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Chitin as a nitrogen source for mycorrhizal fungi
Mycol. Res. 94 ( 7 ) :993-1008 (1990) Printed in Great Britain
SHORT COMMUNICATIONS
Chitin as a nitrogen source for mycorrhizaI fungi
J. R. LEAKE A N D D. J. R E A D Department
of
Animal and Plant Biology, University of Shefield, Shefield S r o ZTN,U.K.
Chitin as a nitrogen source for mycorrhizal fungi. Mycological Research
94 (7):993-995 (1990).
Three ericoid and two ectomycorrhizal fungi were tested for their abilities to utilize chitin as a sole source of nitrogen. All of the ericoid fungi readily degraded chitin. The ecto-fungi, in contrast, used it only sparingly. The results extend the range of polymeric N sources known to be utilized by mycorrhizal fungi and imply that some rnycorrhizal fungi may be involved in recycling of N from this structural component of hyphal walls in soil. The possible role of chitinase activity in influencing fungal interactions in heathland ecosystems is discussed. Key words: Ericoid mycorrhiza, Ectomycorrhiza, Chitin, Chitinase, Nitrogen nutrition, Heathland Recent studies have increased our awareness of the potential of mycorrhizal fungi to provide access to organic sources of nitrogen (Bajwa & Read, 1986; Abuzinadah & Read, 1986; Leake & Read, 1989). These studies have used pure proteins as substrates and there is now a need to determine the ability of these fungi to mobilize other naturally occurring organic macro-molecules which could represent significant sources of nitrogen in soils. One such compound is chitin, a homopolymer of N-acetylglucosamine units linked by P(1-4) glucosidic bonds, which forms around 10% by weight of fungal cell walls and typically contains 6 % nitrogen. While it has been shown that the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Keman can use N-acetyl glucosamine as a sole N source (Bajwa & Read, 1986), the ability of mycorrhizal fungi to use the polymer has not been examined. Because of its importance as a component of the fungal wall it is likely that chitin will be present in considerable quantities, especially in ecosystems which are dominated by plants supporting prolific mycorrhizal mycelial networks. The ability of three ericoid endophytes (two isolates of Hyrnenoscyphus ericae (100 and 101) and one of Oidiodendron griseum Robak), and of two ectomycorrhizal fungi (Paxillus involutus (Batsch) Fr. and Rhizopogon roseolus Fr.) to use chitin was determined by growing the fungi in liquid cultures containing pure chitin as the sole source of nitrogen. The substrate used was purified crystalline a chitin (Sigma Chemical Company) obtained from crustacean shells. O n the basis of Xray powder analysis it has been concluded that this material is structurally similar to that found in fungal cell walls (Aronson, 1965 ; Bumett, 1979). The basal nutrient solution was full-strength MelinNorkrans (MN) solution (Norkrans, 1949) from which the yeast extract and nitrogen source were omitted, but to which was added carbon in the form of D-glucose at a concentration of 10 g I-'. This solution was divided into two equal volumes,
one of which received chitin at a concentration of 0.5 g 1-' while the other remained nitrogen-free. These solutions were adjusted to pH 4.0 and autoclaved. Each solution was dispensed aseptically in 20 ml aliquots into a series of sterile 250 ml Erlenmeyer flasks, thus providing 10 mg chitin per flask in the plus-chitin treatment. Discs of the fungi were cut with a 5 mm diam corkborer from the actively growing colony margins of isolates maintained on plates of half-strength M N agar. These discs were transferred to fresh plates 5 d prior to inoculation of the culture flasks in order to initiate rapid growth of the fungi. They were then carefully floated on the surface of the culture solutions. Harvests were taken 8, 15, 22 and 30 d after inoculation. At every harvest there were three replicate flasks of each fungus. Harvested mycelium was washed in distilled water to remove loosely adhering chitin fragments and ovendried at 80 OC for 24 h prior to weighing. All of the fungi gave higher yields when chitin was present as the nitrogen source than when there was no nitrogen. The ability of the ectomycorrhizal fungi to utilize chitin was, however, very limited. In the case of Paxillus involutus (Fig. I d ) the fungus formed a superficial mat covering the solutions containing chitin whilst on the zero-nitrogen control, growth was submerged and entirely associated with the inoculum disc. A similar but less-marked effect was observed for the growth form of Rhizopogon roseolus (Fig. le). In contrast, the ericoid endophytes were highly effective at utilizing chitin, in each case achieving yields approximately tenfold higher than the weight of added chitin in each flask. However, the rates of growth of the two Hymenoscyphus isolates differed considerably. Both H. ericae (101) (Fig. l a ) and Oidiodendron griseum (Fig. Ic) rapidly colonized the chitin and formed a dense fungal mat which covered the base of the flasks. By the second harvest microscopic examination of these fungal mats confirmed the disappearance of all traces of chitin particles.
