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Soil Vampyrellid amoebae that cause small perforations in conidia of Cochliobolus sativus
Sod Bid Biochem. Vol 12, pp. 159 to 167 0 Pergamon Press Ltd 1980. Printed m Great
0038-0717/80/0301-0159SO2.00/0
Britain
SOIL VAMPYRELLID AMOEBAE THAT CAUSE SMALL PERFORATIONS IN CONIDIA OF COCHLIOBOLUS SATIVUS T. R. ANDERSON+ and Z. A. PATRICK Department
of Botany,
University
of Toronto,
Toronto,
Ontario,
M5S 1Al. Canada
(Accepted 26 September 1979) Summary-The parasitic habits of two mycophagous amoebae, members of the Vampyrellidae isolated from soil, were studied under laboratory conditions. The amoeboid organisms resembled Theratromyxa weheri and VampyrelLa vorax. Both organisms lysed conidia of Cochkoholus satiuus and chlamydospores of Thielauiopsis basicolu within digestive cysts. Perforations 1 pm dia or less were observed in walls of the lysed fungus spores. The mycophagous Theratromyxa sp. and l! uorax were differentiated in laboratory culture chambers on the basis of morphology, encystmenf excystment and diameter of perforations produced in walls of conidia of C. sativus. Both organisms differed from a previously-described species of Vampyrella which causes large perforations and annular depressions in spore walls.
INTRODUCTION
MATERIALS AND METHODS
Isolation of mycophagous amoebae
It was reported that pigmented spores of certain plant pathogenic fungi were parasitized by a mycophagous soil amoeba belonging to the protozoan family, Vampyrellidae Doflein (Anderson and Patrick, 1977; 1978; Old, 1977a,b). While feeding, distinct annular depres-
sions and perforations 1-7 /*rn in dia in fungus spore walls were produced. Although the identity of the causal amoeba is still controversial, our observations indicate that it is similar to Vumpyrella Iateritia (Leidy, 1879). It has been suggested that the mycoparasitic amoeba may have potential for biological control of soilborne plant pathogens (Anderson and Patrick, 1978; Old, 1977a). Recently, two additional mycophagous amoebae in the family Vampyrellidae were isolated from field soil. These amoebae also perforated and lysed conidia of Cochliobolus sativus Ito and Kurib Dreschs ex Dastur, and chlamydospores of Thielauiopsis basicola (Berk. and Br.) Ferr. However, the recently-isolated amoebae exhibited characteristics different from the mycophagous organism described previously (Anderson and Patrick, 1978). They resemble Theratromyxa weberi (Zwillenberg, 1953) and Vampyrella uorax (Cienkowski, 1865, 1876). The organisms produced perforations 1 pm dia or less in fungus spores during the feeding process. The perforations were similar to those reported by Clough and Patrick (1976) and Old and Patrick (1976), in conidia of C. sutivus and chlamydospores of 7: basicola incubated in natural field soil. These investigators suggested that the perforations were caused by lytic soil bacteria, However, based on our present studies, it appears that the perforations were caused by mycophagous amoebae. We describe. details of the feeding habits of the two species of amoeba that produce small perforations in fungus spores, their distribution in soil, and
cultural
characteristics.
* Present address: Agriculture Canada, tion. Harrow, Ontario. NOR lG0, Canada.
Research
Sta159
Soil dilution plates containing 100 and 10 mg field soil and 5 ml distilled water (DW) were baited with conidia of C. sativus (5 x lo4 conidia per plate) and incubated at 20°C. After 2-3 weeks, conidia in the plates were observed by means of an inverted microscope (Reichert) at x 100 magnification. Digestive cysts of the mycophagous amoebae that surrounded partially-lysed conidia were removed by means of a pipette and washed 3 times in sterile 10% soil extract (SE) (Anderson and Patrick, 1978). Single-cyst isolates were cultured in 10% SE at 2O”C, in test tubes and Petri dishes using conidia of C. sutiuus or chlamydospores of 7: basicola as food source. Fixation and staining Mixtures of amoebae and conidia from 1 week old single-cyst cultures were placed on microscope slides. After 24-48 h incubation in moist chambers, active amoebae were fixed by addition of several drops of Carnoy’s fluid (Maloney, 1973). Slides with fixed amoebae were immersed in 70% ethyl alcohol for 24 h. Amoebae were stained in Delafield’s haematoxylin for 24 h and destained with tap water (Mackinnon and Hawes, 1961). Mycoparasitic vorax
activities of Theratromyxa
sp.
and V.
Experiments to determine the rate of spore lysis and increase in number of the two vampyrellid amoebae were performed in tissue culture chambers (Micro Test II, Falcon brand). A single digestive cyst was added to each culture chamber containing several drops of 10% SE and 50&1ooO conidia of C. satiuus. After incubation at 20°C the number of amoebae and lysed conidia were determined using an inverted microscope at x 100 and x 250. Conidia were considered lysed if they were void of contents and surrounded by a digestive cyst wall or if
T. R. ANDERSON and Z. A. PATRICK
160
Table
I. Mycophagous
Vampyrellidae
in soils from various
locations
Organism
Location
Ontario Vineland cultivated field Vineland sod Simcoe tomato field Delhi tobacco field Harriston garden Hastings garden Tomato greenhouse Alberta Edmonton wheat field California Salinas vegetable field Hawaii Waimanalo orchard sod ,/ Number
of organisms
g-
’ Observed in soil dilution
Theratromyza sp.
C’trmpJwlla c0ru.x
13” I
+h +
_I +
2 11 5 8 +
+ -
_
NEd NE +
NEd NE +
4.5
+
-
0
_
+
1.5
_
_
’ of dry soil. determined by soil bioassays (Anderson and Patrick, 1978). plates.
k Not observed in soil dilution plates d Not evaluated.
the conidia experiments
were enclosed within a digestive were replicated IO times.
Conidia of C. .satiuus, produced on 207: VX juice agar. were suspended in distilled water and filtered through 44 pm mesh. The conidia were collected and washed several times on 20 pm mesh before filling the culture chambers. Chlamydospores of 7: husicola were prepared in a similar manner. The stages in the life cycle of each organism were studied in plastic Petri dishes (35 x 10mm) containing 5 ml lO”/~,,SE. Cyst diameters were measured at the greatest width. Observations of undisturbed cultures were made with an inverted microscope using phase contrast microscopy. Material for scanning electron microscopy was air-dried without fixation and coated with gold-palladium. Specimens were observed in a Cambridge Stereoscan MK II scanning electron microscope. The cultural characteristics and stages in the life cycle of Theratromy.wt sp. and V. c0ru.y were compared to those of K luteritia (Anderson and Patrick. 1978). For such comparisons, single cyst isolates of each organism were cultured in l@x, SE in plastic Petri dishes under similar conditions.
Table
2. Effect of incubation
REWLTS
cyst. The
Isolation vorax
of mycophagous
Theratromyxa
sp. und V.
