Journal of' General Microbiology (1 986), 132, 118 1-1 187.

Printed in Great Britain

1181

Water Potential, Growth and Cellulolysis of Fungi Involved in Decomposition of Cereal Residues
By N . M A G A N * A N D J . M . L Y N C H Glasshouse Crops Research Institute, Littlehampton, West Sussex BNI 7 6LP, U K (Received 29 October 1985 ;retised 1I December 1985)

The effect of water potential and temperature on growth and cellulolysis of 10 soil fungi which colonize cereal crop residues was determined in tlitro. On 2% (w/v) milled straw agar all species grew in the range - 0.7 to - 7-0 MPa at both 10 and 20 "C. Growth rates differed depending on the solute type used to control water potential. In general, but with the exception of the Penicillium spp., growth decreased with decreasing water potential. Hyphal growth on sterile straw internode segments was much less than that on 2% straw agar. Fusarium culmorum and Trichoderma harzianum colonized straw pieces best at high potential ( - 0.7 MPa) while only F. culmorum and the Penicillium spp. grew at low potential (-7.0 MPa). Trichoderma spp., Gliocladium spp. and Chaetomium globosum cleared cellulose agar most rapidly, depth of clearing decreasing with water potential in the range -0.7 to -2.8 MPa at 20 "C. The relationship between dry weight loss of inoculated cellulose filter paper and water potential was more variable.

INTRODUCTION

Cereal straw residues remaining after harvest are colonized by a wide variety of fungi often classified as primary 'sugar' fungi and secondary cellulose and lignin decomposers (Garrett, 1970).As cellulose and hemicellulose are the major components of straw, contributing up to 75 % of the dry matter (Harper & Lynch, 1981), those species able to utilize these constituents play a particularly active role in decomposition. The major abiotic factors which determine both primary and secondary colonization and rate of decomposition are the water potential of the substrate, temperature and perhaps pH and gas composition (Griffin, 1972; Swift et al., 1979). Fungi colonizing wheat straw have been shown to belong predominantly to the genera Fusarium, Mucor and Penicillium with species of Trichoderma, Gliocladium, Aspergillus, Pythium and Cephalosporium among others isolated (Sadavisan, 1939; Walker, 1941). More recently Harper & Lynch (1985) studied a range of fungi which colonize straw and found that Trichoderma spp., Sordaria alcina and Chaetomium globosum were particularly active in colonizing straw and decomposing cellulose in vitro. However, in all these studies, the water relations of the soil/straw system and the influence these may have on growth and cellulose decomposition were not considered. This is particularly important in understanding the possible interaction between groups of fungi competing for the straw substrate. Some Penicillium species grow optimally at slightly reduced water potentials down to - 2.8 MPa (Hocking & Pitt, 1979) while Aspergillus species, e.g. the A . glaucus group, grow optimally at - 7.0 to - 14-0MPa (Ayerst, 1969; Magan & Lacey, 1984~). These groups of fungi may therefore under some circumstances dominate the substrate (Chen & Griffin, 1966). More attention has perhaps been given to the water relations of plant pathogens such as Fusarium culmorum and Cephalosporium gramineum, which colonize and survive on crop residues, than to those involved in decomposition (Cook & Duniway, 1981). Although water potential and temperature have been shown to affect the rate of decomposition of milled straw (Bartholomew & Norman, 1946; Sain & Broadbent, 1975; Roper,
0001-3043 0 1986 SGM

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N . MAGAN A N D J . M. LYNCH

Water potential (-MPa) Fig. 1. Relationship between water potential and water content (adsorption) for wheat straw (cv Norman) previously dried at 45 "C.

