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Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi
Soil i?io/. Eiochem. Vol. IS, No. I, pp. 17-24, Printed in Great Britain. All rights reserved
AMOEBAE WHICH FEED
1983 Copyright
FROM A TAKE-ALL SUPPRESSIVE ON GAEUMANNOMYCES GRAbfINIS AND OTHER SOIL FUNGI S. CHAKRABORTY,
Department
K.
M.
OLD’
of Forest
SOIL TRITICI
and J. H. WARCUP
of Plant Pathology.
‘Division
0038-0717/83/010017-08$03,00/O c 1983 Pergamon Press Ltd
Waite Agricultural Research Institute, Glen Osmond, South Australia Research, C.S.I.R.O.. P.O. Box 4064, Canberra, A.C.T. 2600, Australia
5064
10 April 1982)
(Accepted
Summary-Amoebae were isolated from soil of the Waite Institute permanent pasture plot which is suppressive to take-all of wheat. Nine species of amoebae belonging to eight genera were tested for their mycophagy against Gaeumunnomyces graminis var. tritici. Cochliobolus satiuus and Phytophthora cinnamomi. Members of the genera, Gephyrumoebu, Mayorella, Saccamoeba, Thecamoeba and an unidentified species of the order Leptomyxida, were mycophagous. Feeding of mycophagous amoebae and their ability to perforate and lyse melanized propagules of fungi are discussed.
permanent
INTRODUCTION
Soil amoebae are known to feed on fungal propagules and at least 6 different amoebae have been shown to be mycophagous (Old, 1977; Old and Darbyshire, (1978; Anderson and Patrick, 1980; Esser et al., 1975; Pussard et al., 1979, 1980). Known mycophagous amoebae range from the relatively small Thecumoeba grdfkra ssp. minor to giant Arachnula or Vam pyrella species. The techniques used to isolate these organisms have generally been selective in nature, i.e. by enriching the soil with a target fungus. Using selective techniques, mycophagous amoebae have been isolated from take-all decline soil (Homma et al., 1979); forest soil (Old and Oros, 1980); garden soil (Old, 1977); vineyard and other ecologically diverse soils (Anderson and Patrick, 1978). Take-all decline, a reduction in the incidence of take-all disease caused by Gaeumannomyces graminis (Sacc.) v. Arx and Olivier var. tritici Walker after continuous monoculture of wheat has been well documented (Hornby, 1979). Decline of take-all has also been observed in some soils without any history of wheat monoculture (naturally suppressive soils). Soil of the permanent pasture plot in the rotation experiment at the Waite Agricultural Research Institute, South Australia, is naturally suppressive to G.g. tritici (G.B. Wildermuth, unpublished Ph.D. thesis, University of Adelaide, 1977). As a part of a programme to determine the nature of the causal agents in this soil, soil amoebae were isolated by a nonselective soil dilution technique. This paper summarizes the results of in vitro feeding trials carried out with nine amoebae belonging to eight different genera and three soil fungi, G.g. tritici, Cochliobolus sativus (Ito et Kurib.) Drechs. ex Dastur and Phytophthora cinnumomi Rands. MATERIALS
Soil was collected
from
AND METHODS
the top O-5 cm layer
of the 17
\HH 15,‘1 H
pasture plot and suspended in sterile modified N&s amdeba saline (AS) (Page, 1976). This suspension was serially diluted (10 ‘, IO ’ and 10-j) with AS and plated on Prescott and James solution with l.O’A Bacto agar (PJA) (Prescott and James, 1955). Individual amoebae migrating on to the agar were transferred to fresh PJA plates inoculated with a suspension of a Klebsiella sp. as a food bacterium (Singh, 1946). Repeated transfers of agar blocks containing a single trophozoite were used to establish cultures of only one type of amoeba. Only amoeba which were common in this soil were used in the feeding trials. Hyphal suspensions of G.g. tritici to be used in the feeding trials were made from mycelial mats grown in 0.4% Malt extract for I week. Suspensions of C. sativus conidia were obtained from Czapek-Dox cultures by flooding the plates with sterile distilled water and straining it through a piece of sterile cheesecloth. Mycelium and chlamydospores of P. cinnamomi were produced by inoculating V8 juice containing 2Opg cholesterol ml-’ (D. D. Darling, unpublished information) with the fungus and incubating for 3 weeks at 25°C in continuous light. Feeding trials were conducted in 9cm diam. Petri plates containing each fungal substrate suspended in AS and adding a suspension of trophozoites and cysts of an amoeba. At the outset of each trial, six or seven 12 mm dia coverslips were placed in each Petri plate. Plates were incubated at 25°C for 3-14 days, after which coverslips bearing the organisms were transferred to Cruickshank Chambers (Sterilin Ltd, Teddington, Middlesex, England) and examined by phase contrast microscopy. For scanning electron microscopy (SEM), coverslips were either air dried or fixed in 3% glutaraldehyde, dehydrated in graded ethanol series, critical-point dried and coated with a layer of gold~palladium. Specimens were examined with a Jeol JSMU3 SEM.
S. CHAKKABOKTY CI ~1. Table
I. Mycophagy
of various
soil amoebae fungi Fungus
Amoeba Acunrhamoeha
sp.
gy
h
_
Ggt CS Ggt CS Ggt
+
CS
+
+
grunifrru
Ggt
+
_
CS
+
+
sp.
Ggt cs
_ _
_
Ggt
sp.
Qt CS PC
sp. sp.
Plutymoc+u
Thrwmorha ssp.
sp.
minor
Thrrumochu
_
NT” NT + + + + _ _
Gqdyumoeha
Succtrmorhu
Unidentified’ Leptomyxtd llnidcntihed Vampyrellid
NT + + _ _ _
+
+
CS
_
_
PC
+ _
+ _
Ggt
_
CS
_
PC “Gueumcmnom~~ce.v pruminis ph/horcl
AMOEBAE
AND THEIR
FEEDING
var.
rririci.
dNot tested
&nam;mi.
BEHAVIOUR
Among the amoebae commonly isolated and cultured from the Waite permanent pasture soil were, Acunthamoeha sp., Echinanzoehu sp.. Platyamoehu sp., Succamoehu sp., Thecamoehu sp., Mayorellu sp., and unidentified leptomyxid and vampyrellid amoebae. Succumoeha, Members of 5 genera, Gephyrumoeha, Muyorellu, Thecumoehu and an unidentified leptomyxid, proved to be mycophagous (Table I). Saccamoeba
sp.
The amoeba is predominantly limax, although lobose pseudopodia are also observed occasionally. In a newly formed pseudopod, and at the advancing margin of a limax amoeba, a hyaline ectoplasmic crescent is usually present. However, the ectoplasmic cap may be absent in limax amoebae under con-
Fig.
I. Limax trophozoite
pathogenic
Perforation (SFM)
Mycophagy
PC’
MuWr&
on three plant
of Saccamoeha
“Cochlioholus
“Chakraborty
’ Phvfo-
suticus.
and
Old (1982).
tinuous locomotion. A characteristic feature of this amoeba is the occurrence of IO-28 rectangular to cuboidal crystals in its cytoplasm (Figs l-3). Crystals vary between 0.75 to 1.5 pm in length. The trophozoites non-cystic
are,
in general,
smaller
than
those
sp. Note the ectoplasmic
crescent
(E) and cytoplasmic
crystals
(arrows). Fig. 2. Sarumoeha Fig. 3. Formless
debris
(arrow)
sp. feeding
extruded
on hypha
of G.R. tri/ic.i.
by SaccomocJhrr sp. after feeding
on hyphal
fragments
of G.g.
triiici.
Fig. 4. Succamoehu Fig. 5. -C. .scrrhus conidium
sp. forming
ruptured
a loop
by Saccumoeha
at one end of a C. surh~
sp., the spheroplast-like
conidium. bodies
(C) arranged
in
a linear fashion.
