasitic on soil amoebae (Drechsler, 7933, 1935, 1947, 1969; Dayal, 1975) and secondly, several of the terricolous amoebae feed on fungi. funong nycophagous arnoebae particular interest has been shown in the genera Az'acfutuLa, VønpyreLLa and Thez'atz'ornyæa (01d, L977a; 01d and Darbyshire, 1978; 01d and Oros, 1980; Anderson and Patrick, 1978, 1980; Homma et aL.,1979) which belong to the fanily

Vanpyrellidae ¡Uon:-gUer g et aL., 1964). A number of other geneïa,

Ihecamoeba (Esser e't; aL., L975; Alabouvette et aL., 1979¡' Pussard et ãL., 1979) , Cashia (Pussard et aL., 1980) and HartmawtelT.a (C.Palzer, personal communication), have also been found to have mycophagous species.

Although soil amoebae were knotvn to feed on yeasts (Oehler, 1916;

Severtzova, 1928; Castellani, 1930; Negroni and Fischer, 1941; Nero ¿Ú aL., 1964; Bunting et aL.,1979), Heal (1963)was first to show that soil amoebae also feed on filarnentous fungi. Drechsler (1936), holever, had reported that the testate amoeba, Geococcus uulgaz"is France and ArceLLa uuLgarLs Ehrenberg, 1938 attacked oospores of Pythium uLtimum Trow, and Ivliller (1963) found a ciliate which fed on the sporangia of futhiun caro Liní anun Matthew s .

The mode of attack varies with the amoebal species. ArachnuLa impatiens was shown to penetrate the conidiat wa1Is of CochLioboLus satiuus by erosion of an annular depression ínto the conidiun wal1 (01d, L977a, 1978). This disc of waI1 materj.al is dislodged, alloling the spore contents to extrude into the anoebal trophozoite. The closely -67 -

related Theratrom4æa. encompasses whole spolres within digestive cysts, the spore wa1l being penetrated by many sna1l holes each about 0.5 urn in dianeter (Anderson and Patrick, 1980; Old and Oros, 1980).

MayoxelLa sp. has been shown to engulf up to five conidia of C.satiuus and retain then within the trophozoite for many hours. Extruded spores are often lysed but little information is available on the feeding rnechanisms of this genus (Anderson and Patrick, 1980). Pussatd et aL. (1979) have described in detail the penetration of hyphae and micro- conidia of Fusariwn oæAspozam Schlecht er¡rend. Snyder ancl Flansen by

Thecamoeba granife.na sub-species wínor. They regaïd this species as being strictly nycophagous, whereas Old and Darbyshire (1978) showed that Arachnula intpatíens has an extrenely broad range of potential prey including bacteria, algae, protozoa and nematodes.

Nine arnoebae belonging to eight different genera which were conmon- ly isolated fron the WPP soil were tested for mycophagy in feeding trials using Ggt, C.satíuus and Phytophthoz,a cinnamomi.

The feeding trials

Feeding trials were conducted, using the three fungi in Petri plates containing the fungal substrate and amoebae suspended in AS.

Myceliun of Ggt I^Ias grown in 0.49o malt extract broth for one week, wash- ed twice with SDIV and rinsed in AS. Mycelium was either left intact or homogenised. Conidial suspensions of C.satiuus were obtained using the nethod described earlier. Myceliun and chlamydospores of P. cinnønomL were produced by inoculating VB juice liquid rnediurn containing 20 VE cholesterol rnl-1 (D.D.Darling, unpublished infornation) with about 50 srnall pieces of agar culture of the fungus and incubating for up to three -68 -

r.¡eeks at 25oC ín continuous light. ThaLli were harvested, washed in

AS and either honogenised or left intact. AS suspension of fungí were added to Petri plates to a depth of about 5 rnm. At the outset of each trial, six or seven 12 nn diameter glass coverslips were placed into each Petri plate. PJA cultures of anoebae r4¡ere flooded with AS and amoebal suspensions were drawn up into sterile pasteur pipettes. Drops of these suspensions r{eïe addecl to the fungal substrates and the cultures incubated for 4-5 days at zsoc.

Detailed observations of the interactions between trophozoites and fungi were made by transferring coverslips bearing these organisms to

Cruickshank chambers and examining by phase contrast nicroscopy. After a feeding period of 3-L4 days, coverslips lvere air dried and mounted on netal stubs for scanning electron microscopic (SEM) observation. Other specirnens, including intact tha1li and spore suspensions that had been incubated with the amoebae were fixed in 3e" glutaraldehyde in 0.lM phosphate buffer overnight at 4oC, dehydr:ated in a graded ethanol series and critical point dried in a Polaron unít using liquid COr. Before

SEM exarnination, all specimens were coated with approx. 900R layer of gold-palladiurn usirtg a Polaron E5100 sputter coating unit (Polaron Equipment Ltd., Herts. England). Specinens were exanined with a Jeol

JSMUS scanning electron microscope.

Feeding activities of the amoebae

Of the amoebae tested, mernbers of five genera, Saccasnoeba, Gephyt'-

ønoeba, Magor,:eLla, Ihecønoeba and the unidentified leptomyxid proved to be nycophagous (Tab1e 10). The feeding behaviour of these amoebae are detailed as follols. -69-

TABLE 10: Mycophagy of various soil amoebae on three plant pathogenic fungi.

Perforation Anoeba Mycophagy Fungus (sEM)

Aeøúhønoeba poLyplnga Ggt Csa Pcb N.T. C

GepVrynønoeba sp. Ggt N.T. N.T. Cs + + Pe + +

MayoreLLa sp. Ggt + Cs +

PLaty atno eba stenop odi a Ggt

Cs

Saecønoefu. sp. Ggt +

Cs + +

Iheeamoefu. gz,anifera Ggt + ssp. minoz, Cs + +

Theeøno eba quadz'i Lín eat a Gg t Cs

Unidentified Gg t + + Leptonyxid Cs

Dn + +

Unidentified Ggt Vampyrellid Cs Pc

a CoehLioboLus satiuus b Phy t ophtLnra eLnnønomi c Not te-sted. -70-

In cultures, the unidentified vampyrellid arnoeba forms an extensive

reticulun encircling nany conidia of C.satiuus (Fig.15) and Ggt hyphae and renains attached for a considerable period of tine. However, no evi-

dence of conidia or hyphae being perforated or affected otherwise has

been observed. SEM observations of fungal propagules after they have

been cultured with the arnoeba have never shown perforations in their

walls. The amoeba feeds on Klebsiella sp.

Ihe unidentí edL d a¡noeba

The anoeba ernploys two different mechanisms when feeding on P.cinnønomL. On'hyphae, sporangia and hyphal srvellings of the fungus feeding is rapid and conpleted in 35-45 nin. Mature chlarnydospores are

digested within large digestive vacuoles over a prolonged periocl, between 27 and 36 h. These processes are quite distinct and are described

s eparately.

When feeding on hyphae the trophozoite contacts the hypha, its movement ceases and then it adheres firmly to the surface. If hyphal

fragnents are encountered these rnay be dragged along the substratum

attached firmly to the posterion fiiopods. When attacking an intact thallus the anoeba stretches itself along the hyphal length, attaching

at any part (Fig.16a). The trophozoite becomes extremely thin and ex- tended, eventually engulfing a portion of the hypha. This proccss takcs

10-15 min, after which the amoeba contracts and masses a::ouncl a snall section of the hypha (Fig.16b). This process of extension and contrac- tion rnay be repeated 2-6 tines. The trophozoite then moves along the hypha and may concentrate on anot-her part of the hypha (Fig.16c) or leave it altogether. At this stage, it can be seen that the portion of -7L-

Fig. 15 Giant reticulum of the unidentified vampyrellid anoeba encircling spores of CochLioLoLus satiuus .

(Scale bar = 20 pm).

-72-

Fig. 16 Feeding activities of the unidentified leptonyxid anoeba on Phytophthora einnomomi.

a A trophozoite has enveloped a length of hypha (arrows) of the fungus. Figs. I6a-d are a sequence of micrographs at the sarne magnification showing the lysis of the hypha.

b fire trophozoite has concentrated around the subterminal portion of the hypha (arrows).

c The trophozoite has rnigrated back along the hypha and is concentrating at a nell location (arrow).

d The hyphal tip now lacks protoplasrnic contents (arrow).

e. Detail of the hyphal tip.

f Trophozoite exerting force to encompass the hyphal tip. Note the physì.cally distorted hypha (arrow) inside the anoeba

g Amoebal cyst within enpty sporangium (arrow).

h. Anoebal cyst within lysed swollen hyphae.

(Scale bar = 20 pn in all Figs.)

-7 3-

hypha where the trophozoite concentrated is conpletely devoid of cyto- plasn (Figs.16d,e). The anoeba exhibits considerable nechanical force and free ends of hyphae rnay be physically distorted to encompass them within the body of the trophozoite (Fig.16f). The arnoeba is very sensitive to bright illunination when feeding and photography and observa- tion nust be carried out r^/ith internittent or low light intensity or feeding will be slowed or cease altogether.

Sporangia, hyphal swellings, or imnature chlamydospores of

P.cínnamonrL are consumed by the amoeba in a similar way to hyphae, except that engulfinent of the propagule nay be incomplete. A similar series of of the trophozoite occuïs and the contents of extensions and "orrtt".tions the sporangium flow into the amoeba. The f-ungal propagule is rendered conpletely devoid of contents in 35-45 nin after feeding begins arrd then the amoeba either moves a!Íay to feed on othel cells or may rnigrate within the sporangium or hyphal swelling and forn a cyst (Figs.169,h). Scanning electron microscopy of fungal nateriaL after prolonged feeding showed that hyphae and other propagules reere perforated by circular holes varying fron 1.0 um to 3.0 um in dianeter (Fig.I7a).

The node of feeding on Ggt hyphae varies according to the ability of the trophozoite to fu1ly surround the hypha. When intact tha11i are used, the anoeba envelopes sections of hypha and undergoes the extensions an

examination shows that holes varying in size frorn 0.3 Um to 2.0 pm are present in the fungal cell rvalls (Fig.17b). Presurnably these are the

sites of attack by which the amoeba gains access to the fungal protoplast.

When the fungal thallus is fragnented before feeding trials, the tropho- -7 4-

Fis.17: Scanning electron micrographs o{-hfpfle é-^; tto^ t"ãding trials with the unidentified lePtornYxid '

a Perforation in a hypha of Phytophthora cinnn¡nomL, b. Perforations in hyaline hyphae of G. graniwLs tY'itíci (arrows).

The bacteria (B) are KLebsieLLa sp'

(Scale bar = 4 un in both Figs')

-75-

zoites ingest whole pieces of hypha. This is conrnonly achieved by twisting and bending the hypha until it is fu1ly accornmodated inside the trophozoite. The trophozoites continue to move acloss the sub- stratum and in as little as 30-45 min the hyphal fragments are no longer recognisable and shortly afterwards undigested residues are extruded fron the trophozoite.

Digestion of a rnature chlarnydospore of P.einnamowL occurs by a trophozoite contacting and conpletely engulfing it. Formation and expulsion of the contents of the numerous small vacuoles within the amoebal protoplast become intense at this stage and result within approx, 20-35 rnin in the fornation of a large fluid filled vacuole containing the chlamydospore. This vacuole is the subsequent site of digestion of the spore. During this initial phase of attack the amoeba is particularly light-sensitive and rounds up if illumination is too intense. Once the digestive vacuole is fully formed (Fig.18a) the anoeba is less sensitive. Ilicroscopical observation of spores within digestive vacuoles show that clelicate protoplasnic threads traverse the space between the nain vacuolar membrane and the spore wal1. These are not resolved adequately in Figs.lBa-e. Contraction of these strands nay be the cause of the rotation of the chlamydospore within the vacuole shown in Figs.18a-e.

The main activity seen within the anoebal protoplast at this time is the continuous formation of vesicles which develop bordering the digestive vacuole (Fig.1Ba) and discharge their contents into it (Fig. 18b). The other prominent feature, the nucleus, is also visible throughout the period of existence of the digestive vacuole (Fig.18d). At no stage did a cyst wa1l develop aro'-rnd the trophozoite in the manner -76-

Fig.18 Digestion of a mature chlamydospore of PVtytophtl.oxa eLnrnmomt by the wridentified leptomyxid a¡noeba. Figs.18a-i are a sequence of rnicrgraphs at the sane nagnification.

a A fully formed digestive vacuole around the chlanydospore. Note the formation of a vesicle (arrow) in the anoebal cytoplasrn.

b As in Fig.l8a but with a mature vesicle about to discharge into the digestive vacuole (arrow). c. Fornation of a pseudopod. 'd The prominent nucleus (N) inside the anoebal protoplasn.

e The digestive vacuole is reduced in volune after about L6-17 h fron its forrnation . f As the digestive vacuole is further re- duced in volume, the amoeba becQmes nore nobile.

Þct The fluid layer between the chlamydospore and the amoebal cytoplasn is lost.

h The chlamydospore (arrow) has lost its structural integrity.

a The trophozoite noves away leaving a trail of fungal cell debris (arrow) .

(Scale bat = 20 un).

-77 -

described fo¡' Iheyatz'onrgæa (Old and Oros, 1980). In fact, the outline

of the arnoeba was in a state of continuous though gradual change, and the trophozoite could be observed to nigrate a small distance on the substratum, by normal pseudopodial' movement (Figs.18c,e).

After about L6-I7 h the diameter of the vacuole gradually reduces (Figs.18e-h). The anoeba resumes more active pseudopod forrnation (Fig.1Bf) and the generally spherical shape is lost. The fluid layer

between the chlamydospore and the anoebal protoplast disappears cornpletely and the contents of the chlamydospore appear disorganised (Figs.18g,h).

Typical trophozoite movement resumes (Fig.18h) and the remains of the digestive vacuole and its contents are conveyed along by the tropho- zoite. Finally, the digestive vacuole becomes cornpletely disrupted and the renaining fungal protoplasm is taken into amoebal cytoplasm. As the trophozoite rapidl.y moves away, a ttail of debris, presumably the un- digested contents of the digestive vacuole, are extruded on to the sub- stratum (Fig.18i).

l{ith conidia of CochLioboLus satíuus the amoeba often encircles then and spoïes also frequently adhere to the postelior of the tropho- zoite, apparently held.by the gloup of filopods. Clumps of five or. nore conidia, short fragnents of conidj.ophores and iruna.ture spores accumulate in this position and rnay be conveyed along by the trophozoite for many hours. Fungal ce1ls are left behind fron tirne to time and others accumulated. However, careful observation of many such trophozoites has never inclicated that the amoeba is attacking these spores and digest- ing their contents. Examination by SEM of specimens from 14-day old -78-

cultures containing conidia and very large numbers of active tropho- zoites has not revealed any perforated conidia.

A particularly interesting characteristic of this arnoeba is its ability to employ two distinct feeding mechanisms in attacking different cell types of a single fungus. The criterion which directs the feeding option is the ability of the trophozoite to fu11y surround the propagule and the nature of the fungal wall. Once a propagule is fully surround- ed, often by bendíng and coiling the hyphae by application of consider- able force, then digestion of the cells is alnost conplete in 30-60 nin. If however, the anoeba attacks an intact thallus and is unable to ful1y enclose the propagrrf", then discrete perforations in the wal1 are rnade and the protoplast only is ingested. Hyphal wallsappeal to be other-

wise 1ittle damaged.

When preparations of mature chlarnydospores (2-5 weeks o1d) are

exposed to the amoeba the feeding rnechanisn is modified further. After conplete encirclement of the chlarnydospore a large digestive vacuole develops and the trophozoite remains almost imlobile for a prolonged period (I7-20 h) during which the entire chlanydospole is digested.

Chlanrydospores are widely regarded as survival structures, more resistant

than hyphae to lysis, but the structural basis of this characteristic is not known. The stinulus to the fornation of the large vacuole by

this amoeba may be related to the susceptibility of the wal1 conponents to elìzymatic digestion.

This anoeba, unlike Av'acVmuLa irnpatíens, Theratxontyæa and

VønpyreLlq sp, is unable to feed on C. satiuus (01c1, 1977a; Anderson and

Patrick, 1978; O1d and Oros, 1980). Ûpportunities for digestion of -79-

these spoïes are comnonly observed as the trophozoites adhere to conidia and convey them for considerable distances across the substratrm'

Hyphae of Ggt in o1d cultures become pigrnented and an attenpt was made to study their susceptibility to attack by this amoeba. Sorne perforated hyphae were for¡nd, but it was not possible to distinguish by SEM exanina- tion, pigrnented hyphae frorn hyaline hyphae sti1l present in the thallus'

Saecønoeba sp.

In this amoeba the type of darnage to hyahae depends on the ability of trophozoites to acconrnodate these inside their bodies. In an intact thallus, attack usually starts at a hyphal tip. When a trophozoite comes in contact rvith a hypha of Ggt' it adheres with its posterior part, extends its body more or less at a right angle to the hypha and then bends back to form a loop. At this stage the trophozoite begins to contract and gradually forrns a rounded mass containing many refringent granules. The portion of the hypha retained within the amoebal cell becomes trvisted and bent at several points. Iugestion of the fungal cells takes about 10-15 min. After a period of approx. 25 - 40 nin the arnoeba moves on leavin$ the empty deforned section of the hypha on rvhich it has been feeding (Fig.19a) . l\rhen hyphal fragments ate used in feeding trials entire segments are acconmodated within trophozoites which surround the hyphae and bend and twist these into shapes which can be ingested. After about 75-20 min, debris, scarcely recognisable as hlphae, is extruded on to the substlatum (Fig.19b). Frequently hyphal fragments are coiled around a trophozoite (Fig.19c) after the amoeba attaches itself to a portion of the fragment. T'his enables the tropho- zoite to feed on a larger area of the hyphae at once. Hyphae lose -80-

Fig.19: Feeding actívities of Srieeamoeba sp. on G. gnann wLs trítiei and CochlioboLus satíuus.

a Trophozoite feeding on hyphae of Ggt. Note the lack of refringency (arrows) in parts of the hyphae.

b. Fornless debris (arrow) extruded by the trophozoite after feeding on hyphal fragnents of Ggt. c. A hyphal fragrnent of Ggt coiled around a trophozoite as a result of attachments by the trophozoite.

d A trophozoite forming a loop at one end of a C.satiuus conidiun.

e Trophozoite (arrow) inside an enpty C.satiuus conidium. f C.satíuus conidiun ruptured by the anoeba; spheroplast-1ike bodies (C) arranged in a linear fashion.

(Scale bar = 20 ¡rn in fal1 Figs.)

-81-

their refringency in about 20-30 min from the tine the trophozoite established coniact.

When feeding on C.satipus conidia, the auroeba establishes firm contact with the spore by forming a loop (Fig.l9d) sinilar to that de- scribed above and stays attached to the spore for about 4 - 6 h as a

round mass containing refringent granules. Sonetirnes the anoeba moves along the surface of the spore and very occasionally resumes the limax form of loconotion with the spore attached to its posterior end. After this prolonged period of cöntact, one or more cells in the coni- dium adjacent to .the amoeba appear paler in colour. The septa of the spore are usually intact at this stage. The amoeba may leave the spore after emptying one or more cel1s. These conidia when examined with the

SEM show perforations, 1.5 - 5.0 pm in diameter, in their walls.

On other occasions the amoeba enters the spore (Fig.19e), pre- sunably through the perforation and stays inside the spore for periods ranging frorn 6 - 24 h. All compartments of the conidiun may be entered and septa are completely disrupted. In other instances the spore is partially invaded and cells rvhich survive attack may extrude from the conidium as spheroplast-1ike bodies (E\g.19f) . The anoeba invariably ruptures the conidiun wall as it emerges.

Gepltyramoeba sp.

The branched trophozoite of this amoeba covers a wide area and this increases its chance of contact with food. Thus a trophozoite can

contact one or mor.e spores of C.satiUus at once. The amoeba stays attached for up to 15 - 20 h (Fig.2ja). The conidium wall is pene- trated by a pseudopod rvhich nay be follor'¡ed by the entire trophozoite -82-

Fig.20: Feeding activities of GepVtyz,ønoeba sp.

a A branched trophozoite in contact with CoehLioboLus satiuus conidia (S). Note the pseudopodial clusters (arrows) at the tip of a pseudopodial branch.

b A trophozoite inside an enpty C.satiuus conidiun. Note the intact conidial wal1 (arrows) .

c Perforated conidiun of C.satiuus.

d Perforations in hyphae of PhytophtVpra einrnmomL.

(Scale bar - 20 un in Figs. a and b; 2pmin Figs. c and d)

-83-

(Fig.20b). The trophozoite feeds on the conidial cells and if all the cells are attacked, the conidiun J.oses its septa and appears darker in colour; otherwise some cells may survive attack and a few septa nay stil1 be visible. Ihe sequence of invasion of a conidium is difficult to observe as the amoebae are extrenely tight sensitive. They respond to bright light by withdrawal of pseudopodia and by rounding up to form an inactive arnoebal mass. Trophozoites leave spores through perfora- tions in the wall and do not disrupt enpty spores. Spores, when exanined with SElr{, show perforation in their walls varying fron 1.0 to 5.2 un in dianeter (Fig.2Oe). Sirnilar perforations are also observed in the wa1ls of chlanydospores and hyphae of P. eirmønomi (Fig.20d) after they have been incubated with the amoeba for 7 - 10 days.

Theeamoeba granifer"a sub-species minor

Feeding on hyphae of Ggt is sinilar to the feeding of this amoeba on EusarLum oæUsporum as described by Pussard et aL. (1979). After en- circling a length of hypha (Fig.2Ia), the anoeba becomes rounded and stays in contact for 10 - 20 nin, after which it moves along the hypha and the fungal cel1 encirclecl appears empty. Small pieces of t. satiuus hyphae which are usually present in conidial suspensions of this fungus are also emptied in the same l{ay (Figs.21b,c).

The amoeba can perforate conidia of. C.satiuus and digest their contents. Depending on the size of a conidiurn, it nay be partly or conpletely engulfed by the trophozoite which stays attached without movement for up to 45 min, at the end of which the conidiurn appears paler in colour. SENÍ observation shols perforations in conidial wa1ls which range between 1.2 - 5.0 ¡rn in diarneter (Fig.zld). After proJ.onged -84-

Fig.2Lz Feeding activities of Thecamoefu granífera sub-species minPr,

a A trophozoite encircling a length of G. gnanrirris tv'itici hYPha .

b A trophozoite encircling a portion of a conidiophore of CoehLiobolus satiuus' c. As Fig.21b but the portion (arrow) of the cónidiophore encirlced by the tropho- zoíte has lost its protoplasnic contents and hence lacks refringencY.

d Electron rnicrograph showing a perforated and disrupted conidium of C-satious.

(Scale bar = 10 u¡n in Figs. â,b and c; 4 um in Fig.d)

-85-

feeding by the amoeba, many c. satitsus conidia become disrupted.

MauoreLLa sp.

