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Behaviour of a plant growth-promoting sterile fungus on agar and roots of rye-grass and wheat
Mycol. Res. 93 (2); 161-166 (1989)
Printed in Great Britain
Behaviour of a plant growth-promoting sterile fungus on agar and roots of rye-grass and wheat
M. M. DEWAN AND K. SIVASITHAMPARAM Soil Science and Plant Nutrition Group, School of Agriculture, The University of Western Australia, Nedlands, Western Australia, 6009
Behaviour of a plant growth-promoting sterile fungus on agar and roots of rye-grass and wheat. Mycological Research 93 (2); 161-166 (1989).
The radial extension of a plant growth-promoting sterile red fungus (SRF) after 3 d on various agars at 25°C was most on wheat meal agar and least on water agar. Maximum growth on potato dextrose agar (PDA) was at 25° and minimum at 30°. The pH optimum for growth on PDA was 5'5 and minimum was 4'0 both after 3 d at 25° or 70 d at 5°. At 5° the SRF produced drops of exudate around the fungal disk in the centre of the plate. These appeared in all pH treatments after 20 d, with the exception of pH 4'0 and 4'5; most appeared between 50-60 d for all treatments, the amount exuded declining after this period. More exudate was evident at pH 7'5 between 50 and 70 d than in any other treatment. The exudate inhibited growth of Gaeumannomyces graminis var. tritici (Ggt) on agar. The SRF colonized roots of wheat (Triticum aestivum cv. Gamenya) more aggressively than those of rye-grass (Lolium rigidum cv. Wimmera) both in sterilized and non-sterilized soil. Ggt had no effect on the progression of the SRF on the roots of wheat or ryegrass when they were co-infested in sterilized and non-sterilized soils. The SRF colonized the entire root system and crown regions of both hosts 4 d after incubation in both soil treatments. The extent of colonization by the SRF, whether alone or with Ggt, was more in non-sterilized than sterilized soil. The SRF protected the roots of wheat and rye-grass from Ggt in sterilized and nonsterilized soil since most plants of wheat and rye-grass died in Ggt alone treatments. Key words: Gaeumannomyces graminis var. tritici, BiocontroL Triticum, Lolium.
A sterile red fungus (SRF) occurring in roots of wheat and ryegrass in Western Australia has been found to increase plant growth and suppress take-all of wheat (Dewan & Sivasithamparam, 1988a). Sterile basidiomycetes are commonly encountered in plant roots (Pugh, 1967) and certain strains, like that reported by de la Cruz & Hubbell (1975) controlling Macrophomina phaseolina (Tassi) Goid. in seedlings of slash pine, and by Speakman & Kruger (1984) controlling take-all in wheat, have been shown to have potential as biocontrol agents. Laetisaria arvalis Burdsall has also been reported to be an effective biological control agent (Burdsall et al., 1980), controlling Pythium ultimum Trow and Rhizodonia solani Kuhn on beets in natural soil (Martin et al., 1984), and reducing the population of R. solani in naturally infested soil (Larsen et al., 1985) and P. ultimum in pasteurized and untreated soil (Martin et al., 1984). L. arvalis was highly effective in controlling P. ultimum in naturally infested soil at IS, 20 and 25°C (Hoch & Abawi, 1979). The SRF appears to hold great promise as an organism capable of promoting plant growth and reducing disease. Little is known of its behaviour in pure culture or on plant roots. The aim of this study was to investigate the effect of culture medium, pH and temperature on growth of the SRF. As copious exudation was noticed on colonies of this fungus in initial studies, observations were also made of the amount
of exudate on colonies grown at various levels of pH and temperature. The ability of the SRF to colonize roots of wheat and rye-grass in the presence and absence of the take-all fungus, Gaeumannomyces graminis Arx & Olivier var. trifici Walker (Ggt), was also investigated in sterilized and nonsterilized field soil.
MA TERIALS AND METHODS Growth of the SRF on different media Twent-five ml each of water agar (WA), com meal agar (CMA), potato dextrose agar (PO A), soil extract agar (SEA) (Sivasithamparam & Parker, 1981) and wheat meal agar (WMA) were poured into 9 cm diam Petri dishes. The WMA was prepared by boiling 200 g of crushed wheat seeds in 500 ml distilled water for 15 min, followed by filtration. To the filtrate was added 20 g dextrose and 17 g agar and the volume was corrected to I I with distilled water before autodaving at 120° for 15 min. Five replicate plates were used for each medium. The centre of each plate was inoculated with a 5 mm diam disk of agar from growing margins of POA cultures of the SRF. The plates were incubated at 25°. Radial growth of the fungus was measured after 3 d.
162
A plant growth-promoting sterile fungus
Effect of pH and temperature on growth of the SRF
Fig. 1. Radial growth of the SRF at 50 on PDA amended to pH 4 (-~-), pH 4'5 (_ .. ~_ ..), pH 5-0 (-A-), pH 5'5 ("'A"'), pH 6'0 (-0-), pH 6-5 (---0---), pH 7-0 (-e-), pH 7-5 oj or pH 8'0 ( - 0 - ) . Bar indicates L.s.o. (P < 0-05).