The presence of Theratrornyxa sp. and I/. t’oras in soil dilution plates was indicated by lysis of conidia of C. suticus surrounded by a thin cyst membrane or the presence of digestive cysts surrrounding partiallylysed conidia. Digestive cysts of both organisms containing one conidium were similar in appearance and it was necessary to cub-culture the cysts to determine which organism was present. Mixed populations of mycoparasitic Theratromyxa, V vorax and I! luterititr were found in some soil samples (Table 1). Soil surveys indicated that Therutromysa was widely distributed in agricultural soils. It was found in soils from cultivated fields in Ontario, in greenhouse soils, and in a sample of wheat field soil from Alberta. C! 1’oru.v was found in field soils from Ontario and California. Therutromyxa and V. vorux did not occur in dilution plates containing less than 0.1 g soil. It appears therefore, that populations of these organisms in soils are relatively low. As shown in Table I. 1/: luteritia, the amoeba which produces the large diameter holes in
at 20 C on rate of Increase of Therutrorn.?.ua number of lysed conidia of Cochlioholus satious
sp.. P’amp_vrel/rr t’oraz and
(Days) Treatment Amoebae Amoebae Amoebae Amoebae
0 only (Tk~rutrom~.~a sp.) + conidia (A) (B) only (licrmp.vrel/tr cortrz) + conidia (A) (B)
1.0” 1.0 1.0
I .o I .o 1.o
I
2
3
I.0
1.5 4.9 6. I
1.7 14.8 20.5
I .o 1.0 1.0
I.5 I .4
I .o
I .o
1.0 1.4
0.9 1.8
6 1.4 167.9 199.9 0.9 0.9 7.4
(A) Number of T/lrratronl~.~u sp. or l’un~p?‘rr/l~~ POT(~.Y (trophoroites + cysts). (B) Number of lysed conidia wlthin digestive cyst walls including partially lysed conidla tive cysts. “ mean of IO replicates.
IO I.6 397.4 746.7 0.9 0.8 2.4
within
diges-
Soil vampyrellid
fungus spore walls. was the most frequently observed member of Vampyrellidae found in the soils that were sampled. Mycoparasitic activities of Theratromyxa vorax iri culture chambers
sp. and V.
Culture chambers containing conidia of C. satious inoculated with a single digestive cyst of Theratromyxa resulted in lysis of an average of 746 conidia per chamber after 10 days (Table 2). The average number of TherutrornJ~xa in these chambers after the same period was 397 per chamber. In control chambers without TheratromJxa, no lysed conidia surrounded by cyst walls were observed. In the chambers without conidia, the average number of Theratromyxa was 1.6 per chamber after 10 days incubation. The diameter of resting cysts was 63.9 k 28 pm in chambers with conidia and 19.6 +_ 5 pm in chambers without conidia. When culture chambers containing conidia of C. sutiaus were inoculated with a single digestive cyst of V. poras, lysis of an average of 2.4 conidia per chamber resulted after 10 days incubation (Table 2). The average number of 1/: vorax per chamber decreased during the same period. In a number of similar experiments, the mycoparasitism and multiplication of I’. vorax was erratic. Most frequently only few conidia were lysed and the amoebae did not multiply. The activity of 1/: uorax was usually better in cultures containing algae, diatoms and flagellates in addition to the conidia. Parusitism of fungus spores by Theratromyxa Digestive cysts of the amoeba were smooth-walled and approximately 65 pm in length but varied with the size of the fungus spore they enclosed (Fig. 3). The digestive cysts exceeded the spore dimensions by 55IOpm on all sides, Digestive cysts normally contained a single conidium of C. satiuus or a chlamydospore chain of 7: basicolu. The contents of a conidium of C. satious or chlamydospore of 7: busicola were digested in 2436 h. During the first 12 h of feeding, the protoplasts of cells within conidia of C. satiuus appeared to shrink (Fig. 3) and the conidia became distorted. The cyst contents underwent plasmotomy before excystment (Fig. 5) and soon the progeny trophozoites emerged through several small holes in the cyst wall. Each digestive cyst produced 5-22 progeny (Fig. 6). The digestive cyst wall deteriorated after IS-20 days in cultures which contained bacteria. After excystment, shallow pits and small perforations 0.28 k 0.05 pm dia were seen in the fungus spore wall with phase contrast and scanning electron microscopy (Fig. 4). Annulations in the spore wall were not observed. In mixed cultures, Therutromyxa was not seen to ingest other protozoa but some digestive cysts were formed which enclosed the desmid, Closterium sp. Trophozoites often adhered to active larvae of Meloidogyne sp. but were unable to immobilize them. Cultures containing nematodes were observed for several days, but active nematodes were not parasitised. Trophozoite
of‘Theratromyxa
Digestive cyst progeny often assumed a spherical form. 44 k 13.7 Aim dia soon after excystment and
amoebae
161
extended up to 130 pm after contacting the bottom of the culture dish. In general. trophozoites were extremely active, elongate and angularly branched. At times they became flattened, leaf-like and remained motionless. Occasionally they became spherical and floated with their filopodia extended. The cytoplasm was granular and contained numerous contractile vesicles I-3 pm dia. Stained trophozoites were multinucleate. The nuclei were spindle-shaped, 5.3 + 0.5 pm. and the karyosomes were 4.3 _t 0.6pm in length. Extremely fine anastomosing filopodia were concentrated at blunt terminal portions of the organism or along the anterior edge of the cresent-shaped individuals. Fission and fusion of trophozoites was observed. Many trophozoites fused to form a large reticulate plasmodium (Fig. 9). Trophozoites of Tkerutromyxa were consumed by a species of Mayorella which also attacked V. latrritia. Ocassionally the Mayorella also lysed spores of C. satitus and -1 basicolu by consuming the spore contents without formation of digestive cysts (Fig. 20). Multiplication, tromyxa
encystment
and excystment
of Thera-
Therutromyxa formed three types of cysts depending on duration in culture and availability of food. Digestive cysts formed in the presence of viable conidia. Trophozoites moved randomly until conidia were located (Fig. 7); they then formed a smooth sheath which completely enclosed each conidium (Fig. 8). Often fusion of two trophozoites was necessary before a conidium was completely engulfed. Resting cysts formed in the absence of suitable food or in older cultures, Trophozoites became spherical or disk-shaped and a smooth cyst wall was formed after approximately 30min. The diameter of resting cysts varied from 30 to more than IOOpm depending on size of the encysting trophozoite. In the absence of food there was excystment and re-encystment or, at times, the contents of original resting cysts contracted to form double-walled resting cysts (Fig. 9). The colour of resting cysts in transmitted light varied from golden to reddish brown. In general, double-walled cysts were darker than single-walled cysts. Resting cysts were also formed directly from digestive cysts. Such resting cysts were enclosed within the original digestive cyst wall and were 32.9 k 9.8 pm dia. Excystment of resting cysts was preceded by plasmotomy and resulted in 5-7 progeny from single-walled resting cysts. Excystment occurred 3-4 days after transfer to fresh medium containing conidia of C. satiuus. Excystment of darker and double-walled cysts required longer incubation. In the presence of abundant conidia, a third type of cyst was formed. This happened when many individual trophozoites fused to form a motionless irregular plasmodium up to I mm dia (Fig. IO). Initially, the plasmodium contained numerous small vacuoles I-5 ,nm dia. Gradually the size of some vacuoles increased up to IOOpm dia and the plasmodium appeared froth-like (Fig. 1I). Plasmotomy occurred in the dense cytoplasm which had accumulated at numerous regions within the plasmodium. Progeny trophozoites were approximately 1@40~m dia and often fused with adjacent individuals before escaping the froth-like remains of the plasmodium (Fig. 11).
