Perforation and lysis of fungal spores in natural soils

So11 Lhol RMX-kern..Vol. 8. pp 285 to 292. Pergamon

PERFORATION

Press 1976. Prmtrd

m Great

Britain

AND LYSIS OF FUNGAL TN NATURAL SOILS

SPORES

K. M. OLD and J. N. F. WONG University of Dundee.

Department

of Biological

(Accepted

Sciences,

15 Nocemher

Dundee,

Scotland

DDI 4HN

1975)

Summary-Conidia of Cochlioholus satious and five other pigmented fungi lysed when incubated in natural soil. Lysis followed perforation of the spore wall by holes of varying dia. Three possible causes of perforation were investigated. namely autolysis, mechanical puncture by soil animals and enzymatic erosion by soil micro-organisms. Results indicated that soil micro-organisms were the likely causal agents although no micro-organism able to perforate conidia has yet been isolated. Colonization of conidia by the soil microflora was studied by electron microscopy. On the basis of these direct observations, possible perforation mechanisms are suggested. Reports of perforation of fungal, plant and bacterial cell walls are briefly summarized and the perforation phenomenon discussed in relation to the biodegradation of pigmented fungal propagules in soils.

Successful plant pathogens must be equipped for survival in the absence of the host. This is particularly true for soil-borne pathogens which usually remain in situ either in the soil or plant remains. Survival is often accomplished by the formation of resting propagules which can remain viable for extended periods. These propagules are commonly some type of spore (Warcup, 1955). Spores in the soil are subjected to many physical and biotic factors which may reduce their numbers. The topic to be discussed here is the role of soil micro-organisms in the naturally occurring biological control of C. saticus in soils, and the extension of these findings to other pigmented fungal species. C. satiuus (Ito & Kurib.) Drechs ex Dastur, often called Hrlmir~thospouium satiouln Pamm., King, and Bakke, is soil-borne and causes a root rot of cereals. The fungus survives in soil as pigmented, septate conidia. The number of viable conidia determines the incidence of disease in succeeding crops (Chinn et al., 1962; Burgess and Griffin, 1968). This aspect of the biology of C. sativus has received a considerable amount of attention over the years. Anwar (1949) showed that the fungus did not persist in plots cropped continuously with barley. despite regular additions of inoculum to the soil, and considered that an antagonistic microflora could limit survival of the fungus. Chinn and Ledingham have produced a series of articles on the spore populations of the fungus in field soils and their relation to incidence of root-rot of wheat. Considerable variation in survival was found in different locations (Chinn et al., 1960; 1962). Viability of conidia declined more rapidly in wet than in dry soils (Chinn and Ledingham, 1958). In their review, Cook and Papendick (1972) discuss the possibility that, in a considerable number of disease situations, pathogens are suppressed in soils with high water potentials as a result of enhanced activity of antagonistic micro-organisms. 285

FACTORS

OF THE SOIL ENVIRONMENT CAUSE

DEATH

OF

THAT

CONIDIA

Germination-lysis

Chinn and Ledingham (1957) showed that amending soil with various organic substrates could reduce spore populations of C. satit’us. Conidia germinated, and the germ tubes quickly lysed. Isolates of the fungus not susceptible to soil fungistasis possessed ‘inherent germinability’ (Chinn and Tinline, 1964) and rapidly disappeared from natural soil. In contrast wild-type conidia, sensitive to soil fungistasis, survived much longer. The germination-lysis mechanism can only be invoked however in situations where the fungistatic properties of natural soil (Dobbs and Hinson, 1953) are overcome and germination occurs. This is rare. The more usual situation is that conidia, once placed into the soil, remain in the non-germinated state. Sturvation

lysis

Energy-rich substrates are in great demand in natural soils. Fungal spores and their stored nutrient reserves are thus potentially a source of food to the soil microflora. Lockwood and co-workers (Lockwood, 1968; Ko and Lockwood, 1970; Lloyd and Lockwood, 1966) have shown that fungal mycelia may lyse because of leakage of nutrients from fungal cells to the nutrient sink, represented by the vast population of bacteria present in soil. It is not clear however whether fungal conidia, particularly those with pigmented cell walls, will readily lyse under these biotic influences. Perforation

lysis

Pigmented fungal propagules are particularly resistant to lysis in the soil. Many soil-borne pathogens utilize pigmented spores, sclerotia or hyphae as survival structures. Bull, (1970) and Kuo and Alexander,

X6

K.

