Soil Biol. Biochem. Vol. 7, pp. 323 to 327. Pergamon Press 1975. Printed in Gre a t Britain.
CELLULOLYSIS RATE A N D COMPETITIVE SAPROPHYTIC C O L O N I Z A T I O N OF WHEAT STRAW BY FOOT-ROT F U N G I S. D. GARRETT
Botany School, University of Cambridge, CB2 3EA England (Accepted 25 January 1975)
Summary--Competitive saprophytic colonization of wheat straw has been studied with five species of cereal foot-rot fungi: Fusarium roseum f.sp cerealis, Curvularia ramosa, Cochliobolus sativus, Gaeuman•nomyces oraminis var tritici, and Cercosporella herpotrichoides. Published records of determinations by the Cambridge method have been surveyed; by this method, usually 100 substrate units of wheat straw are buried in a series of inoculum-soil mixtures, in which a pure culture of the inoculant fungus (on 3% maizemeal in sand) is progressively diluted with increasing proportions of a natural (i.e. unsterilized) soil. The competitive saprophytic ability of a fungal species for straw colonization has been expressed by its C50 value, i.e. the highest percentage of unsterilized soil in an inoculum-soil mixture that still permits colonization by the inoculant fungus of 50 per cent of the total number of straws in a test sample. Because the C50 value may be affected both by temperature during the incubation period and also by nitrogen and/or glucose supplementation of the straws before burial, the records taken for analysis here have been restricted to experimental series in which straws had received no nutrient-supplementation before burial, and in which temperature during the month's incubation had been maintained within the range 20 + 3°C. Correlations have been sought between C50 values for the five fungi and some specific characteristics likely to affect competitive saprophytic colonization of wheat straw or of any similar mature plant tissue. Only two such characteristics have shown a positive correlation (significant at the 5% level) with C50 value; these are (I) straw-penetration rate and (2) cellulolysis rate, as determined by rate of loss in dry weight by filter-paper cultures. Straw-penetration rate is very closely correlated (significant at 0.1% level) with cellulolysis rate, suggesting that speed of penetration of mature cell-walls depends on rate of enzymic degradation of the wall around the apices of penetrating hyphae; this relationship appears to have escaped adequate recognition and definition until now. Confirmatory evidence for these conclusions is furnished by the saprophytic behaviour of Rhizoctonia solani in colonizing other kinds of mature-plant tissue from natural inoculum in the soil; a single reported trial by the Cambridge method gave a C50 value of 99 and with this is associated a high cellulolysis rate in axenic culture.
INTRODUCTION
Fusarium roseum (Lk. ex Fr.) emend. Snyd. & Hans. Pathogenic root-infecting fungi are potentially cap- f.sp. cerealis (Cke.) Snyd. & Hans. cv. 'Culmorum'. able of two kinds of saprophytic behaviour. Firstly, Hereafter referred to as F. roseum [syn. F. culmorum they may compete with obligate saprophytes, and (W.G.Sm.) Sacc.]. Curvularia ramosa (Bain.) Boedijn. with some other root-infecting fungi, for colonization Cochliobolus sativus (Ito & Kuribay.) Drechsl. ex of a corpus of dead plant tissue lying in or on the soil. The degree of success in such competitive sapro- Dastur (stat. conid. Helminthosporium sativum P.K. & phytic colonization attained by a particular fungus B.). Gaeumannomyces 9rarninis (Sacc.) Arx & Olivier at a given locus in the soil depends upon: (1) the competitive saprophytic ability of the fungal species var. tritici Walker. Hereafter referred to as G. 9raminis for colonization of the particular substrate; (2) its in- [syn. Ophiobolus 9raminis (Sacc.) Sacc.]. Cercosporella herpotrichoides Fron. oculum potential at surface of the substrate; and (3) the total soil environment, including microbial population (Garrett, 1970, chap. 5). Secondly, most rootThese fungal pathogens are responsible for the infecting fungi are capable of saprophytic survival, major cereal foot-rots, including take-all (G. graminis~ sometimes prolonged, in the dead host tissues invaded common root rot (C. sativus and Fusarium spp.), and during the parasitic phase. In experimental work with eyespot lodging (C. herpotrichoides). C. ramosa is a some of these fungi, precautions must be taken to less important pathogen and is not well known outdistinguish between saprophytic survival and dor- side Australia at the present time (Butler, 1961). The mant survival in the form of spores or sclerotia. present paper reports conclusions derived from a Amongst cereal foot-rot fungi, Fusarium roseum f.sp quantitative analysis of results from various expercerealis is known to be a vigorous saprophytic col- iments on competitive saprophytic colonization of onizer of baits of fresh wheat straw buried in field wheat straw by these five fungal pathogens, to which soil (Sadasivan, 1939; Walker, 1941). At Cambridge, some workers outside Cambridge have also contriover a period of 20 years, we have studied competitive buted. It follows a similar analysis of data on saprosaprophytic colonization of straw by a laboratory phytic survival in wheat straw by the same five fungal method, employing the following five fungal species: species (Garrett, 1972). 323