As a trophozoite colnes in contact with a C'satiu¿¿s conidium it stops and encircles the spore. Ingested spores may be carried for long periods and ejected by the trophozoite without any apparent darnage' On other occasions, the amoeba after ingesting one or more C. satiOus conidia gradually assumes a mo1.e or less spherical shape with a number of prominent vacuoles in the cell cytoplasrn (Fig.22a). The r¡acuoles discharge their contents and a fluid layer appears between the spore and the arnoebaf cytopfasm to form a large vacuole (Fig.22b). This vacuole resembles the digestive vacuole formed by the unidentified leptomyxid the amoeba while feeding on chlanyclospores of P. cinnamomi. After vacuole is fully forrned around the conidium the trophozoite nay continue its usual locomotion (Figs .22c,d). The spore can be seen to rotate randomly inside the vacuole and often the vacuolar membrane invaginates

and touches the spore (Fig. 22c). the shape and the diameter of the vacuole, therefore, changes continuously. This is unlike the digestive vacuole forned b1' the leptomyxid amoeba rvhich stays more or less const- ant in size and shape for a considerable period of time. After about 6 - 8 h, the sept,a in the Spole aÏe no longer visible (Fig.22e), and the conidium also appears paler in colour. The amoeba however, carries the spore inside for a much longer period of tine even after the disappearance of the septa (up to 10 h). Finalty the spore is ejected' have not sEM observations of spores taken fron feeding cultures however, provided evidence of perforation. Bacteri-al populations in cultures

have been very high and a film of bacterial ce1ls and nucilage obscured surface detail of the coniclia. -86-

Fíg.222 Feeding activities of MayoreLLa sp.

a A trophozoite with a Cochliobolus satiuus conidiun inside one of the many prominent vacuoles in its cytoplasm.

b The same conidium inside a large vacuole fonned by the dissolution of the smal1 vacuoles. c-d. The trophozoite continues its usual pseudopodial loconotion with the conidiun inside.

e The conidium after 8 h from the fornation of the vacuole. Note the lack of septum in the conidium.

f Trophozoite with engulfed G. gratninis tritiei hypha. Note the twisting and bending the hypha has undergone.

(Scale bar = 20 pn in all Figs.)

-87 -

When macerated hyphae of Ggt are used in feeding trials, Mayoz'eLLa engulfs portions of hyphae by twisting and bending these. fragments (Fig.zzf). Hyphal fragments are retained inside for up to 6 h and ejected fragnents lack refringency in some areas. lbwever, the mechan- ism of feeding is not clear.

Species of MayoreLLa have been reported to ingest and transport fungal spores (Heal , L963; Anderson and Patrick, 1980). Heal regarded feeding as unlikely and Anderson and Patrick suggested nycophagy and of MayoreLla on other amoebae. Observations here support the contention that Mayoy,eLLa can lyse spores of C.satiuus contained within large vacuoles in the trophozoi.te. Disorganisation of septa and protoplasts of coniclia within vacuoles has been recorded. Further rvork is needed to deterrnine whether cell wal1 perforation occurs during this period. The ability of MayoreLLa to feed on other amoebae is confirned.

In ¡nixed cultures of soil amoebae containing ÌulagoreLLa ttophozoites, cysts of other amoebae, especially the readily identi fj-ed Aeanthamoeba cysts, have been seen within vacuoles of MayorelLa.

Feedins of mycophagous amoebae on other soil fungi

Two different isolates of Rhizoctonía soLanL (IVARH/6 and F91) and one isolate of a PhíaLophoz,a sp. (PR-II) were tested as food for five nycophagous amoebae using feeding tlials sirnilar to those described above. After allowing a feeding period of 10 days, cultures l'¡ere fixed, pro-

cessed and examined with the SEII{ for perforations in hyphal wa1ls.

Perforations r4re¡e observed in hyphae of PLrLaLophoYa sp. with T.gnanifera sub-species mLnor and Gephyranoeba sp. and in hyphae of

RfuLzoctonia soLani (Ir91) with Sacccanoeba sp. and the unidentified lepto-

rnyxid (Table 11) . -88-

TABLE 11: Perforation in hyphal waLls of PhiaLopVnta sp. and Rhizoctonía soLani caused by rnycophagous amoebae.

A¡noeba so ra sp. WARH/6 F91 PR-II

* Ihecatnoeba granifera + ssp. m|nor ** Saecamoeba sp. N.T + N.T.

Unidentified + leptomyxid

Gephyranoeba sp. +

MayoreLLa sp.

*- No perforations observed; + perforations observed; ** N.T. Not tested. -89-

F91 is an isolate of the strain that causes bare patch of wheat and IVARH/6 has strongly pigrnented hyphae even when they are young.

Discuss ion

Although the mechanisrn of feeding varies with the anoebal species and the fungi involved, nycophagous amoebae generally enploy the follow- ing basic steps in fungal feeding:

1. Attachment of trophozoites to fungal propagules, whj,ch appears

to be a matter of chance. There seems to be no strong chemotaxy

or thigrnotaxy. In all genera so far studied, amoebae nay engulf

spores or hyphae which are potentially food, only to rnove away

leaving the propagules unharmed.

2. Engulfment of the propagule, partially or completely.

The giant trophozoites of ArachnuLa, VønpyreLLa and Thez'atromyæa are able to conpletely engulf spores or sections of the fungal

thallus (Oid, L977a; Anderson and Patrick, 1978, t9B0; 01d and Oros, 1980). The smaller Saccønoeba and Thecønoeba attach to restricted parts of hyphae or spol:e lva1ls and penetrate through to the protoplast.

3 . Digest ion, which rna y be by rnotile trophozoites which penetrate

ce11 walls as in Av,achnuLa, T\rccamoeba, Saccamoeba, Gephynamoeba and the unidentified leptomyxid, or by digestion of fungal cells

within specialised food vacuoles. lhese may form within cysts,

for example, ArachnuLa inrpatiens forms digestive cysts after a feeding per:iod. A large central vacuole forms in the cyst within which fungal celL residues are digested as completely as possible. -90-

ln TheratnomAæa, the whole process of wal1 penetration and digestion of spore contents proceeds within a cyst. The unidentified lepto- nyxid forrns large vacuoles within which complete chlanydospores of PVtytophthora eínnamomí are reduced to fornless debris. Mayorella

also forms food vacuoles within trophozoites but indications so far are that C.satiuus spores remain largely intact and only the spore contents are digested.

The unidentified leptomyxid anoeba did not lyse pigmented conidia of C.satiuus despite the opportunities that existed during its close association with the spores.

fn nany fungi the brownish to black pigment, melanin, confers resistance to mícrobial lysis (Lockwood, 1960; Lingappa et aL., 1963; Bartnicki-Garcia and Reyes , 1964; Kuo and Alexander, 1967). The ability of a number of genera of soil amoebae to lyse pigmented fungal propagules is particularly significant and has been discuss- ed by 01d and Patrick (1979). With the denonstration of lysis of

C.satiuu.s conidia by Saccamoeba, Thecamoeba, Gephyz.amoeba and MayoreLLa the nunber of genera able to attack pigmented fungal

cells j-s increased to seven, although the taxonorny of some of these

amoebae remains in doubt.

The tine taken by different amoebae to lyse fungal propagules by different feeding mechanisms varies markedly. T.gz,anifer"a sub-species minor emptied a conidiun of C.satiuus in less than t h, whereas A.irnpatíens took appïox. 4.5 - 6 h (Old , IITB). The unidentified lepto- myxid took about 30 nin to lyse P. cinnønonrL hyphae but needed 22 - 36 h to digest chlanydospores of the same fungus. Conidia of C.satiuus were contained within MayoreT.La trophozoites for more than 16 h during which time rupture of internal septa and protoplast disorganisation occurred. -91-

IV, R0LE 0F S0l L AIT0EBAE I N SUPPRESS I0N 0F Gqr

A. SOIL AMOEBAE AND SAPROPHYTIC SURVIVAL OF GqT

That antogonistic microorganisrns nay have a role in the decline of virulence of field inoculum of foot rot fungi has been known for some time (Sirnmonds, 1928; Broadfoot, 1931; Winter, 1940). Soils may differ in their intrinsic 'biological antagonismt and thus con- tribute to the variation in take-all from soil to soil (Garrett, 1937); while others nay develop higher populations of antagonistic micro- organisms and suppress Ggt following the addition of organic naterials (Garrett , 7934; Fellows and Ficke, 1934); annonium nitrogen (Smiley and Cook, 1973); nonoculture of wheat (TAD, Slope and Cox, 7964; Cox, 1965) or cultivation of non-hosts (Zogg, 1969, I972). Whether sone or all of these conditions of disease suppression are related and therefore have sinilar underlying causes is not knoun and it j-s unde- sirable to presume honology between suppression in pots and various kinds of suppression in the field (Hornby, i979).

Itralker (1975) distinguished six conditions of take-a11 suppression and concluded that the term TAD should be restricted to suppression in the field. Gerlagh (1968) recognised two types of take-all suppression, rgeneralt, which is the interaction between the pathogen and increasing nurnbers of non-specific organisrns, and tspecific', which followed wheat rnonoculture rvith severe incidence of take-al1. Accepting these

concepts, Cook and Rovira (1976) characterised general and specific

suppression and Rovira and IVildermuth (1981) suggested that the main characteristic rvhich distinguishes these suppressions is whether they can be transferred into natural or furnigated soil and pointed out that -92-

TAD should be included in specific suppression. These authors out- lined three causes for transferable suppression:

(a) continuous wheat with take-alI (to include TAD soil);

(b) addition of Ggt nycelia to soil i (c) addition of other fungi to soil, e.8., GibbereLla zeae,

G. grønLnís var. graminís.

However, since the developnent of an antagonisn to the parasite in the absence of the host is not necessarily the same as natural TAD (Brown et aL., 1973); the term tcontrived suppressiveness' has been recommended for,laboratory nodels untit corroborated by field studies

(Schroth and Hancock, 1981).

Suppression sinilar to specific suppression in its character- istic.s has been obtained by adding soils from fallorv, wheat' pasture and continuous pasture rotations to funigated soil and the induction of transferable suppression in a soil under pasture indicates that the wheat plant per se is not necessary in the induction of a transferable suppression (Itrildermuth, 1980) . This suggests that the suppressive factors are active in the saprophytic as well as the parasitic stage of the pathogen. Considering this, Palti (198I) rightly defined suppression as rthe resistance offered by certain soils to the survival, saprophytic and pathogenic activity of phytopathogenic fungit. The definition of Rovira and l\rildernuth (1981) for suppressive soi1, i.e., rsoils with a micro-flora which reduces the level of the disease caused by the take-aI1 fungusr does not consider the saprophytic survival of the fungus which is often reduced in a suppressive soil (Pope and Jackson, I973; lVildernuth, 1977) and the possible role of micro-fauna -93-

(Honrna et aL., 1979) in suppression. In postulating the mechanism for specific suppression, Rovira and Wildermuth (1981) however, suggested that the Inajor site of specific suppression is associated with plant residues which carry both the pathogen and the suppressive microorganisms.... r.

According to Shipton (1981), since the parasitic and saprophytic modes of nutrition are so interdependent and inter-related in the field, with alternation from one to the other, it would be unrealistic, if not misleading, to consider either behaviour in isolation.

Several environmental and nutritional factors influence the survival of the fungus. Availability of rritrogen is probably the single nost important factor, and the fungus dies early in N-deficient soil

(Garrett, 1938; Butler, 1953, 1959; Chambers and Flentje, 1969; Scott, 1969; lVeste and Thrower, 1971). l\later potential, texture and temperature of soi1, in general, indirectly influence the saprophytic survival of Ggt through their effects on rni.crobial activity and anta- gonisn. The fungus otherrvise is not appreciably affected by extrernes of temperatures (-29oC to 71oC, Fellows, 1941) or moisture (30e" to SOeo saturation, Garrett, 1938) alone, but is rapidly elinínated in wet hot soil (-10 to -20 kPa and 35oC, lr{acNish, 7973). Anong other factoïs, according to Lal (1939), mycelium of the fungus disappeared(rapidly f',rm acid soils (pH 4.8 to 5.0) than in sand or alkaline soils (pH up to 8.0).

However, Zogg (1959) found that neither pH (5.9 to 8.1) nor humus content or -soil type altered the rate of elinination of inoculum; decline in surviúal was related to cropping history and greater numbers of microorganisms (Garrett, 1958). -94-

Survival of Ggt decreases independently of fertility levels with increasing nunbers of consecutive crops of rvheat (MacNish, 1976) oI barley (Snith, 1979). Cunninghan (1975) found evidence of, loss of conpetitive saprophytic ability in isolates from long sequences of wheat or wheat and barley as conpared to those fron the first, second and third crops after perrnanent pasture. Intensified developnent and activity of rnicroflora (Vojinovic, I973) causes reduced survival of inoculun (Gerlagh, 1968) and results in fer,rer infective particles in

TAD soils (Pope and Jackson, I973). Shipton (1981) considers that ttre phenonenon of TAD can at least in part be explained by the effects on saprophytic survival and associated soil rnicroflora. Natural soils with specific suppression properties also reduce the density of pig- mented hyphae buried in these soils (lVildermuth, L977) and hyphal lysis is more pronounced in the suppressive soils.

To study the role of soil amoebae in the saprophytic survival of Ggt in suppressive and non-suppressive soi1s, rnycelia were buried in soí1s and associated amoebae were isolated.

The nethod used

The menlbrane filter burial technique (01d, I977b) described earlier was used. Blencled hyphal suspensions of Ggt were buried in the suppressive WPP and the non-suppressive lvlB soi1. CS was used as control. Soils were collected from the top 0-5 cn of the profile and sieved through a 2 mn sieve. The IVPP and MB soils were moistened to

-8 kPa and -2 kPa respectively. CS was noistenecl to 15% noisture

(oven dry wt basis). Three replicate membranes lvere retrieved from each soil at weekly intervals and each membrane rvas cut in to 4 quart.ers -95-

One quarter was used for SEM observations; the second for measuring hyphal density by light microscopy and the other two v¡ere used for studying the survival of Ggt and the association of anoebae with the fungus. For SEI'Í , membranes were processed using the nethod describecl earlier.

To estinate hyphal density, filters were stained with I% Iacto- phenol cotton blue and examined at 400X. Density was deternined using the line intercept nethod (Rovira et aL., 1974). lhe mean length of myceliun per unit area (L) was calculated by counting the ntunber of intercepts between hyphae and two orthogonal lines on an eyepiece graticule and by using the equation:

L - mN/2H where, N is the number of intercepts and H is the total length of lines on the graticule.

The third rnenbrane quarter was further divided into 4 approx.

1 nn squares and these were plated on NDY/6 incorporated with 100 ug streptomycin n1-1 and 10 ¡rg tetracycline r1-1. Percentage survival of Ggt was calculated from the number of pieces shorving growth of the fungus after incubation at 25oC for 48 h. The renaining four:th mem- brane quarter was sinilarly subdivided and plated on PJA, incubated at

25oC for two weeks and examined for the growth of amoebae.

Amoebae and s ic survival of hae l-n suppressive and non-suppress 1VESoa 1

The fungus survived longer in the non-suppressive MB soil than in the suppressive WPP soil (Fig.23) . After 4 weeks no mernbrane -96-

Fig.23 Saprophytic survival of G.gz,øninis tz,itiei in suppressive lVaite pernanent pasture and non-suppressive Murray Bridge soil.

^H Sterilized coarse sand.

H lvfurray Bridge soil . H Waite permanent pasture soil roo a-^

o fJ 80 A OL f AA o óo o trl (9 É 40 ztrl U OL lrl o- 20 o

o

o 234 5 7 WEEKS AFTER BURIAL -97 -

Tecovered fron the wPP soil had viable fungal hyphae, whereas the fungus survived for up to 6 weeks in the MB soil. rn all 5 soils, the density of pigrnented hyphae changed with tine (Fig.2Ð. Re- gression analysis showed that the rate of change in density hras significantly (P<0.01) different in the three soils. For the initial 4 weeks the density increased at a sinirar rate in all soils but the¡eafter both MB and cs had progressively higher densities, whereas, density of pigmented hyphae in the wpp soil reduced significantly.

SEM observations of the recovered filters confirn the reduction of hyphal density after 4 weeks in the suppressive wpp soi1. After B weeks mernbranes'recovered from wpp showed nuch lower densities (Fig.25a) than those fron MB (Fig.2sb) and cs (Fie.2sc). Exrensive erosion of hyphal walls (Fig.25d) rvas observed in specirnens fron all soils, though only after 3 weeks of buriar in cs. Discrete perfora- tions were observed in hyphae recoved fron both wpp and lvlB soils

(Fig.25e). These were quite distinct in their inorphology from the general erosion of hyphal walls observed in almost all specimens. The size of perforations varied fron 0.6 to J.5 prn in dianeter. No perfor- ations were observed in hyphae recovered fron cS. Hyphae recovered from MB soil had fewer perforations and aftcr 8 weeks buriai 1vs¡s healthier than those from the IVpp soi1. However, no quantitative estirnate of the nunber of perforations was rnade.

several soil arnoebae including mycophagous species were found associated with hyphae recovered fron these soj_ls (Tab1 e r2). The non-mycophagous Acanthamoeba and Echinamoeba were obtained from al1

3 soils, the mycophagous MctyorelLa, saccønoeba, Thecamoeba granifera sub-species m'inor and the leptomyxid anoeba were not isolated from the non-suppressive soil. -98-

Fig.24z Density of pignented hyphae of G. grøninís tr|tíei on nillipore filters buried in suppressive Waite perrnanent pasture and non-suppressive Murray Bridge soil.

Sterilized coarse sand . H Murray Bridge soil. H Waite permanent pasture soil.

I Standard error. € 50 ç E E l¡l T À I oo ô Flrl zlrl I È oTL 50 t zttl tr¡ ô o12345ó78 WEEKS AFTER BURIAL -99-

Fig. 25 Morphology and density of hyphae of G.grøninis tv"Ltiei after 8 weeks in suppressive lVaite permanent pasture and non-suppressive Murray Bridge soi1.

a Morphology and density of hyphae after 8 weeks in the suppressive l{PP soil.

b It{orphology and density of hyphae after B weeks in the non*suppressive IvfB soil . c Morphology anC density of hyphae after 8 weeks in sterilízed coarse sand. d Hyphae shorving extensive erosion of their walls. These hyphae ürere recovered from sterilized coarse sand after 6 weeks. e Discrete perforations in hyphal wal1s re- covered fron the Waite permanent pasture soi1.

(Scale bar = 2 un for all Figs.) -- {t -100-

TABLE 12 Percentage occurrence of amoebae witt¡ Ggt hyphae buried in soils.

lVeeks Anoeba Soil after burial 1 ) 34 5 6 I

Dfycophagous Leptonyxids Il¡PP 8 0 L7 8 0 L7 L7 MB 0 0 0 0 0 0 0 CS 0 0 0 0 0 8 8

MayoreLLa spp. WPP 0 8 0 0 0 8 33 MB 0 0 0 0 0 0 0 CS 0 0 0 0 0 0 0

Saecamoebd spp WPP 0 0 0 8 0 I 0 MB 0 0 0 0 0 0 0 CS 0 0 0 0 0 0 0

T.granifera ssp. WPP 0 77 0 0 0 0 0 MLNOT MB 0 0 0 0 0 0 0 CS 0 0 0 0 0 0 0

Non-nycophagous WPP t7 75 33 8 8 25 50 Acant?tønoeba spp MB 0 0 0 0 25 8 25 CS L7 0 25 75 100 100 95

Echinønoeba spp. WPP 8 1 7 8 0 0 0 25 MB 0 0 0 0 25 I 58 CS 0 0 8 0 0 0 0

Platyatnoeba spp. I|IPP 8 25 77 t7 0 77 75 MB 0 0 0 0 0 0 L7 CS 0 0 0 0 0 0 0

Tteeatnoeba spp. I{PP 0 25 I B 0 8 8 MB 0 0 0 0 0 0 L7 CS 0 0 0 0 0 0 0

Unidentified WPP 25 5B 33 50 8 5B I MB 0 0 0 25 50 0 0 CS 0 0 0 0 0 0 0

Varrpyrellids }VPP 0 2S 0 T7 0 58 33 MB 0 0 0 0 17 B 25 CS 0 0 0 0 0 0 0 -101- Wi\lTE INSTITUTE LISRARY

Saprophytic survival of Ggt was reduced in the suppressive Wpp soil. In both WPP and MB soils there was an inc:rea.se in the density of pigmented hyphae during the first 4 weeks but thereafter, hyphal density in the WPP soil was reduced significantly although both lr{B and CS had progressively higher densities.

In Garrettts (1938) experiments, two heavy textured clay loams encouraged a high rate of inoculum decline in contrast to a najority of infertile soils. Although this is thought to be due to greater numbers of microorganisms rather than to physical effects of aeration etc. and pH, humus content, or soil classification (Zogg, 1g5g); soil nutrient levels, on the other hand, including those of N (Garrett, 1938; Butler,

1953, 1959; Chanbers and Flentje, 1_969; Scott, 1969; tVeste and

Thrower, 1977) and P (chang, 1939; Butler, 1961) are known to influence the saprophytic survivar of the fungus. since the MB soil is lighter in texture and hence lower in nutrients, a second non-suppressive soil fron the permanent continuous wheat rotation (cw) was included in a sinilar experiment using the nembrane filter burial technique. Both

WPP and CIV are red-brown earths with sinilar pH, textural and nutritional

(C, N and P) characteristics (Steward and Oacles , 1972).

Amoebae and sa ic survival of e].n IV P CIV It{B and

Ggt was buried in these soils and five replicate rnembranes were retrieved at 2,4,6,8,72,74 and 20 weeks after burial. At rec.oveay, densities of both hyaline and pigmented hyphae were estimated, survival of the fungus studierl ancl amoebal association with the fungus deterrnined. -LOz-

Though only for two more weeks in the CItl soi1, Ggt in general, survived longer in the non-suppressive soils than in the suppressive

WPP soil (Fig.26a). In the WPP soil, the fungus survived for less than 4 weeks. Among the non-suppressive soils , Ggt survived for up to

less than 12 weeks in the light-textured lr{B soil, whereas for less than

6 weeks in the heavier CW soil.

In all soils there hras a reduction in the density of total hyphae over tirne (Fig.26b); although the rate of decline was significantly (P.0.01) different. For hyaline hyphae, the rate of decline of their

density was sinilar in the I{PP and CI{ soils and very little hyphae were left on rnembranes after 6 weeks of burial (Fig.26c). Fitted regression

lines of the densities of hyaline hyphae over tirne was linear in CS and curvilinear with initial faster rates of decline in all other soils.

Densities of pignented hyphae increased initially and thereafter declined in a1l soils at significantly (p.0.01) different rares (Fig.26d). Interestingly enough, the regression line for the density of. pignented

hyphae in the CW soil was linear, whereas those in the other three soils welre curvilinear with an initial faster rate of pigmentation. 'Iherefore,

the rate of destruction of hy'aline hyphae was sinilar in IVPP and Cltl soiJ.s,

but that of the pígrnented hyphae was significantly higher in the lt¡PP soil

than in the CIV soi1,

Initially, up tc 4 weeks, higher associations of mycophagous

arnoebae were recorded with Ggt hyphae recovered frorn the I{PP soil, but none after B weeks (Tables 15 and 14). Tliis higher association of nyco- phagous amocbac coincided rvith live hyphae of the fungus in the WPP soil

and mycophagous amoebae were not isolated after 8 week-s of burial when viable hyphae of the fungus could not be detected. No such relationship -103-

Fíg.262 Density and survival of G.gr'øninis tr"itiei h¡ryhae in suppressive lVaite permanent pasture and non-suppressive continuous wheat and Murray Bridge soils.

H Itlaite pernanent pasture soil . Á.---â Continuous wheat soil. Murray Bridge soil. o-o Sterilized coarse sand.

â., Saprophytic survival of the fungus in soils. b. Density of total hyphae. c. Density of hyaline hyphae. d. Density of pigrnented hyphae. !