M. 0~11 and J. N. F. WONG

(1967) have shown that melanins located in cell walls can inhibit the activity of cell-wall-lysing enzymes. Old and Robertson (1970b) showed that pigmented isolates of C. .saric~s resisted cell-wall-lysing enzymes b) virtue of the presence of a pigmented layer in the cell wall, and survived for prolonged periods in natural soil. whereas hyaline isolates were susceptible to enzymes and survived for only 2 weeks in soil. Thus the pigmented conidia of C. scrticusappear well adapted for prolonged survival in agricultural soils. Yet reports that persistence varies with soil and location suggest the existence of mechanisms potentially capable of eradicating the fungus from natural soil. Old (1967. 1969) described such a mechanism which appeared to overcome the barrier offered by the pigmented outer layer of wild type conidia of C. sutiws. Conidia were incubated at 25’C for 50 days on the surface of seven soils of varying type and pH. In five of the soils viability declined from 7@904; on day I to 7-50”;) by day 50. In these five soils, death was accompanied by the appearance of perforations in a substantial proportion of the conidia. In two natural soils and in all pasteurized soils (IO0 C moist heat:45 min) viability of conidia did not decline, and no perforations were observed. Experiments of this nature have been carried out many times since with incubation times up to 100 days, with similar results. Most studies have been carried out in the laboratory. but perforated conidia also have been recovered when spores were buried in the field (Old. 1969).

1969). but only with the SEM. When lysing conidia were sectioned and observed with the transmission electron microscope (TEM) they were extensively occupied by bacteria present in the cell lumina and the microfibrillar matrices of the cell walls (Fig. 3). In advanced states of lysis the entire contents of the spore were digested. leaving the outer shell-like pigmented layer of the cell wall as the sole remaining component. A variety of bacterial types were observed within the lysing conidia (Old and Robertson. 1970; Wong and Old, 1974). Rods and spheroidal bacteria were most common but could not bc identified. Sections of filamentous cells. probably actinomycetes, were also seen. One unique bacterium recognizable by its helically lobed morphology (Fig. 4) was observed regularly in sectioned conidia (Old and Wong. 1972). These have been called Apdmcterim po/!s/,llooitlurn by Nikitin and Vasilyeva (196X). This bacterial type was also observed by SEM on spore surfaces (Wong and Old. 1974). Membrane bound vesicles between 20 60nm in dia were also frequently observed in conidia colonized by bacteria (Wang and Old, 1974). These appear to be budded from the surface of bacterial cells (Fig. 5) and may have some function in the enrymatic Iysis of the fungal cell wall. These observations indicated that a variety of bacteria were able to colonize the lumina and non-pigmented layers of the spore wall. but it is not known how the perforations in the spore wall are made. IN\‘ESTIGATION

MOHPHOLOGY

OF LYSED ARD

PERFORATED

OF THE ACEPiT CAUSING

PERFORATIOV

OF COhIDIA

CONIDIA

Conidia were incubated on soil in Petri dishes, recovcrcd at intervals and observed with the scanning and transmission electron microscopes (Old and Robertson, 1969; Old and Robertson. 1970a; Wong and Old. 1974). The sequence of their colonization by the soil microflora, and the development of perforations, have been described in some detail. After 2 weeks incubation, an increasing number of conidia wcrc empty and septa were no longer clearly distinguishable. Perforations could be seen with the light microscope and their frequency increased during the subsequent weeks of incubation. At 100 days, up to 75”,, of the conidia were lysed and 50’:; or more were perforated by one or several holes (Fig. I). The actual numbers of conidia showing these symptoms varied with the experiment. but the sequence of events remained the same. Observation with the scanning electron microscope (SEM), confirmed the nature of the perforations and revealed the prescncc of annular depressions (Fig. I) of approximately the same dia. as the perforations (size range 2-4,um). The area within the annular depression was often eroded to form a pit-shaped depression. it is thus considered that the process of perforation involves first the formation of an annular depression in the spore wall, and then complete crosion of the enclosed area to produce a hole (Fig. 2). Usually. 1~4 perforations were visible per conidium but larger numbers were occasionally present. In addition to these perforations. holes of approximately 0.5 Ltrn could sometimes be seen (Old and Robertson.