324
S.D. GARRETT ESTIMATION OF COMPETITIVE SAPROPHYTIC COLONIZATION
A quantitative method for assessment of success in competitive saprophytic colonization of wheat straw by cereal foot-rot fungi was first developed by Butler (1953a) and Lucas (1955) in collaboration with me, and was subsequently employed at Cambridge by Macer (1961) and Deacon (1973). The technique has become known as the 'Cambridge method' (Garrett, 1970, chap. 5) and has been used by Gerlagh (1968) in the Netherlands and by Burgess and Griffin (1967) in Australia; it has been adapted for use with Rhizoctoaia solani Ktihn by Zarka (1963). By this method, substrate units of wheat-straw are buried in a series of inoculnm-soil mixtures, in which a pure culture of the inoculant fungus (on 3% maizemeal in sand) is progressively diluted with increasing proportions of a natural (i.e. unsterilized) soil. The percentage of straws colonized by the inoculant fungus is assessed after an incubation period of 4 weeks at laboratory temperature (range 16-22°C; mean 19 + l°C) in each of the inoculum-soil mixtures. The full range of mixtures contains percentages of inoculum as follows: 100 (pure culture alone), 98, 90, 50, 10, 2 and 0 (soil alone). The wheat-straw units are cut to a length of ca. 3"8 cm, each with a node at the lower end, subtending an encircling leaf-sheath; 50 autoclaved straws are buried in ca. 200 ml of inoculum-soil mixture and the standard test sample is 100 straws from two containers of each inoculum dilution. The data for analysis have been taken from the six papers cited above, but with the proviso that experiments, or parts of experiments, had been standardized in respect of the following (1) incubation period of straws in the inoculum-soil mixtures of 4 weeks 1 month at a mean temperature of 20 + 3°C and (2) straws not treated before burial with any nutrients; in some experiments, straws had been soaked in solutions of nitrate and/or glucose. The reasons for these restrictions are as follows. Burgess and Griffin (1967) found that, for the 4 fungal species they studied (Fusarium roseum, Gibberella zeae, Cochliobolus sativus and C. spicifer), percentage of straws saprophytically colonized generally appeared to increase with a reduction in incubation temperature from 3020 down to 10°C. A similar effect of temperature was reported by Gerlagh (1968) in an experiment with Gaeumannomyces graminis. This temperature-effect has been re-investigated by Deacon (1973), who has shown it to be largely an artefact of the Cambridge method, at least for G. 9raminis and Cercosporella herpotrichoides. He found that a reduction in the incubation period at 21°C from 28 to 9 days produced an increase in the apparent percentage of straws colonized; this increase was of an order comparable to that produced by keeping the incubation period at 28 days but lowering the incubation temperature to 10°C. So Deacon concluded that in some of the straws, perhaps incompletely colonized by the ,inoculant fungus at the outset, the primary colonizer was quite quickly replaced by other soil microorganisms, thus resulting in an apparent decline in percentage straws colonized as the incubation period was increased from 9 to 28 days at 21°C. Lowering the incubation temperature from 21 to 10°C was postulated as slowing down rate of replacement of the primary fungal colonizer by other -
microorganisms, thus producing an apparent increase in percentage straws colonized after 28 days at the lower temperature. Deacon's analysis of this situation has also explained some observations by Butler (1953a), which had hitherto been difficult to interpret except in rather vague terms. Butler had found that pretreating the straws with nitrate-nitrogen increased the saprophytic success of Gaeumannomyces 9raminis but reduced that of Cochliobolus sativus. It has been firmly established that an ample supply of nitrogen prolongs saprophytic survival in colonized straw by G. graminis but shortens survival by C. sativus (Garrett, 1972). Butler's observations on these two opposed effects of nitrogen pretreatment of straw can now be explained as effects on early trends in saprophytic survival of the two fungi, between initial colonization of the straws and the time of assessment, after the 4 week incubation period in the inoculumsoil mixtures. From each set of data abstracted for this survey, percentage of straws colonized by the inoculant fungus has been plotted against inoculum dilution; from this plot the highest percentage of soil that still permits