PDA was amended with tartaric acid or sodium hydroxide (NaOH) to adjust the pH to 4-0, 4-5, 5-0, 5-5, 6-0, 6-5, 7-0, 7-5 or 8-0_ Five plates (9 cm diam), each containing 25 ml of PDA adjusted to each pH, were used at each temperature. A 5 mm diam disk of agar from growing margins of a culture of the SRF on PDA was placed in the centre of each plate at 5, 10, 15,20,25 or 30°_ Radial growth of the SRF was recorded after 3 d_
(--- e __ 50
40
Effect of pH on growth and exudation at 5° Five replicate plates (14 cm diam) each with 50 ml PDA were used at each pH of 4-0, 4-5, 5-0, 5-5. 6-0, 6-5, 7'0, 7-5 or 8-0. A 5 mm diam plug of agar from the growing margins of a culture of the SRF on PDA was placed in the centre of each plate_ The plates were incubated at 5 ± 1°. Measurements of radial growth and exudation were made at ten daily intervals. The exudates were collected using a 1 ml syringe and the volume of the material estimated_ As colony growth at 5° was very slow, estimation of exudation was extended up to 70 d_ The inhibitory effect of the exudation on Ggt was tested by placing 0-1 ml of the fluid in each of the two shallow holes (8 mm diam) made 1 cm away from the growing margin of a 3-d-old culture of Ggt on ! strength PDA. Inhibition of growth of the fungal colony was noted after 24 h_
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Colonization of roots of wheat and rye-grass in soil by the SRF
o 10
The soil from Lancelin. Western Australia was a brown sand, pH = 5'9, clay = 2-0%, organic carbon = 0-83 %, Fe 2 0 a = 0-54%, Al 2 0 a = 0-11 % and total copper = 1-5 ~g/g (Brennan et ai_, 1980). One hundred g of this soil within each test tube (2-3 x 29 cm) was autoclaved at 120° for 50 min on three consecutive days. The soil within the tube was approximately 18 cm deep_ The moisture of soil within the tube was maintained at 70 % of its water holding capacity (WHC). Two cm diam disks of agar from growing margins of PDA cultures of the SRF or of Ggt were used as fungal inocula_ These disks were placed 1 cm below surface of soil within tubes. The treatments were: SRF alone (uninoculated disk above a SRF disk), SRF with Ggt (Ggt disk below a SRF
20
30
40
Time (days)
50
pH
5 10
IS 20 25 30
Mean (pH) L.S.o_
(P
.0'-
4'0 0-5 2-6 5-0 6-8 8-0 0-3 3-9
4-5 1-5 5-0
5-5
6'0
6-5
1-6
. 1'8
6'0
6'4 14-0
1'4 5-0 10-0
1-0 4-4 8-4 17-0 27-0 1-0 9-8
5-0
11-0
13-0
]6-0 24-0 0-6 9-7
20'0 30-0 0-9 11'9
21'8 31'8 1'4 12-9
70
disk) and Ggt alone (Ggt disk below an uninoculated agar disk)_ A germinated seed of wheat (Triticum aestivum L. cv. Gamenya) or rye-grass (Latium rigidum L. cv_ Wimmera) was put on the top of the disks in each test tube. The germinated seed and the disks were covered by 1 cm of sterilized or nonsterilized soil. The tubes were sealed with Parafilm:g to reduce evaporation. The test tubes were incubated in an illuminated growth chamber at 15 ± 2°_ Two d after germination. when the coleoptile came in contact with the parafilm seal. a small
Table 1. Effects of pH and temperature on radial growth (mm) of the sterile red fungus after 3 d incubation on PDA Temper. ature (DC)
60
19'4 29-2 1-2 11-0
< 0-05) for pH = 0-7; for temperature = 0-6; or pH x temperature =
7-0 0-7 4'0 8'0
15-0 25-0
0-8 8-9 0-9.
7-5 0-5 3-0 7-0 13'2 22-6 0-6 7-8
8-0 0-0 2-0
5-0 8-4 21-0 0-4 6'1
Mean (temp.) 1'0 4-3 9-0 15'3 24-3 0-8 9-1
M. M. Dewan and K. Sivasithamparam hole was made with a needle to facilitate the passage of the coleoptile through the seal. The roots of wheat and rye-grass were washed free of soil after 4, 8, 12, 16 or 20 d, blotted dry, cut into short lengths (ca 0'5 cm) and plated onto PDA with streptomycin. The extent of colonization of the roots of wheat and rye-grass by the SRF or Ggt was measured after 14 h. The experiment was repeated in non-sterilized Lancelin soil.
RESULTS Growth of the SRF on different agar media Radial growth of the SRF after 3 d was most on WMA (32'2 mm), and was in descending order CMA (28'2 mm), PDA (27'2 mm), SEA (25'4 mm), WA (19'4 mm).
Fig. 2. Drops of exudation prod;"ced by the SRF around the inoculum disk in the centre of a PDA plate. Fig. 3. Inhibitory effect of exudation drops from the SRF on growth of Gaeumannomyces graminis var. trifici (Ggt) on agar.
163
Effect of pH and temperature on growth of the SRF Maximum growth of the SRF on PDA was at 25° and minimum at 30°. The pH optimum on PDA was at 5'5 and minimum at 4'0. The interaction between pH and temperature was significant (Table 1).
Effect of pH on growth and production of exudation by the SRF at 5° Most differences between treatments became evident only after 60 d growth (Fig. 1). After 70 d most growth had occurred at pH 5'5, and least at pH 4'0. At 5° the SRF produced drops of exudate around the fungal disk in the centre of the plate (Fig. 2). They were found to have an inhibitory effect on growth of Ggt on agar (Fig. 3). The exudations appeared in all pH treatments after 20 d, with the exception of pH 4'0 and 4'5. The amount exuded increased with time, the maximum production (as assessed at 10 d intervals) occurring between 50-60 d for all pH treatments; the amount exuded declining after this period. More exudation was produced at pH 7'5 between 50 and 70 d than in any other treatments (Fig. 4).