DENSITY OF TOTAL HYPHAE (mm.mm-2) % SURVIVAL õ o o

@

o

ñ

€ E m m @ @ õ ^an 3L JL Þ a¡ -l DENSITY OF PIGMENTED HYPHAE (mm.mm-2) (mm.mñ2) m DENSIIY OF HYALINE HYPHAE Ð s è cfD 2 t-

@ @

ul I \ OLt+ -104-

TABLE 13: Percentage occurrence of amoebae wrth Ggt hyphae buried in suppressive (WPP) and non-suppressive (CW, MB, CS) soils.

Weeks after burial Anoeba Soil 246 8L21420

Mycophagous Leptornyxids WPP 25 25 0 6 0 0 ,0 CW 0 0 0 0 0 0 0 MB 0 0 0 t2 6 0 0 CS 0 0 0 0 18 0 18

MayoreLLa spp. WPP 0 L2 0 6 0 0 0 CW t2 0 0 6 0 6 L2 MB 0 18 0 0 0 0 0 CS 0 0 0 0 0 0 0

Saecønoeba spp. WPP 0 6 0 0 0 0 0 CW 0 0 0 0 0 0 0 MB 0 0 6 0 0 6 0 CS 0 0 0 0 6 0 2S

Iheeønoeba WPP 2S 25 0 12 0 0 0 granifera ssp. CW 25 0 6 0 L2 6 37 rtinor MB 37 0 6 3l 0 0 72 CS 0 0 0 6 4s 25 5t

Non-mycophagous AeantVnmoeba spp. WPP 3l L2 6 0 0 s7 5/ CW t2 0 6 37 0 6 43 MB B1 81 62 100 75 6B B1 CS 100 100 B1 6B 93 95 L00

EelrLnønoeba spp. WPP 25 18 0 L2 0 28 0 CW 72 0 t2 6 0 1B LB MB 6 43 43 1B 1 ) 25 6 CS 0 25 12 3T 0 25 25

PLatyønoeba spp. WPP 37 25 6 6 0 2T 6 CW T2 0 6 31 18 6 25 MB B1 50 81 B1 31, 68 75 CS 0 31 25 6 0 6 6

Thecønoeba spp. WPP 6 7,2 0 0 0 0 0 clri 0 0 0 T2 0 6 L2 MB' 0 0 -0 72 0 0 0 CS 0 0 0 6 1 2 50 0

Vanpyrellids WPP L2 2S 0 6 0 2T 0 CW 18 0 0 0 1B 6 0 MB B1 87 62 B1 62 62 37 CS 0 0 6 0 0 0 0 Unidentified ItlPP 37 43 6 6 0 42 7S ctrl 43 0 3T 37 43 56 50 MB 62 87 62 6B 62 62 68 CS 6 0 56 68 43 43 43 I

TABLE 14: Sunnary of amoebae isolated fron Ggt }:rypl:rae buried in suppressive (WPP) and non- suppressive (CI^¡, MB and CS) soils. (Percentage of I sq.nn nillipore menbrane plated)

I{eeks after burial

2 4 ó 8 t2 L4 20 Soil AaMbAM AM AM AM AM AM I

(.fi I WPP 19 25 20 27 10 53 00200 t3 0

CW L3 18 00 610t30 915115 20 18

MB 55 1035032340102525292 28 4

CS 10015018018321402410 2S 3l

ê = all anoebae b nycophagous annoebae. -106-

was observed in the non-suppressive CW and MB soils, A lower percent- age of nycophagous amoebae, compared to that in the WPP soil, v¡as found associated with the fungus in the CIII and MB soils throughout the experi- nental period of 20 weeks; even though no viable Ggt was recovered from after 6 weeks in CIV and 12 rveeks in MB soil.

These results suggest that soil type nay be of sone irnportance but by itself can not fu1Iy explain the reduced saprophytic survival of the fungus in the suppressive ülPP soil. As in other soils (Garrett, 1938; Zogg, 1959), the saprophytic survival of the ftngus in these soils is related to cropping history and greater number of microorganisms which may directly or indirectly be influenced by the soil physical factors, since the biological and physical settings of the soil are inseparable rvhen considering the behaviour of soil microbes (Stotzky and Rem, 1966). The differential survival and reduction of hyphal densities of the fungus in the othenvise physically sinilar I{PP and CW soils further suggest that soil suppressiveness was associated with the reduced survival of the fungus in the IVPP soil. Furthermore, the abundance and activities of mycophagoLrs amoebae coincided with the decline in densities and survival of the fungal hyphae in the suppressive

WPP soi1. In TAD soils also, irrtensified developnent and activity of nicroflora cause reducecl survival of inoculum and result in fewer infective particles (Gerlagh, 1968; Pope and Jackson, I973;

Vojinovj c , 1973).

Saprophytic survival of Ggt ín sterile soils arnended with suspensions of nycoph.agous amoebae

Mycelia of Ggt were buried in sterile (autoclaved for t h for 3 consecutive days at 727oC) IVPP and MB soils containing a mixture of -to7 -

suspensions (about 10 ml ptat"-l¡ of three nycophagous amoebae, Saecønoeba sp., Gephyr,ønoeba sp. and the unidentified leptonyxid. In an initial experinent, Iheeønoeba granifez,a sub-species minor was in- cluded with these three amoebae, but as the Fusaríun-Llke fungus associated with this arnoeba almost always overgrew Ggt on NDY/6 plates, this was excluded from the test. Controls were maintained with sterilized soils containing suspensions of the food bacterirnn,

KLebsíeLLa sp., sterilized soil only and unsterile WPP and MB soiI, Three replicate membranes were retrieved I12,4 and 8 weeks after burial and I mn squares were plated on NDY/6.

Results show that there was a slight decrease in the survival of the fungus after 2 and 4 weeks of burial in sterile WPP soil in the presence of the amoebae (Table 15). In the non-suppressive soil however, there L\¡as no loss of viability even after B rveeks in the presence of the anoel¡ae. The bacteriu¡n did not affect the survival of the fungus but the fungus failed to survive in either of the sterile soils after 8 weeks. Reduction in the saprophytic survival as before, was observed in the unsterile WPP and MB soi1s.

Steam sterilization alters both the organic and mineral fractions of soils (Eno and Popenoe, 1964; Sonneveld, 1979). The first colonisers can increase in pr:eviously sterilized soil to high Levels because of the release of nutrients and reduced conpetition (Johnson and Curl , 1972).

In fumigated soil horvever, there is a slower recolonisation by Ggt as measured by its incidence on wheat roots, compared to that in the un- treated soil (Warcup, 1976). Ludwig and Henry (1943) reinfested sterile soi-l with Ggt; vr4l.en recontaminated by additions of sna1l amounts of field soi1, the total nicroflora including some antagonists of the take- all fungus quickly increased in density to a higher leve1 than i.n - 108-

TABLE 15: Saprophytic survival of Ggt in sterile suppressive (WPP) and non-suppressi.ve (MB) soils amended with suspensions of nycophagous amoebae (eo survival).

Soils Weeks after WPP MB burial unstr.a Str.b Kleb.t lrli*.d Unstr. str. Kleb. Mix

1 85 100 70 95 80 100 100 100

2 80 0 100 85 100 9s 100 100

4 50 4s 100 55 BO 0 100 100

8 5 0 100 95 90 060 100

a = Unsterilised b = Sterilised c = Sterilised soil + suspension of KLebsieLLa sp. d = Sterilised soil + mixed suspension of Saceønoeba sp., Gephyratnoeba sp. and the unidentified leptomyxid. - 109-

unsterile soi1. Effects of altered physical. and chenical status of the

sterile soils and absence of other nicroorganisms may have a bearing on

the interactions between the take-all fungus and the nycophagous amoebae

tested; since the precarious balance of microbial interactions in many cases only work in biologically balanced soils (Baker and cook, L974; Bowen and Rovira, 1976).

Ability of mycophagous amoebae to invade infected wheat roots

Darbyshire and Greaves (1971) found that the bacteriophagous soil amoeba AcantVwnoeba palestinensis (Reich, 1953) invaded the epidermis and outer cortical tissues of pea roots lvhen inoculated with a Pseudo- mona,s sp. Since no amoebae t'/ere found inside root,s inoculated only with.4.paLestinensis, the invasion was explained as a passive entry of trophozoites inside roots following the food bacteríun. A report on the presence of Leptomyæa retieuLata Goodey, 1915 var. hwruLí Mclennan, 1930

inside tissues of hop plants was published by Mclennan (19s0). Tuzet

and Roquerol (1961 , 1962) also recorded the presence of amoebae inside roots of rice plants.

Besides killing ectotrophic hyphae on live roots, invasion of in- fectecl wheat Toots by mycophagous amoebae in natural soil may enable amoebae to perforate and lyse the fungal hyphae inside host residues and thereby reduce the saprophytic inoculum. An attempt h/as therefore made to test if mycophagou-s amoebae hrere capable of invading infected wheat root s .

Four rr¡eeks oId seminal roots of whcat infected wíth Cgt lvere obtained from a bioassay and abor.rt 2 crn pieces were incubated at 25oC with a nixed -110-

AS suspension of GepLtyr,ønoeba sp., Saecønoeba sp., Theeanoeba granifena sub-species minon and the unidentified leptomyxid. After 3 weeks roots were surface sterilised with one of the following sterilants, washed twice in SDltl and plated on PJA for growth of amoebae, Alternatively, infected wheat roots were obtained fron a bioassay in which a nixture of suspensions of these amoebae were added to CS andwheatwas grown for

4 weeks. Pieces of these roots were surface sterilised and plated as previously. Surface sterilants used were:

-1 1). Actidione 200 ¡rg n1 for 5 min; 2). HECIZ,0.1% solution for 5 nin;

3). AgNOr 0.1% solution for 5 min and washed with 0.5% NaCl;

4). C2HSOH 70% solution for 5 nin;

s). Milton solution (sodium hypochlorite Ieo and sodium

chloride L6.Seo, Milton Pharmaceutical Cornpany, Sydney) fu1l strength for 5 nin.

Controls were washed with SDItr only.

The unidentified leptomyxid was isolated from roots treated with all of the sterilants (Tab1e 16) and Saccønoeba sp. was absent only in

0.1% AgNO, treated roots. T.granifera sub-species minor and

GepVtyt'ønoeba sp. were not isolated from roots treated wi-tln 70% C2H50H,

0.1% HgCl, or 0.1% ACNO'. Since ttre Saccønoeba sp. is non-cystic, the results can not be explained on the basis of higher resistance of cysts towards these chemicals. The results suggest that mycophagous amoebae were able to invade wheat roots infected rvith the take-all fungus. -111-

TABLE 16: Percent recovery of anoebae from surface sterilised wheat roots infected with take-a11.

Anoeba recovered Sterilant Unldent].fi ed Gephyrønoeba T.grant fera Saceønoeba Leptonyxid sp. ssp. minon sp.

Actidione 100 60 90 60

Milton 10 5 5 5 c2HsoH 10 0 0 10

HgCL, 16 0 0 5

AgNOa 33 0 0 0

Control (SDW) 100 85 95 95 -LLz-

Discussion

The najority of saprophytic inoculun of Ggt originates from pre- viously infected host tissue (Garrett, 1956, 1970); although the possibilities of free-living mycelium has certainly been indicated (Fellows and Ficke, 1959; Warcup, 1957); While alive in plant debris, it is able to respond to the presence of growing roots by linited. growÊh in the rhizosphere (Brown and Hornby, I97I; Pope and Jackson, 7973; Wildermuth, 7977). The frrngus can also grow in soil in the absence of wheat roots (Brown and Hornby, 7977; Wildernuth et aL., 1979). Thus, root-inoculum contact (Garrett, 1956) is not always necessary for colonisation of Toots and the fungus can colonise wheat Toots from a zone 9-I4 *r u*"i from roots in 28 days (Wildennuth, Ig77). The rate of grorvth of hyphae through soil was calculated to be about 0.4 - 0.6 nn -1 day ', which is less than the rate of ectotroplìic growth on roots (2.0 - _1 4.4 mn day -, l\rildernuth, L977).

Growth of Ggt is initiated by hyaline hyphae which nay later become pignented. After burial, within one week, hyphae becarne pigmented in all soils, the rate of pigmentation varying with the soíl. Pigrnentation of hyphae protects them in soils and the fungus present in naturally- infested soil apparently survives longer than in artificially-colonised strah/ because of the presence of dark hyphae (lrlacNish, 1976; Srnith, 1979).

Cunninghanr (1975) also'fowrd that dark hyphae had strong saprophytic capacity. This pigment, melanin or rnelanin-like cornpounds in fungal cel1-wal1s, imparts resistance to microbial lysis (Lockwood, 1960;

Lingappa et aL., 1963; Bartnicki-Garcia and Reyes, 1964; Kuo and

Alexander, 1967). Melanin extracted fron hyphae of Ggt is known to inhibit specific lysis of the fungus by StneptornAces LauenduLae (Waksm. and Curt.) IVaksm and Henr. and less lysis has been observed in darker ísolates (Tschudi and Kern, 1979). -113-

Of the two soils, IIIPP and CW, with sinilar physical and chemical properties used in my experiments, the suppressive WPP soil showed a faster rate of destruction of pigmented hyphae than the CItr soi1. The density of hyaline hyphae, on the other hand, was reduced at a similar but nuch faster rate than that of the pignented hyphae in both of these soils. One difference between these soils, therefore lies in the rate of destruction of pigmented hyphae which are responsible for the sur- vival of the fungus. Considering the growth of the fungus from an inoculun source in soil to the host root, propagules with large ïeserves nay grow several centimetres to the root (Wildermuth, 1977; Brown and Hornby, 1971) and'this period of pre-colonisation is apparently the time when conpetition with PhiaLopVora takes place (Deacon, 1976), and

Pseudomorns fLuot'escens Nligula restricts its growth (cook and Rovira,

7976). The demonstration that soil amoebae can produce cellulase and chitinase rnakes thern capable of lysing hyaline hyphae of Ggt (Tracey, f955). On the other hand, to rny knowledge, soil amoebae are the only group of organisms capable of lysing nelanised fungal propagules (01d, 1977a; Anderson and Patrick, 1978, 1980).

The IVPP soil sholed a higher assoc.iationof mycophagous amoebae with Ggt hyphae for the initial 4 tveeks when the fungus was alive in this soi1.

The walls of many hyphae were perforated. Sirnilar perforations caused by nycophagous amoebae have been recorded before in hyphal wa1Is of the fungus recovered from a TAD soil (Honna et aL., 1979). -774-

B POPULATIONS OF AMOEBAE IN SUPPRESSIVE AND NON-SUPPRESSIVE SOILS

Several of the theories proposed to explain TAD invoke the development of an antagonistic microflora which suppresses the fungus. Vojinovic (1973) reported selective stinulation by infected wheat roots of rod-shaped non-sporing bacteria in TAD soils; Cook and Rovira (1976) and Smiley (i979) found a highly specific group of fluorescent pseudo- monads i Zogg and Jaggi (I974) report.ed an actinonycete with intense hypholytic activity; and Slope et aL. (1978) reported the occurrence of PhiaLophoz.a z,adíeieoLa Cain var. grøninieoLa Deacon predoninantly in

TAD soils. Brown et aL. (1973) suggested that TAD operated through changes in the soil microflora which ¡nodified the root environment and limited the disease nutritionally.

These reports suggest that the diversity and nagnitude of micro- bial populations in suppressive and non-strppressive soils are different and an attenpt was therefore made to study the composition and population of mycophagous and other amoebae in suppressive and non-suppressive soil.

The dilution culture technique originated by Cutler (1920) and developed by Singh (I946a, 1955) has been wiclely used for estimating numbers of nakecl amoebae in soils (Heal , I9TI). Singh (.Ig46a) estimated the method to be 64-73% efficient. Darbyshire et aL. (1974) introduced the microtiter dilution plate as a modification to the dilution culture method of Singh (1955); this technique is rapid, requires feler plates and is suitable for soil protozoa in general. Using a selective rnediurn, Pussard et aL. (1979) estinated the population of the strictly nyco- phagous Thecønoeba graniiera sub-species nrinor and cornpared the efficiency of this rsoil rveights technique'with that of the dilution -115-

culture nethod of Singh (1955). They considered their technique appropriate for arnoebae like ?. granifera sub-species nrLnor, which have a population density of less than 100 arnoebae g-1 dry wt of soil (Alabouvette et aL., 1981) and which. are strictly nycophagous in nature.

The rnycophagous amoebae reported in this thesis and elsewhere (Old, L977a; Anderson and Patrick, L978, 1980) are able to feed on several other organisrns in addition to fungi

Soils

0f the seven najor rgreat soil groupsr of the Australian wheat growing zones, solonised brown soils cover the widest areas , nearly 27% (McGarity, 1975). Two wheat fields, one from Balaklava (Avon) and the other from near Murray Bridge (MB), South Australia, v¡ere selected to represent two sub-groups of this major soil group. The third soil was the red-brown earth, WPP (Northcote, 1981). The I{PP soil is suppre- síve to Ggt (lVildermuth, L977) and the other tlo are non-suppressive (Table 4, page 18). Classification, particle size distribution, pH, cropping history and noisture characteristic curve of each soil have been recorded earlier (pages 10 and 11).

Fields were subdivided into grids; approx. 10m x 10m for Avon and MB and 2m x 2m for'WPP, and about 2 kg of soil from the top 0-5 cn was collecteci fron each of the 20 intercepts. Sarnples were stored in sealed plastic bags at room temperature- Soil noisture contents of the soils at sanpling were:

Ãvon - I.7e" MB - 0.6% (oven dry wt. basis). I'li'PP - 2.2ro -116-

Consideration of nethods

One ain of the present investigation was to deternine the popu- lations of different genera of soil amoebae and thereby calculate the population of rnycophagous genera in these soils. The modified soil dilution technique of Cutler (L920) (Cutler et aL., L922) offers the opportunity of both examining and estirnating the population of different genera on agar plates. However, as Singh (1955) pointed out, Cutlerrs ¡nethod has three disadvantages: (1) insufficient replications;

(2) likelihood of missing some of the protozoan species when the whole surface of the Petri dish has to be exanJ-ned, and (3) the random selection of food organisms which may happen to grow on the plate - this is particularly relevant as many soil bacteria are not only inedible to protozoa but produce secretions toxic to thern (Singh, 1945). With Singhrs (1955) ring nethod, holever, identification of anoebae is difficult rvhen they are present inside a thick bacterial suspension; on the other hand, identification is relatively easier on a cl,ear agar surface as can be obtained with Cutler's '(1920) nethod.

The soil dilution nethod of Cutler et aL. (L922) was therefore used with rnodifications. A suspension of the food bacterium, KLebsieLLd sp., hlas spread on PJA and five replicates of each of the 1O-fold dilutions were plated. Two-fold'and four-fo1d dilution series are however, more accurate than ten-fold series (Singh, I946a, 1955; Stevens, 195B), and

Singh (1946a) recornmended the use of 15 1eve1s of two-fold series with 8 replicates at each level. This woulcl involve the examination of I20 plates per soil sample if the dilution culture method (Cutler et aL., 7922) is used. -ll7 -

In nineral soils prîotozoa can be suspended in soil dilutions by shaking (Singh, L946a; Darbyshire and Greaves, L967) but in litter or organic soils protozoa nay be trapped j¡the larger organic particles.

Heal (1971) found that estimates of numbers of amoebae in litter in- creased four-fold with light homogenising which disintegrated the litter.

To study the distribution of amoebae in various fractions of the IVPP

soil, 2 g each fron three sarnples !{as suspended in 100 m1 AS and sieved

through a 60 nesh sieve. Several particles larger than 500 ¡.rm in size were plated on PJA + KLebsieLla plates at a rate of two particles _1 plate '. The remaining soil suspension.was allowed to settle for about

5 nin and 0.5 n1 of the supernatant was plated on further PJA + KLebsieLLa plates. Portions of the sedinent were sinilarly plated

using a wire loop. Ten replicate plates of each treatment were examined

for amoebae after two weeks at 25oC. AeantLwnoefu. and PLatyamoeba were

isolated fron all fractions; the unidentified leptornyxid was absent

fron tlre 60 nesh fraction and TVteeønoeba and the unidentified vanpy- rellid amoeba wer:e not isolated fron the soil suspension (Table L7).

In cletermining the populations of amoebae, the soil suspensions were

therefore, agitated for about 2-3 nin using a rVor:texr shaker and immediately plated with a wide-nouthed pipette to include all fractions.

Pussard et aL (1979) usecl neornycin (10-100 pg *1-1) and rifamycine _1 (100 ug m1 -) for the selective isolation of a strictly nycophagous arnoeba In ny studies, concentrations of 10,50,100 or 200 pg m1-1 of neomycin did not have any selective effect on either nycophagous or non- nycophagous amoebae as a group. The selective effect of rifanycine was strrcliecl by using 10,50,100 and 200 vg n1-1 of this antibiotic incorpora-

ted PJA nedi.um to assess the population of nycophagous and other amoebae -118-

TABLE 17: Isolation of amoebae from various fractions of the WPP soil (Percentage of fragrnents plated).

Fraction Amoeba 60 Mesh Soi I suspension Sedinent A*BC ABC ABC

AeantV¿ønoeba spp. 75 100 100 80 100 60 100 80 100

Platyamoeba spp. 62 33 100 20 40 BO 20 60 20

Theeanoeba spp. t2 77 77 0 0 0 20 40 0

Leptomyxid 00 0 20 20 0 40 20 20

Vanpyrellid 12 33 0 0 0 0 20 40 40

* soil saurple. - 119-

fron three sarnples of the IVPP soil by the modified soil dilution culture technique (Cutler et aL., L922).

Five g soil was suspended in 50 ml AS in a conical flask, shaken and I ml of this suspension was diluted with 9 ml AS in a test tube using a wide-mouthed pipette and the tube shaken. In this way, the original soil r,ras serially diluted to 10-1 , 70-2 and 10-3. Inmediately after agitation, 50 pI of each dilution was plated on each of five replicate PJA + KLebsieLLa plates incorporated with rifanycine. Control plates were without antibiotic. Plates were exarnined for amoebae after

2 weeks at 25oC. , Populations vrere estirnated by the nost probable nunber technique (Halvorson and ZiegIer, 1953) using the values given by Taylor (1962) to correspond to the nurnber of plates showing positive growth. Populations v/ere expressed in terms of number g-1 dty wt of soil.

Results show that all concentrations of rifanycine significantly reduced the populations of total arnoebae (Tab1e 18). Highest popula- tions of nycophagous amoebae were obtained ín the control and 50 Ug tl-1 tt"atment of the antibiotic; but this decreased significantly in all other concentrations.

It appears that rifarnycine does not have a selective effect on all nycophagous amoebae. However, the conbined effect of neornycin and

rifamycine in 1% nalt extract agar nedium as used by Pussard et aL. (1979) was not studied.

Non-selective rnedia give wider range of species but srnaller nurnbers

than selective nedia (Dixon, 1937; Stout, 1956) and it -is assumed that -L20-

TABLE 18: Population estimates of amoebae fron the WPP soil obtained by plating samples on rifanycine incorporated PJA nediun.

Rifamycine _1 Amoeba (ug nl ^) Control 10 50 100 200

Mycophagous 338 s49 249 83 541

0thers 5947 1696 810 703 817s

Total 6285 2245 1 0s9 786 8776

Least significant difference:

Total amoebae Mycophagous amoebae P = 0.05 944 4 1 24 6 P = 0.01 L440 5 1 90 0 -L27-

non-selective nedia provide representative samples of the species active in soil (Heal, L970); the use of a solid agar nedium, however, select amoebae rather than free-swirnrning ciliates.