Autolysis. mechanical puncture by soil animals or enzymatic lysis by micro-organisms are all possible causes of perforation.

Lloyd and Lockwood (1966) showed that mycelia of several fungi lysed if subjected to nutrient stress. particularly if in combination with cxposurc to antibiotics. This method did not induce perforation of conidia of C. satitus (Old. 1969). SEM studies had shown that perforation was commonly associated with the development of annular depressions in the spore surface. It is difficult to invoke autolytic enzymes originating from within the spore protoplast as giving rise to such a pattern of erosion. It is conceivable that microfibrillar clcments particularly susccptible to enzymatic digestion could be located in the outer spore wall in circular configurations. Invcstigation of this possibility was hampered by the lack of a commercially-available enl-ymc able to Iyse pigmented fungal cell walls. This was solved using a hyaline isolate of C. satins. Spore colour is thought to be a single gene difference (Tinline and Dickson, 1958): it was thus assumed that the disposition of microfibrillar clcmcnts would he the same for hyaline and pigmented isolates. Conidia were treated with cold KOH. chitinase. laminarinase and pronase in various combinations after the technique of Hunsley and Burnett (1970). These treatments were selected because they will digest the main cell wall components of most euascomycete fungi. Treatment with KOH followed by laminarinase eroded the outer cell

5 Plate

I. Scanning Fis.

Fig. 7. Two

(SEW)

1. Lysmg annular

Fig. 2. Section

and transmissi~m

comdium

with

depressions

l&w

(‘TEM1 cioctrori ann~dar

and pcrforation

micrographs

depressions which

of lysing

and perbrations

has penetrated

Fig. 3. Section Fig. 5. Section

to the spore

of Iysing collidill~~ wit/l bacteria in the spore lumen (L) and the clcctran = dcnsc surkc layer (SL) of the spore wall

Now

of a

ol’ a hciicnll!-lobed

(HL)

hactcrium

in the wall

of a

conidla

(arrows)(

in

the

ctf C‘. ,~u;IY~,,. x

I X001. I x 4100).

lumen sport

xail

(SW)

( x 11,400). l)sinp spore ( x ..+5.000).

hxtcrium in the wall ol‘ a I>sing spore. Note the membrane-hound apparently being budded OH from the bacterium ( x 32.000).

vesicles (arrows)

2ss

K. M. 01.1, and J. N. F. WONG

uall Iaycrs and the arrangement of fibrils were clear]) \isible b! TEM observation after shadowing with pold palladium (Wong. unpublished data). In no case \ICI-c annular or circular arrangements of microfibrils obscrLcd. It therefore seems unlikely that perforation of conidia is an autolytic phenomenon. 1lECH4\1C41.

Pl’\CTtIRF

BY

SOIL

AUM4LS

Rcisinger (1972) showed that mitts could puncture conidia of H. apicif>mr~~ and allow subsequent coloni/ation by soil bacteria. Protozoa or nematodes could conceivably accomplish the same effect. A suspension of saprophytic soil nematodes added to autoclaved soil fail4 to ULISC perforation of conidia (Old. 1969). but the cl~ance exists that the right animal was not added to the soil. A more satisfactory approach was da iscd using ‘Nucleporr’ filter membranes of various pot-c G/es. When conidia \vere deposited onto the membrane and placed into natural soil. Iysis and perforation rcCldil! occurred. A second membrane was placed J)LCI’ the first and the margin sealed with vacuum grease bcforc the membranes were placed into the ~011.An! organisms gaining access to the spores thus had to pass through the pores in the membranes. Expcrlmcnts of this nature were carried out usin+ port si/cs of 5.0 /nn. I .O /ltn and 0.2 Ltm. Although It was possible that small animals could penetrate the 5.0 /cm pot-cs. the other two pore sizes would exclude them. After 10 days incubation in natural soil perforated conidia wcrc observed in both the 5.0 pm and I.0 Atrn pore size treatments. 111 the 0.2 pm treatment although some micro-organisms penetrated to the conidia. no perforations were observed. This provides unequivocal evidence that soil animals are not the ca~~sal apent. I;\%\

\li\TlC

I.1 SIS RI’