colonization of 50 per cent of the total number of straws buried has been read off, and is designated as the C50 value (Table 1). Table 1 shows that C50 values for Fusarium roseum and Curvularia ramosa all stand at 98, the highest inoculum dilution that had been tested. For F. roseum this high C50 value is compatible with other evidence that it is a vigorous competitive saprophyte in colonization of fresh wheat straw buried in soil (Sadasivan, 1939; Walker, 1941). But if the straw has become precolonized by air-borne fungi before burial, as when harvest weather has been wet or ploughing under the soil has been long delayed, then such "weathered" straw is a much less available substrate for F. roseum (Cook and Bruehl, 1968). The range of C50 values so far obtained for Cercosporella herpotrichoides is quite narrow, but the corresponding ranges for Cochliobolus sativus and Gaeumannomyces yraminis are rather wide. The chief source of variability resides in the soil type used for inoculum dilution, but such variation can provide additional information about the factors involved in saprophytic competition as further data become available. Other things being equal, we should expect that a soil with a high organic matter and total nitrogen content would have a larger microbial population than a soil with a lower organic content, and would be likely to give lower C50 values, because of a higher degree of microbial competition. We have used Kettering soil, a loam collected from under old grassland, with 5~o organic matter and 0"33~o total N, and with pH around 6.0. With this soil, C50 values for G. 9raminis varied from 2 to 50. Gerlagh (1968) has compared two polder soils in the Netherlands in a single experiment with G. 9raminis by the Cambridge Table 1. Highest percentage of unsterilized soil permitting colonization of 50~oof the total number of straws by the inoculant fungus (C50 value)
io.
~ bll~L,~au
4
2
5
e==em ,t.= C50 ~3,~e
/,11 9a
Bot,h 9e
25-95
2-,5o
2-9
98
98
47
23
4
MM,a C50 ~
a
4
Saprophytic behaviour of foot-rot fungi method. At an incubation temperature of 23°C, a C50 value of 20 was obtained with one soil type, but for another soil, described as more "antagonistic" to G. #raminis on the evidence of other kinds of experiment, a.C50 value of only 5 was obtained. At an incubation temperature of 17°C, the difference in C50 values between the two soils was still wider. Employing Kettering soil as the inoculum diluent with Cochliobolus sativus, we obtained C50 values ranging from 28 to 44. But Burgess and Griffin (1967) obtained colonization of 100~ of straws at their highest inoculum dilution of 95~o soil, indicating a C50 value of >95 at an incubation temperature of 20°C. Their soil, from South Queensland, is described as a semi-arid, brown soil of heavy texture at pH 7-7; they ascribe their high C50 value for C. sativus to the fact that the organic matter content of their soil was lower than that of Kettering soil, as well as differing from it in respect of texture, pH, etc. ASSESSMENT
OF
FACTORS
SAPROPHYTIC
CONTRIBUTING SUCCESS
TO
"
Straw-penetration rate Macer (1961) was the first to realize that strawpenetration rate was likely to be more closely associated with success in saprophytic colonization of straw than was surface growth rate of a fungus as measured on a colony growing on nutrient agar. He therefore determined straw-penetration rate by these five fungi (listed in Tables 1 and 2) under axenic conditions. This was assessed from the time taken by each fungus to grow through pieces of autoclaved wheat-straw (culm surrounded by leaf-sheath subtended by inferior node), from the outside of the sheath to the lumen of the culm, into which had been inserted a 2"5 cm length of narrow culm-straw. The autoclaved straws were buried in pur~-culture inoculum on 3~o maizemeal in sand, and time taken to complete penetration at 22.5°C was estimated by planting out the culm inserts from 45 replicate straws inoculated with each fungus at 12-h intervals, until all straws had been completely penetrated at the final sampling for each fungus. From Macer's table of data, I have calculated the relative rates of straw penetration by each of the five fungi, based on the time taken to penetrate 50 per cent of the straws; these rates, expressed in convenient arbitrary units based on reciprocals of straw-penetration time, are given in Table 2, together with Macer's measurements of surface growth rate over potato-dextrose agar at 22.5°C for the same fungal isolates. Inspection of Table 2 shows the absence of any correlation between surface growth rate and strawpenetration rate amongst the five fungi, which is confirmed by calculation of the correlation coefficient for association between the two variables; r = 0"3786, which is not significant. Table 2. Surface growth rate and straw-penetration rate at 22.5°C P~.a~
f~q'loe IWo~Os m,t, ( m / U ~)
10.75
~ t A c m m r * (Lv~J.t~,,~ w.t',.)