Fig. 6. A branched
trophozoite of Gephyrumoehu sp. in contact with C. surirus pseudopodial cluster (arrows) at the tip of the branches.
Fig. 7. Stained Fig.
8. Cysts
nucleus
of Grphyumoeha germination,
Fig. 9. A trophozoite
of Geph_vrumorha
sp. with the distinct
sp. before a young
of Gephyramoehu
of the
species of Succumoehu listed by Page (1976) or Bovee (1972). their dimensions ranging between 24 and 45 pm in length and 8 to 33 pm in breadth. No cysts have been observed in cultures. The single nucleus and its dense nucleolus can be observed with phase contrast microscopy in live amoebae, but the abundance of cytoplasmic crystals may sometimes obscure this. Nuclei measure between 3.2 to 8.8 pm dia and nucleoli from 1.6 to 7.2 pm. The type of damage to hyphae depends on the ability of trophozoites to accommodate these inside their bodies. In an intact thallus, attack usually starts at a hyphal tip. When the trophozoite comes in contact with a hypha of G.g. tritici. it adheres with its
showing germination, trophozoite (T) emerges
nucleolus
(S). Note
the
(arrow).
the single nucleus from the cyst.
sp. inside an empty c‘. .YLI~~~W conidium. wall (arrows).
spores
(arrows).
Note the intact
On spore
Mycophagous
amoebae
from
Plate
take-all
I
suppressive
soil
19
S. CHAKRABORTVef al.
20
700j~rn’, and branch more profusely (Fig. 6) with branches generally of uniform width to give a ribbonlike appearance as has been observed by Pussard and Pons (1976c) with GeF~yra~~eba delicatula Goodey, 1915. A limax form has not been observed for this amoeba. The size of a trophozoite varies depending on the number of anastomoses it has undergone. We have observed a range of 80-750~~ in length, and 50-130 pm in breadth in liquid medium. In phase contrast, the cytoplasm of live amoebae appears to be devoid of any prominent organelles. The distinction between the hyaline ectoplasm and the finely granular endoplasm is only \/isible at high magnification. Pulsating vacuoles. l-30 or more per individual, with a diameter of approx. I .5-17 pm are the only cytoplasmic structures visible in live trophozoites (Fig. 6). Nuclei can only be observed in stained specimens. Young trophozoites formed by the excystment of uninucleate cysts are uninucl~dt~ but soon develop into multinucleated individuals by anastomosis. The number of nuclei in a trophozoite varied from I to 25 or more. The nucleus is spherical to oval in shape, with a diameter of 1.6-4.1 pm, and contains one darkly stained nucleolus which varies from 0%2.5pm dia (Fig. 7). The tips of pseudopodial branches often have a cluster of lobose pseudopodia (Fig. 6). The amoeba has a very slow rate of movement. It forms thin-walled spherical, oval or almost oblong cysts (Fig. 8) 12.8-36.6 pm dia, which resemble those formed by many other amoebae. Cysts are generally uninucleate. Of 100 cysts examined, only 6 had more than I nucleus (up to 4 in number). The cyst nucleus is specially prominent before germination (Fig. 8). Based on trophozoite and cyst morphology, this amoeba appears to belong to the family Gephyramoebidae (Pussard and Pons, 1976a), and we have tentatively placed it in the genus G~~~zyrff~~~~~ Goodey, 1915. The amoeba is similar to G~p~lyr~~~o~~~ delicatula Goodey, I91 5 in appearance as described by Pussard and Pons (1976~). but differs in being multinucleate and anastomosing. More work and comparison of cultures are needed to clarify its taxonomic position. The branched trophozoite covers a wide area and increases its chance of contact with food. Thus a trophozoite can contact one or more spores of C. satizus at once. The amoeba stays firmly attached for up to 15-20 h (Fig. 6). The conidium wall is pene-
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 graduatly forms a rounded mass containing many refringent granules. The portion of the hypha retained within the amoeba1 cell becomes twisted and bent at several points. Ingestion of the fungal cells takes about 10-l 5 min. After a period of approx. 25-40min the amoeba moves on leaving the empty deformed section of the hypha on which it has been feeding (Fig. 2). When hyphal fragments are used in feeding trials entire segments are accommodated within trophozoites which surround the hyphae and bend and twist them into shapes which can be ingested. After about 15-20 min. debris, scarcely recognizable as hyphae, is extruded on to the substratum (Fig. 3). When feeding on C. satins conidia. the amoeba establishes firm contact with the spore by forming a loop (Fig. 4) similar to that described above and stays attached to the spore for about 4-6 h as a round mass containing refringent granules. Sometimes the amoeba moves along the surface of the spore and very occasionally resumes the limax form of locomotion with the spore attached to its posterior end. After this prolonged period of contact, one or more ceils in the conidium 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 cells. These conidia when examined with the scanning electron microscope show perforations, 1.5-7.0 pm dia, in their walls. On other occasions the amoeba enters the spore. presumably through the perforation. The amoeba stays inside the spore for a variable period of time ranging from 6 to 24 h. All compartments of the conidium may be entered and septa are completely disrupted. In other instances the spore is partially invaded. The amoeba invariably ruptures the conidium wall as it emerges. Portions of the fungal cell which survived attack then may extrude from the conidium as spheroplast-like bodies (Fig. 5). LI
Gephyramoeba
.rp.
The trophozoites are more branched and vary widely in tomosis between individuals. rather limited and tends to be medium trophozoites extend
or less filamentous and their size due to anasOn agar, branching is dichotomous. In liquid over wider area, up to
Fig. 10. Conidium of C. sativzcs perforated by Geph?;rumoeba sp. Fig.
il.
A trophozoite Fig. Fig.
Fig.
12. Trophozoite 13. Perforation
of T. gruni/>ra ssp. minor encircling in C. sariaus conidium
14. Trophozoite of Mqwellu sp. showing pseudopodia. Note the clear ectoplasm
Fig. 15. Conidium Fig.
of T. ~~Q~~~~u ssp. minor showing the contractile ectoplasmic region (E) at the anterior end.
of C. suti~s
caused
vacuole
a hypha
(CV) and
of G..g, wirici.
by T. ~r~ln~/tira ssp. minor.
nucleus (N), mamilliform (M) and digitiform (EC) and the granular endoplasm (En).
inside the large vacuole formed by ~~~t~r~/l~ of the spore is cleariy visible.
Fig.
18. Reticulum formed
G.R. rritici hypha. undergone.
by the vampyreilid
Note the twisting amoeba
encircling
and bending conidia
(D)
sp. The single septum
16. The Same conidium of C. sntizu.~ inside Ma,~~rrllu sp. after 8 h from the formation vacuole. The conidium lacks septum and the contents are disorganised.
Fig. 17. Mr~_~~~rellosp. with engulfed
broad
of the
the hypha
of C‘. sc~ti~s.
has
Mycophagous
amoebae
from
take-all
Plate
2
suppressive
soil
21
22
S.