Populations of annoebae in the soils

Populations of different genera in these soils were estimated by the modified soil dilution technique (cuter et aL., lg22) described earlier. rn deterrnining populations by genus , GepVtyratnoeba and the unidentified leptomyxid r4rere grouped together as teptonyxids and included with Saccønoeba and I.gnanifena sub-species minoz, to calculate the population of rnycophagous amoebae. A number of species of

MayoreLla, apparently distinguishable by trophozoite dinensions, were recorded in some soil sanples. So far only one species of this genus has been found to be mycophagous and as it was not possible to distinguish the mycophagous species on soil p1a.tes, Mayorellas were not included in the nycophagous group, T.granifena sub-species minor,, due to the relative ease of its identification, could be reco::ded separately fron othet Thecamoeba species.

Data on the rnean generic populations are presented in Table 19; populations per sanple are provided in Appendix rrA. The populations of both mycophagous and other amoebae were significantly (P<0.01) higher in the suppressive WPP soil than in the non-suppressive MB and Avon soils; whereas, population differences betlveen the non-suppressive soils were not. The cumulative population of leptornyxids, r.granifera sub-species minor and Saccønoeba was between 40 and 7796 in the t{PP soil,

0-540 in the Avon soil and 0-180 in the MB soi1. The frequency of occurrence of the rnycophagous genera in the soil sarnples was higher in -L22-

TABLE 19: Populations of various genera of anoebae in l{PP' Avon and MB soils (mean of 20 samples).

Number g-1 dry wt of soil Anoeba It¡PP Avon MB

Aeanthønoeba spp. 1360 L280 467

Ec\rLnamoeba spp. 4609 380 677

Leptonyxids 99 10s 48

MayoreLLa spp. 19 18 39

PLatyamoefu tpp. 807 518 6s1

Saecønoeba spp. 220 4 2

Iheeqnoebd spp. 92 52 s2 T,granLfera ssp. 7L L4 23 mirnz,

Unidentified 6002 280 443

Vanpyrellids 200 53 L32

Total I32BO 2709 2537

Mycophagous 394 724 73

Least significant difference Mycophagous Total P = 0.01 189. 95 5899 P = 0.05 L32.8r 4724 -L23-

the suppressive WPP soil than in the non-suppressive soils (Fig.27).

The non-nycophagous genera, except fot Mayoz,eLLa, on the other hand.. were distributed throughout the majority of sanples of all three soi1s.

Populations of Gqt in the survey soils

Being a root-inhabiting fungus, Ggt occupies the host tissue during its parasitic phase (Garrett, 1956, IgTO) and usually saprophytic inoculun originates in this manner (Lucas, 1955) . lrlatural inoculun in soils is capable of causing infection to a wheat crop after two years sunmer fallow or two crops of oats or clover (Broadfoot, 1934b; Russel , I934i Ciynr,", 1935). In sone fields, MacNish and Dodnan (Ig73) found that over a year there was only a small drop in the incidence of Ggt on stubble. Shipton (1972) found the fungus surviving in soil under a non-susceptible break crop for up to 66 rnonths. A range of grass species, cultivated and rvild, ca¡ì support the gnowth of the fungus (Russel, 1934; Padwick, 1935; Garrett, 1941) and the susceptibility of a grass species is related to the saprophytic perpetuation of the fungus (Chanbers and Flentje, 1968; Chanbers, I97I). The fungus is associated especially r4¡ith organic fragnents (Gams and Domsch, 1967) larger than 420 um (Flornby, 1968), and a seedling baiting technique has been used to estimate the number of infective units (Hornby, 1971).

Populations of Ggt ftom one sarnple of each of the survey soils was assessed using the seedling baiting technique with nlodification.s.

I{heat seeds of the cultivar Flalberd were washed with distilled r^rater and surfaced sterilised with fu11 strength ltlilton solution for 10 nin.

Seeds were washed twic.e in SDW and placed on noist filter paper in Petri plates ancl incubated for 60 h at 15oC for gernination. PVC conduits -724-

Fig.27 Distribution of genera of anoebae in sarnples of suppressive and non-suppressive soi1s.

W = Waite permanent pasture soi1. A = Avon soi1.

M = Murray Bridge soi1. No. of Samples 5to t5 20

Acanthamoeba w spp. i

Echinamoeba w spp. i NON-MYCOPHAGOUS Mayorella w spp. #

Platyamoeba w spp. ,;

Thecamoeba w SPP. J

Unidentified A M

Vampyrellids A M

Leptomyxids A M

Saccamoeba w spp. ; M YCOP HAGOUS

Thecamoeba w granif era A ssp. minor n,r 5tct52c -t2s-

(2.7 cn internal dianeter and L2.5 cn long) were half-filIed with CS.

About 10 g of a test soil was added on top of the CS in each PVC conduit and one pre-germinated seed was sown. Seeds were covered with a thin Tayer of CS and watered rvith tap water. One hundred conduits, distributed anong three netal trays were used for each soil sarnple.

After 4 weeks, plants were harvested, roots washed and examined for Ggt.

The percentage of infected seedlings in these soils ürere:

WPP - 2.0 MB - 1.0 Avon - 3.0.

Of the three soils, WPP had not had a wheat crop for the last 27 years. Although some grasses can maintain the fungus (Russel, 1934; Padwick, 1935; Gamett, I94I), the population does not reach a high 1eve1 due to the low cornpetitive saprophytic ability (Garrett, L970) of the fungus, Avon had a direct drilled wheat crop followed by pasture in 1979 and ltiB soil had wheat in 1979 after thro years of pasture pre- ceded by barley (page 10).

The I'JPP soil is finer in texture and has a higher water-holding capacity than the Avon and MB soils. The higher populations of both nycophagous and other amoebae in this soil nay be partly due to these physical fa-ctors

Effect of soil moisture on amoebal activity

The activity of mycophagous and other amoebae at five different soil noisture tensions ruas studied in the WPP and MB soils using the membrane filter burial technique (01d, I977b) described earlier.

Activity tvas the ability of amoebae to ¡nove through the 5.0 un pores of the nuclepore filters which could be detectecl by their isolation from the internal millipore filter. -126-

Soils passed through a 2 mm sieve and noistened to appïox.

-1 kPa were placed in plastic conduits (6.5 cm long and S.5 cn internal dianeter). t'fillipore filters deposited with honogenised hyphal suspensions of Ggt were buried in moist soils ¿¡d suctions of 1,s,10,

100 and 1500 kPa were applied for tlo weeks.

Five replicate membranes h¡eïe retrieved from each tTeatment,.

Each membrane was cut into B approx. 1 sq nm pieces and four of these were plates on PJA + KlebsieLla plates and incubated for the grorvth of

anoebae. The rest of the mernbrane squaïes were plated on NDy/6

rnediun incorporated with 100 ug streptomycin m1-1 10 ug tetracycline "rr,1 -1 n1 and the percentage survivar of Ggt was calculated from the number

of pieces shorving grohrtlì of the fungus after 48 h at 2SoC

Amoebal activity was detected within theTange of -1 kpa to -100

kPa in the lvPP soil and between -5 kPa to -100 kpa in the MB soil (Table 20). No amoebae grew from rnembranes recovered from either soil naintained at -1500 kPa tension. Recovery of mycophagous amoebae was affected at -100 kPa in the t\¡PP soil although not statistically signi- ficant. Ggt survived oveï a Tange of soil moisture tensions in both soils (-1 to -100 kPa, Table 20). In the MB soi1, the fungus survived equally well at all tensions except at -1500 kPa; whereas, in the l{pp soil, maxinum survival was recorded at -100 kpa and a significant

(P<0.01) reduction was observed at all other tensions. Survival of Ggt l^¡as apparently inversely correlated rvith activity of nrycophagous amoebae in both soils and an íncrease in recovery of the mycophagous amoebae alrnost alt'rays corresponded with a reduced survival of the fungus (TabIe 20). -127 -

TABLE 20: Association of amoebae with Ggt hyphae and survival of the fungus in the WPP and MB soils maintained at various soil rnoisture tensions for 2 weeks.

Moisture A¡noebal association* Ggt survival* tension Ar1 amoebae Mycophagous (kPa) WPP MB WPP MB WPP MB

T 40.0A** 0.0 *** L3,7C 0. 0 *** 408 l5H

-5 37 .04 29.38 20.2C 6.3D 5F 35H

-10 34.0A 28 .08 19.lC 17 .8D 30E 25H

-100 26.04 28.0B I .9C 7.tD 100G 50H

_1500*** 0.0 0.0 0.0 0.0 0 0

** Percentage of 1 sq run nillipore rnembrane plated Values followed by the same letter in a column do not díffer *** significantly (P = 0.01) according to X2 test Not included in analysis. -728-

Water retention of soils is governed by soil texture (Salter and hrilliams, 1965). The WPP soil with its heavier sandy-loan texture retains ¡nore available moisture than the sandy MB soil at aII tensions. However, the diffeïences in the recovery of arnoebae at suctions of -5 to -100 kPa were not statistically significant between these soi1s.

Darbyshire (1975) found that at a suction of -50 kPa, no nulti- plication of the ciliate (coþoda steíni Maupas) population occurred and largest populations were obtained in saturated soi1s. cook and Homma (1979) found that mycophagous amoebae, however, were unable to perforate fungal propagules in saturated soils, and perforations occumed betrr,een rnoisture tensions of -5 and -20 kPa. My results show that rnycophagous and other amoebae were active in both soils at higher tensions of up to -100 kPa and they were detectably inactive at near saturation in the coarse textured lr{B soiI.

Survival of arnoebae under extTenes of rnoistuïe stress

To study the effects of moisture stress on their viability, seven different amoebae were deposited on sterile filter papers (Page, r967a) and kept in the laboratory for one year. Equal amounts of AS sus- pensions of the test arnoebae were deposited on wedges of sterile filter paper, placed inside sealed Petri plates and stored at room temperature and hunidity. To test thei:: viability, 10 wedges of filter paper for each amoeba tvere placed on PJA + KLebsieLLa medium, moistened with a drop of AS and examined for amoebal growth after trvo weeks at zsoc. -129-

Three out of seven anoebae, AeantVømoeba poLypfugo, PLatyønoeba stenopodia and the unidentified leptomyxid survived these conditions for one year (Table 21). The non-cystic Saeeønoeba and an unidentified

amoeba (Un.1); and cystic Gephyramoeba and the unidentified vampy::ellid

did not survive. These results show that cysts of different anoebae

react differently torvards environmental conditions (Bodenheimer and

Reich, L933; Page, 7967a). Taylor (1960) recovered viable Amoeba spp. fron dried cultures. Ability to withstand desiccation and temperature

fluctuations would enable an organism to be air-borne and ensure a wider distribution of the species (Gis1en, 1940; Pennak, 1953). Puschkarew (1913) estinated that there were about 2.5 viable pïotozoan cysts per cubic n of air and obtained 13 species, rnainly sna11 amoebae, by exposing containers of sterile nedia to air. Schlichting (1961,

1969) obtained three different species of amoebae from the atnosphere using sinilar techniques.

Populations of amoebae in suppressive and non- S ressive soils s'ith similar soil texture

Further data on populations of soil amoebae were obtained fron two oLher plots with similar texture from the rotation experiment at the Waite Institute. Of these, the pasture-pasture-rvheat (PPW) is suppressive and the continuous r\'heat (CW) is non-suppressive to take-all (Wildermuth, 1977). Both are red-brown earths and were under wheat.

Plots l\Iere sampled as previously and populations of rnycophagous and other amoebae assessed fron 10 samples. The population 1eve1s of both mycophagous and other arnoebae rrere significantly (P.0.05) higher in the suppressive PPIV soil than in the non-suppressive CIV soil (Table 22). Mycopliagous amoebal populations ranged from 40 - 1555 g-7 dry wt in the

PPW soil and 0 - I34 g-1 ,lty wt in the CIV soi1. -130-

TABLE 21: Viability of various amoebae after one year at room temperature and humidity.

Anoeba Grorvth on PJA + KlebsieLLa

Cystic species

Acanthøno eb a p o Ly p Vta.g a +

Gephyratnoeba sp.

PLaty amo eba s tenop odi a +

Unidentified 1 eptonyxid +

Unidentified varnpyrel 1 id

Non-cystic species

Saeeønoeba sp.

Unidentified amoeba (Un. 1) -131-

TABLE 22t Generic populations of arnoebae in suppressive (PPW) and non-suppressive (CItl) wheat soils. (Mean of 10 samples).

Mean nurnber g -l Amoeba dry wt of soil CW PPW

AeantLtarnoeba spp. 1108 77L9 Ee\tLnønoeba spo. 522 497 Leptonyxids 2L 53 MayoreLLa spp. 2t 51 Platyønoeba spp. 434 364 Saccønoeba spp. t2 77 Theeamoeba spp. 35 6s T.granifera ssp. 29 M'LNOT 63

Vampyrel 1 ids 2S 60 Unidentified 666 2706

Total population 287 3 5049* Mycophagous 62 195*

Significantly (p.0.05) hi.gher than the corresponding values in CW soil. -132-

Suitability of the survey soils as a nedium for amoebal growth

fn an attempt to study the effect of soil tlT)e on the growth and nultiplication of amoebae, three nycophagous amoebae, the unidentified leptomyxid, Saccamoeba sp. and T.granife::a sub-species minor were re- introduced into sterilised WPP, Avon and MB soils and their populations nonitored over a period of 8 weeks. Soils sieved through 2 mn sieve were moistened to -8 kPa. One hundred g of noist soil was dispensed in 250 m1 conical flasks and. autoclaved at L27oC for I h. Stock cultures of these arnoebae h¡ere suspended in AS and 0.5 nl of the mixed suspension containing approx. 32000 n1-1 of the unidentified leptornyxid,

5600 rnl-1 of Saccønoeba sp. and 260 nL-L of r.gz,anifera sub-species minor was added to each f1ask. The flasks were shaken to distribute the suspension, sealed with tAlfoilr and incubated in darkness at 25oC.

Populations of the three amoebae were assessed from three replicate flasks for each soil at 0,7,2,4 and B weeks after reintroduction.

Factorial analysis of the data showed that the populations of

SaccØnoeba and T.gz,anife.ra sub-species nrLnon declined in all soils over the period of 8 weeks (Table 23). Populations of the unidentified leptornyxid shorqed an initial increase followed by a gradual decline in all three soi1s. Populations of all amoebae declined at a significantly higher rate in the MB soil and this was followed by the Avon and the l\¡PP soil. The population of the accompanying food organisns , KLebsieLLa sp. and the FusarLwn sp. rúere not stuclied during these periods.

Discussion

1 The number of amoebae g dry wt of soil varied fron 900 to 55400 in the three soi1s. This is sornervhat lower than the range of 1000 to -1 53-

TABLE 23: Populations of three nycophagous arnoebae after their reintroduction into sterilised suppressive (WPP) and non-suppressive (N{B and Avon) soils.

Population (No. g dry wt of soil) Amoeba Soil Initial Weeks after reintroduction 7248

Unidentified WPP 327 6 3868s 32300 4s070 15277 Leptomyxid Avon 4698 323L4 32374 17 403 5952 MB 315 0 6s96 9824 3992 1978

Saceønoeba sp. WPP 7605 6s44 4337 6572 t97 Avon 7I37 655 7L7 s7 0 MB 10105 350 742 0 0

I.graniferd ssp. WPP 29s 61 L7 256 767 minov, Avon 86 76 0 3T 0 MB 77 0 0 0 t4

Factorial analysis Unident i fied T.granifez'a Variance ratios Saecønoeba sp. I eptornyxid ssp. mLnor

Soi.1 20.339** 4.606* g. 946** Time L0.822** 7 "849* ), 3 -262* Soil x Tine 2.842* 1.164ns 1.047ns **significant at P = 0.01 * significant at P = 0.05 ns, non-significant . -134-

_1 1690,000 g - dry wt obtained by others using sinilar techniques

(Heal, 797I). As soil samples were stored at roon temperature and humidity for up to 18 months before population estination, the effects of storage on the populations of soil amoebae were studied. Fresh samples were collected from approx. the sarne sites of the MB soil and populations ü/ere assessed from two of these samples using the sane technique. There was no significant difference in arnoebal populations between these and those of the old sanples collected fron the same sites (TabIe 24). Sinilar results were obtained by Fanthan and

Paterson (1924) and Cutler and Dixon (1927) r.iho obtained víab1e amoebae from soils stored for three years and longer.

In these studies, the total population rvas calculated by adding the populations of various genera which could be easily iclentified. This did not take into account the number of species of each genus in these soils; also in nany samples, the unidentified group included more than one type of amoeba. The metlÌod therefore, underestirnated the total population but was useful in determining the populations of different genera and my estinates on the population of the rnycophagous I.granifer.ø sub-species tnínoz, are comparable to those obtai.ned by Alabouvette et aL. (1981) using selective techniques.

Populations of rnycophagous amoebae are generally lower than sorne of tlre other cornmon soil amoebae like AcantLwnoeba. The population of I'. granifera sub-species tnLrlÐy, ilt soils is usually less than 100 1 individuals g- - and higher dens:-ties (15,000 to 30,000 g')-1 are obtained in heat-treatecl and fungus-enrichecl soils (Alabouvette et aL., 198f). Using the most probable number technique, Ander:son and Patrick (1980) - 135-

TABLE 24: Populations of soil amoebae in freshly collected and 18 nonth old sanples of MB soi1.

Sanple 1 Sanple 2 A¡noeba Fresh 01d Fresh 01d

Aeanthamoeba spp. 440 460 220 440 EelrLnø¡noeba spp. s20 280 s40 340 Leptonyxids 90 0 0 40

MayoreLLa spp. 40 0 0 40 PLatyatnoefu spp. 186 660 736 280 Ihecønoeba spp. 40 90 90 0

Unidentified 280 90 186 220

Vanpyrellids 80 80 90 0

Total 1676 1660 1.262 1 360

Mycophagous 90 0 0 40 -136-

estimated the population of the mycophagous VanrpyreLLa LaterLtia Leidy, 1879 in various garden soils as between 1 and 13 g-1 dty *a of soil. The cumulative population of the rnycophagous leptomyxids, r.gz'anifer"a sub-species nrinor and saecønoeba was between 40 and 7796 -1* _1 g dry wt in the IVPP soil; 0 - 540 g-^ dry wt in the Avon soil and -1 0 - 180 g - dry wt in the MB soil.

Terrestrial protozoa are regarded as fresh\.ater species that invaded land with varying degrees of success (Stout, 1963; Schonborn, f964). The sane species is often found ín both aquatic and terrestrial habitats and rnany species of amoebae in soil a¡e distinctly snaller in size than their aquatic forms (Fantharn and Paterson, rg26; chaudhuri, f929). Their sma11 size a11ows them to exploit restricted spaces in soil where rnoisture is retained (Jongerius, 1957; Banforth, IgTS).

The effects of soil moisture on amoebae have been studied by a nunber of workers (Koch, 1916; Cutler et aL., L922; Cutler and Dixon, 1927;

Losina-Losinsky and Martinov, 1930; .Bonnet, Ig64; Nicoljuk, 1964;

YoIz, L972) and the general conclusion is that soil amoebae are active within a range between 20% to 60% of the water holding capacity of most soils. Active anoebae tJere detected betrveen moistuïe tensi.ons of -5 and -100 kPa in two soils of different textures in rny studies.

Results presented here suggest that soil texture, water holding capacity and soil tfpe are not a najor influence on popul.ation levels of nycophagous and other amoebae. stout (1968) failed to deteïnine any direct influe¡rce of soil texture on the anoebal fauna and Cutler and Crump (1935) could not correlate changes in amoebal populations of field soils with ternperature and moisture, although such environrnental factors had some effect on the grorvth and activity of other soil protozoa. -L37 -

It is almost impossible to correlate the distribution of protozoan speci-es with major soil ty¡les or most macroenvironnental factors (Darbyshire, 1975).

When reintroduced into sterilised survey soils with the accompany- ing food organisms, the nycophagous amoebae produc,ed larger final populations in the wPP soil than in the other two soils. Perhaps the single important factor which regulates the soil amoebal population is the quantity of available food organisrns (Sandon, L92T; Singh, 1945, !946a, 1948). Although physical characters of soil like texture, water holding capacity and soil trpe do not directly influence anoebal fauna (sandon, L927; stout, 1968), the effects of these on the food organisns rnay be important in the regulation of population build up in these soils.

Bryant et aL. (19S2) studied interactions between populations of

Acanthamoeba poLyphaga and Pseudomonns paucinoLrLLis Holmes et aL. 1977 in soil microcoslns with fluctuating moisture content. They found that in constantly moist soil amoebal grazing limited bacterial numbers, whereas, in soil alternately wetted and dried, this effect was inter- nittent; although the amoebal population v/as almost entirely encysted in dry soi1, their total population was not affected by three dryings.

Sniley (1979) recovered highly antagonistic Pseudomonads from a suppressive wheat-monoculture soil but not from a non-suppressive soi1. Slope et aL. (1978) found that PhiaLopLnra ra&icicoLa var. gratnini,cola, the fungus inhibitory to Ggt, was abundant on wheat after grass where it seemed to delay the onset of take-all by one year. Higher popula- tions of mycophagous amoebae in the suppressive soils support the general contention that in suppressive soils there is an abundance of - 138-

organisms which are antagonistic, inhibitory, andfor conpetítors of Ggt and this results in the reduced saprophytic survival of the fungus (Pope and Jackson, 1973; ltlilderrnuth, 1977). -159-

c ASSOCIATION OF AMOEBAE IN WHEAT RHIZOSPHERES IN SUP PRESSIVE AND NON-SUPPRESSIVE SOILS

In L904 Hiltner coined the terntRhizospärerto describe the zone of soil within the sphere of influence of legtune roots. This original

concept has been extended considerably and now includes non-leguminous plants, microbes and a very wide range of both inorganic and organic naterials released from roots (Rovira and McDougall, 7967; Darbyshire

and Greaves, 7973; Rovira and Davey, 1974; Rovira, 1979). The extent of the rhizosphere, which is related to the release and diffusion of components from the root, is dependant on soil conditions, plant species and their age and various other environmental conditions

(Papavizas and Davey, 1961; Rovira and Davey, L974; Brown, 1975).

Generally the rhizosphere supports a higher population of micro- organisrns and this is clue to thetrhizosphere effecttwhich is defined as the ratio of the number of microorganisrns in the rhizosphere soil to the nurnber in the corresponding root-free soil (R/S ratio). Starky (1938) and Linford (1942) were the first to demonstrate the presence of protozoa in rhizospheres of plants. Rouatt et aL. (1960) determi.ned the R/S r:atio for the najor groups of soil microorganisms in the rhizosphere of spring wheat and these are presented below:

-1 Number d wt soil 0rganism Rhizosp eIe 1 zosp ere R/S ratio soi 1 (R) soil (S)

6*. Bacteria 1200 x 10 53 x 106 23:1 6* Actinornycetes 46x10 7xIo6 7zt 5^ Fungi L2xt0 1x10 .5 L2z! 2* 2 Protozoa 24xI0 10x10 2zI 3 Algae 5x10 27 x70J 0.22L tr Significantly (P=0.01) higher than the non-rhj-zosphere soi1. -140-

Geltzer (1963) reported a large accumulation of Haz'trnanneLLa sp., Amoeba sp., MayoreLla sp., NaegLer"La sp. and an unidentified sma1l amoeba in rhizospheres of wheat, flax and peas. Darbyshire (1966) found that the populations of both flagellates and a¡noebae were higher in the rhizosphere soi-l of LoLíwn penenne L. than those in interrow soi1. Large differences between rhizosphere and unplanted soil popu- lations of amoebae, flagellates and bacteria were found during flower- ing of both an annual (Sinqsis aLba L.) and a perennial plant (TnifoLiun repens L.) by Darbyshire and Greaves (1967). From an early stage in plant growth, the populations of these organisns were signi- ficantly higher in the rhizosphere soil, whereas ciliates were only found in lorv nurnbers in both rhizosphere and unplanted soils. gicsók (1956) reported that certain species of protözoa were only found in the rhizosphere; however, Darbyshire and Greaves (1967) concluded that the protozoan genera found in the rhizosphere and unplanted soil were identical and no genus was specific to the rhizosphere of mustard, clover or ryegrass.