1985), little information is available on the effect these factors may have on the ability of different soil fungi to grow on straw substrates and to utilize cellulose. This paper presents information on the effect of water potential and temperature on growth and cellulolysis of a range of soil fungi in uitro.
METHODS

Fungal isolates. The isolates used in this study were isolated from decomposing crop residues in soil and were Acremonium persicinum (Nicot) Gams (IMI 284720), Chaetomium globosum Kunze ex Stend. (IMI 286935), Gfiocfadiumroseum Bain (R190), Gliocfadiumuirens Miller (R193), Fusarium culmorum (W. G. Sm.) Sacc. (R191), Penicilfiumjanthinellum Biourge (IMI 284724), P . hordei Stolk ( P . hirsutum Dierckx) (IMI 284723), Trichoderma harzianum Rifai (IMI 275950), T. hgibrachiatum Rifai (IMI 284728), and T. viride Pers. :Fr. (IMI 298375). Those cultures with IMI numbers are held by the Commonwealth Mycological Institute (CMI) and those with R numbers are in the GCRI culture collection. Waterpotentialandgrowth. Freshly harvested straw (winter wheat cv. Norman, C : N ratio 130: 1) was milled to pass through a 0.5 mm mesh and used to prepare 2% straw agar (2%, w/v, milled straw and 2%, w/v, technical agar no. 3: Oxoid) modified osmotically with glycerol (Dallyn & Fox, 1980)and KCl (Campbell & Gardner, 1971)in the water potential range -0.7 to - 14.0 MPa. Straw agar (pH 5.5) in 9 cm Petri dishes was inoculated centrally with 4 mm discs of test fungi, taken from the margins of 7-10-d-old colonies on potato dextrose agar. Peniciffiumspp. were inoculated as a fine loop ofspore suspension (Pitt, 1979).Plates were stacked and sealed in clean polyethylene bags and then incubated at 10 and 20 "C for 7-28 d depending on the water potential. Growth was measured along two diameters at right angles to each other, usually at daily intervals. Experiments were done twice with three replicates on each occasion. Radial growth rates in mm d-' at different combinations of water potential and temperature were calculated for each test species. The standard errors of mean radial growth rates were calculated. Internode sections of straw (cv. Norman, 5-3cm long and 2.5-3 mm wide) were cut carefully to avoid splitting and dried at 45 "C to constant weight (approx. 2.5% water content, wet weight basis). Known amounts of straw were autoclaved at 121 "C for 30 min. Known volumes of sterile distilled water were carefully added to groups of sterile straws in 250 ml flasks, as calculated from a straw-water adsorption curve (Fig. 1) to obtain water potentials of -0-7, -2.8 and -7.0 MPa. Flasks were sealed and kept at 5 "C for 24 h with regular mixing to allow equilibration. Sterile straw internodes were placed carefully in three compartments of a 9 cm Petri dish divided into four sections. The fourth contained 2% water agar modified with KCl to the same water potential as the straw to help maintain an accurate potential. A 4 mm disc, 0.5-1 mm thick, of a test fungus was inoculated onto the upper surface of one end of the straw segment. Petri plates were sealed with masking tape or Parafilm and incubated at 20 "C. The Petri dishes were examined daily for 7 d and then as required for a maximum of 14 d. Growth was measured along the length of the straw segments using a dissecting microscope. For each species the experiment was done with five replicate Petri plates for each water potential (total of 15 straw segments). The standard error of the mean growth achieved was calculated. Water potential and celfulofysis.The ability of fungi to utilize cellulose was examined by two methods. (a) The clearing of 2% ball-milled cellulose powder (Whatman C F 1 1,72 h) in a mineral salts medium (Dalton & Postgate, 1969) modified with glycerol to -0.7, - 1.4 and -2.8 MPa. The medium was adjusted to pH 6 with 0.1 M-HCI.

Water relations of' soil fungi

1183

4-

P.janthinellum

P. hordei

1I

2.8

5.6

8.4 11.2 2.8 Water potential (-MPa)

5.6

8.4

11.2

Fig. 2. Effect of water potential on radial growth (mm d-I) of Trichoderma harzianum, Chaetomium globosum, Penicillium janthinellum and P. hordei on 2 % (w/v) straw agar. Water potential was controlled osmotically with glycerol (a)and KCl (0). Bars represent SE.