CHAKKABORTY
trated by a pseudopod which may be followed by the entire trophozoite (Fig. 9). The contents of the spore may be completely lysed or part of the protoplast may survive attack. The sequence of invasion of the conidium is very dithcult to observe as the amoebae are extremely light sensitive. They respond to bright illumination by withdrawal of pseudopodia and by rounding up to form an inactive amoeba1 mass. Trophozoites leave spores through perforations in the wall and do not disrupt empty spores. Spores, when examined with SEM. show perforation in their walls varying from 1.0-3.2 pm dia (Fig. 10). Similar perforations are also observed in the walls of chlamydospores and hyphal swellings of P. cinnamomi after they have been incubated with the amoeba for 7710 days. Thecamoeba
granifera
.~.rp. minor
Trophozoites are more or less elliptical in shape measuring between 24.440.8 pm in length and 15524.5 ,nm in breadth. The endoplasm is characteristically granular with refringent granules which appear yellow or brown in colour, the intensity of colour varying considerably between individuals. A small trophozoite may have few or no granules, and as it feeds on fungal propagules the granules increase in number. Pussard et al. (1979) demonstrated that these granules are mostly lipids which have been accumulated by the amoeba by its feeding on fungal cells. The hyaline ectoplasm of the trophozoite is broader at the anterior end when in continuous locomotion (Fig. I I). There is usually one pulsating vacuole l-6 ,nm dia in the posterior part of the body. Locomotion is rapid and may be I .5 pm set ‘. In a live trophozoite the nucleus is rendered inconspicuous by the abundance of lipid granules. When stained, nuclei measure between 8 to 12 pm dia. Cysts have not been observed. Feeding on hyphae of G.x. tritici is similar to feeding on Fusurium oq~sporum Schlecht amend. Synder and Hansen (Pussard et ul., 1979). After encircling a length of hypha (Fig. 12), the amoeba becomes rounded and stays in contact for 1O-20 min, after which it moves along the hypha and the fungal cell encircled appears empty. Small pieces of C. satizw hyphae which are usually present in conidial suspensions of this fungus are also emptied in the same way. The amoeba can perforate conidia of C. .saticu.y and digest their contents. Depending on the size of a conidium, it may be partly or completely engulfed by a trophozoite which stays attached without movement for up to 45 min, at the end of which the conidium appears paler in colour. SEM observation shows perforations in conidial walls which range between 1.2-5.0 pm dia (Fig. 13). After prolonged feeding by the amoeba, many C. .suticus conidia become disrupted. Mayorella
.sp.
Trophozoites are more or less elongated and flattened. In live trophozoites the single nucleus, 4-8 /lrn dia appears as the prominent cytological structure and is more or less spherical in shape with a rounded nucleolus measuring 2.555.5 nm dia (Fig. 14). The number of large contractile vacuoles,
et
al.
2-l I nm dia may vary from I to 20 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 hyaline ectoplasmic region is present in the pseudopodial projections and also appears as a fine film all around the cell. Pseudopodia are usually conical mamilliform but can be digitiform and sometimes quite extended with a rounded end (Fig. 14). The rate of locomotion varies from 0.5 to 1.8pm set ‘. Often the ridge formed on mamilliform pseudopodia extend beyond the pseudopodial length and can be seen as more or less parallel streaks along the entire length of the trophozoite. Trophozoites measure from 50 to 130 pm in length and 20 to lOO,um in breadth. Trophozoites of this amoeba resemble Myvorellu penurdi Page, 1972 (Page, 1976) except for the morphology of floating forms. Floating forms of M. penurdi are irregularly rounded but those observed here of this amoeba have radiating slender pseudopodia. As a trophozoite comes in contact of a C. sutizus conidium it stops and encircles the spore. Ingested spores may be carried for long periods and ejected by the trophozoite without apparent damage. This behaviour of Muyorellu has been reported by Anderson and Patrick (1980). Apart from the usual spore carriage, as seen by these authors, we have observed the conidium being ingested and the amoeba gradually assuming a spherical shape. This is followed by the appearance of a fluid layer between the spore and the amoeba1 cytoplasm to form a large vacuole (Fig. 15). This vacuole resembles the digestive vacuole formed by the leptomyxid amoeba while feeding on chlamydospores of P. cinnumomi (Chakraborty and Old, 1982). After the vacuole is fully formed around the conidium (Fig. 15) the trophozoite may continue its usual locomotion. The spore can be seen to rotate randomly inside the vacuole and often the vacuolar wall invaginates and touches the spore. The shape and the diameter of the vacuole, therefore, changes continuously. This is unlike the digestive vacuole formed by the Leptomyxrd amoeba which stays more or less constant in size and shape for a considerable period of time. After about 668 h, the septa in the spore are no longer visible (Fig. 16) and the conidium also appears paler in colour. The amoeba however, carries the spore inside for a much longer period of time even after the disappearance of the septa (up to 10 h). Finally the spore is ejected. Scanning electron microscopic observations of spores taken from feeding cultures have not so far provided evidence of perforation. Bacterial populations in cultures have been very high and a tiim of bacterial cells and mucilage obscured surface detail of the conidia. When macerated hyphae ot G.g. tritici are used m feeding trials, Muyorellu engulfs portions of hyphae by twisting and bending these fragments (Fig. 17). Hyphal fragments are retained inside for up to 6 h and ejected fragments lack refringency in some areas. However, the mechanism of hyphal feeding is not clear. DISCUSSION
The technique
used here to isolate soil amoebae
Mycophagous
amoebae
from
produced a number of mycophagous and nonmycophagous genera. The previously described mycophages, Arachnula (Old and Darbyshire, 1978), Vamp_vrella (Anderson and Patrick. 1978) and Theratromyxa (Old and Oros, 1980, Anderson and Patrick 1980) were not isolated. Of the amoebae tested, members of 5 genera proved to be mycophagous. Of these, only Thecamoeha was known to have this ability, as Pussard et al. (1979) had shown that T. granifera ssp. minor fed on Fusarium oxysporum and other hyaline fungi. We found this species of amoeba also can feed on pigmented conidia of C. .saticus. In many fungi the brownish to black pigment, melanin confers resistance to microbial lysis (Lockwood, 1960; Lingappa et al., 1963; Bartnicki-Garcia and Reyes, 1964; Kuo and Alexander, 1967). The ability of a number of amoeba1 genera to lyse pigmented fungal propagules is particularly significant, as discussed by Old and Patrick (1979). With the demonstration here of lysis of C. satiws conidia by Saccamocda, Thecamoeha, Gephvramoeha, and Mayorella the number of genera able to attack pigmented fungal cells is increased to 7, although the taxonomy of some of these amoebae remains in doubt. A major difficulty lies with the separation of GephJtramoeba from Leptomyla by Pussard and Pons (1976a) based on the number of nuclei per trophozoite. Chakraborty and Old (1982) described a leptomyxid which fed on P. cinnamomi propagules and hyaline cells of G.g. tritici but failed to perforate and lyse C. satiws conidia. In most respects this amoeba resembles Leptomyxa,flahellata (Pussard and Pons. 1976b) but trouhozoites are clearlv uninucleate. By Pussard ‘and PO&’ (1976a) criterion this would exclude the isolate from the genus LeDtomvxa. In this work. isolates of an amoeba *resembling GephJ,ramoeba had up to 25 or more nuclei per trophozoite, excluding it from this genus. Clearly, comparison of cultures and some revision of the taxonomy of the Leptomyxida is necessary. The demonstration that amoebae contact and engulf fungal propagules is not in itself evidence of feeding. Amoebae resembling Leptomyxa reticulata failed to perforate Chalara elegans Nag Raj and Kendrick ( = Thielaciopsis basicola) and C. .sutiws (Anderson and Patrick, 1978; Old and Oros, 1980). The amoeba shown in Fig. 18 is commonly found in the suppressive permanent pasture soil. It is probably a member of the family Vampyrellideae (Honigberg rt cd.. 1964) and it forms an extensive reticulum in cultures encircling many conidia of C. saticus and also enveloping G.g. tritici hyphae. The trophozoite is quite unlike L. reticulata and so far no evidence of perforation or lysis of either fungus has been observed. Species of Muyorella have been reported to ingest and transport fungal spores (Heal, 1963; Anderson and Patrick, 1980). Heal regarded feeding as unlikely and Anderson and Patrick suggested mycophagy and parasitism of Muyorella on other amoebae. Observations here support the contention that MaJ,orella can lyse spores of C. suticus contained within large vacuoles in the trophozoite. Disorganization of septa and protoplasts of conidia contained within vacuoles has been recorded. Further work is needed to deter-
take-all
suppressive
23
soil
mine whether cell wall perforation occurs during this period. The ability of Mayorella to feed on other amoebae is confirmed. In mixed cultures of soil amoebae containing Mavorella trophozoites, cysts of other amoebae, especially the readily identified Acanthamoebu cysts, have been seen within vacuoles of Mayorella.