Fig. 4. Production of exudate by the SRF (assessed at 10 d intervals)
at 5° on PDA amended to pH 4'0 (-1::',.-), pH 4'5 (--- 1::',. ---), pH 5'0 (-A-), pH 5'5 (---A---), pH 6'0 (-0-), pH 6'5 (--- 0 ---), pH 7'0 (-e-), pH 7'5 (--- e ---) or pH 8'0 (-0-). Bar indicates 1.S.D. (P < 0'05). 0·6
0·5
0·\
10
20
30
40 50 Time (days)
60
70
A plant growth-promoting sterile fungus
164
Fig. 5. Extent of colonization of roots of wheat and rye-grass by the SRF in non-sterilized soil. Soil infested with the SRF only and planted with wheat (-l::,-) or rye-grass (---l::,---). Soil infested with the SRF and Gaeumannomyces graminis var. tritici and planted with wheat (-A-) or rye-grass (- - - A ---). The extent of colonization by Ggt of roots of wheat ( - 0 - ) or rye-grass (---0---) growing in soil infested with the SRF and Ggt. The soil infested with Ggt only and planted with wheat (-e-) or rye-grass (--- e ---) served as control. Bar indicates L.S.D. (P < 0'05).
Fig. 6. Extent of colonization of roots of wheat and rye-grass by the SRF in sterilized soil. Symbols as in Fig. 5. /:;.
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Colonization of roots of wheat and rye-grass in soil by the SRF The SRF colonized roots of wheat more aggressively than those of rye-grass both in sterilized and non-sterilized soil. The extent of colonization by the SRF was more in nonsterilized soil than in sterilized soil whether SRF was alone or with Ggt (Figs 5,6). The SRF protected the roots of wheat and rye-grass from Ggt in sterilized and non-sterilized soil. since most plants of wheat and rye-grass died in Ggt alone treatments. The SRF colonized the entire root system of wheat and ryegrass in sterilized and non-sterilized soil, 4 d after inoculation. At 8 d the SRF was found to colonize only up to 50 % of the lengths of the root systems of both hosts (Fig. 7). By day 20, however, the full lengths of the roots of wheat and rye-grass were fully colonized by the fungus (Fig. 8). Microscopic examination of roots showed that the SRF invaded the root cortex of wheat and rye-grass but rarely the stele. The lumen of all cells of the cortex colonized were filled by hyphae of the fungus. The SRF also colonized the crown regions of wheat and rye-grass, and extended into the sheath tissues of wheat 0'5-1'0 em above the soil.
The SRF is capable of fast growth at a wide range of pH and temperatures. It promotes growth of wheat and reduces rootrot caused by Ggt (Dewan & Sivasithamparam, 1988a). It is shown in a subsequent paper that it protects wheat and ryegrass from infection by Ggt at - 0'0015 or 0'001 MPa at 15 or 20° (Dewan & Sivasithamparam, 1989a), conditions which are conducive to take-all (Henry, 1932; Sivasithamparam & Parker, 1981). This makes it a potentially effective biological control agent. Unlike certain Trichoderma species which produce antibiotics at low pH (Dennis & Webster, 1971), the SRF is able to produce exudation inhibitory to Ggt across a wide range of pH. The optimum temperature and pH for the growth of the SRF appear to be similar to those for most fungi (MooreLandecker, 1982) and very close to those recorded commonly for basidiomycetes (Lilly & Barnett, 1951). There may be a stimulatory effect of other organisms on the growth of the SRF in the soil, since its growth was faster in non-sterilized soil than in sterilized soil. This would be very unusual behaviour which was not noted with any of the 80 other soil fungi screened in a large study of which this investigation is a part (Dewan & Sivasithamparam, 1988 b, c; 1989 b; unpubl.). This effect, however, may also be due to the slowing down of colonization of roots by the SRF in sterilized soil by substances released during the sterilization procedure. The significant protection by the SRF of wheat and rye-grass roots
M. M. Dewan and K. Sivasithamparam
165
Fig. 7. Extent of roots of wheat colonized by the SRF after 8 d in soil infested with the SRF and Ggt. The plate was incubated for 14 h at 25°. Portions of root indicated with the letter 'c' were colonized by a fungal contaminant and not the SRF. Fig. 8. Root system of wheat grown in soil infested with the SRF and Ggt showing recovery of the SRF from the entire length of the roots after 20 d. Discolouration of roots indicates soil adhered to mycelia rather than root necrosis.
from Ggt may have resulted from the rapid invasion by the SRF of the cortical tissues in advance of Ggt, thus providing a physical and/or chemical barrier. This protection may also have resulted from the triggering of the host defence mechanisms by initial infection of the SRF in a manner similar to that of Phialophora graminicola Walker (Deacon, 1981). The SRF colonized roots rapidly. This rate of colonization is quite remarkable and far more rapid and extensive than that reported for Trichoderma spp. (Ahmad & Baker, 1987). As the inoculum was restricted to the top I em of soil, the extent of colonization was related to the rate of root growth. At 4 d, when the length of root of wheat did not exceed 2'5 em, the fungus was able to colonize the full length. The failure of the fungus to colonize the roots entirely at 8 d may be related to the rapid rate of root growth between 4 and 8 d which resulted in the roots attaining a length of 13 em. By 20 d, however, as all roots had reached the bottom of the tube, the fungus may have had sufficient time to colonize the full length of the root system.