10.0
eex'vaxR~ ~ a a / ~ t a l M ~ w ~ m ~ imt.t~ ~ 4.80 10.4
~.tO 10.~
hK..~2ahetma
8.(0
1 .~j
6.4
4,6
325
Table 3. Mean percentage loss in dry weight of filter-paper cultures after 7 weeks at 22.5°C I~mm~Imll o=++*.tRIJ
11o. Slolltlm In ~
4 6.~-8.5
1
c++++.l.'t+bo.l~-~ ~
6 5.4~.4
5 :e.l-~J.I;
4 0.8-1.1
Cellulolysis rate A t t h e time of Macer's (1961) study of saprophytic colonization, no information seemed to be available on cellulolytic ability of any of these five cereal footrot fungi. In a series of studies, I provided this information (Garrett, 1963, 1966, 1967, 1971). This work was originally designed to furnish a possible explanation of why these fungi varied widely in their response to nitrogen in terms of their longevity of saprophytic survival in wheat-straw; this hope was eventually realized in terms of an explanation that accommodates all the known facts (Garrett, 1972). Not until recently, however, have I discovered that cellulolytic ability appears to be closely connected with strawpenetration rate, and that both are associated with success in competitive saprophytic colonization, as expressed by the C50 value. Cellulolysis rates of the five fungi have been assessed by determining percentage loss in dry weight of filter-paper wads inoculated with each fungus and incubated for 7 weeks at 22.5°C. The method was standardized and full details are given in Garrett (1966), with one exception overlooked at the time; this is that the agar disc used to inoculate a filterpaper wad has always been placed at the perimeter of the wad, and not centrally. This allows quicker and easier detachment of the agar disc from the inoculating needle than does a central placement, but it is essential to note that any variation in inoculum placement will affect reproducibility of results with this method, especially with fungi having a slow rate of growth, such as Cercosporella herpotrichoides. A summary of results is given in Table 3, which is reproduced from Table 10 in Garrett (1970, p. 155), with the addition of standard errors of the mean for all isolates tested of each fungus, calculated from analyses of variance for each trial. The data given in Table 3 are all from my own determinations, which guarantees uniformity of procedure even in minor details. Working with five other isolates of Cercosporella herpotrichoides, Deacon (1973) obtained a mean loss of 0.79~ in dry weight of filter-paper cultures incubated for 7 weeks at 21°C, employing the same mineral nutrient solution as mine with K2HPO4, at pH 7.2. When he substituted KH2PO4 as the potash phosphate source, buffering at pH 5"4, then mean loss in filter-paper dry weight for the same five fungal isolates was rather higher, at 1-20~o. Working in collaboration with me and employing a selection from my isolates of these five fungi, Bhargava (1972) demonstrated a close correlation (r = 0"918, significant at the 0.1~ level) between rates of decomposition of filter-paper cellulose and of wheatstraw, respectively, after the standard incubation period of 7 weeks at 22.5°C. The connection between x, representing percentage loss in dry weight of
326
S.D. GARRETT
wheat-straw cultures of the five fungi, and y, representing loss in filter-paper cultures, is given by the equation x = y + 6-26. The value 6'26 represents the percentage loss in dry weight of a wheat-straw culture that is caused by a fungal isolate unable to decompose filter-paper cellulose, and was exemplified by one isolate of Cercosporella herpotrichoides with a negligible degree of cellulolytic ability. DISCUSSION
Macer (1961) was the first to suggest that strawpenetration rate might be an important component of competitive saprophytic ability for colonization of this substrate, and he made an assessment of this rate for these five fungal species (Table 2). Since that time, more C50 values for colonization of wheat straw by these fungi have become available (Table 1), and so I have had to revise my previous estimates (Garrett, 1970, Table 5; 1972, Table 2). Even on preyious estimates of C50 values for these fungi, a possible relationship between these values and straw-penetration rates could be discerned; more recently I have discovered a relationship between straw-penetration and cellulolysis rates. A test for the closeness of association between these three sets of variables is provided by the correlation coefficient, r, and so values of r, together with the degree of their statistical significance, are set out in Table 4. Table 4 shows firstly that the correlation between straw-penetration rate and cellulolysis rate is a highly significant one, at the 0"1~o level. The only surprising aspect of this correlation is my failure to notice it earlier. Wheat straw is a dead plant tissue, even before autoclaving, and thus offers no active host resistance to invasion by fungi. The only