It is now possible to summarize the steps in feeding by soil amoebae on fungal propagules. (1) Attachment of trophozoites to fungal propagules appears to be a matter of chance. There seems to be no strong chemotaxy or thigmotaxy. In all genera so far observed, amoebae may engulf spores or hyphae which are potentially food, only to move away leaving the propagules unharmed. (2) Engdjnent of the propagule, partially or completely. The giant trophozoites of Arachnula, Vampyrella and Theratromyxa are able to completely engulf spores or sections of the fungal thallus (Old, 1977; Old and Oros, 1980; Anderson and Patrick, 1978, 1980). The smaller Succamoeba and Thecamoebu attach to restricted parts of hyphae or spore walls and penetrate through to the protoplast. (3) Digestion, which may be by motile trophozoites which penetrate cell walls as in Arachnula, Thecamoeba, Saccamoeba, Gep~~~vramoeba and the unidentified leptomyxid (Chakraborty and Old, 1982) or by digestion of fungal cells within specialized food vacuoles. These may form within cysts, for example, Arachnula impatiens forms digestive cysts after a feeding period. A large central vacuole forms in the cyst within which fungal cell residues are digested as completely as possible. In Theratromyxa, the whole process of wall penetration and digestion of spore contents proceeds within a cyst. The leptomyxid amoeba of Chakraborty and Old (1982) forms very large vacuoles within which complete chlamydospores of P. cinnamomi are reduced to formless debris. Mayorella also forms food vacuoles within trophozoites but indications so far are that C. satiws spores remain largely intact and only the spore contents are digested. The time taken by different amoebae to lyse fungal propagules by different feeding mechanisms varies markedly. T. gran@a ssp. minor emptied a conidium of C. saticus in less than I h, whereas A. impatiens took approx. 4.5-6 h (Old, 1978). The unidentified leptomyxid took about 30 min to lyse P. cinnamomi hyphae but needed 22-36 h to digest chlamydospores of the same fungus. Conidia of C. saticus were contained within Mayorella trophozoites for more than 16 h during which time rupture of internal septa and protoplast disorganization occurred. Information on the effect of mycophagous amoebae on populations of soil fungi is lacking. Perforation in hyphae of G.g. tritici in a suppressive soil by vampyrellid amoebae has been shown by Homma et ul. (1979). The evidence presented here shows that 3 of the amoeba1 species isolated from a naturally suppressive soil feed on the take-all fungus, but their role in suppression is not known. Ackno&dgement-We Forest Research. micrographs.
thank Mr J. M. Oros, Division of CSIRO.
for
printing
the
photo-
S. CHAKRABORTYet ul.
24 REFERENCES
pathogenic IRI-789 _ _,
Anderson T. R. and Patrick Z. A, (1978) Mycophagous amoeboid organisms from soil that perforate spores of Thielariopsis hasicola and Cochliobolus saticus. Phylopathology 68, 161 X-1626. Anderson T. R. and Patrick Z. A. (1980) Soil vampyrellid amoebae that cause small perforations in conidia of Cochlioholus 159-167.
sutiws.
Soil
Biology
&
Biochemistry
12,
Bartnicki-Garcia S. and Reyes E. (1964) Chemistry of spore wall differentiation in Mucor rouxii. Archiaes ofBiochemistry and Bioph.vsics 108, 1255 133. Bovee E. C. (1972) The lobose amebas. IV. A key to the order Granulopodia Bovee and Jahn 1966, and descriptions of some new and little-known species in the order. Archires ,fur Protistenkunde 114, 37 l-403. Chakraborty S. and Old K. M. (1982) Mycophagous soil amoeba: interactions with three ulant pathogenic fungi. Soil Biology,
& Biochemistry
Esser R. P., Ridings Ingestion of fungus
14, 2477255.
-
-
W. H. and Sobers E. K. (1975) spores by Protozoa. Proceedings of
the Soil and Crop Science Society
of
Florida
34, 206298.
Heal 0. W. (1963) Soil fungi as food for amoebae. In Soil Organisms (J. Doeksen and J. van der Drift. Eds), pp. 289-297. North-Holland, Amsterdam. Homma Y., Sitton J. W., Cook R. J. and Old K. M. (1979) Perforation and destruction of pigmented hyphae of Gaeumunnomyces graminis by vampyrellid amoebae from Pacific North-west wheat held soils. Phvtonathologv , <1, 69. 1118~1122. Honigberg B. M., Balamuth W.. Bovee E. C., Corliss J. O., Godjics M., Hall R. P., Kudo R. R., Levine N. D., Loeblich A. R. Jr. Weiser J. and Weinrich D. M. (1964) A revised classification of the phylum Protozoa. Journul qf Protozoology 11, 7720. Hornby D. (1979) Take-all decline: a theorist’s paradise. In Soil-Borne Pathogens (B. Schippers and W. Gams, Eds), pp. 133-156. Academic Press, London. Kuo M. J. and Alexander M. (1967) Inhibition of the lysis of fungi by melanins. Journal of Bacteriology 94,624629. Lingappa Y., Sussman A. S. and Bernstein I. A. (1963) Effect of light and media upon growth and melanin formation in Aureobasidium pullut&ts (De Bary) Am. ‘
( = Pullulariu Applicatu
Lockwood
pullulans). 20, I0991 28.
J.
L.
(1960)
Mycopathologia
Lysis
of
et
mycelium
Mycologiu
of
piant-
fungi
by natural
soil.
Phytopathology
50,
Old K. M. (1977) Giant soil amoebae cause perforation and lysis of spores of Cochliobolus snticus. Trunsactions of the Brirish Mycological Society 68, 277728 I. Old K. M. (1978) Fine structure of perforation ot Cochlioholus satinus conidia by giant amoeba. Soil Biology & Biochemisfry 10, 5099516. Old K. M. and Darbyshire J. F. (1978) Soil fungi as food for giant amoebae. Soil Biology & Biochemistry 10, 933100. Old K. M. and Oros J. M. (1980) Mycophagous amoebae in Australian forest soils. Soil Biology & Biochemi.vtry 12, 1699175.
Old K. M. and Patrick Z. A. (1979) Giant soil amoebae, potential biocontrol agents. In Soil-Borne Puthogens (B. Schippers and W. Gams, Eds), pp. 617-628. Academic Press, London. Page F. C. (1976) An Illustrated Ke.v to Freshwater and Soil Amoebae. Freshwater Biological Association. Scientific Publication No. 34. Prescott D. M. and James T. W. (1955) Culturing of Amoeba proteus on Tetrahymena. E,xperimental Cell Research 8, 256258.
Pussard
M. and Pons R. (1976a) Etude des genres Lepto(Protozoa. Sarcodina). I Lepto1915. Protistologica 12, 15lll68. Pussard M. and Pons R. (1976b) Etude des genres Leptomyxa et Gephyramoeba (Protozoa, Sarcodina). II Leptomwa ,jabellata Goodey I91 5. Proti.rtologica 12, myxa et Gephyramoeba myxa reticulata Goodey
307-3 19.
Pussard
M. and Pons R. (1976~) Etude des genres Leptomyxa et Gephyramoeba (Protozoa, Sarcodina). III Gephyramoebu delicatulu Goodey, 19 15. Protistologica 12, 351-383.
Pussard M., Alabouvette C. and Pons R. (1979) Etude preliminaire dune amibe mycophage Thecamoeba granifera ssp. minor (Thecamoebidae. Amoebida). Protistologica 15, 139-149. Pussard M., Alabouvette C.. Lemaitre I. and Pons R. (1980) Une nouvelle amibe mycophage endogee Cashia mycophaga n. sp. (Hartmannellidae, Amoebida). Protistologica 16, 433-45 I.
Singh B. N. (1946) A method of estimating the number of soil Protozoa, especially amoebae, based on their differential feeding on bacteria. Annals of AppliedBiology 33, 112~119.