In the colonization studies, large glass tubes (20 em) had to be used to minimize contamination in treatments involving sterilized soil. It must be noted that only length of roots, rather than number of roots colonized, was assessed. Although the confinement of all roots within 18 em depth of soil did not simulate field conditions, it certainly gave a clear indication of the rate of colonization of the roots by the SRF. In similar colonization studies, Sclerotium sp., Macrophomina phaseolina (Tassi) Goid. (Dewan & Sivasithamparam, unpubl.), species of Trichoderma (Dewan & Sivasithamparam, 1988 b) and Penicillium (Dewan & Sivasithamparam, 1989 b) failed to colonize the full length of roots, even after 20 d. The significant reduction of take-all by the SRF may also be due to the ability of this fungus to effectively and rapidly colonize the crown region which is critical in relation to severe infections by Ggt. Exudations are commonly produced by fungi in culture. Some species of Penicillium, especially when grown on Czapek yeast agar at 25°, produce exudates. In this genus, colour of
A plant growth-promoting sterile fungus the exudates is charaderistic of a particular species and can be a useful taxonomic tool (Raper & Thorn, 1968; Pitt, 1979). The exudates of the SRF, however, were straw-coloured and were copiously produced especially under low temperatures which are unsuitable for colony growth on agar. The chemical nature of this exudate is currently being investigated. The ecological significance of these exudates is not clear, but the physiological basis of this phenomenon may be relevant to understanding the nature and importance of the antibiotic(s) produced by this fungus.
We wish to thank Mr Terry Armitage and Mrs Sabita Barua for their valuable assistance, and John Bridson for assistance in statistical analysis. One of us (M. M. D.) was supported by a generous grant from Ministry of High Education, Government of Iraq.
REFERENCES
166 DEWAN, M. M. & SIVASITHAMPARAM, K. (1988 b). Identity and frequency of occurrence of Trichoderma spp. in roots of wheat and rye-grass in Western Australia and their effect on root-rot caused by Gaeumannomyces graminis var. tritici. Plant and Soil 109, 93-101.
DEWAN, M. M. & SIVASITHAMPARAM, K. (1988 c). Pythiu~ spp. in roots of wheat and rye-grass in Western Australia and their effect on root-rot caused by Gaeumannomyces graminis var. trifici. Soil Biology and Biochemistry 20, 801-808. DEWAN, M. M. & SIVASITHAMPARAM, K. (1989a). Growth promotion of rotation crop species by a sterile fungus from wheat and the effect of soil temperature and moisture on its suppression of take-all. Mycological Research 93, 156-160. DEWAN, M. M. & SIVASITHAMPARAM, K. (1989b). Occurrence of species of Aspergillus and Penicillium in roots of wheat and ryegrass and their effect on root rot caused by Gaeumannomyces graminis var. tritici. Australian Journal of Botany (In the Press.) HENRY, A. W. (1932). The influence of soil temperature and soil sterilization on the reaction of wheat seedlings to Ophiobolus graminis. Canadian Journal of Research 7, 198-203. HOCH, H. C. & ABAWI, G. S. (1979). Biological control of Pythium root rot of table beets with Corticium sp. Phytopathology 69, 417-419.
AHMAD, J. S. & BAKER, R. (1987). Rhizosphere competence of Trichoderma harzianum. Phytopathology 77, 182-189. BRENNAN, R. F., GARTRELL, J. W. & ROBSON, A. D. (1980). Reaction of copper with soil affecting its availability to plants. I. Effect of soil type and time. Australian Journal of Soil Research 18, 447-459.
BURDSALL, H. H., HOCH, H. c.. BOOSALIS, M. G. & SETLIFF, E. C. (1980). Laetisaria arvalis (Aphyllophorales, Corticiaceae): a possible biological control agent for Rhizoctonia solani and Pythium species. Mycologia 72, 728-736. DEACON, J. W. (1981). Ecological relationships with other fungi. In Biology and Control of Take-All (ed. M. J. C. Asher & P. J. Shipton), pp. 75-101. London, U.K.:' Academic Press. DE LA CRUZ, R. E. & HUBBELL, D. H. (1975). Biological control of the charcoal root rot fungus Macrophomina phaseolina on slash pine seedlings by a hyperparasite. Soil Biology and Biochemistry 7, 25-30.
DENNIS, C. & WEBSTER, J. (1971). Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Transactions of the British Mycological Society 57,25-39. DEWAN, M. M. & SIVASITHAMPARAM, K. (1988a). A plant growth promoting sterile fungus from wheat and rye-grass roots with a potential for suppressing take-all. Transactions of the British Mycological Society 91, 687-692. (Received for publication 19 April 1988)
LARSEN, H. J" BOOSALIS, M. G. & KERR, R. D. (1985). Temporary depression of Rhizoctonia solani field population by soil amendment with Laetisaria arvalis. Plant Disease 69, 347-350. LILLY, V. G. & BARNETT, H. L. (1951). Physiology of the Fungi. McGraw-Hill Book Company Inc. MARTIN, S. B., ABAWI, G. S. & HOCH, H. C. (1984). Influence of the antagonist Laetisaria arvalis on infection of table beet by Phoma belae. Phytopathology 74, 1092-1096. MOORE-LANDECKER. E. (1982). Fundamentals of the Fungi. Englewood Cliffs, New Jersey. Prentice-Hall, Inc. PITT, J. I. (1979). The Genus Penicillium and Its Teleomorphic States, Eupenicillium and Talaromyces. Sydney: Academic Press. PUGH, G. J. F. (1967). Root colonisation by fungi. In Progress in Soil Biology (ed. O. Graff & J. E. Stachell), pp. 21-26. Amsterdam, The Netherlands: North Holland. RAPER, K. B. & THOM, C. (1968). A Manual of the Penicillia. New York: Hafner Publishing Company. SIVASITHAMPARAM, K. & PARKER. C. A. (1981). Physiology and nutrition in culture. In Biology and Control of Take-All (ed. M. j. C. Asher & P. J. Shipton), pp. 125-150. London and New York: Academic Press. SPEAKMAN, J. B. & KRUGER, W. (1984). Control of Gaeumannomyces graminis var. tritici by a sterile, black mycelial fungus. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 91, 391-395.