Accumulation of large numbers of an organism in the rhizosphere is an indication that these play a role in the inter-relationships betrveen plant Toots, the soil environment and other microorganisms in the immediate vicinity.of the roots. The role of soil anoebae as rlrizosphere organisms is largely unknown. E1liot et aL. (1979) found that in the rhizosphere of BouteLona gnaeilis (H.B.K.) Lag. ex Steud., a species of grass, gnotobiotic associations of anoeba (Acanttnrnoeba sp.) and bacteria (Pseudomona.s sp.) nineralised signifícantly nore (50.-100%) Nfl4-N than those with bacteria alone. Similar higher mineral-. isation of inorganic phosphorus and armnonium nitrogen in microcosms contairring amoebae and bacteria were reported by Colernan et aL. (L977). -t4L-

Several of the hypotheses advanced to explain suppression propose the developnent and/or increased activities of ¡nicroflora antagonistic to Ggt in rd-reat rhizospheres of suppressive soils (Vojinovic, 1972; Pope and Jackson, 1973; Cook and Rovira, 1976; Hornby and Brown, 1977). Soil anoebae have been found associated with Ggt in its saprophytic phase (Honma et aL., 1979) and it is likely that both these organisrns occupy sinilar ecological niches, fn the caSe of Ggt, the two types of existence, saprophytic and parasitic, are inter-related. Soil amoebae to be closely associated rvith Ggt in its parasitic phase, will have to conpete with several other microorganisrns in the rhizosphere, which is considered as a zone of hígh microbial activities (Rovira, 1956; Rouatt et aL., 1960; Rovira and Davey, Ig74).

Association of rnycophagous and other amoebae in wheat rhizospheres and rhizoplanes in suppressive and nqn-suppressive soils rvas determined during two wheat crops and that of the harvested wheat rows during an inter-crop fa11ow using one non-suppressive and two suppressive soils. Soil noisture and the occurrence of Ggt, assessed throughout this period, ü/ere correlated with the populations of nycophagous and other amoebae to detennine the inter-relationships between these variables.

Associat.ion of soil amoebae rvith wheat roots - a survey

As a prelininary experiment, the association of soil amoebae r'rith wheat roots was studied using 9 - 13 weeks old plants obtained fron six widely separated locations in South Australia. The sample sites were selected at random by the S.A. Departnent of Agriculture for a survey on the severity of wheat diseases, and plants with adhering soil were obtained from them. Details on the cropping history and soil characterístics of the sanple sites are presented in Table 25. TABLE 25: Cropping history and soil characteristics of the six soils.

Available Total N Salinity ms - 25oc Area Soil type Cropping pH (Hundred of) history P (ppn) K (ppm) (conductiriity) I/ 5

7 9 O.TL2 Pinkawil linie sandy loam PWPIVP* L7 360 0. 049 I Þ Stirling sandy loan WPPPB 38 550 0. 136 B 5 0.282 N I Ikpunda clay loan PPBIf 40 476 0. 153 6 2 0.242 Carina sandy loan PBWPB 29 3s0 0. 053 8 6 0.747 Yaninee sandy loam PBÌ\'IVP 44 77s 0. 105 B 4 0. 188 Minnipa sandy loam WPOWP 43 440 0.07 4 I 6 0.186

* P = pasture; W = wheat; B = barleyi 0 = oat (Courtesy - Dr. A. Mayfield, S.A. Dept. of Agriculture). -L43-

Roots were washed clear of adhering soil particles and about 2 cm pieces of the seninal roots cut from an area I cn away fron the seed.

These pieces were washed twice i.n SDI{ and plated on PJA + KlebsieLLa plates. Four replicates, each with two pieces of root, weÌe incuba- ted at 25oC for two weeks and examined for annoebae. Several different amoebae were isolated fron these sanples. Most genera' except

Aeanthønoeba and the leptonyxids, were present in wheat rhizoplanes in all sites (Table 26).

It is evident that soil amoebae are a part of the microbial component of the. wheat root surface in the soils studied.

Population dynamics of amoebae in suppressive and non-suppressive soils

I{heat rhizosphere populations of nycophagous and other amoebae were estirnated during the 19Bl season and for up to 10 weeks after sowing in 1982 from two suppressive and one non-suppressive soil of the permanent rotation trials at the lVaite Agricultural Research Institute. For the inter-crop fa11ow, anoebal populations were estimated frorn the soi-t of the harvested wheat rows.

Selection of soils and sanpling

The selection of the three soils was based on their suppressive properties; the pasture-pastule-wheat (PPW) rotation has a high degree of suppressiveness, the pea-wheat (PelV) rotation with a rnoderate suppressiveness and the continuous-wheat (ChI) without any detectable suppressiveness to the disease in pot bioassays (page 18, Table 4). -144-

TABLE 26 Percent recovery of amoebae from wheat roots collected fron six locations in South Australia.

SITES Amoeba Pinka- Stirling Yaninee Carina Minnipa Kapunda willinie

AeantVtønoeba spp. 50 0 0 2S 12.5 72.5

Platyamoeba spp. 37 .5 75 100 87.5 87. s 37.5

Ihecønoebd spp. 50 37 .5 100 100 50 100

Leptonyxids 37 .S 37.5 0 2S 50 25

Vampyrellids 100 72.5 t2.s 25 37.s 25

Unidentified 62.5 87. 5 87.5 s7 .s 100 50 -14s-

A number of nethods are available for sampling and estinating population of organisrns in rhizosphere soils and these have been re- viewed by Johnson and Curl (L972), Darbyshire and Greaves (1973), Rovira and Davey (1974); but because of the heterogeneous nature of the rhizosphere no precise and sinple sarnpling nethod will suffice under every plant and soil condition. I used the traditional plate count nethod of Katznelson (1946) with modifications.

Each plots was subdivided into grids, approx. 2m x 2m and wheat plants in a rorv with soil to a depth of about l0 cn were lifted using a spade from three randomly selected intercepts for each plot at each sampling. Roots were shaken, often after breaking large soil clods by hand, to discard the surplus soil from around roots. The firmly ad- hering soil together with roots constituted the rhizosphere soi1.

Populations of amoebae were estinated from two sanples of each rotation using the modified soil dilution technique described earlier. As before, Gephyramoeba and the unidentified leptomyxid were grouped to- gether and included with Saccønoeba and TVtecamoeba granifet"a sub-species minor to calculate the population of rnycophagous amoebae. Often sus- pensions rve::e lightly honogenised to break large fragrnents of roots.

The PPIV and PelV rotations are maintained in more than one plot so that each crop i, in one plot each year and wheat rhizo- "epr"rented sphere and rhizoplane samples in 1982 for these rotations came from plotsadjacent to the onesused in 1981 samplings. During the inter- croþ fallorv, samples from the top 0 - 10 cn layer of harvested wheat rows were collected from three randomly selected intercepts of each plot using a 4 cn internal dianeter soil auger. Sarnples were collected at nonthly intervals and populations of arnoebae determined. -146-

Populations of mycophagous and other amoebaç in the PPW PelV and CW soils

Populations of amoebae were determined fron rhizosphere soil of thè three rotations throughout the 1981 wheat season and for up to 10 weeks after sowing in 1982. The non-suppressive CW soil was however, not included in determinations until B weeks after sowing in the 1981 season. Data.on the populations of various genera of anoebae are presented in Appendix IIB.

Populations of mycophagous amoebae were consistently higher in the rhizospheres, of plants fron the suppressive PPW and PeW plots than in the non-suppressive CW (Fig.28). Populations of all other amoebae were also higher in the suppressive soils throughout most part of the growing season except during the boot stage of the crop in 1981, when it was higher in the CW than in the other two soils (Fig.28) . In the suppressive PPW soil, population peaks of mycophagous amoebae tvere observed at boot stage in 1981 and 32 leaf stage in 1982 season. In

PelV soil, relatively smaller peaks could be detected at early til1erì-ng and boot stages. In the non-suppressive CIV soil however, there lvas no apparent peak and thepopulations of rnycophagous amoebae were generally higher during the entire crop season starting fronl the tillering phase.

Populations of all other amoebae reached their peaks during the boot stage in all soils.

After harvesting, there vras a drop in the populations of myco- phagous amoebae in alt soils; pcpulation g-1 in these soils wete 824 and 92 in PPIV, I72 and 7I2 in PelV and 108 and 92 in CI\l, three weeks before and three tveeks after harvest respectively. However, samples collected aftel harvest can not be regarded as rhizosphere samples. -147-

Fig.28: Populations of soil anoebae during thro wheat crops and the i.nter-crop fallow in the suppressive pasture-pasture-wheat (O), pea-wheat (Ä) and non-suÞpressive continuous-wheat (O) soi1.

Mycophagous amoebae.

All other amoebae. =tõ 42 tn o 3.8 +J 3 3 (- ! -tgt 3.0 .o o C 2 6

C. o 2 o o .Fo .o. o a )è 1.8 o o 1.t, o c,.) 1.0 o o J WHEAT 0 WHEAT INTER - CROP FALLOW ' 34 56 I 10 3456I 10 13 17 20 wøøks oftør sowing WøøKS afler sowing

Aug Aug I søp oct Nov I Dec | .,ron I røb I vor I apr I ruroy I JUN ¡ul

1 1 I leof 3Lleof jointing oJ., 3L eorly 2 tilløring tillering -148-

The recovery of nycophagous amoebae was drastically reduced during the inter-crop fa1low in the non-suppressive soil (Fig.2S); in comparison, populations weïe nuch higher in the suppressive ppW

and Pelv soils during this period. Population of all other amoebae

was slightly reduced in all soils during the inter-crop fa11ow although, the reduction was far less conparecl to that of the nyco- phagous amoebae. Lowest poprrlations of both nycophagous and other amoebae were observed cluring February-March period in all soi1s.

Anoebae in wheat rhi lane in su TESS]-VE non-suppTess].ve so ls

The association of rnycophagous and other amoebae on the rhizo-

planes of wheat grolr,n in suppressive PPIV and PelV and non-suppressive

cl{ soils was determined throughout the lg81 season and for up to 10

weeks after sorving in 1982.

About 2-3 cnt pieces of the seminal roots from an area 1 cm ahray fron the seed were plated on PJA + KlebsieLLa prates using the root

plating netliod descrjbed earlier. Ten replicate pieces of root r.Ieïe

plated from each soil at each sarnpling and the associati.on of anoebae

was determined by recording tlie arnoebal genera on the PJA pJ ates after

an incubation of t-wo rveeks at 25oC. .-- :.

Results on the occurrence of various genera on wheat rhizoplanes

in these soils are presented in Appendix IIC. Figure 29 summarises

the association of rnycophagous and other arnoebae with rvheat roots in

PPI\I, CW ancl PelV soils during the 1981 and 1982 seasons. There was

generally a higher association of rnycophagous amoebae with wheat roots

in the suppressive PPI{ and PelV soils than in the non-suppressive CW soil

(Fig.29). The PPIV soi1, with its higher degree of suppressiveness, - 149-

Fig. 29: Wheat rhizoplane amoebae during two croPs in suppressive (PPl\t and Peht) and non- suppressive (CW) soils,

@ Mycophagous anoebae

F-l All other amoebae. Rhizoplane amoebae Weeks Rhizoptane amoebae af ter (% (oÁ roots ptated) sowrng roots ptated ) 50 40 30 20 ro to 20 30 40 50 3 PPW PEW CW 4 PPW PEW CW 5 PPW _PEW cw ó PPW PEW CW B PPW PEW CW ro PPW PEW CW l3 PPW PEW CW 17 PPW PEW CW 20 PPW PEW CW

t981 t982 -150-

showed a consistently higher rhizoplane population of both nycophagous

and other arnoebae with peaks around tll'e 3Lz leaf stage of the crop

(4-5 weeks after sorving). Both PeW and CIV soils shorved a more or less

sinilar degree of assoc.iation of amoebae other than nycophagous during

the initial period of up to 10 lveeks after sowing but thereafter, more

amoebae were found in wheat rhizoplanes in the PelV than in the CW soil.

Correlation between soil noisture, population of Ggt and amoebae in the suppressive an

Correlations between populations of amoebae, Ggb and soil moisture were calculated in an attempt to deter¡nine the basis for the higher populations of nycophagous and other amoebae in the suppressive PPW and Pelll soils.

At each sampling, the presence of Ggt was assessed and soil rnoisture determined from the three samples of each plot (Table 27).

During the 1981 crop season, roots were exarnined fo:: take-a11 and 2 crn pieces from infected roots were plated on NDY/6 j-ncorporated with 100 ug streptomycin m1-1 urd 10 ug tetracycline m1-1. For the 1982 season and the inter-crop fa1low horvever, bulk soil rvas used in the seedling baiting technique (Hornby, 1971) clescribed earlier to assess the presence of the fungus. Portions of infected roots from the 1982 samples were also plated to compare the two techniques. Little corre- lation bet\veen the tr'¡o techniques was found and the seedling baiting technique always gave a higher assessment of the fungus.

Data on the soil rnoisture, Ggt recovery and populations of myco- phagous and other anoebae were correlated by calculating Pearsonrs correlati on coefficients. Results shol that at the |eo level of TABLE 272 Soil noisture ang Ggt populations during two wheat seasons and inter-crop fallow in suppressive (PPW and PeW) and non-suppressive (CW) soi1s.

Weeks after sowing SoiL noisture (%)a Weeks after sowing Wheat season,1981 Inter-crop fal1ow Wheat season, 1982 Soi I 3 4 5 6 8 10 15 t7 20 _Jan f eU Uar npr Play .lun 5456810

PPIIJ 20.5 2L.7 20.4 18.0 ls.2 L6.2 B.ó 14.8 4.9 3.L 3.I 2.0 7.0 16.6 77.0 18.4 L7.7 18.1 L6.2 73.7 L7.0 PelV 79.2 20.2 18.8 15.6 LS.2 L7.s 7.3 77.3 4.2 2.5 3.0 2.4 4.t 16.3 77 .0 16.5 15.9 16.9 14.7 72.4 L5.3

CW No determination 13.5 il.6 6.4 L3.6 3.6 2.5 2.9 ¿.5 5.0 15.9 16.9 17 .0 16.7 t7 .4 14.8 13. s t5.7 Ggt population &)b

PPW 6 6 0.0 0.0 6.6 6.6 6.6 0.0 0.0 0. 0 15. 5 34.L 15. I 45.6 26.4 58. s 55. 3 43.0 75.6 70.0 .r. (/l PeW 26 0 0.0 0.0 6.6 6.6 0. 0 L3.3 6.6 0. 0 1.5 10.9 1,2.6 L2.9 9.6 29.7 72.4 26.9 20.6 40. 0 33.4 39.2

CW No determination 6.6 6.6 0. 0 73 .3 0. 0 3.7 13. B 72.9 9.5 11.3 33.2 24.9 29.4 30.4 35.3 42.6 38. 1

3= Oven wt basis ö= dry Percentâge of infected roots for 1981 season and percentage of inoculum units in bulk soil, as detected by the seedling baiting technique, for 1982 season and inter-crop fallow. -ls2-

prcobability, populations of nycoplìagous arnoebae correlated with soil moisture and Ggt recovery in the non-suppressive CW soil and only with soil moisture in the suppressive PeW soil (Table 28). In the suppressive PPIV soil however, no correlation was observed between soil moisture and amoebal populations.

These results indicate that the population build-up of the myco- phagous amoebae in the CIV and PPIV soil was dependant on the population of Ggt. This is also supported by the fact that during the inter- crop fa1low, when population of the fungus was detectably lower in these soils, a lower population of the nycophagous anoebae occttrred in these soils. The reduction in the population of mycophagous anoebae was higher in the non-suppressive CIV soil than in the PPll soi1, wl'rich in turn also showed a generally lower population of Ggt during the inter-crop fallow (TabIe 27). Geltzer (1963) also found that amoebae nultiplied in the rhizospheres of six plant species only when there was an ac.tive development of the food organisrns, bacteria in that case, oll then.

Nicoljuk (1964) found the largest numbers of individuals and species in irri.gated soils and suggested that Protozoan populations are greatly influenced by soil moisture. Darbyshire (1966) found highest populations of amoebae and flagellates in warm and moderately wet rhizosphere soils. In ny studies, the lack of a higher positive correlation between soil moisture and populations of both mtcophagous and other amoebae in the suppressive PPll¡ indicates that soil rnoisture alone can not explain the higher populations of these amoebae in this soil throughout the growing seasons. -1s5-

TABLE 28: Correlation coefficients between populations of nycophagous and other annoebae, soil moisture and Ggt 'recovery in the suppressive (PPI{ and PeW) and non-suppressive (CW) soils.

Population of Population of moisture I'lycophagous Soil other amoebae (e") arnoebae

Population of other amoebae

Pell¡ 0.43 (P=0.03)* PPItj 0.36 (P=0.06) cl'r 0.26 (P=0.16)

Soi 1 It{oisture (e")

PeW 0. 68 (P=0. 01) 0. 53 (P=0. 09) PPhI 0.23 (P=0. 16) -0.002 (P=0.49) CW 0. 54 (P=0. 01) 0.10 (P=0.36) Ggt recovery (e")

PelV 0.29 (P= o. 11) -0.02 (P=0 .4s) 0.25 (P=0.14) PP}\I 0.40 (P= 0.04) 0.2s (P=0 .L4) o .77 (P=0.23) CW 0. s7 (P= 0. 01) -0. 07 (P=0 .38) 0.69 (P=0.002)

* Denotes the probability level of significance. - 154-

Darbyshire et aL. (7977) studied the inter-relationships of bacteria, protozoa and the take-al1 fungus associated with the roots of growing barley and of the post-harvest stubble. During the growth of the crop, the bacterial and the trophic protozoan populations gradually increased in all soils near the roots and these increases were acconpanied by changes in the levels of NHO and N0, nitrogen.

Rovira and Wildermuth (1981) reported a nassive proliferation of Gran-negative, non-sporing, rod-shaped bacteria on wheat roots following infection by Ggt and proposed that this was the forerunner to TAD. Plant roots in general, stimulate certain groups of nicro- organisms and several reports suggest that sone spore-forrning BaciLLus spp. and Gran-positive cocci are depressed in the rhizosphere (C1ark, 1940; Lochhead, 1940). Conversely, Gram-negative, non-sporing bacteria such as Agrobactez"tum and Pseudomonds spp. nay account for as rnuch as 40 to 50% of the rhizosphere populations of bacteria (Vagnerova et aL., 1960; Rouatt and Katznelson, 1961). Therefore, the suggestion that the proliferation of a certain type of bacterium in the rhizosphere is the forerunner to TAD appears to be inconclusive.

Data on the populations of anloebae in this section could ncjt be statistically analysed for significance due to the lack of adequate replications. Even with two replicates in each plot, a total of 90 plates had to be exa¡nined to estimate amoebal populations at each sampli.ng. The results horvever, indicate that the populations of myco- phagous amoebae are consistently hígher in the suppressive soils than in the non-suppressive soil studied. lvlore detailed investigatiorÌs are needed to test these interesting findings on the association of myco- phagous alnoebae in w}reat rhizospheres of suppressive and non-suppressive soils and their j.nter-relationships rvith other soil nicroorganisms, especially bacteria and plant pathogenic fungi. -155-

D REDUCTION OF TAKE-ALL BY MYCOPHAGOUS AMOEBAE IN POT BI OAS SAYS

Suppression of the take-alI disease of wheat is one of the best- known and most-studiedcases of a naturally occurring biological control of a soil-borne plant pathogen (Baker and Cook, I974; l{alker, 1975; Cook, 1981; Papavizas and Lewis, 1981; Rovira and l{ildermuth,

1981). Although the precise mechanism of control is still uncertain, the denonstration that the suppressive factors are sensitive to heat

(Gerlagh, 1968; Shipton et aL., 1973; Cook and Rovira, 1976) and some forms of nitrogen (Brown et aL., 7973) and also that suppression can be re-established by the addition of I% antagonistic soil to funigated or steamed soil (Shipton et aL., 1973; l{ildermuth, L977) or partly re-establisheC by the re-colonization of funrigated soil by air-borne contaminants (Baker and Cook, L974; page 206) suggests that the suppressive factors are microbial in nature. This has led to the formulation of several hypotheses to explain suppression (Hornby, 1979). Rovira and It¡ildernuth (1981) surnmarised these hypotheses as follols:

1 Increased nicrobial activity associated with the pathogen and tlre infecied host (Gerlagh, 1968; Vojinovic, IgJ2,

I973; Shipton, 1975).

2 Increased microbial activity associated rvith the pathogen

in the soil arvay fron the root (Zogg and Jaggi, 1974).

3 Changes in the virulence of the pathogen with the j.nvolve- nent of virus-1ike partíc1es (Lapierre et aL., 1970).

4 Developnent of a mic::oflora in the rhizosphere of lvheat grorving in suppressive soil which inhibits the trophic

grorvth of the fungus to the w.heat- root (Pope and Jackson,

1e73) . -156-

5 Selective stirnulation by infected wheat roots of rod-shaped, non-sporing bacteria (Vojinovic, L972) or a highly specific

group of fluorescent Pseudomonads (Cook and Rovira, 1976).

6 A change in the general micro-flora of TAD soil which alters

the ratio of NHf-N to NO;-N in the rhizosphere (Hornby and

Brown, 1977).

7 An effect upon thc emergence of hyphae fron the infectious propagule and subsequent darkening of the hyphae (Wildernuth et aL., 1979).

The lack of understanding of the rnechanisms of suppression has not barred workers from attempting to control the disease using specific antagonists or conpetitors of the take-aI1 fungus (Cook, 1981; Papavizas and Lewis, 1981). Successful biological control of Ggt ín the field was obtained by introducing BaetLlus myeoides Flugge

(Campbell and Faull, 1979); GaeumannornAces grøninis (Lemaire et aL.,

1977); G.gz,amLnis vat. graminis and PhiaLophora z,a.dicicoLa Caín (Wong and Southwell, 1980). Up t.o 46v" control of take-all in field plots were obtained by introducing hypo-virulent strai.ns of. Ggt (Lemaire et aL., I97I, 1976; Tivoli et aL.,7974).

Apart froni these field studies, Rovira and lVildermuth (1981) demonstrated the inhibition of Ggt on agar and protection of wheat roots by cultures of fluorescen'l Pseuclononads, and Sivasithamparam et aL. (1975) discovered a volatile factor inducing t-ransmissible self-

Perpetuating lysis o1' Ggt. , However, the relevance of inhibition of the fungus on agar plates is open to question and Sivasithamparam and

Parker (1978) could not shov¿ any correlation between the antagonism -157 -

towards Ggt on agar by five isolates each of actinomycetes, bacteria and fluorescent Pseudornonads and the ability of these organisns to protect wheat seedlings in sterile sand with added Ggt. Cook (1981) considers that Pseudomonas fLuot,escens intrcduced with wheat seed at sowing has potential to reduce damage fron take-al1 in fie1d.

The possibility of using mycophagous amoebae for the control of soil-borne plant pathogens has been indicated (Hornma et aL., 1979; 01d and Patrick, 1979). Experiments were conducted to test if nycophagous amoebae c.ou1d effectively reduce the severity of take-aI1 and thereby protect wheat seedlings during the initial four weeks of plant growth in a controlled environment.