AMOEBAE WHICH FEED
1983 Copyright
FROM A TAKE-ALL SUPPRESSIVE ON GAEUMANNOMYCES GRAbfINIS AND OTHER SOIL FUNGI S. CHAKRABORTY,
Department
K.
M.
OLD’
of Forest
SOIL TRITICI
and J. H. WARCUP
of Plant Pathology.
‘Division
0038-0717/83/010017-08$03,00/O c 1983 Pergamon Press Ltd
Waite Agricultural Research Institute, Glen Osmond, South Australia Research, C.S.I.R.O.. P.O. Box 4064, Canberra, A.C.T. 2600, Australia
5064
10 April 1982)
(Accepted
Summary-Amoebae were isolated from soil of the Waite Institute permanent pasture plot which is suppressive to take-all of wheat. Nine species of amoebae belonging to eight genera were tested for their mycophagy against Gaeumunnomyces graminis var. tritici. Cochliobolus satiuus and Phytophthora cinnamomi. Members of the genera, Gephyrumoebu, Mayorella, Saccamoeba, Thecamoeba and an unidentified species of the order Leptomyxida, were mycophagous. Feeding of mycophagous amoebae and their ability to perforate and lyse melanized propagules of fungi are discussed.
permanent
INTRODUCTION
Soil amoebae are known to feed on fungal propagules and at least 6 different amoebae have been shown to be mycophagous (Old, 1977; Old and Darbyshire, (1978; Anderson and Patrick, 1980; Esser et al., 1975; Pussard et al., 1979, 1980). Known mycophagous amoebae range from the relatively small Thecumoeba grdfkra ssp. minor to giant Arachnula or Vam pyrella species. The techniques used to isolate these organisms have generally been selective in nature, i.e. by enriching the soil with a target fungus. Using selective techniques, mycophagous amoebae have been isolated from take-all decline soil (Homma et al., 1979); forest soil (Old and Oros, 1980); garden soil (Old, 1977); vineyard and other ecologically diverse soils (Anderson and Patrick, 1978). Take-all decline, a reduction in the incidence of take-all disease caused by Gaeumannomyces graminis (Sacc.) v. Arx and Olivier var. tritici Walker after continuous monoculture of wheat has been well documented (Hornby, 1979). Decline of take-all has also been observed in some soils without any history of wheat monoculture (naturally suppressive soils). Soil of the permanent pasture plot in the rotation experiment at the Waite Agricultural Research Institute, South Australia, is naturally suppressive to G.g. tritici (G.B. Wildermuth, unpublished Ph.D. thesis, University of Adelaide, 1977). As a part of a programme to determine the nature of the causal agents in this soil, soil amoebae were isolated by a nonselective soil dilution technique. This paper summarizes the results of in vitro feeding trials carried out with nine amoebae belonging to eight different genera and three soil fungi, G.g. tritici, Cochliobolus sativus (Ito et Kurib.) Drechs. ex Dastur and Phytophthora cinnumomi Rands. MATERIALS
Soil was collected
from
AND METHODS
the top O-5 cm layer
of the 17
\HH 15,‘1 H
pasture plot and suspended in sterile modified N&s amdeba saline (AS) (Page, 1976). This suspension was serially diluted (10 ‘, IO ’ and 10-j) with AS and plated on Prescott and James solution with l.O’A Bacto agar (PJA) (Prescott and James, 1955). Individual amoebae migrating on to the agar were transferred to fresh PJA plates inoculated with a suspension of a Klebsiella sp. as a food bacterium (Singh, 1946). Repeated transfers of agar blocks containing a single trophozoite were used to establish cultures of only one type of amoeba. Only amoeba which were common in this soil were used in the feeding trials. Hyphal suspensions of G.g. tritici to be used in the feeding trials were made from mycelial mats grown in 0.4% Malt extract for I week. Suspensions of C. sativus conidia were obtained from Czapek-Dox cultures by flooding the plates with sterile distilled water and straining it through a piece of sterile cheesecloth. Mycelium and chlamydospores of P. cinnamomi were produced by inoculating V8 juice containing 2Opg cholesterol ml-’ (D. D. Darling, unpublished information) with the fungus and incubating for 3 weeks at 25°C in continuous light. Feeding trials were conducted in 9cm diam. Petri plates containing each fungal substrate suspended in AS and adding a suspension of trophozoites and cysts of an amoeba. At the outset of each trial, six or seven 12 mm dia coverslips were placed in each Petri plate. Plates were incubated at 25°C for 3-14 days, after which coverslips bearing the organisms were transferred to Cruickshank Chambers (Sterilin Ltd, Teddington, Middlesex, England) and examined by phase contrast microscopy. For scanning electron microscopy (SEM), coverslips were either air dried or fixed in 3% glutaraldehyde, dehydrated in graded ethanol series, critical-point dried and coated with a layer of gold~palladium. Specimens were examined with a Jeol JSMU3 SEM.
S. CHAKKABOKTY CI ~1. Table
I. Mycophagy
of various
soil amoebae fungi Fungus
Amoeba Acunrhamoeha
sp.
gy
h
_
Ggt CS Ggt CS Ggt
+
CS
+
+
grunifrru
Ggt
+
_
CS
+
+
sp.
Ggt cs
_ _
_
Ggt
sp.
Qt CS PC
sp. sp.
Plutymoc+u
Thrwmorha ssp.
sp.
minor
Thrrumochu
_
NT” NT + + + + _ _
Gqdyumoeha
Succtrmorhu
Unidentified’ Leptomyxtd llnidcntihed Vampyrellid
NT + + _ _ _
+
+
CS
_
_
PC
+ _
+ _
Ggt
_
CS
_
PC “Gueumcmnom~~ce.v pruminis ph/horcl
AMOEBAE
AND THEIR
FEEDING
var.
rririci.
dNot tested
&nam;mi.
BEHAVIOUR
Among the amoebae commonly isolated and cultured from the Waite permanent pasture soil were, Acunthamoeha sp., Echinanzoehu sp.. Platyamoehu sp., Succamoehu sp., Thecamoehu sp., Mayorellu sp., and unidentified leptomyxid and vampyrellid amoebae. Succumoeha, Members of 5 genera, Gephyrumoeha, Muyorellu, Thecumoehu and an unidentified leptomyxid, proved to be mycophagous (Table I). Saccamoeba
sp.
The amoeba is predominantly limax, although lobose pseudopodia are also observed occasionally. In a newly formed pseudopod, and at the advancing margin of a limax amoeba, a hyaline ectoplasmic crescent is usually present. However, the ectoplasmic cap may be absent in limax amoebae under con-
Fig.
I. Limax trophozoite
pathogenic
Perforation (SFM)
Mycophagy
PC’
MuWr&
on three plant
of Saccamoeha
“Cochlioholus
“Chakraborty
’ Phvfo-
suticus.
and
Old (1982).
tinuous locomotion. A characteristic feature of this amoeba is the occurrence of IO-28 rectangular to cuboidal crystals in its cytoplasm (Figs l-3). Crystals vary between 0.75 to 1.5 pm in length. The trophozoites non-cystic
are,
in general,
smaller
than
those
sp. Note the ectoplasmic
crescent
(E) and cytoplasmic
crystals
(arrows). Fig. 2. Sarumoeha Fig. 3. Formless
debris
(arrow)
sp. feeding
extruded
on hypha
of G.R. tri/ic.i.
by SaccomocJhrr sp. after feeding
on hyphal
fragments
of G.g.
triiici.
Fig. 4. Succamoehu Fig. 5. -C. .scrrhus conidium
sp. forming
ruptured
a loop
by Saccumoeha
at one end of a C. surh~
sp., the spheroplast-like
conidium. bodies
(C) arranged
in
a linear fashion.
Fig. 6. A branched
trophozoite of Gephyrumoehu sp. in contact with C. surirus pseudopodial cluster (arrows) at the tip of the branches.