Printed in Great Britain
Behaviour of a plant growth-promoting sterile fungus on agar and roots of rye-grass and wheat
M. M. DEWAN AND K. SIVASITHAMPARAM Soil Science and Plant Nutrition Group, School of Agriculture, The University of Western Australia, Nedlands, Western Australia, 6009
Behaviour of a plant growth-promoting sterile fungus on agar and roots of rye-grass and wheat. Mycological Research 93 (2); 161-166 (1989).
The radial extension of a plant growth-promoting sterile red fungus (SRF) after 3 d on various agars at 25°C was most on wheat meal agar and least on water agar. Maximum growth on potato dextrose agar (PDA) was at 25° and minimum at 30°. The pH optimum for growth on PDA was 5'5 and minimum was 4'0 both after 3 d at 25° or 70 d at 5°. At 5° the SRF produced drops of exudate around the fungal disk in the centre of the plate. These appeared in all pH treatments after 20 d, with the exception of pH 4'0 and 4'5; most appeared between 50-60 d for all treatments, the amount exuded declining after this period. More exudate was evident at pH 7'5 between 50 and 70 d than in any other treatment. The exudate inhibited growth of Gaeumannomyces graminis var. tritici (Ggt) on agar. The SRF colonized roots of wheat (Triticum aestivum cv. Gamenya) more aggressively than those of rye-grass (Lolium rigidum cv. Wimmera) both in sterilized and non-sterilized soil. Ggt had no effect on the progression of the SRF on the roots of wheat or ryegrass when they were co-infested in sterilized and non-sterilized soils. The SRF colonized the entire root system and crown regions of both hosts 4 d after incubation in both soil treatments. The extent of colonization by the SRF, whether alone or with Ggt, was more in non-sterilized than sterilized soil. The SRF protected the roots of wheat and rye-grass from Ggt in sterilized and nonsterilized soil since most plants of wheat and rye-grass died in Ggt alone treatments. Key words: Gaeumannomyces graminis var. tritici, BiocontroL Triticum, Lolium.
A sterile red fungus (SRF) occurring in roots of wheat and ryegrass in Western Australia has been found to increase plant growth and suppress take-all of wheat (Dewan & Sivasithamparam, 1988a). Sterile basidiomycetes are commonly encountered in plant roots (Pugh, 1967) and certain strains, like that reported by de la Cruz & Hubbell (1975) controlling Macrophomina phaseolina (Tassi) Goid. in seedlings of slash pine, and by Speakman & Kruger (1984) controlling take-all in wheat, have been shown to have potential as biocontrol agents. Laetisaria arvalis Burdsall has also been reported to be an effective biological control agent (Burdsall et al., 1980), controlling Pythium ultimum Trow and Rhizodonia solani Kuhn on beets in natural soil (Martin et al., 1984), and reducing the population of R. solani in naturally infested soil (Larsen et al., 1985) and P. ultimum in pasteurized and untreated soil (Martin et al., 1984). L. arvalis was highly effective in controlling P. ultimum in naturally infested soil at IS, 20 and 25°C (Hoch & Abawi, 1979). The SRF appears to hold great promise as an organism capable of promoting plant growth and reducing disease. Little is known of its behaviour in pure culture or on plant roots. The aim of this study was to investigate the effect of culture medium, pH and temperature on growth of the SRF. As copious exudation was noticed on colonies of this fungus in initial studies, observations were also made of the amount
of exudate on colonies grown at various levels of pH and temperature. The ability of the SRF to colonize roots of wheat and rye-grass in the presence and absence of the take-all fungus, Gaeumannomyces graminis Arx & Olivier var. trifici Walker (Ggt), was also investigated in sterilized and nonsterilized field soil.
MA TERIALS AND METHODS Growth of the SRF on different media Twent-five ml each of water agar (WA), com meal agar (CMA), potato dextrose agar (PO A), soil extract agar (SEA) (Sivasithamparam & Parker, 1981) and wheat meal agar (WMA) were poured into 9 cm diam Petri dishes. The WMA was prepared by boiling 200 g of crushed wheat seeds in 500 ml distilled water for 15 min, followed by filtration. To the filtrate was added 20 g dextrose and 17 g agar and the volume was corrected to I I with distilled water before autodaving at 120° for 15 min. Five replicate plates were used for each medium. The centre of each plate was inoculated with a 5 mm diam disk of agar from growing margins of POA cultures of the SRF. The plates were incubated at 25°. Radial growth of the fungus was measured after 3 d.
162
A plant growth-promoting sterile fungus
Effect of pH and temperature on growth of the SRF
Fig. 1. Radial growth of the SRF at 50 on PDA amended to pH 4 (-~-), pH 4'5 (_ .. ~_ ..), pH 5-0 (-A-), pH 5'5 ("'A"'), pH 6'0 (-0-), pH 6-5 (---0---), pH 7-0 (-e-), pH 7-5 oj or pH 8'0 ( - 0 - ) . Bar indicates L.s.o. (P < 0-05).