The depth of clearing of the cellulose agar in test tubes (15 x 1.5 cm) was determined after incubation at 20 "C for four weeks. The experiment was done twice with three replicates on each occasion. (b) Loss in dry weight of filter paper (five Whatman no. 3, 7.5 cm) by the method of Garrett (1963) as modified by Forbes & Dickinson (1977). The water potential of the mineral salts medium was altered to -0.7, - 1-4 and -2.8 MPa with NaCl (Scott, 1957). Glycerol was substituted with NaCl in this experiment to maintain a C :N ratioof 180, as used originally by Garrett (1963). The medium was adjusted to pH 6 as before. The filter papers in 250 ml flasks were inoculated with 4 mm agar plugs from the margin of growing fungi. The flasks were incubated at 20 "C for four weeks. Wads of filter papers were then dried to constant weight at 80 "Cand compared with that of control wads of the same water potential. The experiment was done twice with three replicates per fungus at each water potential. Water potential measurement. The water potentials of agar media, mineral salts solutions and straw samples, to determine the moisture adsorption curve, were all checked with a 100 channel automated thermocouple psychrometer (Stevens & Acock, 1976) for high water potentials and a Sinascope (Sina, Switzerland) at low water potentials.
RESULTS

Fig. 2 shows the effect of water potential on the growth of four of the test fungi. Some differences in growth were apparent between media with the water potential amended by glycerol and KCI. Growth was slightly greater when KCl was used as solute at high water potentials while at low water potentials growth on glycerol-amended media was greater.

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N . MAGAN A N D J . M . LYNCH

Table 1. Relationship between water potential and radial growth rate at I0 and 20 "C on 2% (wlu) straw agar modiJied with glycerol
Radial growth rate (mm d - ' )

Water potential (-MPa) . . . 0.7 Species 2.3 A . persicinum 1.7 C. globosum 2-9 F . culmorum 1.2 G . roseum G . uirens 1.3 1.2 P . hordei 1.1 P . janthinellum T. harzianum 3.5 T. longibrachiatum 2.6 T. r?iride 3.2 0.23 Pooled SE

10 "C
A

20 "C 9.8
NG NG

2.8 1.7 1.4 2.3 0.3 0.9 1.7 1.4 2.4 1.5 1-4 0.20

7.0 0.3 0.2 1.0 0.1 0.1 1.0 0.2 0.6 0.7
NG

14.0
NG NG NG NG NG

>

0.7 4.9 4.6 7.8 3.0 2.4 2.9 1.7 9.3 8.4 0.67

2.8 3.5 3.5 4.8 1.6 1-4 2-7 1.9 7.2 6.1 4.6 0.68

7-0 0.7 0.7 1.7 0.2 0.3 2.0 1-4 1.9 1.4 0.6 0.41

9.8 0.4 0.1 0-8

NG

14.0
NG NG NG NG

0.4

0.2

0.8 0.2

NG NG

0.6 0-1

NG NG NG

NG NG NG

11.5

0.15
NG.

0.08

0-03

0.3 1.6 0.3 0.8 0.2 0.1 0-10

1.0 0.2

NG NG NG

0.07

N o growth.

Table 2. Efect of water potential on colonization of sterile internode straw segments after 14 d at 20 "C with, in parentheses, mean growth rates in mm d-'
Water potential (-MPa). Colonization (mm
SE)
. I

Species
A . persicinum C. globosum F. culmorum G . roseum G . virens P . hordei P . janthinellum T. harzianum T, longibrachiatum T, viride

..

0.7 0.7 (0.7) 2.2 (1.7) 10.9 (7.0) 1.8 (0.6) 0.1 (0.4) 1.7 (1.6) 2.9 (2.1) 10.5 (5.1) 1.6 (2-3) 1.0 (1.4)

2.8 3.0 f 0.7 (0-2) 7.2 f 1.3 (0.5) 36.0 f 6.4 (2.6) 3.7 f 0.8 (0.2) 2.8 f 0.7 (0.2) 18-0 f 1-O(l.3) 18.1 f 3.1 (1.3) 5.5 f 0.3 (0.4) 4.9 f 1.3 (0.4) 6.0 f 1.0 (0.4)
1.0