SOIL

MICRO-ORGANISMS

Isolations wcrc made from lysing and perforated conidia (Old. 1969) and tested for their ability to protiucc perforations. So far. more than 50 isolates of soil bacteria and streptomycetes have been inoculated Into autoclaved soil. and conidia added; none have rcproduccd the perforation symptom. Soil has been amended aith organic substances to stimulate differcni components of the microflora (Old 1969). None (;I’ !hc
reduced the collapse of both fungal and bacterial cells during desiccation, and good SEM images were obtained of the surface microflora of lysing conidia. The CPD method preserved the morphology of bacteria; HL forms and bacteria with long filamentous appendages were thus observed (Fig. 6). Actinomycetes were often seen on spore surfaces and frequently formed loops of cells which adhered firmly to the cell wall (Figs. 7-9). Based on observations of the surface microflora hypotheses have been dcvizcd to explain the formation of the two distinct types of perforation, namely those up to 0.5/Lrn dia and those 2-~4pm in dia which apparentI\ develop from annular depressions in the spore wall. The smaller holes are approximately the width of a single bacterial cell. Bacteria have often been observed embedded or firmly affixed end-on to the spore wall (Figs. IO. I I). Such attachment of bacteria to surfaces has been shown by Marshall (1972) to be a result of physical attraction and the secretion of bridging polymers. Once firmly attached to the conidium it is possible that by activity of surface bound enzymes such bacteria could directly penetrate the spore wall. There are precedents for this manner of penetration. Btlt~llo~ihrio species parasitic on bacteria penetrate through discrete holes in the cell wall (Starr and Baigcnt. 1966). Old and Nicolson (1975) have observed perforations in the walls of epidermal and cortical cells of grass roots recovered from the field (Fig. 12) and have shown electron micrographs of bacteria occupying perforations and channels in the walls of lysing cells. Development of holes in the conidium wall from annular depressions is much more difficult to explain. The loop-forming actinomycetes are of particular interest however. because the loops are approximately the same size as the annular depressions formed during perforation of spore walls. Of I30 loops measured at random the mean dia was 3.4~~m. These are the onI4 micro-organisms so far observed on spore surfaces which could. by the activity of enzymes bound to the cell surface. cause erosion of the conidium surface in the annular pattern described above. This however is only a tentative hypothesis. Dr. S. T. Williams (personal communication) has suggested that the loops may rcprcscnt stages in the formation of spore chains and as such are structures unlikely to be engaged in active Iysis of the spore sufacc. Invcstigations of the causal agents and mechanism of spore perforation arc continuing. PERFORATIOA

OF ‘TH4h

SPORES

OF

SPECIES

OTHER

C.. S,lT11’15

A variety of pigmented species other than C. .sati~~ have been incubated in natural soils and subsequently observed for perforations. These include Curruluriu /Jl.OfldX’I.N~~L.

Stc’,ll/~~l!‘//iZ//f1

dUldl.itiC11111.

,dtC’,_Ml,iU

and Str~c~/~~hor~~,.c sp. Conidia of these species were incubated both on the surface of natural or steamed soil. and on buried filter membranes. The results arc shown III Table I. All of these species bccamc pcrforatcd and lyscd. C’onidia of C/~dospo~iurtl sp. also became perforated after 90 days incubation in natural soil. Holes developing in S. rlrndriticum tcwuis.

Perforation

Plate 2. SEM of surface Fig. 6. Bacteria Fig. 7. Spore

with

enveloped

long

of fungal

microflora

spores

of lysing conidia

filamentous appendages (arrows) associated in the spore surface (X 8.000).

by actinomycete

filaments.

Note

289

in soil

the tendency

of C. ,sutilu,s. with

an annular

of the filaments

depression to fragment

(x 2000). Fig. 8. Spore Fig. 9. Loop

with actinomycete formed

cells on the surface. Note (x 2150).

the loop-shaped

by actinomycete hypha (arrowcd). The dia of the loop same as that of a perforation (P)( x 5650).

configuration is approximately

(arrow) the

K. M. OLD and J. N. F. WONG

290

Plate 3. SEM of surfaces

of conidia

(Figs.

I I and I?) and epidermal

10.

cells of Anu~iplrilu

urruirria

(L) Link. Fig. IO. Conidium Fig.