Fig. 7. Stained Fig.
8. Cysts
nucleus
of Grphyumoeha germination,
Fig. 9. A trophozoite
of Geph_vrumorha
sp. with the distinct
sp. before a young
of Gephyramoehu
of the
species of Succumoehu listed by Page (1976) or Bovee (1972). their dimensions ranging between 24 and 45 pm in length and 8 to 33 pm in breadth. No cysts have been observed in cultures. The single nucleus and its dense nucleolus can be observed with phase contrast microscopy in live amoebae, but the abundance of cytoplasmic crystals may sometimes obscure this. Nuclei measure between 3.2 to 8.8 pm dia and nucleoli from 1.6 to 7.2 pm. The type of damage to hyphae depends on the ability of trophozoites to accommodate these inside their bodies. In an intact thallus, attack usually starts at a hyphal tip. When the trophozoite comes in contact with a hypha of G.g. tritici. it adheres with its
showing germination, trophozoite (T) emerges
nucleolus
(S). Note
the
(arrow).
the single nucleus from the cyst.
sp. inside an empty c‘. .YLI~~~W conidium. wall (arrows).
spores
(arrows).
Note the intact
On spore
Mycophagous
amoebae
from
Plate
take-all
I
suppressive
soil
19
S. CHAKRABORTVef al.
20
700j~rn’, and branch more profusely (Fig. 6) with branches generally of uniform width to give a ribbonlike appearance as has been observed by Pussard and Pons (1976c) with GeF~yra~~eba delicatula Goodey, 1915. A limax form has not been observed for this amoeba. The size of a trophozoite varies depending on the number of anastomoses it has undergone. We have observed a range of 80-750~~ in length, and 50-130 pm in breadth in liquid medium. In phase contrast, the cytoplasm of live amoebae appears to be devoid of any prominent organelles. The distinction between the hyaline ectoplasm and the finely granular endoplasm is only \/isible at high magnification. Pulsating vacuoles. l-30 or more per individual, with a diameter of approx. I .5-17 pm are the only cytoplasmic structures visible in live trophozoites (Fig. 6). Nuclei can only be observed in stained specimens. Young trophozoites formed by the excystment of uninucleate cysts are uninucl~dt~ but soon develop into multinucleated individuals by anastomosis. The number of nuclei in a trophozoite varied from I to 25 or more. The nucleus is spherical to oval in shape, with a diameter of 1.6-4.1 pm, and contains one darkly stained nucleolus which varies from 0%2.5pm dia (Fig. 7). The tips of pseudopodial branches often have a cluster of lobose pseudopodia (Fig. 6). The amoeba has a very slow rate of movement. It forms thin-walled spherical, oval or almost oblong cysts (Fig. 8) 12.8-36.6 pm dia, which resemble those formed by many other amoebae. Cysts are generally uninucleate. Of 100 cysts examined, only 6 had more than I nucleus (up to 4 in number). The cyst nucleus is specially prominent before germination (Fig. 8). Based on trophozoite and cyst morphology, this amoeba appears to belong to the family Gephyramoebidae (Pussard and Pons, 1976a), and we have tentatively placed it in the genus G~~~zyrff~~~~~ Goodey, 1915. The amoeba is similar to G~p~lyr~~~o~~~ delicatula Goodey, I91 5 in appearance as described by Pussard and Pons (1976~). but differs in being multinucleate and anastomosing. More work and comparison of cultures are needed to clarify its taxonomic position. The branched trophozoite covers a wide area and increases its chance of contact with food. Thus a trophozoite can contact one or more spores of C. satizus at once. The amoeba stays firmly attached for up to 15-20 h (Fig. 6). The conidium wall is pene-
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 graduatly forms a rounded mass containing many refringent granules. The portion of the hypha retained within the amoeba1 cell becomes twisted and bent at several points. Ingestion of the fungal cells takes about 10-l 5 min. After a period of approx. 25-40min the amoeba moves on leaving the empty deformed section of the hypha on which it has been feeding (Fig. 2). When hyphal fragments are used in feeding trials entire segments are accommodated within trophozoites which surround the hyphae and bend and twist them into shapes which can be ingested. After about 15-20 min. debris, scarcely recognizable as hyphae, is extruded on to the substratum (Fig. 3). When feeding on C. satins conidia. the amoeba establishes firm contact with the spore by forming a loop (Fig. 4) similar to that described above and stays attached to the spore for about 4-6 h as a round mass containing refringent granules. Sometimes the amoeba moves along the surface of the spore and very occasionally resumes the limax form of locomotion with the spore attached to its posterior end. After this prolonged period of contact, one or more ceils in the conidium 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 cells. These conidia when examined with the scanning electron microscope show perforations, 1.5-7.0 pm dia, in their walls. On other occasions the amoeba enters the spore. presumably through the perforation. The amoeba stays inside the spore for a variable period of time ranging from 6 to 24 h. All compartments of the conidium may be entered and septa are completely disrupted. In other instances the spore is partially invaded. The amoeba invariably ruptures the conidium wall as it emerges. Portions of the fungal cell which survived attack then may extrude from the conidium as spheroplast-like bodies (Fig. 5). LI
Gephyramoeba
.rp.
The trophozoites are more branched and vary widely in tomosis between individuals. rather limited and tends to be medium trophozoites extend
or less filamentous and their size due to anasOn agar, branching is dichotomous. In liquid over wider area, up to
Fig. 10. Conidium of C. sativzcs perforated by Geph?;rumoeba sp. Fig.
il.
A trophozoite Fig. Fig.
Fig.
12. Trophozoite 13. Perforation
of T. gruni/>ra ssp. minor encircling in C. sariaus conidium
14. Trophozoite of Mqwellu sp. showing pseudopodia. Note the clear ectoplasm
Fig. 15. Conidium Fig.
of T. ~~Q~~~~u ssp. minor showing the contractile ectoplasmic region (E) at the anterior end.
of C. suti~s
caused
vacuole
a hypha
(CV) and
of G..g, wirici.
by T. ~r~ln~/tira ssp. minor.
nucleus (N), mamilliform (M) and digitiform (EC) and the granular endoplasm (En).
inside the large vacuole formed by ~~~t~r~/l~ of the spore is cleariy visible.
Fig.
18. Reticulum formed
G.R. rritici hypha. undergone.
by the vampyreilid
Note the twisting amoeba
encircling
and bending conidia
(D)
sp. The single septum
16. The Same conidium of C. sntizu.~ inside Ma,~~rrllu sp. after 8 h from the formation vacuole. The conidium lacks septum and the contents are disorganised.
Fig. 17. Mr~_~~~rellosp. with engulfed
broad
of the
the hypha
of C‘. sc~ti~s.
has
Mycophagous
amoebae
from
take-all
Plate
2
suppressive
soil
21
22
S.