T. R. ANDERSON and Z. A. PATRICK
162
Oiqeslive
cysf
8’” ,&
Trophoroi
Trophozoites
tes
within
cyst
wall
Plosrnodium
Stages
in
the
life
cycle
(diagrammatic
Fig. I. Schematic
representation
of
mycophaqous
Theratromyxa
sp.
representolion)
of the various
The process of plasmodium and froth-like cyst formation and release of progeny required 2-3 days. During emergence, progeny became spherical with radiating filopodia and floated in the culture medium. After contact with a sohd surface, ~~le~~fro~z~.~u became elongate and branched. In the absence of food fusion of progeny was followed by encystment. Some of the observed stages in the life cycle of Theratromyxa are shown in Fig. 1.
After engulfing a conidium of C. satiz~s, a trophozoite of V: uorax often continued to move randomly, collecting additional conidia before forming a digesfive cyst (Fig. 13). The number of conidia within digestive cysts varied from one to severa (Fig. 15). Digestive cysts were smooth walled 70.4 & 4 pm dia and were often surrounded by culture debris. In soil dilution plates, digestive cysts were often found associated with organic matter and soil particles. It took 24-36 h to digest conidia within the cyst. After excystment, numerous perforations 0.42 + 0.2 ftrn dia and pits 1.1 & 0.2 pm dia, were visible in walls of lysed conidia within the empty cysts
stages in the life cycle of Titcrat~on~,wa
sp
(Figs 16, 17). Small perforations were frequently seen at the base of surface pits (Fig. 17). Annulations in the conidial walls similar to those produced by I/: lareritia (Fig. 18) were not seen. In soil dil~ltion plates and in mixed cultures. digestive cysts of V: uorax often contained small diatoms and flagellates as well as conidia of C. snticus or chlamydospores of 7: basic&. It is not known if these additional organisms are essential in the nutrition of 1/ UOI(IX.However, it was difficult to maintain the organism in cultures that contained only bacteria and conidia of C. sutivus. Trophozoite
cd V. vorax
Recently-excysted trophozoites varied in size from 60pm dia to more than 3~~rn in length. The cytoplasm was hyahne, granular and contained numerous contractile vesicles that were less than 5 pm dia. The most frequent form assumed by an active trophozoite was crescent or fan-shaped (Fig. 12). While floating, I/ mrax became globular or reniform and appeared to move by means of filopodia which formed anteriorly and retracted posteriorly. I/ VOYCIX was also seen to form an elongated cord-like structure
Soil vampyrellid
with anastomosing and branched filopodia that were concentrated at spatulate terminal portions. Trophozoites avoided strong light and remained in the vicinity of clumps of organic matter in soil dilution plates. Multiplication,
encystment and excystment of V. vorax
Trophozoites frequently formed digestive or resting cysts. With prolonged encystment, the organism contracted further to form a double-walled resting cyst. Resting cysts of I! uorax were vacuolated or contained a large central vacuole. Solid material was frequently seen within such vacuoles, but the nature of this material is unknown. Excystment of resting cysts was promoted by diluting the culture medium with 10% SE and adding fresh conidia of C. sativus. Under such conditions excystment occurred in approximately 24 h at 20°C. A single progeny emerged through large perforations, S-10pm dia. in the cyst wall. Excystment from digestive cysts was preceded by plasmotomy. The cyst cytoplasm divided at the midpoint and the resulting two progeny emerged from opposite ends of the cyst (Fig. 14). On one occasion, cyst progeny fused before excystment; this resulted in release of a single trophozoite. Some of the stages in the life cycle,of I/: vorax are shown in Fig. 2.
DigesIive (contoininq
0
amoebae
163
Comparison of Theratromyxa,
V. vorax and V. lateritia
Although the morphology of each mycophagous member of Vampyrellidae was variable, active trophozoites exhibited their specific characteristic forms in laboratory culture chambers. Theratromyxn was elongate, branching and angular, with fine filopodia concentrated at blunt extremities (Fig. 6) I! uorax was crescent, or globular-reniform with filopodia concentrated along the anterior edge (Fig. 12) while I/: lateritia was elongate and branched with filopodia concentrated at terminal spatulate areas of the organism. Vumpyrella lateritia was often indistinguishable from Y uorux, especially when the latter became elongate and branched. All three amoebae produced “pinhead” pseudopodia. Trophozoites from a recently-isolated single-cyst culture of K lateritia frequently produced large peripheral vacuoles and “pinhead” pseudopodia (Fig. 19) similar to those described by Leidy (1879) and Lloyd (1926). This characteristic became less apparent after three to four sub-cultures. “Pinhead” pseudopodia were also produced occasionally by Theratromyxa and frequently by K vorax. Digestive cysts which contained conidia were formed by Therutromyxa and f! vorax, but not by % luteritiu. Digestive cysts of Therutromyxu rarely con-
cyst spores1
Lysed spore wlthin cell wall
Stages
in
the
life
cycle
of
(diogrommotic
Fig. 2. Schematic
representation
of the various
mycophogous representation
Vampyrella
vorox
1
stages in the life-cycle of Vampyrella uorax Cnk.
T. R.
164
ANDERSON and
tained more than one conidium while those of i/: L’OIXSoften contained several. Excystment from digestive cysts of Theratromy.~u. C’:corux and I/: lateritia resulted in the formation of $22, 2 and I progeny. respectively. Single-walled
Z. A.
PATRICK
resting cysts of 7%erutromy.w, 1/. cora.x and CI laferiritr released 5-7, 1 and 1 progeny respectively. The frothlike modified cysts produced by Therutrom)\-a were not formed by the other amoebae. Differences in the parasitic activities of the three
Figs 3--t 1. Stages in the life cycle of Theratrom~ua Fig. 3. Lysed and partially Fig. 4. Scanning
electron
lysed conidia micrograph
of C. salirus wlthin x 600.
(SEM) showing
digestive
perforations
Fig. 5. Plastomotomy progeny
Fig. 7. Fusion
trophozoites of progeny
Fig. X. Formation Fig. 9 Fusion
of trophoroites
Fig. IO. Stationary Fig.
of digestive
cyst cytoplasm
escaping
from digestive X 375.
trophozoites
of digestive
under crowded double-walled
before excystment. cyst containing
on the surface
cyst which completely
of T/~rtrrro~n~,\ct.
(arrows) 0.3 Ltrn dia in comdlum x 1300.
.surims lysed by Tkrrcrrromy\-tr.
Fig. 6. Angular
sp.
cysts (arrows)
x 375.
lysed comdium
of viable conidium. conidium.
x 375.
conditions forming irregular resting cyst (arrow). x 360.
branching
plasmodium.
(arro\$s) escaping plasmodium
the foam-like modified in Fig. IO. x 400.
cyst formed
(arrow).
x -375.
encloses
vacuolate plasmodium resulting from extensive fusion of numerous Note digestive cyst over plasmodium (arrow). x 260.
I I Progcnq tropho/oltes
of C
Note
trophoroltes.
from the stotlonar)
Soil vampyrellid mycophagous vampyrellid amoeba were exhibited by lysed conidia of C. sc4ticu.s. Conidia lysed by Therutromyxu and 1/: t’orax were enclosed within empty digestive cysts while those lysed by I! lateritio were never seen within such cysts. Conidia lysed by Theratromyxa became flattened and distorted during excystment. In contrast, conidia lysed by I/: corax generally retained
Fig. Fig. 14. Excystment
165
their
shape. Perforations in walls of conidia of C. caused by Therutromyxa, V. t1oru.x and V. latrritiu were 0.2-0.3, 0.221.0 and I-7,rrm dia respectively
sutivus
(Figs
4, 16,
18).Perforations
caused
trophozoite
of V wru.v approaching
13. Digestive cyst containmg
of digestive
Fig. 16. SEM showing Fig. 17. SEM of conidium
conidia
conidia
perforations
of C. SLIII‘L’US, x 375
of C. ~tiaus.
cyst resulting in progeny trophoroites ends of the cyst, x 375.