PDA was amended with tartaric acid or sodium hydroxide (NaOH) to adjust the pH to 4-0, 4-5, 5-0, 5-5, 6-0, 6-5, 7-0, 7-5 or 8-0_ Five plates (9 cm diam), each containing 25 ml of PDA adjusted to each pH, were used at each temperature. A 5 mm diam disk of agar from growing margins of a culture of the SRF on PDA was placed in the centre of each plate at 5, 10, 15,20,25 or 30°_ Radial growth of the SRF was recorded after 3 d_
(--- e __ 50
40
Effect of pH on growth and exudation at 5° Five replicate plates (14 cm diam) each with 50 ml PDA were used at each pH of 4-0, 4-5, 5-0, 5-5. 6-0, 6-5, 7'0, 7-5 or 8-0. A 5 mm diam plug of agar from the growing margins of a culture of the SRF on PDA was placed in the centre of each plate_ The plates were incubated at 5 ± 1°. Measurements of radial growth and exudation were made at ten daily intervals. The exudates were collected using a 1 ml syringe and the volume of the material estimated_ As colony growth at 5° was very slow, estimation of exudation was extended up to 70 d_ The inhibitory effect of the exudation on Ggt was tested by placing 0-1 ml of the fluid in each of the two shallow holes (8 mm diam) made 1 cm away from the growing margin of a 3-d-old culture of Ggt on ! strength PDA. Inhibition of growth of the fungal colony was noted after 24 h_
E -5 30 ~
.,
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o
-5 20 ~
2
c
10
Colonization of roots of wheat and rye-grass in soil by the SRF
o 10
The soil from Lancelin. Western Australia was a brown sand, pH = 5'9, clay = 2-0%, organic carbon = 0-83 %, Fe 2 0 a = 0-54%, Al 2 0 a = 0-11 % and total copper = 1-5 ~g/g (Brennan et ai_, 1980). One hundred g of this soil within each test tube (2-3 x 29 cm) was autoclaved at 120° for 50 min on three consecutive days. The soil within the tube was approximately 18 cm deep_ The moisture of soil within the tube was maintained at 70 % of its water holding capacity (WHC). Two cm diam disks of agar from growing margins of PDA cultures of the SRF or of Ggt were used as fungal inocula_ These disks were placed 1 cm below surface of soil within tubes. The treatments were: SRF alone (uninoculated disk above a SRF disk), SRF with Ggt (Ggt disk below a SRF
20
30
40
Time (days)
50
pH
5 10
IS 20 25 30
Mean (pH) L.S.o_
(P
.0'-
4'0 0-5 2-6 5-0 6-8 8-0 0-3 3-9
4-5 1-5 5-0
5-5
6'0
6-5
1-6
. 1'8
6'0
6'4 14-0
1'4 5-0 10-0
1-0 4-4 8-4 17-0 27-0 1-0 9-8
5-0
11-0
13-0
]6-0 24-0 0-6 9-7
20'0 30-0 0-9 11'9
21'8 31'8 1'4 12-9
70
disk) and Ggt alone (Ggt disk below an uninoculated agar disk)_ A germinated seed of wheat (Triticum aestivum L. cv. Gamenya) or rye-grass (Latium rigidum L. cv_ Wimmera) was put on the top of the disks in each test tube. The germinated seed and the disks were covered by 1 cm of sterilized or nonsterilized soil. The tubes were sealed with Parafilm:g to reduce evaporation. The test tubes were incubated in an illuminated growth chamber at 15 ± 2°_ Two d after germination. when the coleoptile came in contact with the parafilm seal. a small
Table 1. Effects of pH and temperature on radial growth (mm) of the sterile red fungus after 3 d incubation on PDA Temper. ature (DC)
60
19'4 29-2 1-2 11-0
< 0-05) for pH = 0-7; for temperature = 0-6; or pH x temperature =
7-0 0-7 4'0 8'0
15-0 25-0
0-8 8-9 0-9.
7-5 0-5 3-0 7-0 13'2 22-6 0-6 7-8
8-0 0-0 2-0
5-0 8-4 21-0 0-4 6'1
Mean (temp.) 1'0 4-3 9-0 15'3 24-3 0-8 9-1
M. M. Dewan and K. Sivasithamparam hole was made with a needle to facilitate the passage of the coleoptile through the seal. The roots of wheat and rye-grass were washed free of soil after 4, 8, 12, 16 or 20 d, blotted dry, cut into short lengths (ca 0'5 cm) and plated onto PDA with streptomycin. The extent of colonization of the roots of wheat and rye-grass by the SRF or Ggt was measured after 14 h. The experiment was repeated in non-sterilized Lancelin soil.
RESULTS Growth of the SRF on different agar media Radial growth of the SRF after 3 d was most on WMA (32'2 mm), and was in descending order CMA (28'2 mm), PDA (27'2 mm), SEA (25'4 mm), WA (19'4 mm).
Fig. 2. Drops of exudation prod;"ced by the SRF around the inoculum disk in the centre of a PDA plate. Fig. 3. Inhibitory effect of exudation drops from the SRF on growth of Gaeumannomyces graminis var. trifici (Ggt) on agar.
163
Effect of pH and temperature on growth of the SRF Maximum growth of the SRF on PDA was at 25° and minimum at 30°. The pH optimum on PDA was at 5'5 and minimum at 4'0. The interaction between pH and temperature was significant (Table 1).
Effect of pH on growth and production of exudation by the SRF at 5° Most differences between treatments became evident only after 60 d growth (Fig. 1). After 70 d most growth had occurred at pH 5'5, and least at pH 4'0. At 5° the SRF produced drops of exudate around the fungal disk in the centre of the plate (Fig. 2). They were found to have an inhibitory effect on growth of Ggt on agar (Fig. 3). The exudations appeared in all pH treatments after 20 d, with the exception of pH 4'0 and 4'5. The amount exuded increased with time, the maximum production (as assessed at 10 d intervals) occurring between 50-60 d for all pH treatments; the amount exuded declining after this period. More exudation was produced at pH 7'5 between 50 and 70 d than in any other treatments (Fig. 4).