7.0 (0.1)
NG

9.7 f 23.9 f 98.5* f 8.5 f 6.2 f 22.2 f 29.4 f 71*0* f 32-8 f 20.1 f
NG,

6.3 f 0.5 10.9 +_ 0.7 3-5 f 0.3 1.0 0.4

1.0
NG NG

(0.5)

(0.8) (0.3) (0.1) (0.1) (<O*l)

* By extrapolation.
Chaetomiumglobosum was an exception to this. All species grew in the range - 0-7to - 7.0 MPa at both 10 and 20C on 2% straw agar (Table l), the growth rate usually decreasing with decreasing water potential. The Trichoderma spp. had the highest growth rates of all species tested between -0.7 and -2.8 MPa. The exceptions were Penicillium hordei (10 "C) and P . janthinellum (10 and 20 "C) where optimum growth occurred at -2.8 MPa when glycerol was used as solute. Growth of all species at 10 "C was less than 50% of that at 20 "C, especially at -0.7 and -2.8 MPa. Growth on sterile straw segments where water potential was controlled matrically was not directly correlated with growth on 2% straw agar (Table 2). Optimum growth was at - 0.7 MPa but was significantly reduced at a water potential of - 2.8 MPa. Only F. culmorum,P. hordei and P . janthinellum grew at a water potential of -7.0 MPa. The cellulose clearing test favoured fungi able to produce extracellular cellulases, for example Trichoderma spp., Gliocladium spp. and C. globosum. Depth of clearing decreased with decreasing water potential (Table 3 ) . Penicillium spp. and F. culmorum demonstrated poor activity in the clearing test over a period of four weeks. Dry weight loss of cellulose filter paper in

No growth.

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1185

Table 3. EJect of water potential on cellulolysis measured by clearing of' cellulose agar and loss in dry weight offilter papers afier 4 weeks incubation at 20 "C
Water Clearing (mm) potential ,* -( ( - M P a ) . . . 0.7 1.4 2.8 Species 3.5 A . persicinurn 5.0 C. globosum 9.0 6-5 5.5 6.0 4.0* 2*0* F . culmorum G. roseum 1-O* 9.0 3.5 G . cirens 2*5* 11.5 8.0 P. hordei 1-5* 6.0 5-0 3*5* P. janthinellum 5.5 T. harzianum 9.5 7.5 5.0 T. longibrachiatum 10.0 8.5 2.5 7.5 4.5* 2*0* T. tliride 1.0 0.9 0.6 Pooled SE Weight loss (mg)
r-*-,

0.7

1.4

2.8 15 40 124 20 100 260 80 64 180 47 13.7

170 220 220 120 264 240 200 60 194 34 28.9

230 40 60 20 170 240 100 50 160 45 16.5

* Partial clearing.

relation to water potential was more variable. C. globosurn, F. culmorum, G . virens and P. hordei caused greatest loss in weight of filter paper at -0.7 MPa while at the lowest water potential tested, -2.8 MPa, P. hordei, T. longibrachiatum and F. culmorum were most active.