Fig. 15. Lysed conidia with small perforations resting cyst within original
x 375
(arrows)
leaving
from opposite
(arrows) within empty digestive cysts of V I‘O~U.S.Note digestive cyst (short arrow). x 375.
0.2 1.O pm dia in conidia
lysed by I/: LYWUX showing
perforation
lysed by K ror‘(.x, x 1500. at base of pit (arrow),
Fig. IX. SEM of conidia lysed by I/: hfrritict showing large perforations (arrow) and annular (short arrow). Note detached disk (white arrow) from conidial wall on adjacent conidium, Fig. 19. Pin-head
by Thrratrompua
appeared to be the result of direct penetration. The perforations caused by I! uorax appeared to result from direct penetration or by the formation of shal-
II- 17. Stages in the life cycle of k’rrrnp~~lla CO~(IY
Figs Fig. 12. Typical
amoebae
pseudopodia (arrows) produced by spherical 1/: lrztrrrfia similar 1’ UV~I.Xand occasionally by Tlrrrrrrror~~~.~~~,x 600.
Fig. 20. Active trophozoite
of parasitic
Mo)ore/ltr
with ingested
conidium
x 3900. depression x 1575.
to those produced
of C. strrirus,
x 375
by
T. R. ANUER~ON and 2. A. PATRICK
166
low pits I pm dia followed by penetration at the base of the pits (Fig. 17). Those caused by 1/: lateritia exhibited annular depressions in the spore walls as well as the large diameter holes described earlier by Anderson and Patrick (1978) and Old (1978) (Fig. 18).
DISCUSSION
The finding of additional species of mycophagous Vampyrellidae in arable soils that were capable of lysing spores of plant pathogenic fungi, further suggests the potential importance of soil protozoa in biological control of soilborne plant diseases. Results indicate that Theratromyxa was relatively common in agricultural soils and was capable of lysing large numbers of conidia of C. safitlus under laboratory conditions; V. corax was not found as often and was more difficult to maintain in the laboratory. It appears, therefore, that Thrrutromyxu is more suitable for applied studies on biological control of soilborne plant pathogens. Theratro~~yxu wrheri Zwillenberg was originally described as a nematophagous amoeba (Weber et al., 19.52: Zwillenberg. 1953). In our studies, Theratromyxu did not digest larvae of Meloidogpne sp. However, conditions for successful parasitism of nematodes may have not existed in our culture chambers. It is also possible that the baiting techniques used for isolating Theratrom~uu favoured the selection of mycophagous organisms and the species we studied was different from the nematophagous one. Thcwtromyxa resembled the original descriptions of nematophagous amoeba in morphology. encystment and excystment. The formation of the digestive cyst surrounding the food source and plasmotomy of digestive cyst contents were similar to the previous descriptions (Sayre, 1973: Weber et al., 1952). Sporefilled cysts described by Zwillenberg (1953) were not seen in our studies but such cysts may be similar to the froth-like modified cysts that we observed, in which numerous trophic progeny were produced. Such progeny were capable of forming small cysts and may have been encysted at the time of Zwillenberg’s (1953) observations. Such progeny often assumed a heliozoid form after excystment and became amoeboid after contact with a flat surface. The origin of such heliozoid forms was not previously known (Zwillenberg, 1953). The mycophagous Therntromyxa sp. also resembled the nematophagous species described by Sayre (1973). We have reported (Anderson and Patrick. IY77) that mycophagous member of Vampyrellidae which formed digestive cysts around fungus spores resembled AruchnuIu imputirns Cnk. After more extensive observations, however, it is more likely that the organism we observed was V: wra.x. In the earlier descriptions (Cienkowski, 1865. 1876), L! wrax was reported to feed by forming a digestive cyst around the food source. Diatoms were arranged within the cyst on the longitudinal axis and on excystment two to four progeny emerged. Specialized reproductive cysts were not described by Cienkowski (1865, I X76). Artrchnula imputier~s is described as being similar to I/: coru.y. but excystment of digestive cysts results in one. rarely two progeny. Also. special reproductive cysts were formed
(Dobell, 1913). In our study, fission of the digestive cyst cytoplasm resulted in the formation of two progeny and the special reproductive cysts were not observed. Although Arachnula and Vumpyrellu were considered by some as being synonymous (Dobell. I9 13). the mycophagous amoeba in our studies resembled the description of 1;. voruy more closely than that of A. impatiens. Perforations in spore walls, similar to those produced by Thrratromyxa and k: crux in culture chambers, have been observed in spores incubated in field soil (Clough and Patrick. 1976; Old and Patrick. 1976). This indicates that one or both organisms ma) be active in field soils. On occasion, large perforations and annulations as well as the much smaller perforations, to.5 pm dia were seen in the same fungal spore (Old and Patrick, 1976). In the earlier studies (Old and Patrick, 1976; Old and Wong. 1976). it was concluded that small perforations were caused by lytic soil bacteria. But our study indicates, it is more likely that such spores were attacked by more than one species of mycophagous amoebae. each of which formed their characteristic perforations in the walls, Thus, it appears that there are at least three different mycoparasitic members of Vampyrellidae that are widely distributed in arable soils. These organisms can be readily distinguished from each other when cultured in the laboratory by a combination of characters consisting of general morphology, type of digestive cysts. methods of excystment and diameter of the perforations they produce in walls of conidia of C. sufivus.
Support in part by Operating Grant to 2. A. Patrick from the National Sciences and Engineering Council of Canada. Acknowledgements-
REFEREYCES
ANDERSON T. R. and PATRICK 2. A. (1977) Parasitism
spores of
of
and Hrlmir~thospr~~ium by soil protozoa. Proc~edin
husk&r
strticum pctrholngical
Society 44, 31 (abstr.). ANDERSON T. R. and PATRICK Z. A. (1978) Mycophagous amoeboid organisms from soil that perforate spores of Thiehiopsis hasicolo and Coch/ioholu.s .S(IIIL’II\.PII r!opchhgy 68, 161 X-l 626. CIENKOWSKI L. (1865) Beitriige zur Kenntnis der Monaden. Archkfiir
n~ikru,skopi.scl~ Anofomi~
1, 223-25.
CIENKOWSKI L. (1876) Uber einige Rhizobopen und Lerwandte Organismen. Archir .fiir nlikroskoprscll Arlcrton~ic, 12, 24- 28. CI.OUC;H K. S. and PATRI(.K Z. A. 11976)Charncterlsttcs 01 the perforating agent of chlamydosporec of 7~irie/m.iclp\i.\ htrsicoltr (Berk & Br.) Ferraris. Soil Biokqy & Birwhmi.\try 8, 473-478. DOBELL C. (1913) Observations on the lift-history of (‘ienkowski’s “Arachnula”. .Archir fiir Fmr~~~enkundc 31. 317-353. LEI~Y J. (1879) Freshwater rhizopods of North America. C’.S. Geologicc~l Surcel of’tl~r Terrrtorirs 12, 253 2X7. LLOYD F. E. (1926) Some features of structure and heha\lor in L’amp~~r~~/h hfwlrio. Scie~w 63. 364 361. MACKINNOI‘: D. L. and HAWKS R. S. J. (1961) .4t1I,ztrotluction to thu Srudx ~$Protozm Clarendon Press. Oxford. MAt.o\f Y R. (1973) Lrrhorcltorj. ‘/‘(~(~/~~iquo\ ill %oo/r~~q\. Wiley. New York.