1I. Conidium

Fig. t2. Epidermal

of C. sutirus of A. rmuis

(Cl with perforation (C)

with bacteria

cells of A. arr%ciriu showing

(P) occupied

(R) adhering

perforations

by bacteria

to the spore wall

similar

to those

found

( x 1900). Fig. 13. Conidia

of Stachyhorry,s

showing

perforations

(P) (x 2150).

(x 7,000). ( x 5650). in fungal

spores

291

Perforation of fungal spores in soil Table I. Lysis and perforation of conidia of five fungi on membranes soil incubated at 25°C and recovered

buried in garden

“/, lysis (l) and percentage of perforated conidia (p) after recovery from soil Day 30

Day 60

1

Day

1

P

1

Day

15

P

1

P

1

P

1

Day 80 P

8 8

0 0

24 7

0 0

63 14

59 0

75 17

60 0

67 38

60 0

3 7

0 0

0 1

0 0

16 0

13 0

39 16

13 0

59 24

14 0

4 3

0 0

5 5

0 0

39 7

11 0

51 24

6 0

36 34

20 0

0 0

-0 0

0 0

0 0

31 0

30 0

88 13

63 0

51 13

42 0

0 3

0 0

0 8

0 0

74 64

41 0

78 39

70 0

79 43

76 0

C. sativus

Natural soil Steamed soil* C. protuberata Natural soil Steamed soil A. tenuis Natural soil Steamed soil S. drndriticum Natural soil Steamed soil Stachyhotrys sp. Natural soil Steamed soil * lOO”C/45 min.

and Stachybotrys sp. conidia were only 1-2 pm in diameter (Fig. 13) compared to the larger holes, 2-4pm in C. satiuus. After 60days most conidia of Stachybotrys were completely disrupted and it was difficult to distinguish perforations from other damage. Annular depressions were observed with C. sativus and C. protuberata but not with the other species. Reisinger and Kilberlus (1973) showed that with Dendryphiella vinosa and H. spiciferum holes were produced in spores cultured with bacteria isolated from mite faeces but no annular depressions were seen on the surface of lysing conidia. Recently in our laboratory, while observing resting spores of Endogone recovered from natural soil, several annular depressions were observed on the bulbous attachment of spores to vegetative hyphae. In addition to these annular depressions, the attachment and spore wall were perforated by holes of approximately 0.5 pm dia. Clough and Patrick (1972) showed that the chlamydospore walls of Thielaviopsis b&cola became perforated, and bacteria colonized spore walls and cell lumina. Perforated spores were recovered from soil samples collected in the field as well as with spores introduced artificially into soil samples. Perforations were from 0.1-0.5 pm dia and developed after incubation of the chlamydospores in natural soil for 4 weeks or more. Following a thorough examination of the occurrence and nature of these perforations they concluded that soil bacteria were probably the causal agents (Clough and Patrick, 1973, 1976). Annular depressions were never observed in the spore wall. It may be significant that actinomycetes were rarely observed on lysing chlamydospores of T. basicolu and were not considered to play a part in perforation (Miss K. M. Clough. personal communication). Thus perforation of pigmented fungai spores in natural soils is a widespread phenomenon and occurs in a variety of species. The morphology and diameter of perforations varies, possibly because there are several causal agents. Our work suggests that both

soil bacteria and actinomycetes can initiate perforation of the spore wall. Any agency which pierces the outer pigmented layer of the spore renders it susceptible to colonization by a variety of soil microorganisms and the protoplast and inner wall layers are readily digested. Since pigmented fungal propagules are resistant to attack by cell-wall-lysing enzymes, it seems reasonable that in the heterogeneous population of the microbiota soil mechanisms should have evolved that breach the pigmented layer and render pigmented fungal propagules susceptible to biodegradation. Where the fungus under attack is a plant pathogen, such naturally-occurring mechanisms hold special significance in reducing disease in the field. The progress and problems in biological control of plant pathogens have been thoroughly reviewed (Baker and Cook, 1974) who make the case for exploitation of naturally occurring biological controls. The work described here provides an insight into some biotic factors affecting survival of C. sativus and may aid in the formulation of biological control measures for this and other soil-borne plant pathogenic fungi.

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292

K. M. OLD and J. N. F. WONC

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