Effect of mycophagous amoebae on the severity of take-al1

A preliminary experiment to determine the effects of nycophagous a¡noebae on the severity of take-al1 was conducted using Penrosers (1980) technique for scoring the susceptibility of wheat cultivars to Ggt.

Plastic pots were fil 1ed with 350 g CS and five 10 nm dianeter discs of a potato clextrose agar culture of Ggt were placed on the surface of the

CS. Discs of potato dextrose agar mediurn only rvere used in control pots. One pre-germinated seed of rvheat was placed on each agar disc and one of the follorving treatrnents applied:

a A suspension (1 nl) of one of the nycophagous Saccamoeba, urridentified leptomyxicl , Gephyz'ønoeba or Thecønoeba granifera

sub-species minor', prepared in AS tvas poured on top of each

wheat seed.

b A mixed suspension (1 nl) of all four rnycophagous amoebae was

poured on top of each wheat seed. - 1.58-

c. Sieved (2 run) IVPP soil (1g) was sprinkled on each wheat seed

d. AS suspension (1 mI) of KLebsieLLa sp. poured on top of each

wheat seed. e. AS suspension (1 tnl) of the acconpanying organisns of

T.gz,awifer"a sub-species nrinoz, poured on top of each wheat

seed.

f. Only AS (1 ml) poured on top of each wheat seed.

Seeds were covered with 50g CS and an additional 50 n1 of distil-led r,Jater was added to each pot. Plants were harvested after two weeks, rated for disease and plant heights determined.

0f the four mycophagous amoebae used, three reduced the disease rating and increased plant height significantly (P<0.01) conpared with the Ggt treatnent (Table 29). Greatest protection was obtained with

Gephyr,ønoeba or with the mixture of amoebae. The accompanying organisrns of I.granifera sub-species minor, consisting of a Fusariwn sp. and bacteria, also significantly reduced the disease rating, rvhereas, the food bacterium, KLebsieLLa sp. did not. Since the accompanying organ- isms were present in suspensions of T.granifera sub-species minoz, oniy, the reduction of disease severity by Gephyrunoeba and Saccønoeba ís inclependant of the effects of the accompanying organisms.

Reduction of take-a11 severity by soil cultures of mycophagous amoebae

The contribution cf nycophagous amoebae in disease suppression can be better understood i.f their effects can be directly cornpared with that of a suppressive soi1. While addition of AS suspensions of nycophagous amoebae selves the purpose of demonstrating their effect on the disease, -1s9-

TABLE 29: Effect of suspensions of nycophagous amoebae on the severity of disease caused by Ggt (nean of 25 plants treatment-r).

Treatnent Disease rating Plant height (cn)

*LGgt + IVPP soil 1.8 Ll.s LGgt + Gephyrønoeba sp, 2.L 71.2 LGgt + T.granifera ssp. mLnoy 2.6 10.8 LGgt + Saecønoeba sp. 2.8 9.7 LGgt + Unidentified leptomyxid 3.L 9.9 LGgt + lrfixture of all four amoebae 2.2 7r.4 LGgt + KLebsíeLLa sp. 5.5 9.7 LGgt + Acconp. org.f 2.6 10. s LGgt 3.3 9.9 PDA disc 0.0 13 .9

Least significant differences P=0. 01 0 .35 1.03 P=0.05 0.25 0.72

* L = Live + Acconp. org. = Accompanying organisms of T.granifera ssp. wLnon. -160-

the effects of a suppressive soil with its added advantage of supplying plant nutrients can not be simply compared with that of AS suspensions.

Mycophagous amoebae hrere therefore cultured in sterilized soils for use in pot bioassays.

Soil cultures of nycophagous amoebae

Sanples of both WPP and Avon soils rvere sieved and noistened to -8 kPa. Moist soils were dispensed in glass Petri plates, wrapped with tAlfoilfand autoclaveð. at I27oC for t h for three consecutive clays. Snall arnounts of AS suspension from PJA cultures of each of the four nycophagous amoebae were added to sterilized soil and incubated at 25oC in darkness. Suspensions of accompanying organisns of T.gz,anifera sub- species nrLnoz, and the food bacterium, KLebsieLLa sp., were added to sterilized soils and sinilarly incubated for controls. After two weeks, grorrth of arnoebae were tested by plating sna1l amounts of soil cultures on PJA + KLebsieLLa plates. All four amoebae were isolated fron both Avon and l'lPP soils, although only cultures in Avon soil were used jn the bioassay. Since the viability of amoebae decreased after about four rveeks in the Avon soil (page 133, Table 23) , only fresh (15- 20 days old) cultures were used.

Effect of soj-l cultures of rnycophagous amoebae on t-he severity of take-a11

The pot bioassay (Shipton et aL., L973) used to test soil suppress- iveness against Ggt was used except that CS was used instead of fumigated soil. Ground oat grain inoculum of Ggt was used. Three controls with dead (autoclaved oat grain inocula) Ggt in which either sterilized Avon soil or soil culture of accompanying organisrns + KLebsíeLLa sp. u/as -161-

added were compared with live Ggt and live Ggt plus the suppressive WPP soil or soil cultures of the mycophagous amoebae, or a rnixture of all four amoebae. Soil cultures of the accompanying organisrns of I.gr.anifez,a sub-species nrLnor and KLebsíeLLa sp., ürere also used with live Ggt. Inoculu:n of Ggt was added at the rate of 0.2% and the suppressive soil/soil culture at the rate of 1%. Five wheat seeds were solün per pot, covered with CS and 50 nl distilled water was added. Plants were harvested after four weeks, rated for disease, heights neasured and dry wt of tops cleternined (Table 30). Portions (about 2 cn) of roots fron the mixture of amoebae tTeatment were plated on PJA +

KlebsieLLø plates to cletermine the association of amoebae with wheat roots.

Soil cultures of Gephynønoeba, Saeca¡noeba and T.granífer"a sub- species nrLnor significantly (P<0.01) reduced disease severity and in- creased plant height (Fig.30) and top dry wt (Table 30). The reduction of disease and the increase in top dry wt in plants where the mixture of antoebae rvere used was conparable to the effect of the suppressive I{PP soil and there was no significant difference between these two treat- ments. Soil cultures of the acconpanying organisms also .significantly reduced disease rating and increased height and top dry wt of plants. Similarity in disease ratings in líve Ggt treatments with and without sterile Avon soil shows that the soil as such did not have any detectable effect on the fungus or the host. All four amoebae were found associated r.¡itl'r roots; of the number of pieces of roots plated, 98% had the uniderrtified leptornyxid, B\eo lnad Gephyrønoeba-. 90% had T,gnanifera sub-species mincr and BTeo had Saccamoeba, -162-

TABLE 30: Disease rating, height and top dry wt of plants grown in sterile coarse sand with soil cultures of amoeba.e and 0.2% Ggt ínoculum (mean of 25 plants treatment-1)

Plant Top dry Treatnent Di seas e height weight rating (cn) (me)

LGgt + IVPP soil 1.9 15.9 25.8 LGgt + Gephyramoeba sp. 2.2 L4.7 24.6 LGgt + ?,granífez?a ssp . nrLnot, 2.O 15. 3 23.0 LGgt + Saccønoeba. sp. 2.8 15.9 22.3 LGgt + Unidentified I eptonyxid 3.8 73.3 19.4 LGgt + Mixture of all four 1.9 ls .6 28.7 arnoebae LGgt + Acconp. org. 2.2 15. 9 23.6 LGgt + Str. Avon soil 4.2 L2.7 16.6 LGgt 4.2 L2.7 L6.3 DGgt + Str. Avon soil 0.0 18.9 29.4 DGgt 0.0 19. s s2.2

Least significant clifferences P=0. 01 o.7L 2.37 4.82 P=0. 05 0.50 1.6s 5.37

*L = Lilr"; D = Deacl; Accomp. org. = Accompanying organisns of I.gz,aníferd ssp. minor + KlebsieLLa sp. -163-

Growth of wheat after 28 days in sterilized coarse sand inoculated with G.gratnLnis tv"itici and soil cultures of mycophagous arnoebae or the suppressive Waite permanent pasture soi1.

1 Líve Ggt. 2 Dead Ggt. 3 Líve Ggt + suppressive soil. 4 Live Ggt + Saccønoeba sp. 5 Líve Ggt + Theeønoeba granifera sub-species nrtnot,. 6 Líve Ggt + Gephyrønoeba sp. 7 Live Ggt + Mixture of four nycophagous amoebae.

I Þ

-164-

Effect of initial ulation size of amoe ae on sever]- tyo ta -al 1

The effect of large initial populations of the nycophagous amoebae on,the severity of take-all was tested in a pot bioassay using two population levels. Five and 50 ml of AS suspension of individual arnoeba was added to 4009 CS containing 0.2% ground oat grain inocula of Ggt; this gave initial population levels of approx. Z g-l and 20 g-1 for Geph¡z,amoeba and 40 g-1 .r,d 400 g-1 of CS for Saeeønoeba anð.

T.gz'anife.na sub-species minoz.. The nixed amoebae treatments consistecl of approx . Z7 g-L and 270 g-1 of CS of these amoebae. The unidentified leptomyxid was not used in this exper.irnent due to its inability to reduce the disease severity in the previous tests.

Higher popr-rlation 1eve1s of all amoebae caused a further reduction in the disease rating (Table 31), however, this was not statistically significant in any treatment. Only in the mixed amoebae tïeatment, a higher initial population resulted in a significant (P<0.01) increase in the heiglit and top dry rvt of plants. Top dry wt of plants rvas arso significantly increased with an increase in the initial population of the accompanying orgatrisms. All mycophagous amoebae, however, were effective in reducing disease rating and increasing plant heights and top clry wt when compa.red to plants in the live Ggt treatments,

The acc n rns of Thecanoeba era su s ecl-es mLnoT

A significant reduction in the disease rating and increase in the plant height and top dry rvt rvas obtained in bioassays using either AS suspensions or soil cultures of the accompanying organisns of T.granifera sub-species minor (Tables 29,30,3I). The accompanying organisms a.r-e a -165-

TABLE 31: Disease rating, height and top dry wt of plants grown in sterile coarse sand with two population densities of nyco- phagous amgebae and 0.2u, Ggt inocula (mean of 25 plants treatnent-').

Anount of Treatment Plant Disease Top dry suspension height rating weight added (nl) (cn) (rng)

*LGgt + WPP soil L4.7 2.0 26.8 LGgt + Gephgr,ønoeba sp. 5 17.0 2.4 29.2 s0 t7 .2 1.8 28.8 LGgt + T,grawLfey'a, ssp. minor 5 L7.7 2 J 24 I 50 18. 1 1 6 26. 8 LGgt + Saceønoeba sp. 5 16 .8 2.6 29.1 50 L7 .L 2.4 27 .l LGgt + Mixture of all three 5 18 .2 1.8 27,9 amoebae 50 19 .8 1.5 33.7 LGgt + IrS 5 12.9 4.3 77 .4 50 L2.6 4.9 16.9 LGgt + Acconp. org. 5 14.t 2.5 22.0 s0 74.8 2.t 2s.8 DGgt + ÃS 5 17.4 0.0 29.4 50 18. 1 0.0 30.0

Least significant difference P=0. 01 1. 69 0.62 4.78 P=0. 05 7.20 0.52 3.39

L = Live; D = Dead; Acconp. org. = Accompanying organisms of T.gz,anífera ssp. minoz, + KLebsielLa sp. -166-

nixture of bacteria and a fungus which produces sickle-shaped two- septate conidia and has been tentatively identified as a Fusaz"Lun sp. several bacteria , íncluding BacilLus (campbell and Fau1l, 1979) and fluorescent Pseudomonas (Cook and Rovira, I976; Rovira and Wildermuth, 1981) and sorne actinonycetes (Zogg and Jaggi , I9T4; Zogg, Lg76; Tschudi and Kern, 1979) have hypholytic properties and species of BaetLlus and Pseudomonqs have been shorvn to reduce the severity of take- all in pot and field experiments. The presence of fluorescent pseudo- rnonads in the culture of the accompanying organisms of T.granifera sub- species minor was examined using a selective mediun.

PJA cultures of the acconpanying organisrns were flooded with SnW and the suspension was serially diluted in a 10-fold dilution series with sDI{. A drop (0.1 mr) of each suspension was spread on petri plates poured with NPcc rnediun (sinon et aL., ,J,973, Appendix I) prepared at pH 7,2. Plates were incubated at i0oc ancl examined for yel1ow-green or blue-white fluorescence in and arouncl the colonies under ultraviolet radiation.

some fluorescent pseudomonads, as detected by the presence of a yel1ow-green fluorescence itr and around the colonies, were present in the accompanying organisns of T.granifez,a sub-species nrinor and tJrese nay have contribut,ed to the reduced severity of take-all. However, as

Gephyramoeba and saccønoeba. a're naintained with a KlebsieLLa sp. and this bacterium itself did not have any detectable effect on the disease (Tab1e 29), the effects of both of these amoebae on take-all severity are independant of the effects of the accompanying orgamisns of

T. gnanifera sub-species nrLnor. -L67 -

Discussion

Three rnycophagous amoebae, Gephryønoeba, SaecØnoeba and I. gz,an|fez,a sub-species nrinor,, were able to protect wheat Toots against take-al1 during the initial four weeks of plant growth. Higher level of protection was obtained when these amoebae were used in conbination and at higher population densities.

Anderson and Patrick (1978) reported that populations of ChaLara eLegans (:TlrieLauiopsis basicoLa) and C.satiuus nay be reduced in pot experiments by additions of cysts of vanpyrellid amoebae. 01d (unpublished, 1979) observed significantly less pre-emergence seedling blight when seeds inoculated with C.satiuus were first inoculated with cysts of A.inrpatiens.

However, the protection offered by nycophagous amoebae in controlled bioassays does not imply that such protection is operative under field conditions. Faull (1978) found that 25% of the isolates of saprophytic bacteria were antagonistic to Ggt on agar plates, 6% were effective in sand, 3e" effective in soil and only I% effective in field trials. Some of the mycophagous annoebae can be grown with a food bacteriurn on low- nutrient media and stored at low tenperatures in diatomaceous earth granule cultures for up to one year. Field trials with cultures of amoebae either i-ntroduced before sowing or with seeds can be expected to provide a better assessnent of their practical value as biological control agents.

01d and Patrick (1979) reviewed the suitabí1ity of mycophagous amoebae, especially vanpyrellids, as biological control agents, They consiclered that the factors that would tend to linit their use would be - 168-

their limited growth at higher soil rnoisture tensions, the possibility that soil amoebae thenselves may constitute a biohazzard (Culbertson,

1971) and their apparent lack of specificity in selection of fungi as food. Another aspect is the ability of soil arnoebae to invade plant tissues (l"fclennan, 1950; Darbyshire and Greaves , 197L) which may result in pathogenic spnptons in host plants. However, observations so far have suggested that the invasion follows the penetration of plant tissues by their food organisms (Darbyshire and Greaves , I97I). - 169-

V, GENERAL DISCUSSION

Several aspects of nycophagy by soil amoebae have been investi- gated in some detail in this study. lVhile AraehnuLa, TheratroTTtAæa.,

VatnpgreLLa, Thecamoeba and Cashia v¡ere known as mycophagous genera, the inclusion of Gephyz,atnoeba, Saccønoeba, MayorelLa and the unidenti- fied leptonyxid has extended this list of genera with nycophagous species to nirie. More research is needed to ful1y appreciate the proportion of genera of soil amoebae which can feed on fungi.

Many investi-gators have considered that the presence of perfora= tions in the fungal ce11-walls is a positive indication of mycophagy by amoebae. While many amoebae nay cause perforations, the results reported here show that perforation rnay not be detectable in all arnoeba-fungal ìnteractions. Although, it is generally believed that amoebae gain access to the cel1 cytoplasm of fungi by dissolving portions of the wal1, subsequent feedíng by amoebae nay often lead to distortion and collapse of the fungal propagule thereby rendering the perforations undectectable. This type of distortion has been observed in hyali.ne hyphae of Ggt after feeding by trophozoites of the Saccønoeba sp. In case of the unidentified leptonyxid feeding on nature chlany- dospore of PTtytop,hthora einnønomi, delicate protoplasmic threads were seen traversing the space between dhe digestive vacuolar membrane and the chlamydospore lvall. However, whethe-r these protoplasnic threads are ca,pable of dissolving holes in the chJ.amydospore waI1 is not known. Only formless debris is extruded from a digestive vacuole at the end of the feeding period. Time-lapse trans¡nission electron microscopic stuclies may help in understanding the exact mecha,nisrn of chlamydospore penetra.tion b¡' this amoeba. -L70-

0f particular interest is the ability of many nycophagous amoebae to perforate and lyse rnelanised fungal propagules. rt is believed that this process of perforation is, at least, partry enzymatic (K.M.old, personal conmunication). Melanins have been regarded as resistant to

enzynatic degradation (Bu11, 1970). The ¡nechanisms of perforation and

lysis of rnelanised wal1s of fungi by nycophagous amoebae deserve. further attention

The take-al1 suppressive soils studied here showed a higher popu-

lation of nycophagous and other amoebae irrespective of the texture and water-holding capacity of the soil. The basis for the developnent of a higher nurnber or rndrlrrduals in these soils is not known. While large populations of food organisrns have been considered essential for

higher amoebal populations in some soils (Sandon, Lg27; Singh, 1945,

L946a,1948), a definite relationship between these has not been shown (Bryant et aL., 1982).

Figure 31, based on a conceptual nodel of the plant rhizosphere

(Bowen, 1979), shows a hypothetical case of wheat root colonisation by

Ggt from an infected host residue. Ir{icrobial interaction with Ggt can be envisaged in the three processes, as suggested by Bowen (rg7g) , uiz:

germination of the propagule, movenent towards the root and growth on the root surface. Beiìrg a root-inhabiting fungus, pathogenic activities during the parasitic phase of Ggt establishes a prioï possession of the host tissue before the death of the host, Once the host-tissue is dead, potential saproph;"tic invaders may challenge its earlier monopoly (Garret-t, 1956, 1970). The threat to the saprophytic survival of the fungus increases apparently as ageing and deconposition of the sapro- phytic inoculum proceeds; thus the snaller fragments lose their infecti- vi.ty more quickly (!brnby, t97S). -L7L-

Eg-:!' A diagrammatic representation of the process of growth of G.gratntwLs tritiei hyphae from an infected host residue throu-gh soil to infect a healthy host root. a

SOIL HEALTHY RESIDUE O-14 mm 1-3 mm ROOT

(o.4- 0.6 mm o"y-l) -172-

Invasion of Ggt-infected host residues by saprobic microbes nay

therefore result in the reduction of the szrprophytic inoculum even

prior to gernination of the propagule ancl mycophagous amoebae have been

isolated from surface sterilised infected wheat roots in ny experiments.

A higher association of nycophagous anobae with Ggt hyphae in a suppressive soil and the presence of perforations in h1'phal walls

suggest that soil arnoebae are a part of the microbial component re- sponsible for the reduction of sapropl-rytic survival of the fungus in the permanent pasture soil.

As emphasized by Skou (1.981), contact between host root and Ggt

is established by trophic gror.rth of the hyphae towards roots rather than by chance; the inportance of alrtagonistic nicroorganisrns during this phase of pre-colonisation can be easily understood.. It is during this period that PhiaLophora spp. anð. Pseudomonas fluoz,esce;ns were reported

to have restricted the grohrth of the fungus (Deacon, 1976; Cook and

Rovira, L976). In my studies, hyphae became pigmentecl at a faster rate in the suppressive pasture soil, presunìably as a result of a response to

competing microorganisns (Deacon, I973). Ivhile many organisrns, inclucl-

ing mycophagous amoebae, can lyse hyaline hyphae, only tnycophagous

amoebae have been shown to perfor:ate and 1y-se pigmented hyphae of the

fungus. Grolth of Ggt in soil is initiated by hyaline hyphae which

later become pigmented (l^Jildermuth, 1977); mycophagous amoebae rvoulcl therefore, be likely organisns to control the fungus at this pre- colonisation stage.

The zone of root exudates (rhizosphere?) is deterrnined by the distances volatile and dissolvecl exu-dates diffuse from roots (Bowen ancl -173-

Rovira, 1976) and often some forrn of selective antagonism and inhibition between organisms occurs within this zone (Rovira, 1956; Rouatt et aL., 1960; Rovira and Davey, 1974). The fact that there is a higher association of nycophagous amoebae with r,rheat roots and a higher rhizos- phere population of soil amoebae occur in suppressive soils indicates that these organisns are also effective in this selective zone of root exudates. Reduction of the severity of take-all i; pot bioassays by nycophagous amoebae furthersupports this fact. However, whether this reduction in disease severity results from a reduction in the ecto- trophic grorvth of the fungus or by the perforation and lysis of pigmented runner,hyphae on the root surface, or both, is not known.

Rovira and Wildermuth (1981), proposing a nechanism for the development of transferrable suppression rvith wheat monocultute, stated that there is a proliferation of non-sporing ::od shaped bacteria on Ggt lesions of wheat roots; a srnall proportion of these bacteria build up on nelú roots ancl hyphae with successive wheat crops and resttlt in less take-all. Although the developnent of suppression was not studied in this work, the scherne proposed by Rovira and Wildernuth (1981) can be used to envisage the rol.e of nycophagous amoebae in suppression in the pasture soil. As with other antagonists of Ggt' mycophagous amoebae associate themselves with the fungus in infected host residues, reduce the saprophytic survival of the fungus, lyse both pigmented and hyaline hyphae during the pre-colonisation and parasitic phase of the fungus and result in the reduction in disease severity.

In proposing this role of mycophagous amoebae in suppressiou, at no stage is it intended to clain that they are the only organisrns re- sponsible for suppressi-on. The clata on associated organisms with -t7 4-

Iheeatnoefu, granifera sub-speci es nrf;non would substantiate this. Other studies also show that several organisms can reduce the severity of take-al1 at least in pot experiments and therefore attenpts to explain suppression on the basis of specific antagonists nay well be futile (Hornby, 1979). It is likely that several different nechanisms are functioning simultaneously. This observation ís also supported by the apparent lack of a high degree of specificity of suppressive soils (Baker and Cook, 7974i Wildermuth, I977).

One practical aspect of the ability of mycophagous arnoebae to reduce take-al1 severity is the hope for their future use as biological control agents against the disease. Two approaches are possible, firstly, nodifications in soil management practices to augment myco- phagous anoebae, and secondly, the introduction of the organisms to soils to st¡ppress the disease. Since natural soils often seen to resist attempts to alter their nicrobial balance by introducing large amounts of pure cultures of antagonists (Garrett, 1956), enhancenent of activities and populations of mycophagous amoebae by modifying soil environment appears to be a better choice. For this further research on the effects of soil environmental conditions on these organisms would be needed. -L7s-

VI, APPENDICES

APPENDIX I Fornulae for nedia and saline solutions

NDY/6 (Warcup, 1955)

NaNO, 0.33 g

wzPo+ 0.16 g

MBSOO, 7HZ0 0.08 g

KC1 0.08 g

FeSOO, 7H20 0.0019

Difco Yeast Extract 0.08 g

Sucrose s.o g

Agar (Davis) 10.0 g Distilled water TL

V-8 Juice agar (Miller, 1955)

V-B juice 200 nl

CaC0, 3.0 g

Agar 20.0 g

Distilled water 800 rnl

2eo Mal-t agat Malt extract (Difco) 2og Agar L2g Distilled water LL -176-

Czapek-Dox agar (Proprietary mediun, Difco Laboratories, Detroit, U.S.A.).