Soil vampyrellid OLD K. M. and WONG J. N. F. (1976) Perforation and lysis of fungal spores in natural soil. Soil Biology & Biochemistry 8, 285-292. OLD K. M. and PATRICK Z. A. (1976) Perforation and lysis of spores of Cochliobolus sativus and Thielauiopsis hasicola in natural soils. Canadian Journal of Botany 54, 2798-2809. OLD K. M. (1977a) Giant soil amoeba cause perforation of conidia of Cochliobolus satiuus. Transactions of the British Mycological Society 68, 277-281. OLD K. M. (1977b) Perforation of conidia of Cochliobolus sarivus by soil amoebae. Acta Phytopathologica Academiae Scientiarum Hungaricae 12, 113-119.
amoebae
167
OLD K. M. (1978) Fine structure of perforation of CocIlliobolus sarivus conidia by giant amoebae. Soil Biology & Biochemisrry 10, 509-516. SAYRE R. M. (1973) Theratromyxa weberi, an amoeba predatory on plant-parasitic nematodes. Journal cf Nrmatology 5, 258-264. WEBER A. P., ZWILLENBERG L. 0. and VAN DER LAAN P. A. (1952) A predacious amoeboid organism destroying larvae of potato root eelworm and other nematodes. Nature 169, 834-835. ZWILLENCBERG L. 0. (1953) Theratromyxa weberi, a new proteomyxean organism from soil. Antonie van Leeuwenhoek. Journal of Microbiology and Serology 19, 101-l 16.
0038-0717/80/0301-0159SO2.00/0
Britain
SOIL VAMPYRELLID AMOEBAE THAT CAUSE SMALL PERFORATIONS IN CONIDIA OF COCHLIOBOLUS SATIVUS T. R. ANDERSON+ and Z. A. PATRICK Department
of Botany,
University
of Toronto,
Toronto,
Ontario,
M5S 1Al. Canada
(Accepted 26 September 1979) Summary-The parasitic habits of two mycophagous amoebae, members of the Vampyrellidae isolated from soil, were studied under laboratory conditions. The amoeboid organisms resembled Theratromyxa weheri and VampyrelLa vorax. Both organisms lysed conidia of Cochkoholus satiuus and chlamydospores of Thielauiopsis basicolu within digestive cysts. Perforations 1 pm dia or less were observed in walls of the lysed fungus spores. The mycophagous Theratromyxa sp. and l! uorax were differentiated in laboratory culture chambers on the basis of morphology, encystmenf excystment and diameter of perforations produced in walls of conidia of C. sativus. Both organisms differed from a previously-described species of Vampyrella which causes large perforations and annular depressions in spore walls.
INTRODUCTION
MATERIALS AND METHODS
Isolation of mycophagous amoebae
It was reported that pigmented spores of certain plant pathogenic fungi were parasitized by a mycophagous soil amoeba belonging to the protozoan family, Vampyrellidae Doflein (Anderson and Patrick, 1977; 1978; Old, 1977a,b). While feeding, distinct annular depres-
sions and perforations 1-7 /*rn in dia in fungus spore walls were produced. Although the identity of the causal amoeba is still controversial, our observations indicate that it is similar to Vumpyrella Iateritia (Leidy, 1879). It has been suggested that the mycoparasitic amoeba may have potential for biological control of soilborne plant pathogens (Anderson and Patrick, 1978; Old, 1977a). Recently, two additional mycophagous amoebae in the family Vampyrellidae were isolated from field soil. These amoebae also perforated and lysed conidia of Cochliobolus sativus Ito and Kurib Dreschs ex Dastur, and chlamydospores of Thielauiopsis basicola (Berk. and Br.) Ferr. However, the recently-isolated amoebae exhibited characteristics different from the mycophagous organism described previously (Anderson and Patrick, 1978). They resemble Theratromyxa weberi (Zwillenberg, 1953) and Vampyrella uorax (Cienkowski, 1865, 1876). The organisms produced perforations 1 pm dia or less in fungus spores during the feeding process. The perforations were similar to those reported by Clough and Patrick (1976) and Old and Patrick (1976), in conidia of C. sutivus and chlamydospores of 7: basicola incubated in natural field soil. These investigators suggested that the perforations were caused by lytic soil bacteria, However, based on our present studies, it appears that the perforations were caused by mycophagous amoebae. We describe. details of the feeding habits of the two species of amoeba that produce small perforations in fungus spores, their distribution in soil, and
cultural
characteristics.
* Present address: Agriculture Canada, tion. Harrow, Ontario. NOR lG0, Canada.
Research
Sta159
Soil dilution plates containing 100 and 10 mg field soil and 5 ml distilled water (DW) were baited with conidia of C. sativus (5 x lo4 conidia per plate) and incubated at 20°C. After 2-3 weeks, conidia in the plates were observed by means of an inverted microscope (Reichert) at x 100 magnification. Digestive cysts of the mycophagous amoebae that surrounded partially-lysed conidia were removed by means of a pipette and washed 3 times in sterile 10% soil extract (SE) (Anderson and Patrick, 1978). Single-cyst isolates were cultured in 10% SE at 2O”C, in test tubes and Petri dishes using conidia of C. sutiuus or chlamydospores of 7: basicola as food source. Fixation and staining Mixtures of amoebae and conidia from 1 week old single-cyst cultures were placed on microscope slides. After 24-48 h incubation in moist chambers, active amoebae were fixed by addition of several drops of Carnoy’s fluid (Maloney, 1973). Slides with fixed amoebae were immersed in 70% ethyl alcohol for 24 h. Amoebae were stained in Delafield’s haematoxylin for 24 h and destained with tap water (Mackinnon and Hawes, 1961). Mycoparasitic vorax
activities of Theratromyxa
sp.
and V.
Experiments to determine the rate of spore lysis and increase in number of the two vampyrellid amoebae were performed in tissue culture chambers (Micro Test II, Falcon brand). A single digestive cyst was added to each culture chamber containing several drops of 10% SE and 50&1ooO conidia of C. satiuus. After incubation at 20°C the number of amoebae and lysed conidia were determined using an inverted microscope at x 100 and x 250. Conidia were considered lysed if they were void of contents and surrounded by a digestive cyst wall or if
T. R. ANDERSON and Z. A. PATRICK
160
Table
I. Mycophagous
Vampyrellidae
in soils from various
locations
Organism
Location
Ontario Vineland cultivated field Vineland sod Simcoe tomato field Delhi tobacco field Harriston garden Hastings garden Tomato greenhouse Alberta Edmonton wheat field California Salinas vegetable field Hawaii Waimanalo orchard sod ,/ Number
of organisms
g-
’ Observed in soil dilution
Theratromyza sp.
C’trmpJwlla c0ru.x
13” I
+h +
_I +
2 11 5 8 +
+ -
_
NEd NE +
NEd NE +
4.5
+
-
0
_
+
1.5
_
_
’ of dry soil. determined by soil bioassays (Anderson and Patrick, 1978). plates.
k Not observed in soil dilution plates d Not evaluated.
the conidia experiments
were enclosed within a digestive were replicated IO times.