Fig. 4. Production of exudate by the SRF (assessed at 10 d intervals)
at 5° on PDA amended to pH 4'0 (-1::',.-), pH 4'5 (--- 1::',. ---), pH 5'0 (-A-), pH 5'5 (---A---), pH 6'0 (-0-), pH 6'5 (--- 0 ---), pH 7'0 (-e-), pH 7'5 (--- e ---) or pH 8'0 (-0-). Bar indicates 1.S.D. (P < 0'05). 0·6
0·5
0·\
10
20
30
40 50 Time (days)
60
70
A plant growth-promoting sterile fungus
164
Fig. 5. Extent of colonization of roots of wheat and rye-grass by the SRF in non-sterilized soil. Soil infested with the SRF only and planted with wheat (-l::,-) or rye-grass (---l::,---). Soil infested with the SRF and Gaeumannomyces graminis var. tritici and planted with wheat (-A-) or rye-grass (- - - A ---). The extent of colonization by Ggt of roots of wheat ( - 0 - ) or rye-grass (---0---) growing in soil infested with the SRF and Ggt. The soil infested with Ggt only and planted with wheat (-e-) or rye-grass (--- e ---) served as control. Bar indicates L.S.D. (P < 0'05).
Fig. 6. Extent of colonization of roots of wheat and rye-grass by the SRF in sterilized soil. Symbols as in Fig. 5. /:;.
100
1/;
90 80
120
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110
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12 Time (days)
16
20
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DISCUSSION 4
8
12 Time (days)
16
20
Colonization of roots of wheat and rye-grass in soil by the SRF The SRF colonized roots of wheat more aggressively than those of rye-grass both in sterilized and non-sterilized soil. The extent of colonization by the SRF was more in nonsterilized soil than in sterilized soil whether SRF was alone or with Ggt (Figs 5,6). The SRF protected the roots of wheat and rye-grass from Ggt in sterilized and non-sterilized soil. since most plants of wheat and rye-grass died in Ggt alone treatments. The SRF colonized the entire root system of wheat and ryegrass in sterilized and non-sterilized soil, 4 d after inoculation. At 8 d the SRF was found to colonize only up to 50 % of the lengths of the root systems of both hosts (Fig. 7). By day 20, however, the full lengths of the roots of wheat and rye-grass were fully colonized by the fungus (Fig. 8). Microscopic examination of roots showed that the SRF invaded the root cortex of wheat and rye-grass but rarely the stele. The lumen of all cells of the cortex colonized were filled by hyphae of the fungus. The SRF also colonized the crown regions of wheat and rye-grass, and extended into the sheath tissues of wheat 0'5-1'0 em above the soil.
The SRF is capable of fast growth at a wide range of pH and temperatures. It promotes growth of wheat and reduces rootrot caused by Ggt (Dewan & Sivasithamparam, 1988a). It is shown in a subsequent paper that it protects wheat and ryegrass from infection by Ggt at - 0'0015 or 0'001 MPa at 15 or 20° (Dewan & Sivasithamparam, 1989a), conditions which are conducive to take-all (Henry, 1932; Sivasithamparam & Parker, 1981). This makes it a potentially effective biological control agent. Unlike certain Trichoderma species which produce antibiotics at low pH (Dennis & Webster, 1971), the SRF is able to produce exudation inhibitory to Ggt across a wide range of pH. The optimum temperature and pH for the growth of the SRF appear to be similar to those for most fungi (MooreLandecker, 1982) and very close to those recorded commonly for basidiomycetes (Lilly & Barnett, 1951). There may be a stimulatory effect of other organisms on the growth of the SRF in the soil, since its growth was faster in non-sterilized soil than in sterilized soil. This would be very unusual behaviour which was not noted with any of the 80 other soil fungi screened in a large study of which this investigation is a part (Dewan & Sivasithamparam, 1988 b, c; 1989 b; unpubl.). This effect, however, may also be due to the slowing down of colonization of roots by the SRF in sterilized soil by substances released during the sterilization procedure. The significant protection by the SRF of wheat and rye-grass roots
M. M. Dewan and K. Sivasithamparam
165
Fig. 7. Extent of roots of wheat colonized by the SRF after 8 d in soil infested with the SRF and Ggt. The plate was incubated for 14 h at 25°. Portions of root indicated with the letter 'c' were colonized by a fungal contaminant and not the SRF. Fig. 8. Root system of wheat grown in soil infested with the SRF and Ggt showing recovery of the SRF from the entire length of the roots after 20 d. Discolouration of roots indicates soil adhered to mycelia rather than root necrosis.
from Ggt may have resulted from the rapid invasion by the SRF of the cortical tissues in advance of Ggt, thus providing a physical and/or chemical barrier. This protection may also have resulted from the triggering of the host defence mechanisms by initial infection of the SRF in a manner similar to that of Phialophora graminicola Walker (Deacon, 1981). The SRF colonized roots rapidly. This rate of colonization is quite remarkable and far more rapid and extensive than that reported for Trichoderma spp. (Ahmad & Baker, 1987). As the inoculum was restricted to the top I em of soil, the extent of colonization was related to the rate of root growth. At 4 d, when the length of root of wheat did not exceed 2'5 em, the fungus was able to colonize the full length. The failure of the fungus to colonize the roots entirely at 8 d may be related to the rapid rate of root growth between 4 and 8 d which resulted in the roots attaining a length of 13 em. By 20 d, however, as all roots had reached the bottom of the tube, the fungus may have had sufficient time to colonize the full length of the root system.