49 g of the following nixture in 1 Z of distilled water Saccharose (Difco) 3og

NaNO, 2g

K2HP04 tg

MgSOO, 7HZ0 0.5 g

KC1 0.5 g

FeSOO, 7HZ0 0.01g Bacto agar 15g

Tryptone-Glucose-Yeast agar (TGY, Cavender and Raper, 1965) Bacto tryptone 5g Glucose 1g Yeast extTact 5g Agar 72g Distilled water TL

Prescott and James t Agar (PJA)

One nI of each of the follorving stock solutions in 997 ml of glass-distilled water makes Prescott and Jarnes I solution (Prescott and Janes, 1955). Ba.cto agar is added at a rate of leo to nake PJA.

Stock solution A CaCIr,2H20 0.433 g

KC1 0.L62 g Glass-disti11ecl water 100 nl -t77 -

Stock solution B K2HP04 O.512 g Glass-distilled water 100 nI

Stock solution C I'fgSOO, 7H20 0.280 g

Glass-distil 1ed vrater 100 n1

Modified Neffts annoeba saline (AS, Page, 7967) A separate stock solution of each of the following conponents is nade by dissolving the amounts given in 100 nl glass-distilled water. Ten nl of each of the stock solution are mixed and the volume is made up to 1 L with glass-distilled water.

1). NaCl L.2 g

2) . MgSOO ,7H20 0. 04 g

3). Na,HPOO 1.42 g

4). CaClr, 2 H20 0.04 g

s). KH2P04 L.36 e

NPCC nedium (Simon et aL., t97S)

The basal medium:

Difco Proteose Peptone No.3 2og Oxoid Ionagar No.1 (Oxoid puri- fied agar-) 129 Glycerol 8nl

K2S04 1.5 g

I'lgS0O, 7H20 1.5 g Distilled water 940 nl -L78-

pH adjusted to 7.2 wítll. 0.1N NaOH before autoclaving for 15 nin and following anti- biotics are added before pouring plates: Penicillin G (Benzopenicillin) 75 units nl-1

Novobiocin (Albanycin) 45 ug m1-1 Cycloheximide (Actidione) 75 pg rnl-1

Chloramphenicol (Chlorornycetin) 10 ug n1-1 APPIìNDIX IIA: Gcneric populations of anoebae in samples of tlìe hraitc pcrnancnt pasture, Avon and Murray Bridge soil

¡iaite peñ¡ílnent pasture Ano D Ë F K M N 0 PQR S T

Ac,tltitznoeba spp. 980 980 660 980 2600 980 158 0 158 0 980 2600 1580 920 980 460 660 1580 4800 980 980 EclLinanoebc spp. 10800 18400 7000 980 184 00 280 0 34 00 340 280 14 00 440 2200 920 1s80 520 440 5600 10800 280 5600 Leptomyxids 40 90 40 0 5,10 90 80 0 40 14ó 40 260 40 80 90 146 90 136 746 90 ì,icyorella spp. 40 40 0 0 4A 0 0 0 0 90 0 0 0 90000 0 090 Platyanoeba spp, 260 440 136 220 540 220 15ó 280 158 0 2200 220 340 1580 5400 540 540 660 l9 00 186 980 Saceanocba spp. 40 660 460 0 260 220 40 40 280 0 146 746 L46 00090 1 580 40 260 llheecnocba spp. 260 I46 90 40 !46 90 90 14ó 156 40 40 0 90 4D40040 280 40 90 T.granifcna ninor 40 90 40 40 80 90 40 40 80 18ó 40 0 40 40000 80 4ù 460 I Unidentified anoebae 14 00 10800 18400 56 00 32000 7 000 18400 420 920 340 220 7000 280 4400 136 736 2600 3400 2200 4400 \¡P Vampyrcllid a:noebae 260 40 260 4ó0 980 460 746 0 80 80 340 L46 40 220 40 146 40 40 90 746 ro I Avon Acanthamoeba spp. 220 340 460 1 0800 2600 660 660 980 460 gg0 zso 158 0 660 920 660 980 óó0 440 340 920 Echirørnoeba spp. 146 36 540 t46 340 40 186 80 136 280 280 80 36 0 4400 220 220 18ó 80 18ó l,cptonyxids 90 0 136 40 80 56 90 440 40 0 460 220 0 146 40 40 90 80 90 0 Ma¡.1orella spp. 0 40 40 0 0 040 0 0 0 40 0 0 90 40 40 40 0 0 Platycanocbe spp. 440 146 460 746 1580 440 280 136 s40 15 80 136 80 540 280 980 460 460 90 Sccaanoeba spp. 0 0 0 0 0 00 0 0 0 0 0 0 040 0 0 0 Titt:etto¿ltn 5p¡,. 40 90 40 90 40 40 0 0 90 0 40 746 156 40 40 40 0 40 0 T, a :' civ,- i fe va rrL no t' 40 0 0 40 0 40 90 0 0 0 40 0 0 0 0 040 0 0 0 Unidcntificd anoebae 746 r86 340 136 420 280 s20 80 i86 80 136 158 0 280 80 340 280 ?80 0 80 186 Vampyrel 1id anocbae 0 40 0 40 36 00 90 0 146 90 40 L46 90 746 90 36 40 36 0 Murray Bri.dge AccLntlLutoeba spp. 660 440 460 980 aa^ ?20 460 980 340 660 660 220 460 340 660 460 90 460 146 440 EchinarccL,a spp, 2600 340 230 19 00 280 440 340 15S0 540 980 340 440 260 660 440 340 440 280 540 520 Lcptonyxids 90 40 0 136 0 0 90 56 0 90 0 0 40 90 90 146 0 0 90 Mayorella spp. 90 40 90 90 40 0 90 40 40 90 0 0 40 0 40 0 0 0 0 90 Platyo,nceba spp. 156 280 146 186 260 980 920 440 220 340 280 r46 540 2600 540 980 2600 660 340 440 Saceanoeba spp. 0 0 0 0 0 0 0 0 0 0 0 0 40 0 0 0 0 0 0 0 Ihecmoeba spp. 90 0 40 90 40 746 40 40 0 40 0 40 40 0 40 146 0 90 80 90 T.gnanilera ninon 40 0 40 40 0 40 40 0 40 0 0 90 0 0 90 40 0 0 0 0 Unidentificd amocbae 980 2?0 540 540 40 136 158 0 80 40 280 260 280 1s 80 440 660 540 280 90 220 80 t46 460 Vanpyrcllid anoebae 260 0 260 9U t0 90 136 14ó 136 260 0 40 146 90 220 80 40 0 -1 APPENDIX IIB Population (no. g d"y tut; of soil amoebxe dtrring two wheat crop and inter-crop fallow in suppressive (PPW and Pew) and non- supPrcssive (ClV) soil,

Pasturc-pf,strrrc-whcat (PPIV) soil lVeeks sorving, 1981 scason Ânoeba after Inter-crop fallow lVeeks aftcr sowi 1982 season 5 4 5 6 I 10 l3 L7 20 Jan Fcb Ì'lar Apr May June

a7^1 Aeanthanoeba spp, 5 020 418 825 154ó 7?7 I 7177 2982 737 2 !142 7924 1.907 915 15 70 315 9 t257 1885 a 111 1rôo 2158 Echirønoeb+ 146 647 530 55v 607 390 775 729 301 Leptonyxids 81 168 85 r27 111 4L 762 0 47 20 430 48 0 155 140 0 78 èt qf llctyorella spp. IùÓ 280 106 74 272 98 7L 66 0 120 00 44L 2L2 101 163 792 PLaiyanocba spp. 73 583 135 180 365 283 262 +òJ 116 515 387 322 1545 7I44 1852 128I ô)ö 370 470 Sc"ccmtoebct spp. 0 57 141 45 76 3t3 338 347 0 2I 20 0 87 t92 110 303 223 154 762 82 lhecmoeba spp. 25 178 147 82 160 256 191 108 t78 41 20 20 69 77 169 293 559 457 667 I 134 351 T.grantfera ntinor 81 149 5ó 48 70 rr0 191 2S1 27 92 20 45 139 ?09 292 248 568 0 1336 466 422 anoebae Unidcntifi.cd 704 204 392 ó58 280 178 339 36 33 431 1 4025 7403 569 1979 5 301 J 999 2327 2405 870 2187 154 3 1 ?a Vampyrellid amoebae 50 r07 75 22s 94 0 238 76 66 0 20 139 77 81 0 0 0 0 23 I Pea-wheat (Peltr) soiì. æ Acanihanoeba spp. 2055 953 3203 7704 3620 2705 2687 2357 1650 7847 148 4 839 2502 77 79 r904 57s 0 642 1276 2099 2738 2469 o L?) I Ecltínanoeba spp. 0 81 0 210 424s 724 1037 793 238 284 172 298 50ó 407 52s 337 293 647 ó6s Lcptomyxids 60 L7S 111 100 109 50 0 76 46 20 66 70 23 2l 23 47 0 s2 182 23 Mal1orelLa 210 lt) 106 53 45 27 73 spp. i 772 0 0 20 20 0 I05 5J 7t 252 46 20 0 1!a Platllatnceba spp. 99 267 133 4328 158 285 186 ))) 218 250 294 525 7434 910 511 1215 1137 342 519 Saocctnoeba spp. 74 301 55 110 776 22 0 99 20 20 0 0 67 2T 48 767 197 747 126 275 139 216 704 109 'Iheactnoeba spp. 219 76 22 70 76 4r 46 20 20 20 53 361 1s5 110 644 205 163 159 115 23 ?.çnani'-fcta níror 56 55 76 73 43 146 t6 46 20 0 20 0 1ó9 179 178 135 238 253 206 UniJcntified anocbae 4668 4590 7,39 165 352 r27 ¿!6 315 8 553 1200 443 1ó4 297 2508 22r8 7784 589 891 4059 16 54 Vampyrellid anocbac 22 153 22 -12 47 +J 96 20 20 66 41 212 155 23 0 0 53 47 Continuous-wheat (Cl{) soil Aca.nthu.eba spp No deteilnination 948 141.1 64r 949 1762 130 3 115 4 27 25 108 3 1729 128I 674 1 537 872 64s 1758 783 Ecltínanoeba spp. 473 1403 ¿Ò5J 8222 742t 346 422 2r7 L39 713 193 77I 408 423 434 254 27L Lcptomyx i ds 23 22 0 43 0 ?0 0 0 2T 23 77r 20 0 lldllarella spp- 67 r26 23 67 0 0 0 27 0 78 135 103 109 10s 101 53 Plai4z,nôebcl spp. 280 203 288 s67 788 128 515 )71 294 559 673 590 450 15 13 oõ4 729 1139 lr Sacec;¡noeba spp, 23 45 69 23 41 0 20 20 0 23 24 72 48 7r2 46 248 r00 Ihecamoeba spp. 707 4S 159 104 47 20 0 20 97 23 2t0 t4r 78 2L7 46 254 177 nLno I. gronífera r 0 50 48 75 67 47 20 0 27 0 87 s4 0 72 23 t07 258 Unidentified arìoebae 196 125 7464 436s 13 54 373 202 662 487 7877 807 336 1787 1091 I 191 746 Vanpyrcl).id anoebae 128 0 63 r.3 0 4l 20 0 20 0 23 111 0 0 0 0 52 23 APPENDIX IIC: Wheat rhizoplane amoebae (percentage root plated) during tr^ro crops in suppressive (PPW and PelV) and non-suppressive (CW) soil.

Pea-wheat (PeW) soil WEEKS AFTER SOWING Amoeba 1981 season 1982 season 34 5 6810 t3 17 20 345 68 10 AcantLønoeba spp. 10 10 30 20 20 2A 0 20 100 10 10 30 10 BO 40 EcLrLnønoeba spp. 00 0 0 80 40 30 30 30 30 10 50 0 30 20 Leptonìyxids 20 50 30 0 0 0 0 0 10 40 0 40 0 0 0 MayoreLLa spp. 200 20 60 0 0 0 0 0 50 0 L0 50 0 0 I l-¡ PLatyønoeba spp. 010 70 40 50 30 60 70 60 BO 50 70 30 80 60 æ ts Saccønoeb¿¿ spp. 20 60 60 70 0 10 0 10 40 30 0 50 40 0 20 I Thecomoeba spp. 020 40 10 0 0 0 10 10 0 10 10 0 .0 0 T.granífexa minoz, 10 50 0 20 0 0 0 0 50 0 0 30 0 20 0 Unidentified anoebae 60 100 90 70 20 30 90 90 90 BO 90 70 70 90 60 Vampyrellid anoebae 300 0 20 10 10 30 50 0 0 10 0 20 0 10 Pasture-pasture-rvh

Aeanthamoeba spp. 70 40 20 10 70 60 60 BO 90 50 40 40 60 50 20 Echinønoeba spp. 0 30 0 20 30 60 30 60 70 40 10 t0 50 30 10 Leptornyxids 0 30 50 0 10 40 0 10 10 50 0 60 20 10 10 MayoreLla spp. 60 60 70 40 40 0 0 0 10 10 0 20 0 0 10 PLatyanoeba spp. 20 40 20 60 50 70 40 60 70 60 70 90 BO BO 50 Saccamoeba spp: 10 70 90 50 20 20 50 30 20 20 20 BO 20 40 50 Ihecamoeba spp. 10 30 30 40 10 20 10 0 0 10 50 40 50 10 20 T"granifera minor 40 50 20 20 30 0 40 0 50 10 40 40 10 20 30 Unidentified anoebae 90 100 100 90 80 60 70 90 100 90 80 90 70 100 BO Vampyrellid anoebae 30 40 20 20 40 70 20 L0 0 10 20 0 0 40 50

continued/. APPENDIX IIC: continued

Continuous-wheat (CW) soil WEEKS AFTER SO$JING funoeba 198l season 1982 season 34s68 0 3 1_7 20 34s 6B

Aeanthønoeba spp. No deter¡nination 30 0 0 10 60 60 40 10 40 10 20 EcVrinamoeba spp. It BO 30 40 30 40 20 50 10 50 20 20 Leptomyxids It 20 0 0 0 0 0 20 0 0 0 0 MayorelLa spp. It 0 0 0 0 0 10 0 0 0 0 0 I tt @ Platyønoeba spp. 90 60 50 50 90 70 60 90 40 60 50 N Saccønoeba spp. lt 10 0 10 0 0 50 50 50 30 10 30 I Theeamoebø spp. il 0 0 0 0 10 0 0 10 0 0 10 T.granífera minov' il 0 0 20 0 10 0 10 0 0 0 0 It Unidentified anoebae 40 50 50 100 100 60 70 100 90 90 BO Vampyrellid arnoebae ?t 40 40 30 20 20 0 0 0 50 20 50 -,183 -

APPENDIX III: Publications

CHAKRABORTY S. and OLD K.M. (1982). Mycophagous soil anoeba : interactions with three plant pathogenic fungi. SoíL BioLogy ød BioehewLstry L4: 247-255.

CHAKRABORTY S., 0D K.M. and WARCUP J.H. (1983). Anoebae from a take-all suppressive soil which feed on Gaeumannomyces gnønLnis tnitici and other soil fungi. SoiL BioLogg and BioeVpmistr.y t5: L7-24.

CHAKRABORTY S. and WARCUP J.H. (1983). Soil amoebae and saprophytic survival of GaeumutnomAees graminis trítici in a suppressive pasture soil. SoiL Biology and Biochemistry L5: (in press).

CHAKRABORTY S. and I1¡ARCUP J.H. (1983). Population of nycophagous and other amoebae in take-a11 suppressive and non- suppressive soi1s. SoiL BioLogy and. Bíocy'pmistry 15: (in press).

CHAKRABORTY S. (1983). Population dynanics of annoebae in soils suppressive and non-suppressive to wheat take-al1. SoiL BioLogy and Bioehemistry 15: (in press). Soíl Biol Biochem. Vol 14, pp 247 to 255,1982 0038-07 r 7/82/030247-09 $03 00/0 Printed in Great Britain. All rights reserved Copyright O 1982 Pergamon Press Ltd

MYCOPHAGOUS SOIL AMOEBA: INTERACTIONS WITH THREE PLANT PATHOGENIC FUNGI

S. CH¡rn¡,nonrv Department of Plant Pathology, Waite Agricultural Research Institute, The University of Adelaide, Glen Osmond, South Australia 5064 and K. M. Oro Division of Forest Research, C.S.I.R.O., Canberra, A.C.T. 2600, Australia

(Accepted 10 September l98l)

Summary-An amoeba of the order Leptomyxida was isolated lrom wheat take-all decline soil and was found to attack and lyse hyphae and spores of Gaeumannomyces graminis var. tritící and Phytophthora cinnamomi. The amoeba enveìoped portions of hyphae of both iungi and penetrated the cell walls by means of fine holes. One-week old chlamydospores and hyphal swellings of P. cinnamoni were also attacked in this way, protoplast lysis being completed within I h. Hyphal fragments which could be ingested by the amoeba were lysed leaving amorphous cell debris. Three-week old chlamydospores of P. cinnqmomi were enclosed within large food vacuoles and completely digested in about 20h. Pigmented conidia ol Cochliobolus sd¡irrs were transported across the substratum for up to several hours but were not perforated or lysed.

INTRODUCTION moeba granífera ssp. minor. They regard this species as Since 1977 there,have been a series of reports con- being strictly mycophagous, whereas Old and cerning the mycophagous activities of soil amoebae. Darbyshire (1978) showed that A. impatiens has an Particular interest has been shown in the genera extremely broad range of potential prey including Arachnula, Vampyrella and Theratromyxa which bacteria, algae, protozoa and nematodes. belong to the family Vampyrellidae in the classifi- The significance of mycophagous amoebae as cation of the protozoa proposed by Honigberg et al. agents for control of soil-borne fungal propagules is (1964), (Old, 1977; Old and Darbyshire, 1978; Old unknown. Old and Patrick (1979) reviewed available and Oros, 1980; Anderson and Patrick, 1978, 1980; knowledge, particularly with respect to vampyrellids Homma et al,, 1979). A number of other genera have and recommended further research on the effects of also been found to have mycophagous species notably amoebal groups on populations of soil borne patho- Thecamoeba (Esser er al., 1975; Allabouvette er a/., gens. A pathogen of particular interest in many tem- 1979; Pussard et al., 1979) and Hartmannella (C. perate regions is Gaeumannomyces graminís (Sacc.) v. Palzer, personal communication). As early as 1936, Arx & Olivier var. tritici Walke¡, the cause of take-all Dreschler reported that the testate amoebae, Geo- disease of cereals. The phenomenon of take-all de- coccus uulgaris and Arcella uulgaris attacked oospores cline, that is, the observed reduction ol disease after o1 Pythium ultimum. The mode of attack varies with repeated wheat crops has been well documented and amoebal species. Arachnula impatiens Cienk. was has attracted the attention ol plant pathologists and shown by Old (1977, 1978) to penetrate the walls of soil microbiologists for more than 15 yr. A number of conidia of Cochlíobolus satit)us (Ito et Kurib.) Drechs. theories as to its cause have emerged and were sum- ex Dastur by erosion of an annular depression into marized by Hornby (1979); several of these invoke the conidium surface. This disc of wall material is the gradual development of an antagonistic micro- dislodged, allowing the spore contents to extrude into flora which suppresses the pathogen. The demon- the amoebal trophozoite. The closely relafed Thera- stration that mycophagous amoebae are widely distri- tromyxa encompasses whole spores within digestive buted in soils offers the opportunity to test whether cysts, the spore wall being penetrated by many small they could have a role in take-all decline (Homma er holes each ca.O.5 pm dia (Old and Oros, 1980; Ander- al., 1919). son and Patrick, 1980). Mayorella species have been During a search for mycophagous amoebae in a shown to engulf up to five conidia of C. sariuus and soil which has been demonstrated to'oe suppressive to retain them within the trophozoite for many hours. G. graminis var. tritici (G. B. Wildermuth, unpub- Extruded spores are often lysed but little information lished Ph.D. Thesis, University of Adelaide, 1977) an is available on the feeding mechanism of this genus amoebal species has been regularly isolated which (Anderson and Patlick, 1980). Pussard et al. (1979) differs markcdly from prcviously-described mycopha- have described in detail the penetration of hyphae gous amoebae, both in its morphology and feeding and microconidia of Fusarium oxyspotum by Theca- mechanism. We describe this unidentified amoeba