Conidia of C. .satiuus, produced on 207: VX juice agar. were suspended in distilled water and filtered through 44 pm mesh. The conidia were collected and washed several times on 20 pm mesh before filling the culture chambers. Chlamydospores of 7: husicola were prepared in a similar manner. The stages in the life cycle of each organism were studied in plastic Petri dishes (35 x 10mm) containing 5 ml lO”/~,,SE. Cyst diameters were measured at the greatest width. Observations of undisturbed cultures were made with an inverted microscope using phase contrast microscopy. Material for scanning electron microscopy was air-dried without fixation and coated with gold-palladium. Specimens were observed in a Cambridge Stereoscan MK II scanning electron microscope. The cultural characteristics and stages in the life cycle of Theratromy.wt sp. and V. c0ru.y were compared to those of K luteritia (Anderson and Patrick. 1978). For such comparisons, single cyst isolates of each organism were cultured in l@x, SE in plastic Petri dishes under similar conditions.
Table
2. Effect of incubation
REWLTS
cyst. The
Isolation vorax
of mycophagous
Theratromyxa
sp. und V.
The presence of Theratrornyxa sp. and I/. t’oras in soil dilution plates was indicated by lysis of conidia of C. suticus surrounded by a thin cyst membrane or the presence of digestive cysts surrrounding partiallylysed conidia. Digestive cysts of both organisms containing one conidium were similar in appearance and it was necessary to cub-culture the cysts to determine which organism was present. Mixed populations of mycoparasitic Theratromyxa, V vorax and I! luterititr were found in some soil samples (Table 1). Soil surveys indicated that Therutromysa was widely distributed in agricultural soils. It was found in soils from cultivated fields in Ontario, in greenhouse soils, and in a sample of wheat field soil from Alberta. C! 1’oru.v was found in field soils from Ontario and California. Therutromyxa and V. vorux did not occur in dilution plates containing less than 0.1 g soil. It appears therefore, that populations of these organisms in soils are relatively low. As shown in Table I. 1/: luteritia, the amoeba which produces the large diameter holes in
at 20 C on rate of Increase of Therutrorn.?.ua number of lysed conidia of Cochlioholus satious
sp.. P’amp_vrel/rr t’oraz and
(Days) Treatment Amoebae Amoebae Amoebae Amoebae
0 only (Tk~rutrom~.~a sp.) + conidia (A) (B) only (licrmp.vrel/tr cortrz) + conidia (A) (B)
1.0” 1.0 1.0
I .o I .o 1.o
I
2
3
I.0
1.5 4.9 6. I
1.7 14.8 20.5
I .o 1.0 1.0
I.5 I .4
I .o
I .o
1.0 1.4
0.9 1.8
6 1.4 167.9 199.9 0.9 0.9 7.4
(A) Number of T/lrratronl~.~u sp. or l’un~p?‘rr/l~~ POT(~.Y (trophoroites + cysts). (B) Number of lysed conidia wlthin digestive cyst walls including partially lysed conidla tive cysts. “ mean of IO replicates.
IO I.6 397.4 746.7 0.9 0.8 2.4
within
diges-
Soil vampyrellid
fungus spore walls. was the most frequently observed member of Vampyrellidae found in the soils that were sampled. Mycoparasitic activities of Theratromyxa vorax iri culture chambers
sp. and V.
Culture chambers containing conidia of C. satious inoculated with a single digestive cyst of Theratromyxa resulted in lysis of an average of 746 conidia per chamber after 10 days (Table 2). The average number of TherutrornJ~xa in these chambers after the same period was 397 per chamber. In control chambers without TheratromJxa, no lysed conidia surrounded by cyst walls were observed. In the chambers without conidia, the average number of Theratromyxa was 1.6 per chamber after 10 days incubation. The diameter of resting cysts was 63.9 k 28 pm in chambers with conidia and 19.6 +_ 5 pm in chambers without conidia. When culture chambers containing conidia of C. sutiaus were inoculated with a single digestive cyst of V. poras, lysis of an average of 2.4 conidia per chamber resulted after 10 days incubation (Table 2). The average number of 1/: vorax per chamber decreased during the same period. In a number of similar experiments, the mycoparasitism and multiplication of I’. vorax was erratic. Most frequently only few conidia were lysed and the amoebae did not multiply. The activity of 1/: uorax was usually better in cultures containing algae, diatoms and flagellates in addition to the conidia. Parusitism of fungus spores by Theratromyxa Digestive cysts of the amoeba were smooth-walled and approximately 65 pm in length but varied with the size of the fungus spore they enclosed (Fig. 3). The digestive cysts exceeded the spore dimensions by 55IOpm on all sides, Digestive cysts normally contained a single conidium of C. satiuus or a chlamydospore chain of 7: basicolu. The contents of a conidium of C. satious or chlamydospore of 7: busicola were digested in 2436 h. During the first 12 h of feeding, the protoplasts of cells within conidia of C. satiuus appeared to shrink (Fig. 3) and the conidia became distorted. The cyst contents underwent plasmotomy before excystment (Fig. 5) and soon the progeny trophozoites emerged through several small holes in the cyst wall. Each digestive cyst produced 5-22 progeny (Fig. 6). The digestive cyst wall deteriorated after IS-20 days in cultures which contained bacteria. After excystment, shallow pits and small perforations 0.28 k 0.05 pm dia were seen in the fungus spore wall with phase contrast and scanning electron microscopy (Fig. 4). Annulations in the spore wall were not observed. In mixed cultures, Therutromyxa was not seen to ingest other protozoa but some digestive cysts were formed which enclosed the desmid, Closterium sp. Trophozoites often adhered to active larvae of Meloidogyne sp. but were unable to immobilize them. Cultures containing nematodes were observed for several days, but active nematodes were not parasitised. Trophozoite
of‘Theratromyxa
Digestive cyst progeny often assumed a spherical form. 44 k 13.7 Aim dia soon after excystment and
amoebae
161
extended up to 130 pm after contacting the bottom of the culture dish. In general. trophozoites were extremely active, elongate and angularly branched. At times they became flattened, leaf-like and remained motionless. Occasionally they became spherical and floated with their filopodia extended. The cytoplasm was granular and contained numerous contractile vesicles I-3 pm dia. Stained trophozoites were multinucleate. The nuclei were spindle-shaped, 5.3 + 0.5 pm. and the karyosomes were 4.3 _t 0.6pm in length. Extremely fine anastomosing filopodia were concentrated at blunt terminal portions of the organism or along the anterior edge of the cresent-shaped individuals. Fission and fusion of trophozoites was observed. Many trophozoites fused to form a large reticulate plasmodium (Fig. 9). Trophozoites of Tkerutromyxa were consumed by a species of Mayorella which also attacked V. latrritia. Ocassionally the Mayorella also lysed spores of C. satitus and -1 basicolu by consuming the spore contents without formation of digestive cysts (Fig. 20). Multiplication, tromyxa
encystment
and excystment
of Thera-
Therutromyxa formed three types of cysts depending on duration in culture and availability of food. Digestive cysts formed in the presence of viable conidia. Trophozoites moved randomly until conidia were located (Fig. 7); they then formed a smooth sheath which completely enclosed each conidium (Fig. 8). Often fusion of two trophozoites was necessary before a conidium was completely engulfed. Resting cysts formed in the absence of suitable food or in older cultures, Trophozoites became spherical or disk-shaped and a smooth cyst wall was formed after approximately 30min. The diameter of resting cysts varied from 30 to more than IOOpm depending on size of the encysting trophozoite. In the absence of food there was excystment and re-encystment or, at times, the contents of original resting cysts contracted to form double-walled resting cysts (Fig. 9). The colour of resting cysts in transmitted light varied from golden to reddish brown. In general, double-walled cysts were darker than single-walled cysts. Resting cysts were also formed directly from digestive cysts. Such resting cysts were enclosed within the original digestive cyst wall and were 32.9 k 9.8 pm dia. Excystment of resting cysts was preceded by plasmotomy and resulted in 5-7 progeny from single-walled resting cysts. Excystment occurred 3-4 days after transfer to fresh medium containing conidia of C. satiuus. Excystment of darker and double-walled cysts required longer incubation. In the presence of abundant conidia, a third type of cyst was formed. This happened when many individual trophozoites fused to form a motionless irregular plasmodium up to I mm dia (Fig. IO). Initially, the plasmodium contained numerous small vacuoles I-5 ,nm dia. Gradually the size of some vacuoles increased up to IOOpm dia and the plasmodium appeared froth-like (Fig. 1I). Plasmotomy occurred in the dense cytoplasm which had accumulated at numerous regions within the plasmodium. Progeny trophozoites were approximately 1@40~m dia and often fused with adjacent individuals before escaping the froth-like remains of the plasmodium (Fig. 11).