CHAKKABORTY
trated by a pseudopod which may be followed by the entire trophozoite (Fig. 9). The contents of the spore may be completely lysed or part of the protoplast may survive attack. The sequence of invasion of the conidium is very dithcult to observe as the amoebae are extremely light sensitive. They respond to bright illumination by withdrawal of pseudopodia and by rounding up to form an inactive amoeba1 mass. Trophozoites leave spores through perforations in the wall and do not disrupt empty spores. Spores, when examined with SEM. show perforation in their walls varying from 1.0-3.2 pm dia (Fig. 10). Similar perforations are also observed in the walls of chlamydospores and hyphal swellings of P. cinnamomi after they have been incubated with the amoeba for 7710 days. Thecamoeba
granifera
.~.rp. minor
Trophozoites are more or less elliptical in shape measuring between 24.440.8 pm in length and 15524.5 ,nm in breadth. The endoplasm is characteristically granular with refringent granules which appear yellow or brown in colour, the intensity of colour varying considerably between individuals. A small trophozoite may have few or no granules, and as it feeds on fungal propagules the granules increase in number. Pussard et al. (1979) demonstrated that these granules are mostly lipids which have been accumulated by the amoeba by its feeding on fungal cells. The hyaline ectoplasm of the trophozoite is broader at the anterior end when in continuous locomotion (Fig. I I). There is usually one pulsating vacuole l-6 ,nm dia in the posterior part of the body. Locomotion is rapid and may be I .5 pm set ‘. In a live trophozoite the nucleus is rendered inconspicuous by the abundance of lipid granules. When stained, nuclei measure between 8 to 12 pm dia. Cysts have not been observed. Feeding on hyphae of G.x. tritici is similar to feeding on Fusurium oq~sporum Schlecht amend. Synder and Hansen (Pussard et ul., 1979). After encircling a length of hypha (Fig. 12), the amoeba becomes rounded and stays in contact for 1O-20 min, after which it moves along the hypha and the fungal cell encircled appears empty. Small pieces of C. satizw hyphae which are usually present in conidial suspensions of this fungus are also emptied in the same way. The amoeba can perforate conidia of C. .saticu.y and digest their contents. Depending on the size of a conidium, it may be partly or completely engulfed by a trophozoite which stays attached without movement for up to 45 min, at the end of which the conidium appears paler in colour. SEM observation shows perforations in conidial walls which range between 1.2-5.0 pm dia (Fig. 13). After prolonged feeding by the amoeba, many C. .suticus conidia become disrupted. Mayorella
.sp.
Trophozoites are more or less elongated and flattened. In live trophozoites the single nucleus, 4-8 /lrn dia appears as the prominent cytological structure and is more or less spherical in shape with a rounded nucleolus measuring 2.555.5 nm dia (Fig. 14). The number of large contractile vacuoles,
et
al.
2-l I nm dia may vary from I to 20 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 hyaline ectoplasmic region is present in the pseudopodial projections and also appears as a fine film all around the cell. Pseudopodia are usually conical mamilliform but can be digitiform and sometimes quite extended with a rounded end (Fig. 14). The rate of locomotion varies from 0.5 to 1.8pm set ‘. Often the ridge formed on mamilliform pseudopodia extend beyond the pseudopodial length and can be seen as more or less parallel streaks along the entire length of the trophozoite. Trophozoites measure from 50 to 130 pm in length and 20 to lOO,um in breadth. Trophozoites of this amoeba resemble Myvorellu penurdi Page, 1972 (Page, 1976) except for the morphology of floating forms. Floating forms of M. penurdi are irregularly rounded but those observed here of this amoeba have radiating slender pseudopodia. As a trophozoite comes in contact of a C. sutizus conidium it stops and encircles the spore. Ingested spores may be carried for long periods and ejected by the trophozoite without apparent damage. This behaviour of Muyorellu has been reported by Anderson and Patrick (1980). Apart from the usual spore carriage, as seen by these authors, we have observed the conidium being ingested and the amoeba gradually assuming a spherical shape. This is followed by the appearance of a fluid layer between the spore and the amoeba1 cytoplasm to form a large vacuole (Fig. 15). This vacuole resembles the digestive vacuole formed by the leptomyxid amoeba while feeding on chlamydospores of P. cinnumomi (Chakraborty and Old, 1982). After the vacuole is fully formed around the conidium (Fig. 15) the trophozoite may continue its usual locomotion. The spore can be seen to rotate randomly inside the vacuole and often the vacuolar wall invaginates and touches the spore. The shape and the diameter of the vacuole, therefore, changes continuously. This is unlike the digestive vacuole formed by the Leptomyxrd amoeba which stays more or less constant in size and shape for a considerable period of time. After about 668 h, the septa in the spore are no longer visible (Fig. 16) and the conidium also appears paler in colour. The amoeba however, carries the spore inside for a much longer period of time even after the disappearance of the septa (up to 10 h). Finally the spore is ejected. Scanning electron microscopic observations of spores taken from feeding cultures have not so far provided evidence of perforation. Bacterial populations in cultures have been very high and a tiim of bacterial cells and mucilage obscured surface detail of the conidia. When macerated hyphae ot G.g. tritici are used m feeding trials, Muyorellu engulfs portions of hyphae by twisting and bending these fragments (Fig. 17). Hyphal fragments are retained inside for up to 6 h and ejected fragments lack refringency in some areas. However, the mechanism of hyphal feeding is not clear. DISCUSSION
The technique
used here to isolate soil amoebae
Mycophagous
amoebae
from
produced a number of mycophagous and nonmycophagous genera. The previously described mycophages, Arachnula (Old and Darbyshire, 1978), Vamp_vrella (Anderson and Patrick. 1978) and Theratromyxa (Old and Oros, 1980, Anderson and Patrick 1980) were not isolated. Of the amoebae tested, members of 5 genera proved to be mycophagous. Of these, only Thecamoeha was known to have this ability, as Pussard et al. (1979) had shown that T. granifera ssp. minor fed on Fusarium oxysporum and other hyaline fungi. We found this species of amoeba also can feed on pigmented conidia of C. .saticus. In many fungi the brownish to black pigment, melanin confers resistance to microbial lysis (Lockwood, 1960; Lingappa et al., 1963; Bartnicki-Garcia and Reyes, 1964; Kuo and Alexander, 1967). The ability of a number of amoeba1 genera to lyse pigmented fungal propagules is particularly significant, as discussed by Old and Patrick (1979). With the demonstration here of lysis of C. satiws conidia by Saccamocda, Thecamoeha, Gephvramoeha, and Mayorella the number of genera able to attack pigmented fungal cells is increased to 7, although the taxonomy of some of these amoebae remains in doubt. A major difficulty lies with the separation of GephJtramoeba from Leptomyla by Pussard and Pons (1976a) based on the number of nuclei per trophozoite. Chakraborty and Old (1982) described a leptomyxid which fed on P. cinnamomi propagules and hyaline cells of G.g. tritici but failed to perforate and lyse C. satiws conidia. In most respects this amoeba resembles Leptomyxa,flahellata (Pussard and Pons. 1976b) but trouhozoites are clearlv uninucleate. By Pussard ‘and PO&’ (1976a) criterion this would exclude the isolate from the genus LeDtomvxa. In this work. isolates of an amoeba *resembling GephJ,ramoeba had up to 25 or more nuclei per trophozoite, excluding it from this genus. Clearly, comparison of cultures and some revision of the taxonomy of the Leptomyxida is necessary. The demonstration that amoebae contact and engulf fungal propagules is not in itself evidence of feeding. Amoebae resembling Leptomyxa reticulata failed to perforate Chalara elegans Nag Raj and Kendrick ( = Thielaciopsis basicola) and C. .sutiws (Anderson and Patrick, 1978; Old and Oros, 1980). The amoeba shown in Fig. 18 is commonly found in the suppressive permanent pasture soil. It is probably a member of the family Vampyrellideae (Honigberg rt cd.. 1964) and it forms an extensive reticulum in cultures encircling many conidia of C. saticus and also enveloping G.g. tritici hyphae. The trophozoite is quite unlike L. reticulata and so far no evidence of perforation or lysis of either fungus has been observed. Species of Muyorella have been reported to ingest and transport fungal spores (Heal, 1963; Anderson and Patrick, 1980). Heal regarded feeding as unlikely and Anderson and Patrick suggested mycophagy and parasitism of Muyorella on other amoebae. Observations here support the contention that MaJ,orella can lyse spores of C. suticus contained within large vacuoles in the trophozoite. Disorganization of septa and protoplasts of conidia contained within vacuoles has been recorded. Further work is needed to deter-
take-all
suppressive
23
soil
mine whether cell wall perforation occurs during this period. The ability of Mayorella to feed on other amoebae is confirmed. In mixed cultures of soil amoebae containing Mavorella trophozoites, cysts of other amoebae, especially the readily identified Acanthamoebu cysts, have been seen within vacuoles of Mayorella.