247 248 S. Cu¡xnesonrv and K M. Or-o

and the manner of its feeding on propagules of three slips were airdlied and mounted on metal stubs for soil-borne plant-pathogenic lungi, G. graninis var'. tli scanning electron microscopic (SEM) examination. tici, C. satiuus and Phytophthora citlnamonti Rands. Other specimens, including intact thalli ol P. cinnct- ntomi and spol'e suspensions of C. .s¿lÍi¿r¿i,s that had MATERIALS AND METHODS been incubated with the amoeba were fixed in 3f" glutaraldehyde in phosphate buffer ovenright at 4"C, 'lhe amoeba was isolated liom soil collected from a dehycirateci rn a gladed ethanol series and critical- permanent rotation trial plot at the \¡y'aite Agricul- point dried in a Polalon unit using liquid CO2. tural Research Institute, Glen Osmond, Australia. Before SEM examination, all specimens were coated The plot has been under continuous pasture for over rvith a layer of gold palladium in a Polaron 85100 20 yr and the soil has been suppressive lo G. graminis sputter coating unit (Polalon Equipment Ltd, Herts., var. ffitici. Soil was collected from the top 0-5 cm of England). Specimens were examined with a JEOL the profile and moistened to - 8kPa. Moist soil was JSMU3 SEM. placed into 15cm Petri plates to a depth ol 7 to 8 mm. Mycelial mats of G. granini.s var. tli¡ici, RESUI,TS obtained fi'om 1 week-old cultures grown in 0.4"/o Tr o p t p Malt Extract broth at 25"C were washed twice with hoz oi e mor holog 1' sterile distilled water (SDW), placed in silk cloth bags The amoebal trophozoite exists in trvo distinct (5 x 5 cm) and buried in moist soil (J. H. Warcup, forms, a branched flattened lorm ranging lrom 80 to pelsonal communication). After 3 weeks at 25"C, 150pm in length and 28 to 95¡rn in breadtlt;(Figs 1 mycelial mats wel'e recovered and homogenized to and 4) ancl a limax fbrm (Fig 2). The change between form a suspension which was streaked on 1.01 Bacto the two forms can be readily observed. The limax agar in Prescotts and James (1955) solution (PJA). form is common in aqueous media which al'e dis- Amoebae which nrigrated from the mycelium on to turbed and can be found at the interface betweeu the the PJA medium were isolated fi'or¡ other arnoebae suspension and the air. Amoeba in non-distulbed cul- by repeated transfels of small blocks of agar bearing tures tend to settle to the bottom oi the dish and single individuals, and gradually freed from bacterial produce the branched form. The latter form is also contamination (Culbertson, 1971). Isolated amoebae comn'ìor1 ol1 agal'rnedia. Litnax folms are narrower at were maintained on PJA bearing Aerobacter aero- the postelior end thau at the anteliol'cnd and measure genes as a food bacterium. Stock cultures o[ G. fi'om 50 to 180 ¡im in length and 20 to 40 ¡rm in width. çTrantinís var. tritici, C. satiuus and P. cintuntonti were Both fornrs are uninucleate, the nucleLrs as seen by rnaintained on Czapek Dox agar and colnmeal agar' PCM tneasures 6 to lO¡un in dia (Figs 1,3,4) and respectively. Mycelium of G. graminis to be used in contains an elongated nucleolus (3 to 5 ¡rrn). The pro- feeding trials was grown on 0.4/" malt extract broth toplast is distinctly divisible into a lryaline ectoplasnr for 1 week, washed twice with SDW and rinsed in and ¿ì granular, compact endoplasm (Fig. 3). The sterile Neffs amoeba saline solution (AS) (Page, 1976). blanched form contains one to nrány vacuoles up to Mycelium was either left intact or homogenized. l6¡rm in dia. The limax form normally has a single Conidia of C. sariua.s were harvested from Czapek large vacuole at the posterior end ol the cell (Fig. 2). Dox cultures by flooding plates with SDW and lightly Locomotion is particularly interesting, and can be 1 rubbing with a sterile loop. Suspensions of conidia as rapid as 5 /¿m s in the branched folms. From the wele filteled thlough sterilc chccse cloth and washed advancing margin of the cell snrall eruptive pseuclo- twice in SDW. Mycelinm and chlamydospores of P. pods (type lobosa) are formed by the ectoplasm into cinnantoni were produced by inoculating V8 juice which the graflular endoplasm flows. As the tropho- liquid medium containing 20 ¡rg cholesterol ml- I zoite advances the posterior malgin lbrms a variabte (D. D. Darling, unpublished infolmation) with ap- number of fine filopods (Fig. 4). Similar' filopods also proximately 50 small pieces of agar bearing P. cinna- are formed when a lobose pseudopod is retracted. ntomi and incubating for up to 3 weeks at 25'C in Pseudopods can be projected in a number of different continuous light. Thalli were harvesteel, washed in directions simultaneously, but ultimately flow in one sterilc AS and either homogenized or leit intact. Feed- of these directions. Tiue polyaxial rnovernerrt llas not ìng trials were conducted, using the three fungi in been observed, Anastomosis between indlvidual trt¡- 9 cm dia Petri plates containing the fungal substrate phozoitcs has not bccn obsclvcd so lal cvcn though suspencled in sterile AS. The saline covered the base crowding is common on agâr cultures. of the Petri plates to a depth of approx. 5 mm. At the During cell division the tlophozoite extends outset of each trial, six or seven 12 mm dia glass linearly with pseudopods adhering to the substratum covelslips wele placed into each Petri plate. at both ends. Two nuclei can be seen clearly in such Stock cultures of the amoeba were flooded with dividing cells. The central portion oi the protoplast sterile AS and suspensions of amoebal cysts were becomes constricted and finally the two daughter cells drawn up into sterile Pasteur pipettes. Drops of these separate. The generation time on PJA medium with ,4. suspensions wele added to the fungal substlate and aerogenes as a food source vaLies fronr 3 to 5 h. the cultures incubated fcr 4 5 days at 25'C. Detailed observations of the interactions between the amoebal Cyst ntorpholoqy tlophozoites and their prey were made by transferring The amoeba forms cysts which are r-rsually spherical coverslips bealing these organisms to Cruikshank in shape (Fig. 12), but this call vary if individuals ale chainbers (Sterilin Ltd, Teddington, Middlesex, crowded, or cysts íorm u,ithin the confines of lysed England) and examining by phase contrast micro- fungal cells. For instance, cysts olten form within scopy (PCM). After feeding for 3 to 14 days, cover_ empiy sporangia of P. cinrunnonti (Fig. 11). Cysts are A new mycophagous amoeba 249

i. { t'' tÅ ..""ì . $tt I .l

Plate l. Fig' 1' Large nattened unt"'io' robopodia (L)' ""ffiäli,""Ti::,ì:J?¿r.iT,:Ji,ii:iii,J:lft,lh" Fig. 2. Tubular limax amoebae. Note the tapered posterior and contractile vacuole (CV). Fig. 3. Form intermediate between branched and limax showing the single nucleus (N), hyaline ectop- lasm (E) and granular endoplasm (EN). 250 S Cs¿.rn¡.nonrv and K. M. Olo

smooth walled and measure 15 30¡rm in dia. The is lendeled completely devoid of contents rn single nucleus can be seen just before excystment. 35 45 min after feeding begins and the amoeba either Rarely up to 3 nuclei have been seen at this stage. A moves a',vay to feed on other cells or may migrate single trophozoite invariably emergos lrom the cyst. within the sporangium or hyphal swelling and form a Excystment can be stimulated by placing cysts into cyst (Fig. 12). Scanning electron microscopy of fungal sterile AS or on PJA bearing A. aerogenes. material after prolonged feeding showed that hyphae, and other propagules were perfolated by circular Feeding mechanism of the amoeba orr P. cinnamomi holes varying from 1,0 to 3.0¡rm dia (Fig. 9). No annular depressions similar to those described by Old The amoeba employs two separate mechanisms (1977) were recorded. when feeding on P. cinnamoml. Penetration and lysis of hyphae, sporangia and hyphal swellings of the Digestion of mature chlam¡,¡l6sp6¡'¿s o/P. cinnamomi fungus is very rapid and is completed in 35 to 45 min. Having contacted the chlamydospore the tropho- Mature chlamydospores are digested within large zoite completely engulÍs it. Formation and expulsion digestive vacuoles over a prolonged period, between of the contents of numerous small vacuoles within the 2l To 36 h. These processes are quite distinct and will amoebal protoplast are intense at this stage. This pro- be described separately. cess results in the formation of a large fluid frlled vacuole containing the chlamydospore within ap- penetration Rapid of hypha proximately 20-35 min. This vacuole is the sub- The trophozoite contacts the hypha, movement sequent site of digestion of the spore. During this in- ceases and it adheres firmly to the cell surläce. Il' itial phase of attack the amoeba is particularly light- hyphal fragments are encountered these may be sensitive and rounds up if illumination is too intense. dragged along the substratum attached firmly to the Once the digestive vacuole is fully formed (Fig. 13) the posterior filopods. When attacking an intact mycelial amoeba is less sensitive. Careful microscopical obser- thallus the amoeba stretches itself along the hyphal vation of spores within digestive vacuoles shows that length, attaching at any part (Fig, 5). The trophozoite delicate protoplasmic th¡eads traverse the space is extremely thin and extended, eventually engulfing a between the main vacuolar membrane and the spore portion of the hypha. This process takes 10 15 min wall. These are not resolved adequately in Figs l3 16. after which the amoeba contracts and masses around Contraction of these strands may be the cause of the a small section of hypha (Fig. 6). This process of rotation of the chlamydospore within the vacuole extension and contraction may be repeated 2 6 times. shown in Figs 13 16. The trophozoite then moves along the hypha, or may The main activity seen within the amoebal proto- leave it altogether. At this stage, it can be seen that plast at this time is the continuous formation of ves- the portion of hypha where the trophozoite concen- icles which develop bordering the digestive vacuole trated is completely devoid of cytoplasm (Fig. 8). The and discharge their contents (Figs 15, 16). At no stage amoeba exhibits considerable mechanical force at this did a cyst wall develop around the trophozoite in the time and free ends of hyphae may be physically dis- manner described lor Theratromyxa (Old and Oros, torted to encompass them within the body of the t¡o- 1980). In fact, the outline of the amoeba was in a state phozoite. The feeding activity of the amoebae is very of continuous though gradual change, and the tro- sensitive to bright illumination. Photography and ob- phozoite could be observed to migrate a small dis- servation rnust be carried out with intermittent or low tance on the substratum, by normal pseudopodial light intensity or feeding will be slowed or cease al- movement. together. After about 16 ro l7 h from formation of the diges- Sporangia and hyphal swellings or immature chla- tive vacuole, the diameter of the vacuole gradually mydospores of P. cinnantonti are deal| with by the reduces (Fig. 17). The amoeba resumes mole active amoeba in a similar way to hyphae, except that pseudopod formation and the generally spherical engulfment of the propagule may be incomplete. A shape is lost. The fluid layer between the chlamydos- similar series of extensions and contractions of the pore and the amoebal protoplast disappears com- trophozoite occurs and ihe contents o[ the sporan- pletely. At this stage the contents of chiamydospore gium flow into the amoebal cell. The fungal propagule appear to be disorganized and granular (Fig. 18).

Plate 2. Fig. 4. Trophozoite oi the mycophagons amoeba showing anterior lobopodia (L), posterior filopodia (F) nucleus (N) and the typical branched morphology. Fig. 5. Amoeba feeding on a hypha of P cinnntomi. The trophozoite has enveloped a length of hypha (arrows). Fig. 6. Trophozoite has concentrated around the subterminal portion oi the hypha (arrows). Fig 7 rrophozoite has new rocarion (arrow)' ''if:'irii:i.1iilås;l¿Jii"i:i,;ï:::'#i:ii:: "'a Fig. 8, Detail of the hyphal tip. Fig 9 Perforated hypha of P cinnamomi Fig. 10. Perlorations in hyaline hypha of C. graninis var. titici (arrows). The bacteria (B) are ,4. 4eroqenes. A new mycophagous amoeba 251

-=--.

sBB l4l'3 F 252 S Csnxnn¡onry and K. M. Oro

Typical trophozoite movement resumes and the DISCUSSION remains of the digestive vacuole and its contents are The amoeba we describe shares with other species, conveyed along by the trophozoite. Finally, the diges- conclusively shown to be mycophagous, the ability to tive vacuole becomes completely disrupted and the digest discrete holes in fungal cell walls. But, a par- remaining fungal protoplasm is taken into the amoe- ticularly interesting characteristic is the ability to bal cytoplasm. As the trophozoite rapidly moves employ two distinct feeding mechanisms in attacking away, a trail of debris, presumably the undigested diflerent cell types of a single fungus. The criterion contents of the digestive vacuole are extruded on to which directs the feeding option is the ability of the the substratum (Fig. 19). trophozoite to fully surround the propagule. If this is achieved, often by bending and coiling the hypha by Feeding of the amoeba on hyphae o/ G. graminis uar. application of considerable force, then digestion of the tritici cells is almost complete in 30 to 60 min. Undigested residues are expelled from the moving trophozoite. If, The mode of feeding varies according to the ability however, the amoeba attacks an of the trophozoite to fully surround the hypha. When intact thallus and is unable to fully enclose the propagule, then discrete intact thalli are used, the amoeba envelops sections of perforations in the wall are made and the protoplast hypha and exhibits the extensions and contractions only is ingested. Hyphal walls appear to be otherwise shown for P. cinnamomi hyphae (Figs 5 7). Protoplas- little damaged. These types feeding have been mic contents of these hyphal cells are lost and sub- of closely observed for P. cinnamomi and hyaline hyphae sequent SEM examination shows that holes varying ol G. graminis var. tritici. in size from 0.3 to 2 are present in the fungal cell rum Hyphae of both these fungi and hyphal swellings walls. Presumably these are the sites of attack by and chlamydospores in 1 week-old cultures of P. cin- which the amoeba gains access to the fungal proto- namomi are rapidly attacked. Protoplast ingestion plast (Fig. l0). When the fungal thallus is fragmented commonly takes only 30 to 60 min. When prep- before feeding trials, the trophozoites ingest whole arations of older chlamydospores (2 to 3 weeks) are pieces of hypha. This is commonly achieved by twist- exposed to the amoeba the feeding mechanism is ing and bending the hypha until it is fully accommo- modified further. After complete encirclement of the dated inside the trophozoite. The trophozoites con- chlamydospore alarge digestive vacuole develops and tinue to move across the substratum and in as little as the trophozoite remains almost immobile for a pro- 3G-45 min the hyphal fragments are no longer recog- longed period (17 to 20h) during which the entire nizable. Shortly afterwards undigested residues are chlamydospore particularly extruded from the trophozoite. is digested. It is signifi- cant that the mature chlamydospore requires much longer for lysis to be completed than do hyphae or Interaction of the amoeba wl¡h C. sativus young chlamydospores. Chlamydospores are widely Thc amoeba commonly encircles conidia of C. regarded as survival structures, more resistant than satiuus and the spores frequently adhere to the pos- hyphae to lysis, but the structural basis for this terior of the trophozoite, apparently held by the characteristic has not been discovered. The stimulus group of filopods. Clumps of 5 or more conidia, short to the formation of the large food vacuole by the fragments of conidiophores and immature spores ac- amoeba may be directly related to the susceptibility of cumulate in this position and may be conveyed along the wall components to enzymatic digestion. This by tbe trophozoite for many hours. Fungal cells are newly-described amoeba, unlike ,4. impatiens, Thera- left behind from time to time and others accumulated. tromyxa and Vampyrella spp (Old, 1977; Old and However, careful observation of many such tropho- Oros, 1980; Anderson and Patrick, 1978) is unable to zoites has never indicated that the amoeba is attack- attack conidia of C. satiuus. Opportunities for diges- ing these spores and digesting their contents. Examin- tion of these spores are commonly observed as the ation of specimens taken from 14-day old oultures trophozoites adhere to conidia and convey them for containing conidia and very large numbers of active considerable distances across the substratum. Yet the trophozoites by SEM has not revealed any perforated fungus remains in an apparently viable condition as conidia. shown by the presence of intact protoplasts and per-

Plate 3. Fig. 11. Amoebal cyst within empty P. cínnamomi sporangium (arrow). Fig. 12. Amoebal cysts within lysed swollen hyphae. Fig. 13. Chlamydospore of P. cinnamon¿ within a digestive vacuole (Figs 13 19 arc a sequence of microglaphs at the same magnification showing lysis of the chlamydospore). Fig. 14 Change of amoebal shape and rotation of the chlamydospore Fig. 15. Digestive vacuole showing a vesicle lorming in the amoebal cytoplasm (arrow). Fig. 16. As Fig 15 but v/ith a mature vesicle about to discharge into the digestive vacuole (arrow). Fig. 17. The digestive vacuole is reduced in volume and the amoeba becomes more mobile. Fig. 18. The chlamydospore (arrow) has lost its structural integrity Fig, 19. The trophozoite moves away leaving a trail of fungal cell debris (arrow). A new mycophagous amoeba 253 254 S Cu,rxR.,reonrv and K M. Or-o

loration-fi'ee walls. Movement ol fungal spores by lies Leptomyxidae and Gephyramoebidae we are amoebae, without apparent damage to therr was de- unable to attlibute our isolates to the species L. /c- scribed by Heal (1963) and has been legular-ly seen by bellata. Further work and comparison of cultures will us for species ol Mayorella. The walls of ('. .safjr¿rs be needed to name this amoeba with certainty. At conidia contain deposits of melanin in their outer. present we are confident in placing the amoeba in the layers (Old and Robeltson, 1970) and may protect the older Leptomyxida. spore from perforation. Melanin has been shown to The mycophagous amoeba of the or-der. Leptomyx- markedly inhibit the activity of glucanases and chiti- ida described here has been frequently isolated from nases which digest major fungal cell wall components the take-all suppressive soil and from a number of (Bull, 1970) Hyphae of G gramini.s in old cultures other sites in South Australia. It is probably found in become pigmented with age and all attempt was made many other soils and regions in common with other to study their susceptibility to attack by the arnoeba. mycophagous genera already studied. Evidence so far Some perforated hyphae were found, but it was not suggests that it may be restricted to feeding on hya- possible to distingrrish, by SEM examination, pig- line fungal propagules and unable to penetr.ate pig- mented hyphae fi'onr the hyaline hyphae still present mented spores. Nevertheless its ability to feed on bac- in the thallus. teria would allow populations of this amoebal species It is extlentely difficult to determine the taxonomic to maintain themselves in moist soils as a component affinities of large freshwatel ancl soil amoebae. Many of the soil ecosystem that may exert an influence on of the early workers who observed these organisms, soil-borne pathogens. It shares many of the attributes usually in n-rixed culturc, described them as new spe- discussed by Old and Patrick (1979) and is untikely to cies and often erected new genera on the basis ol lend itself to ready manipulation as a biocontrol differences in their specimens. The axenic cultur.e of agent. Even so, the study of mycophagy in soil amoe- large amoebae is vely demanding, and type cultures bae deserves the closest attention as a factor influenc- are largely unavailable ior comparison. Zwillenberg ing the population dvnamics oi soil fungi. (1953) in describing a new species of Theratonyxu consideLed 19 genera as possible synonyms. The Acktntt,ledgentents We thank genela leceiving detailed modern tleatlnent are Lep- Dr J. H, Warcup for read- ing the rnanuscript, and Mr J, M. Oros printing the tomlt¡¡¡ (Pussard and Pons, l976a,t:), Geph¡t¡¡¡¡7ss6¡¡ lor photomicrographs. S.C. thanks Professor H. Wallace, (Ptrssard and Pons, 1976c), Arachnula (Old and Dar- R ChaiLman, Department of Plant Pathology, Waìte Agricul- byshire, l98O), Vumpylel/a (Anderson Patrick, and tural Research Institute, for making a research grant avail- 1978, 1980) and Tlterotron rt,.ra (Sayre, 1973; Anderson abie for this work and Patlick, 1980; Old and Olos, 1980). The amoeba we desclibe here does not fit any oi the species de- scriptions which we have encountered, howevel it REFERENCES clearly has close af'fìnities wttll Leptotil),-ra, especially ALLnnouvnrru C, Pussrno M and PoNs R (1979) Isola- L. (Goodey, 1915). In 1976, Pussa¡d and flabellara tion and characterization of a rnycophagous amoeba Pons considered that genus the Leptontyxu is so dif- Thecantoebagronifera subsp nrinor ln Soil-Borne Putho- ferent from the other members of the order Proteo- gens (B. Schippers and W Gams, Eds), pp 629 633. Aca- myxida that a nciv order Leptontyxida was necessary. demic Press, London This new order includes amoebae forming thin AnornsoN T, R. and Plrnlcr Z. (1978) Mycophagous spleading rcticlrlatc trophozoites witl'r polyaxial loco- amoeboid organisms from soil that^. perforate spores of motion. Pser-rdopods arc ol indeterminate shape of the Tltielatiopsís bosicola and Cochlíobohrs sd¡i¿ìus P/l)'¡o- type lobosa, the ectoplasm is poorly developed. No puthology 68, 1618 1626. A;sornsor T. R and Plrnrcx Z. A. (1980) Soil varnpylellid flagellate stage is present ir.r the life cycle and no lruc- amoebae th¿rt cause smzrll perforations in conidia of tifications are knorvn. The order contains fami- two Cochlioholus satirris. Soii Biology 6 Biochctttist¡, 12, lies, Leptomyxidae ancl Gephylamoebidae, dis- ts9-t67. tinguished on the basis ol mrLltinucleate troplìozoites Bull A T, (1970) Inhibitjon of polysacchalases by nrela- in the former. aud uninucleate tro¡rhozoites in the lat- rrin: enzyme jnhibition in relatior.r to nycolysis Arclùtes tel family. Leptam.ixo .flabelkrta Coodey was retaiired of Biotlrcntistry untl Biaph¡sic:; 137,345 356, witlriri tlre same genrÌs as L. reticulttta, the type spe- Cul¡enrso¡¡ C G (1971) Pathogenicity of soil amoebae. cies, (Pussard ancl Pons, 1976b) despitc significant dif- Anttuol Reuien, of Microhiolog¡, 25, ) jl 254 ferences in trophozoite rnolphology. ht L. flabellatct, trophozoites are branched lather than reticulate and locornotion is not polyaxial Another significant characteristic is the production ol tubular limax forms when growing in a water film. In all these character- istics onl amoeba closely resembles L. flabellata. GoooEy T. (1915) A preliminary communication on three Photomicrographs new Proteomyxan presented by Pussard and Pons Rhizopods from soil Archit:es .füt (1976b) clearly show a postelior" lringe of filopods as Proti:renkunde 35, 80 102, (1963) illustrated in Fig. 1 The principal diflerencc appcârs Herl O W Soil l'ungì as food for amoebae In Sct/ Organi.sns (J. to be in the nuclear nuntber. L fllbeilata is reported Doekscn and J. van der Drjft, EcÍs), pp 289 291. North-Holland, Amsterdam. to have froln 1 to 48 nuclei pel trophozoite. We have Houu¡ oK R. J. consistently observed a singlc nucleus in the tr.opho- Pcrlo jon 9) zoite Up to three ltuclet ol ol. we¡e seen in cysis in our (irtpilt by vanr cultures. m Pacifi field soj g, As nuclcar number is basis [or sepalatìng the fami- 1l18 A new mycophagous amoeba 255

HoNrcn¡nc B. M., BrlnlturH W., Bovse E. C., Conrtss in Australian forest soils. Soíl Blology & Biochemístry J. O., Goo¡lcs M., H¡,t-l R. P., Kuoo R. R., LEvIN¡ N. 12,169 175. D., LorwrcH A. R. Jn, WEISER J. and W¡INrIcs D. M. Pnc¡ F. C. (1976) An Illustrtúetl Key to Freshwater arul Soìl (1964) A revised classification ol the phylum Protozoa. Amoebae. Freshwater Biological Association. Scientific Journal of Protozoology ll,7 20. Publication No. 34. (1955) Culturing of Hor¡¡nv D. (1979) Take-all decUne: a theorists paradise. In Pnescorr D. M. and J¡vss T V/. Cell Soil-Borne Pathogens (8. Schippers and Vy'. Gams, Eds), Amoeba proteus on Tetrohymena. Ex¡tetintental pp. 133 156. Academic Press, London. Research 8,25Ç258. des getes Lepto' Olo K. M. (1977) Giant soil amoebae cause perforalion Pussnno M. and Pous R. (1976a) Etude Lepto- and lysis of spores o[ Cochliobolus satiuus. Transactions myxa el Gephyranroeba (Protozoa, Satcodina)' I' ret¡culatct Goodey 1915. Prot¡stologica 12' of the Brítish Mycologícal Society 68,277 281. ^yro Or-o K. M. (1978) Fine structure of perforations of Cochlio- 15 1-1 68. (1976b) des genres Lepto- bolus satiuus conidia by giant amoebae, Soil Biology & Pussrno M and Por'¡s R. Etude (Protozoa, Sorcodina). Lepto' Biochemistry 10, 509 516. myxa el Gephyramoeba lI' 319' Oro K. M. and Ros¡nrsoN W. M. (1970) Effects ol lytic myxa flabellataGoodey 1915. Protistologica 12, 307 des ger'res Lepto- enzymes and natural soil on the fine structure of conidia Pussnno M. and Pons R. (1976c) Etude of Cochlíobolus satiuus. Transactions of the Brítish Myco- myxa ef Gephyt'amoeba (Ptotozoa, Sarcodina)'. lll' logical Society 54, 343-350. G'ephyramoeba delicatula Goodey, 1915. Protistologica Or¡ K. M. and Drnsvsutnn J. F. (1978) Soil fungi as food 12, 351 383. for giant amoebae. Soil Bîology & Biochemistry 10, Pussnno M., Alln¡ouverrE C. and Pors R. (1979) Etude gtani- 93-100. preliminaire d'une amibe mycophage Thecamoebo P rotistologica and P.crR.tcrZ. A,.(1919) Giant soil amoebae, j"r o minor (T hecomoebidcte, amoebida) Oro K. M. ""p. potential biocontrol agents. In Soil-Botne Pathogens (8. 15, 139 149. (1973) Thertttrontyxn weberi an amoeba pre- Schippers and W. Gams, Eds), pp. 617-628. Academic S,q.váe R. M. plant parasitic nematodes. Journal of Nemato- Press, London. datory on Orr K. M. and D¡nsvsHIRE J. F. (1980) Arachnula impa- Iogy 5,258-264. new tiens Cienk., a mycophagous giant amoeba lrom soil. ZwILLËNBERc L. O. (1953) Theratromyra webetí, a proteomyxan organism from soil. Atltonie uan Leeuwen- P r o t ísto Io g ica 16, 27 7 -287 . Or-o K. M. and Onos J. M. (1980) Mycophagous amoebae hoek, Journal of Microbiology and Serology l9' 101-116'

- 184-

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