T. R. ANDERSON and Z. A. PATRICK
162
Oiqeslive
cysf
8’” ,&
Trophoroi
Trophozoites
tes
within
cyst
wall
Plosrnodium
Stages
in
the
life
cycle
(diagrammatic
Fig. I. Schematic
representation
of
mycophaqous
Theratromyxa
sp.
representolion)
of the various
The process of plasmodium and froth-like cyst formation and release of progeny required 2-3 days. During emergence, progeny became spherical with radiating filopodia and floated in the culture medium. After contact with a sohd surface, ~~le~~fro~z~.~u became elongate and branched. In the absence of food fusion of progeny was followed by encystment. Some of the observed stages in the life cycle of Theratromyxa are shown in Fig. 1.
After engulfing a conidium of C. satiz~s, a trophozoite of V: uorax often continued to move randomly, collecting additional conidia before forming a digesfive cyst (Fig. 13). The number of conidia within digestive cysts varied from one to severa (Fig. 15). Digestive cysts were smooth walled 70.4 & 4 pm dia and were often surrounded by culture debris. In soil dilution plates, digestive cysts were often found associated with organic matter and soil particles. It took 24-36 h to digest conidia within the cyst. After excystment, numerous perforations 0.42 + 0.2 ftrn dia and pits 1.1 & 0.2 pm dia, were visible in walls of lysed conidia within the empty cysts
stages in the life cycle of Titcrat~on~,wa
sp
(Figs 16, 17). Small perforations were frequently seen at the base of surface pits (Fig. 17). Annulations in the conidial walls similar to those produced by I/: lareritia (Fig. 18) were not seen. In soil dil~ltion plates and in mixed cultures. digestive cysts of V: uorax often contained small diatoms and flagellates as well as conidia of C. snticus or chlamydospores of 7: basic&. It is not known if these additional organisms are essential in the nutrition of 1/ UOI(IX.However, it was difficult to maintain the organism in cultures that contained only bacteria and conidia of C. sutivus. Trophozoite
cd V. vorax
Recently-excysted trophozoites varied in size from 60pm dia to more than 3~~rn in length. The cytoplasm was hyahne, granular and contained numerous contractile vesicles that were less than 5 pm dia. The most frequent form assumed by an active trophozoite was crescent or fan-shaped (Fig. 12). While floating, I/ mrax became globular or reniform and appeared to move by means of filopodia which formed anteriorly and retracted posteriorly. I/ VOYCIX was also seen to form an elongated cord-like structure
Soil vampyrellid
with anastomosing and branched filopodia that were concentrated at spatulate terminal portions. Trophozoites avoided strong light and remained in the vicinity of clumps of organic matter in soil dilution plates. Multiplication,
encystment and excystment of V. vorax
Trophozoites frequently formed digestive or resting cysts. With prolonged encystment, the organism contracted further to form a double-walled resting cyst. Resting cysts of I! uorax were vacuolated or contained a large central vacuole. Solid material was frequently seen within such vacuoles, but the nature of this material is unknown. Excystment of resting cysts was promoted by diluting the culture medium with 10% SE and adding fresh conidia of C. sativus. Under such conditions excystment occurred in approximately 24 h at 20°C. A single progeny emerged through large perforations, S-10pm dia. in the cyst wall. Excystment from digestive cysts was preceded by plasmotomy. The cyst cytoplasm divided at the midpoint and the resulting two progeny emerged from opposite ends of the cyst (Fig. 14). On one occasion, cyst progeny fused before excystment; this resulted in release of a single trophozoite. Some of the stages in the life cycle,of I/: vorax are shown in Fig. 2.
DigesIive (contoininq
0
amoebae
163
Comparison of Theratromyxa,
V. vorax and V. lateritia
Although the morphology of each mycophagous member of Vampyrellidae was variable, active trophozoites exhibited their specific characteristic forms in laboratory culture chambers. Theratromyxn was elongate, branching and angular, with fine filopodia concentrated at blunt extremities (Fig. 6) I! uorax was crescent, or globular-reniform with filopodia concentrated along the anterior edge (Fig. 12) while I/: lateritia was elongate and branched with filopodia concentrated at terminal spatulate areas of the organism. Vumpyrella lateritia was often indistinguishable from Y uorux, especially when the latter became elongate and branched. All three amoebae produced “pinhead” pseudopodia. Trophozoites from a recently-isolated single-cyst culture of K lateritia frequently produced large peripheral vacuoles and “pinhead” pseudopodia (Fig. 19) similar to those described by Leidy (1879) and Lloyd (1926). This characteristic became less apparent after three to four sub-cultures. “Pinhead” pseudopodia were also produced occasionally by Theratromyxa and frequently by K vorax. Digestive cysts which contained conidia were formed by Therutromyxa and f! vorax, but not by % luteritiu. Digestive cysts of Therutromyxu rarely con-
cyst spores1
Lysed spore wlthin cell wall
Stages
in
the
life
cycle
of
(diogrommotic
Fig. 2. Schematic
representation
of the various
mycophogous representation
Vampyrella
vorox
1
stages in the life-cycle of Vampyrella uorax Cnk.
T. R.
164
ANDERSON and
tained more than one conidium while those of i/: L’OIXSoften contained several. Excystment from digestive cysts of Theratromy.~u. C’:corux and I/: lateritia resulted in the formation of $22, 2 and I progeny. respectively. Single-walled
Z. A.
PATRICK
resting cysts of 7%erutromy.w, 1/. cora.x and CI laferiritr released 5-7, 1 and 1 progeny respectively. The frothlike modified cysts produced by Therutrom)\-a were not formed by the other amoebae. Differences in the parasitic activities of the three
Figs 3--t 1. Stages in the life cycle of Theratrom~ua Fig. 3. Lysed and partially Fig. 4. Scanning
electron
lysed conidia micrograph
of C. salirus wlthin x 600.