It is now possible to summarize the steps in feeding by soil amoebae on fungal propagules. (1) Attachment of trophozoites to fungal propagules appears to be a matter of chance. There seems to be no strong chemotaxy or thigmotaxy. In all genera so far observed, amoebae may engulf spores or hyphae which are potentially food, only to move away leaving the propagules unharmed. (2) Engdjnent of the propagule, partially or completely. The giant trophozoites of Arachnula, Vampyrella and Theratromyxa are able to completely engulf spores or sections of the fungal thallus (Old, 1977; Old and Oros, 1980; Anderson and Patrick, 1978, 1980). The smaller Succamoeba and Thecamoebu attach to restricted parts of hyphae or spore walls and penetrate through to the protoplast. (3) Digestion, which may be by motile trophozoites which penetrate cell walls as in Arachnula, Thecamoeba, Saccamoeba, Gep~~~vramoeba and the unidentified leptomyxid (Chakraborty and Old, 1982) or by digestion of fungal cells within specialized food vacuoles. These may form within cysts, for example, Arachnula impatiens forms digestive cysts after a feeding period. A large central vacuole forms in the cyst within which fungal cell residues are digested as completely as possible. In Theratromyxa, the whole process of wall penetration and digestion of spore contents proceeds within a cyst. The leptomyxid amoeba of Chakraborty and Old (1982) forms very large vacuoles within which complete chlamydospores of P. cinnamomi are reduced to formless debris. Mayorella also forms food vacuoles within trophozoites but indications so far are that C. satiws spores remain largely intact and only the spore contents are digested. The time taken by different amoebae to lyse fungal propagules by different feeding mechanisms varies markedly. T. gran@a ssp. minor emptied a conidium of C. saticus in less than I h, whereas A. impatiens took approx. 4.5-6 h (Old, 1978). The unidentified leptomyxid took about 30 min to lyse P. cinnamomi hyphae but needed 22-36 h to digest chlamydospores of the same fungus. Conidia of C. saticus were contained within Mayorella trophozoites for more than 16 h during which time rupture of internal septa and protoplast disorganization occurred. Information on the effect of mycophagous amoebae on populations of soil fungi is lacking. Perforation in hyphae of G.g. tritici in a suppressive soil by vampyrellid amoebae has been shown by Homma et ul. (1979). The evidence presented here shows that 3 of the amoeba1 species isolated from a naturally suppressive soil feed on the take-all fungus, but their role in suppression is not known. Ackno&dgement-We Forest Research. micrographs.
thank Mr J. M. Oros, Division of CSIRO.
for
printing
the
photo-
S. CHAKRABORTYet ul.
24 REFERENCES
pathogenic IRI-789 _ _,
Anderson T. R. and Patrick Z. A, (1978) Mycophagous amoeboid organisms from soil that perforate spores of Thielariopsis hasicola and Cochliobolus saticus. Phylopathology 68, 161 X-1626. Anderson T. R. and Patrick Z. A. (1980) Soil vampyrellid amoebae that cause small perforations in conidia of Cochlioholus 159-167.
sutiws.
Soil
Biology
&
Biochemistry
12,
Bartnicki-Garcia S. and Reyes E. (1964) Chemistry of spore wall differentiation in Mucor rouxii. Archiaes ofBiochemistry and Bioph.vsics 108, 1255 133. Bovee E. C. (1972) The lobose amebas. IV. A key to the order Granulopodia Bovee and Jahn 1966, and descriptions of some new and little-known species in the order. Archires ,fur Protistenkunde 114, 37 l-403. Chakraborty S. and Old K. M. (1982) Mycophagous soil amoeba: interactions with three ulant pathogenic fungi. Soil Biology,
& Biochemistry
Esser R. P., Ridings Ingestion of fungus
14, 2477255.
-
-
W. H. and Sobers E. K. (1975) spores by Protozoa. Proceedings of
the Soil and Crop Science Society
of
Florida
34, 206298.
Heal 0. W. (1963) Soil fungi as food for amoebae. In Soil Organisms (J. Doeksen and J. van der Drift. Eds), pp. 289-297. North-Holland, Amsterdam. Homma Y., Sitton J. W., Cook R. J. and Old K. M. (1979) Perforation and destruction of pigmented hyphae of Gaeumunnomyces graminis by vampyrellid amoebae from Pacific North-west wheat held soils. Phvtonathologv , <1, 69. 1118~1122. Honigberg B. M., Balamuth W.. Bovee E. C., Corliss J. O., Godjics M., Hall R. P., Kudo R. R., Levine N. D., Loeblich A. R. Jr. Weiser J. and Weinrich D. M. (1964) A revised classification of the phylum Protozoa. Journul qf Protozoology 11, 7720. Hornby D. (1979) Take-all decline: a theorist’s paradise. In Soil-Borne Pathogens (B. Schippers and W. Gams, Eds), pp. 133-156. Academic Press, London. Kuo M. J. and Alexander M. (1967) Inhibition of the lysis of fungi by melanins. Journal of Bacteriology 94,624629. Lingappa Y., Sussman A. S. and Bernstein I. A. (1963) Effect of light and media upon growth and melanin formation in Aureobasidium pullut&ts (De Bary) Am. ‘
( = Pullulariu Applicatu
Lockwood
pullulans). 20, I0991 28.
J.
L.
(1960)
Mycopathologia
Lysis
of
et
mycelium
Mycologiu
of
piant-
fungi
by natural
soil.
Phytopathology
50,
Old K. M. (1977) Giant soil amoebae cause perforation and lysis of spores of Cochliobolus snticus. Trunsactions of the Brirish Mycological Society 68, 277728 I. Old K. M. (1978) Fine structure of perforation ot Cochlioholus satinus conidia by giant amoeba. Soil Biology & Biochemisfry 10, 5099516. Old K. M. and Darbyshire J. F. (1978) Soil fungi as food for giant amoebae. Soil Biology & Biochemistry 10, 933100. Old K. M. and Oros J. M. (1980) Mycophagous amoebae in Australian forest soils. Soil Biology & Biochemi.vtry 12, 1699175.
Old K. M. and Patrick Z. A. (1979) Giant soil amoebae, potential biocontrol agents. In Soil-Borne Puthogens (B. Schippers and W. Gams, Eds), pp. 617-628. Academic Press, London. Page F. C. (1976) An Illustrated Ke.v to Freshwater and Soil Amoebae. Freshwater Biological Association. Scientific Publication No. 34. Prescott D. M. and James T. W. (1955) Culturing of Amoeba proteus on Tetrahymena. E,xperimental Cell Research 8, 256258.
Pussard
M. and Pons R. (1976a) Etude des genres Lepto(Protozoa. Sarcodina). I Lepto1915. Protistologica 12, 15lll68. Pussard M. and Pons R. (1976b) Etude des genres Leptomyxa et Gephyramoeba (Protozoa, Sarcodina). II Leptomwa ,jabellata Goodey I91 5. Proti.rtologica 12, myxa et Gephyramoeba myxa reticulata Goodey
307-3 19.
Pussard
M. and Pons R. (1976~) Etude des genres Leptomyxa et Gephyramoeba (Protozoa, Sarcodina). III Gephyramoebu delicatulu Goodey, 19 15. Protistologica 12, 351-383.
Pussard M., Alabouvette C. and Pons R. (1979) Etude preliminaire dune amibe mycophage Thecamoeba granifera ssp. minor (Thecamoebidae. Amoebida). Protistologica 15, 139-149. Pussard M., Alabouvette C.. Lemaitre I. and Pons R. (1980) Une nouvelle amibe mycophage endogee Cashia mycophaga n. sp. (Hartmannellidae, Amoebida). Protistologica 16, 433-45 I.
Singh B. N. (1946) A method of estimating the number of soil Protozoa, especially amoebae, based on their differential feeding on bacteria. Annals of AppliedBiology 33, 112~119.