In the colonization studies, large glass tubes (20 em) had to be used to minimize contamination in treatments involving sterilized soil. It must be noted that only length of roots, rather than number of roots colonized, was assessed. Although the confinement of all roots within 18 em depth of soil did not simulate field conditions, it certainly gave a clear indication of the rate of colonization of the roots by the SRF. In similar colonization studies, Sclerotium sp., Macrophomina phaseolina (Tassi) Goid. (Dewan & Sivasithamparam, unpubl.), species of Trichoderma (Dewan & Sivasithamparam, 1988 b) and Penicillium (Dewan & Sivasithamparam, 1989 b) failed to colonize the full length of roots, even after 20 d. The significant reduction of take-all by the SRF may also be due to the ability of this fungus to effectively and rapidly colonize the crown region which is critical in relation to severe infections by Ggt. Exudations are commonly produced by fungi in culture. Some species of Penicillium, especially when grown on Czapek yeast agar at 25°, produce exudates. In this genus, colour of
A plant growth-promoting sterile fungus the exudates is charaderistic of a particular species and can be a useful taxonomic tool (Raper & Thorn, 1968; Pitt, 1979). The exudates of the SRF, however, were straw-coloured and were copiously produced especially under low temperatures which are unsuitable for colony growth on agar. The chemical nature of this exudate is currently being investigated. The ecological significance of these exudates is not clear, but the physiological basis of this phenomenon may be relevant to understanding the nature and importance of the antibiotic(s) produced by this fungus.
We wish to thank Mr Terry Armitage and Mrs Sabita Barua for their valuable assistance, and John Bridson for assistance in statistical analysis. One of us (M. M. D.) was supported by a generous grant from Ministry of High Education, Government of Iraq.
REFERENCES
166 DEWAN, M. M. & SIVASITHAMPARAM, K. (1988 b). Identity and frequency of occurrence of Trichoderma spp. in roots of wheat and rye-grass in Western Australia and their effect on root-rot caused by Gaeumannomyces graminis var. tritici. Plant and Soil 109, 93-101.
DEWAN, M. M. & SIVASITHAMPARAM, K. (1988 c). Pythiu~ spp. in roots of wheat and rye-grass in Western Australia and their effect on root-rot caused by Gaeumannomyces graminis var. trifici. Soil Biology and Biochemistry 20, 801-808. DEWAN, M. M. & SIVASITHAMPARAM, K. (1989a). Growth promotion of rotation crop species by a sterile fungus from wheat and the effect of soil temperature and moisture on its suppression of take-all. Mycological Research 93, 156-160. DEWAN, M. M. & SIVASITHAMPARAM, K. (1989b). Occurrence of species of Aspergillus and Penicillium in roots of wheat and ryegrass and their effect on root rot caused by Gaeumannomyces graminis var. tritici. Australian Journal of Botany (In the Press.) HENRY, A. W. (1932). The influence of soil temperature and soil sterilization on the reaction of wheat seedlings to Ophiobolus graminis. Canadian Journal of Research 7, 198-203. HOCH, H. C. & ABAWI, G. S. (1979). Biological control of Pythium root rot of table beets with Corticium sp. Phytopathology 69, 417-419.
AHMAD, J. S. & BAKER, R. (1987). Rhizosphere competence of Trichoderma harzianum. Phytopathology 77, 182-189. BRENNAN, R. F., GARTRELL, J. W. & ROBSON, A. D. (1980). Reaction of copper with soil affecting its availability to plants. I. Effect of soil type and time. Australian Journal of Soil Research 18, 447-459.
BURDSALL, H. H., HOCH, H. c.. BOOSALIS, M. G. & SETLIFF, E. C. (1980). Laetisaria arvalis (Aphyllophorales, Corticiaceae): a possible biological control agent for Rhizoctonia solani and Pythium species. Mycologia 72, 728-736. DEACON, J. W. (1981). Ecological relationships with other fungi. In Biology and Control of Take-All (ed. M. J. C. Asher & P. J. Shipton), pp. 75-101. London, U.K.:' Academic Press. DE LA CRUZ, R. E. & HUBBELL, D. H. (1975). Biological control of the charcoal root rot fungus Macrophomina phaseolina on slash pine seedlings by a hyperparasite. Soil Biology and Biochemistry 7, 25-30.
DENNIS, C. & WEBSTER, J. (1971). Antagonistic properties of species-groups of Trichoderma. I. Production of non-volatile antibiotics. Transactions of the British Mycological Society 57,25-39. DEWAN, M. M. & SIVASITHAMPARAM, K. (1988a). A plant growth promoting sterile fungus from wheat and rye-grass roots with a potential for suppressing take-all. Transactions of the British Mycological Society 91, 687-692. (Received for publication 19 April 1988)
LARSEN, H. J" BOOSALIS, M. G. & KERR, R. D. (1985). Temporary depression of Rhizoctonia solani field population by soil amendment with Laetisaria arvalis. Plant Disease 69, 347-350. LILLY, V. G. & BARNETT, H. L. (1951). Physiology of the Fungi. McGraw-Hill Book Company Inc. MARTIN, S. B., ABAWI, G. S. & HOCH, H. C. (1984). Influence of the antagonist Laetisaria arvalis on infection of table beet by Phoma belae. Phytopathology 74, 1092-1096. MOORE-LANDECKER. E. (1982). Fundamentals of the Fungi. Englewood Cliffs, New Jersey. Prentice-Hall, Inc. PITT, J. I. (1979). The Genus Penicillium and Its Teleomorphic States, Eupenicillium and Talaromyces. Sydney: Academic Press. PUGH, G. J. F. (1967). Root colonisation by fungi. In Progress in Soil Biology (ed. O. Graff & J. E. Stachell), pp. 21-26. Amsterdam, The Netherlands: North Holland. RAPER, K. B. & THOM, C. (1968). A Manual of the Penicillia. New York: Hafner Publishing Company. SIVASITHAMPARAM, K. & PARKER. C. A. (1981). Physiology and nutrition in culture. In Biology and Control of Take-All (ed. M. j. C. Asher & P. J. Shipton), pp. 125-150. London and New York: Academic Press. SPEAKMAN, J. B. & KRUGER, W. (1984). Control of Gaeumannomyces graminis var. tritici by a sterile, black mycelial fungus. Zeitschrift fur Pflanzenkrankheiten und Pflanzenschutz 91, 391-395.