(SEM) showing
digestive
perforations
Fig. 5. Plastomotomy progeny
Fig. 7. Fusion
trophozoites of progeny
Fig. X. Formation Fig. 9 Fusion
of trophoroites
Fig. IO. Stationary Fig.
of digestive
cyst cytoplasm
escaping
from digestive X 375.
trophozoites
of digestive
under crowded double-walled
before excystment. cyst containing
on the surface
cyst which completely
of T/~rtrrro~n~,\ct.
(arrows) 0.3 Ltrn dia in comdlum x 1300.
.surims lysed by Tkrrcrrromy\-tr.
Fig. 6. Angular
sp.
cysts (arrows)
x 375.
lysed comdium
of viable conidium. conidium.
x 375.
conditions forming irregular resting cyst (arrow). x 360.
branching
plasmodium.
(arro\$s) escaping plasmodium
the foam-like modified in Fig. IO. x 400.
cyst formed
(arrow).
x -375.
encloses
vacuolate plasmodium resulting from extensive fusion of numerous Note digestive cyst over plasmodium (arrow). x 260.
I I Progcnq tropho/oltes
of C
Note
trophoroltes.
from the stotlonar)
Soil vampyrellid mycophagous vampyrellid amoeba were exhibited by lysed conidia of C. sc4ticu.s. Conidia lysed by Therutromyxu and 1/: t’orax were enclosed within empty digestive cysts while those lysed by I! lateritio were never seen within such cysts. Conidia lysed by Theratromyxa became flattened and distorted during excystment. In contrast, conidia lysed by I/: corax generally retained
Fig. Fig. 14. Excystment
165
their
shape. Perforations in walls of conidia of C. caused by Therutromyxa, V. t1oru.x and V. latrritiu were 0.2-0.3, 0.221.0 and I-7,rrm dia respectively
sutivus
(Figs
4, 16,
18).Perforations
caused
trophozoite
of V wru.v approaching
13. Digestive cyst containmg
of digestive
Fig. 16. SEM showing Fig. 17. SEM of conidium
conidia
conidia
perforations
of C. SLIII‘L’US, x 375
of C. ~tiaus.
cyst resulting in progeny trophoroites ends of the cyst, x 375.
Fig. 15. Lysed conidia with small perforations resting cyst within original
x 375
(arrows)
leaving
from opposite
(arrows) within empty digestive cysts of V I‘O~U.S.Note digestive cyst (short arrow). x 375.
0.2 1.O pm dia in conidia
lysed by I/: LYWUX showing
perforation
lysed by K ror‘(.x, x 1500. at base of pit (arrow),
Fig. IX. SEM of conidia lysed by I/: hfrritict showing large perforations (arrow) and annular (short arrow). Note detached disk (white arrow) from conidial wall on adjacent conidium, Fig. 19. Pin-head
by Thrratrompua
appeared to be the result of direct penetration. The perforations caused by I! uorax appeared to result from direct penetration or by the formation of shal-
II- 17. Stages in the life cycle of k’rrrnp~~lla CO~(IY
Figs Fig. 12. Typical
amoebae
pseudopodia (arrows) produced by spherical 1/: lrztrrrfia similar 1’ UV~I.Xand occasionally by Tlrrrrrrror~~~.~~~,x 600.
Fig. 20. Active trophozoite
of parasitic
Mo)ore/ltr
with ingested
conidium
x 3900. depression x 1575.
to those produced
of C. strrirus,
x 375
by
T. R. ANUER~ON and 2. A. PATRICK
166
low pits I pm dia followed by penetration at the base of the pits (Fig. 17). Those caused by 1/: lateritia exhibited annular depressions in the spore walls as well as the large diameter holes described earlier by Anderson and Patrick (1978) and Old (1978) (Fig. 18).
DISCUSSION
The finding of additional species of mycophagous Vampyrellidae in arable soils that were capable of lysing spores of plant pathogenic fungi, further suggests the potential importance of soil protozoa in biological control of soilborne plant diseases. Results indicate that Theratromyxa was relatively common in agricultural soils and was capable of lysing large numbers of conidia of C. safitlus under laboratory conditions; V. corax was not found as often and was more difficult to maintain in the laboratory. It appears, therefore, that Thrrutromyxu is more suitable for applied studies on biological control of soilborne plant pathogens. Theratro~~yxu wrheri Zwillenberg was originally described as a nematophagous amoeba (Weber et al., 19.52: Zwillenberg. 1953). In our studies, Theratromyxu did not digest larvae of Meloidogpne sp. However, conditions for successful parasitism of nematodes may have not existed in our culture chambers. It is also possible that the baiting techniques used for isolating Theratrom~uu favoured the selection of mycophagous organisms and the species we studied was different from the nematophagous one. Thcwtromyxa resembled the original descriptions of nematophagous amoeba in morphology. encystment and excystment. The formation of the digestive cyst surrounding the food source and plasmotomy of digestive cyst contents were similar to the previous descriptions (Sayre, 1973: Weber et al., 1952). Sporefilled cysts described by Zwillenberg (1953) were not seen in our studies but such cysts may be similar to the froth-like modified cysts that we observed, in which numerous trophic progeny were produced. Such progeny were capable of forming small cysts and may have been encysted at the time of Zwillenberg’s (1953) observations. Such progeny often assumed a heliozoid form after excystment and became amoeboid after contact with a flat surface. The origin of such heliozoid forms was not previously known (Zwillenberg, 1953). The mycophagous Therntromyxa sp. also resembled the nematophagous species described by Sayre (1973). We have reported (Anderson and Patrick. IY77) that mycophagous member of Vampyrellidae which formed digestive cysts around fungus spores resembled AruchnuIu imputirns Cnk. After more extensive observations, however, it is more likely that the organism we observed was V: wra.x. In the earlier descriptions (Cienkowski, 1865. 1876), L! wrax was reported to feed by forming a digestive cyst around the food source. Diatoms were arranged within the cyst on the longitudinal axis and on excystment two to four progeny emerged. Specialized reproductive cysts were not described by Cienkowski (1865, I X76). Artrchnula imputier~s is described as being similar to I/: coru.y. but excystment of digestive cysts results in one. rarely two progeny. Also. special reproductive cysts were formed
(Dobell, 1913). In our study, fission of the digestive cyst cytoplasm resulted in the formation of two progeny and the special reproductive cysts were not observed. Although Arachnula and Vumpyrellu were considered by some as being synonymous (Dobell. I9 13). the mycophagous amoeba in our studies resembled the description of 1;. voruy more closely than that of A. impatiens. Perforations in spore walls, similar to those produced by Thrratromyxa and k: crux in culture chambers, have been observed in spores incubated in field soil (Clough and Patrick. 1976; Old and Patrick. 1976). This indicates that one or both organisms ma) be active in field soils. On occasion, large perforations and annulations as well as the much smaller perforations, to.5 pm dia were seen in the same fungal spore (Old and Patrick, 1976). In the earlier studies (Old and Patrick, 1976; Old and Wong. 1976). it was concluded that small perforations were caused by lytic soil bacteria. But our study indicates, it is more likely that such spores were attacked by more than one species of mycophagous amoebae. each of which formed their characteristic perforations in the walls, Thus, it appears that there are at least three different mycoparasitic members of Vampyrellidae that are widely distributed in arable soils. These organisms can be readily distinguished from each other when cultured in the laboratory by a combination of characters consisting of general morphology, type of digestive cysts. methods of excystment and diameter of the perforations they produce in walls of conidia of C. sufivus.
Support in part by Operating Grant to 2. A. Patrick from the National Sciences and Engineering Council of Canada. Acknowledgements-
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