resistance offered to hyphal penetration is therefore the mechanical resistance of the cellulose cell-walls, some of which are lignified. Enzymic degradation of the cell wall around the apical region of a penetrating hypha will therefore facilitate and hasten cell-wall penetration. The microscopical aspects of this process have been reviewed and interpreted by Liese (1970). So it now seems likely that cellulolysis rate is the chief determinant of straw-penetration rate. Table 4 also shows correlations, significant at the 5~o level, between C50 value and straw-penetration rate, and between C50 value and cellulolysis rate. Because the link between cellulolysis rate and strawpenetration rate is so close, it seems probable that the association between cellulolysis rate and C50 value is mediated by straw-penetration rate. We are not entitled to expect that the correlation between either straw-penetration rate or cellulolysis rate and C50 value should be any closer than this, because competitive saprophytic ability for colonization of the straw substrate must have a number of components Table 4. Significance of correlations between straw-penetration rate, cellulolysis rate and C50 value for the five fungal species m,Ct'~ ot W l u e et •
m~.m~
~twlM,a i%a'iW-l~llr~/l~t/~ alxl ~llulollmdJ ~ I
0.9741
A¢ 0.1~ 1 ~ 1
~e~qen a ~ a e - l m l ~ t l o n
0.8651
At ~,~ ~ i
x~te ~
c5o ~ l u e
in any species of fungus. Tolerance of fungistatic growth products liberated by other microorganisms is one such component of saproph3;tic ability. At the outset of this work on saprophytic competition, Butler (1953b) demonstrated that the vigorous saprophyte Curvularia ramosa possessed a higher tolerance to most of the antibiotics he tested in vitro than had Cochliobolus sativus, a taxonomically similar and possibly closely related species. Recognition of this factor in competition also implies acceptance of the converse proposition, i.e. that a fungus producing a fungistatic growth product may be able to depress the activity of competing microorganisms and hence to gain an advantage. Convincing evidence that production of an antibiotic can prolong saprophytic survival has been furnished by Bruehk Millar and Cunfer (1969), working with Cephalosporium 9ramineum Nisikado & Ikata, which causes a vascular wilt, known as stripe disease, of winter wheat. This fungus produces a wide-spectrum, antifungal antibiotic. In a comparison between wild-type isolates and laboratory mutants that had lost the ability to produce the antibiotic, Bruehl et al. (1969) demonstrated that the wildtype isolates survived for longer in colonized wheat straws buried in soil, and were better able to exclude invasion by other fungi, than did the laboratory mutants. They therefore concluded that antibiotic production is an ability possessing survival value, and must have been conserved by natural selection in the wild-type population. Despite this complexity of saprophytic competition, it now seems that speed of tissue penetration, mediated by cellulolysis rate, is an essential component of competitive saprophytic ability for invasion of dead, mature plant tissues lying in or on the soil. This explains at least in part the sucess of Rhizoctonia solani as a pioneer colonizer of old dead stems of buck~vheat, cotton and cereals buried in soils naturally infested by this fungus (Papavizas and Davey, 1961, 1962; Sneh, Katan, Henis and Wahl, 1966). Zarka (1963) obtained a more precise estimate of competitive saprophytic colonization by R. solani through his use of the Cambridge method; as substrate units, he employed l cm segments of dried mature stems of Corchorus olitorius L.. with a replication of 100 segments for each inoculum dilution. A pure culture of R. solani on 1°/,, maizemeal in sand was progressively diluted with a sandy loam (pH 6.1) containing 3"6~o organic matter. The C50 value given by his data exceeds 99, after an incubation period in the inoculum-soil mixtures of 10 days at 18-19°C, which was the maximum period tested. For this ecological niche, R. solani is equipped by a high cellulolysis rate; I found that it caused 9-10°,~i loss in dry weight of filter-paper cultures after 6 weeks at 22°C (Garrett, 1962). These observations on R. solani thus suggest that the relationship between cellulolysis rate and success in saprophytic competition, demonstrated in this paper for a group of cereal foot-rot fungi, may have a wider application. Acknowledoements--I wish to thank all my former research associates who have collaborated in this project and particularly Dr. F. C. Butler, now Senior Deputy DirectorGeneral of the Department of Agriculture, N.S.W., whose energy and initiative contributed so much to the launching of this enterprise.
Saprophytic behaviour of foot-rot fungi REFERENCES
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