DISCUSSION

Fungal colonization and decomposition of crop residues in soil occurs over a much wider range of water potential than that permitting crop growth, which becomes limited at about - 1.5 MPa, the so-called permanent wilting point (Griffin, 1972). Some decomposition of cereal straw has been detected at between - 19.6 and -28.0 MPa although active decomposition of rice straw occurred at - 14.0 MPa (Bartholomew & Norman, 1946; Sain & Broadbent, 1975). Most of the fungal species examined in this study grew optimally at a water potential of - 0.7 to - 1.4 MPa at both 10 and 20 "C, growth being markedly reduced below -2.8 MPa. Interactions between reduced water potential and temperature resulted in the greatest reduction in growth rate. In natural environments such interactions often determine the microbial communities colonizing the straw substrate. The higher growth rates obtained when KCl rather than glycerol was used to control water potential have been previously observed (Brownell & Schneider, 1984) and may be due to K + ions in the medium being accumulated by the microbial cells and perhaps serving as a compatible solute to assist in adjusting to water stress. However, high concentrations of the salt (low water potentials) can inhibit enzyme activity, e.g. isocitrate dehydrogenase, in some fungi (Luard, 1983).The glycerol in amended media may also have been utilized by some of the test fungi to help overcome water stress. Polyols, particularly glycerol, have been shown to function as compatible solutes, being accumulated by yeasts such as Saccharomyces rouxii (Brown, 1978) and by filamentous fungi such as Penicillium chrysogenum and Chrysosporium firstidium (Luard, 1982), conferring protection to enzymes at low water potentials. Much narrower ranges for growth of C. globosum and T. viride (minima of -8.4 and - 5.6 MPa respectively) were obtained on clean glass than were obtained in this study on 2% straw agar (Kouyeas, 1964). Nutrient status may influence the water potential range for growth (Griffin, 1972) and the latter medium is perhaps a more realistic in vitro substrate. More information is available on F. culmorum isolates causing seedling diseases and ear blight of wheat, growth occurring down to - 8.3 to - 11.2 MPa and - 14-0MPa, respectively (Cook et af., 1972; Magan & Lacey, 19843) and for P. hordei and P. janthinellum, which can grow over much wider ranges of water potential down to -21.0 MPa and lower (Pitt, 1973; Magan &

1186

N. MAGAN A N D J. M. LYNCH

Lacey, 1984a). Very little information is available on the water relations of the other fungi examined in this study (Domsch et al., 1980). The importance of considering growth in vitro on substrates as close as possible to that encountered in the natural environment was emphasized by the results obtained on the water relations of growth on sterile straw segments. Growth of most test species was considerably less than that obtained on straw agar, although competition from the natural microflora which would normally occur on the straw was absent. This may partly be due to the water potential in straw segments being controlled matrically while that in straw agar is controlled osmotically. The matric water potential range for growth of pathogens such as Alternaria alternata and Phytophthora cinnamomi in soil is less than the osmotic water potential range (Adebayo & Harris, 1971) while Cook et al. (1972) found little difference for I;. culmorum. Again, only P.culmorum and the Penicillium spp. grew on straw segments at a water potential of - 7.0 MPa. Although the Penicillium spp. often grow relatively slowly at high water potential (-0.7 MPa), at slightly reduced substrate water potential they may be at a competitive advantage. Although the cellulose clearing test suggested that optimum cellulolysis by Trichoderma, Gliocladium spp. and C. globosum occurred at a water potential of -0.7 MPa, the loss in dry weight of cellulose filter paper suggested that some fungi, particularly Penicillium spp., may be just as active at slightly reduced water potentials. Although the effects of some environmental factors such as temperature, gas composition and pH on cellulolysis of some fungi has been determined (Walsh & Stewart, 1971; Forbes & Dickinson, 1977), information on the effect of water availability is scarce. Aspergillus terreus hydrolyses cellulose best at its maximum water holding capacity, 400%, while at 300-100% cellulolysis is slower (Kundu et a/., 1983). Unfortunately, the relationship between water content and water potential of the cellulose was not determined in their study. Penicillium and Aspergillus spp. have the ability to degrade plant residues with a large cellulose/hemicellulose component (Chatterjee & Nandi, 1981). They therefore may be important components of the microbial community colonizing cereal straw (Kouyeas, 1964). Garrett (1975) suggested that successful saprophytic colonization of straw by a fungal species is dependent on the growth rate (metabolic activity) and ability to degrade cellulose while others have suggested that pectinase and xylanase activity may be just as important (Boothby & Magreola, 1984). Growth, nutrient utilization and competition between species may be markedly influenced by interaction between water potential and temperature (Magan & Lacey, 1984~). Individual species may be affected in different ways, influencing the colonization of the straw substrate. Although in vitro studies cannot alone satisfactorily explain the role of different species in decomposition, knowledge of interactions of these factors, growth and nutrient utilization may assist in understanding their ecological role.
We are grateful to Dr R. I. Grange for advice and use of the thermocouple psychrometer and to Nina Jenkins for technical assistance.
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