Short Communications
994
Fig. 1. Mycelial dry weight yield of ericoid and ectomycorrhizal fungi when grown for 30 d with chitin as sole source of nitrogen (m) and in control treatments with no nitrogen (0). Vertical bars give L.S.D.P = 005. (a) H . er-icae 101 ( h ) H. ericae 100 120,
-
-0
I
( c ) 0.griseum
40
1 /
( d ) P. involutus
capability, have melanized walls. This may reduce their susceptibility to autolysis and provide them with an advantage when growing with mycelia of the ectomycorrhizal type which, as in the case of the two fungi employed in this study, normally have hyaline or only lightly melanized walls. The preponderance of dark-walled hyphae was recognized by Muller (1884) as being a characteristic feature of 'mor' humus soils. It is, however, evident that since some of the chitinolytic fungi which are characteristic of heathland soil, including Morfierella and M u c o r species, do not have melanized walls, additional factors must contribute to the provision of resistance to the enzymes. We envisage that the chitinolytic capability of the large population of ericoid mycorrhizal fungi in heathland ecosystems will be a potent factor contributing to the success of their host plants. Direct benefits will arise as a result of mobilization of nitrogen, and indirect advantages will be gained if chitinase activity leads to inhibition of ectomycorrhizal fungi and hence to the weakening or exclusion of tree species which are dependent upon such fungi. Further studies are investigating chitin and other fractionated components of fungal mycelia as N sources for mycorrhizal fungi and their host plants.
( e ) R. roseolus
We gratefully acknowledge the financial support of the Natural Environment Research Council and the skilled technical assistance of Mr R. Bradley.
Time (days)
REFERENCES The H. ericae (100) (Fig. 1b) isolate initially grew very slowly and formed discrete subcolonies which took until the final harvest to merge into a single mycelium. Measurement of residual glucose concentration in the culture filtrates at the final harvest by the method of Nelson (1944) confirmed that the cessation of growth of the two most active endophytes was due to exhaustion of the glucose carbon source. Previous studies have demonstrated an ability of bacteria and actinomycetes to degrade chitin (Veldkamp, 1955; Ames, Mihara & Bayne, 1989). There have been relatively few reports of such activity in fungi, though Skinner & Dravis (1937) isolated 42 species of fungi capable of decomposing chitin. BHIth & Soderstrom (1980) examined the ability of 60 non-mycorrhizal fungi isolated from acidic coniferous forest soils to decompose chitin. They found that while the majority were unable to degrade the polymer, a few, notably members of the genera Morfierella, Verticilliurn and Mucor, had some chitinolytic capability. Both Morfierella and Mucor are ubiquitous in heathland soils (Thomton, 1956; Sewell, 1959). Since in mor humus most of the nitrogen is present in polymeric organic forms (Stribley & Read, 1974), there will be selection for fungi which have an ability to mobilize nitrogen from such sources, and which are themselves resistant to the hydrolytic enzymes required in these processes. Melanization of hyphal walls is known to contribute to this resistance (Bloomfield & Alexander, 1967). It is of interest that the ericoid fungi, shown here to have significantly chitinolytic
ABUZINADAH, R. A. & READ, D. J. (1986).The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. I. Utilization of peptides and proteins by ectomycorrhizal Fungi. New Phytologist 103, 481-493. AMES, R. N., MIHARA, K. L. & BAYNE, H. G. (1989). Chitin decomposing actinomycetes associated with a vesicular-arbuscular mycorrhizal fungus from a calcareous soil. New Phyfologist 111, 67-71. ARONSON, J. M. (1965). The cell wall. In The Fungi 1 (ed. G. C. Ainsworth & A. S. Sussman). New York: Academic Press. B ~ ~ T E. H& , SODERSTROM, B. (1980). Degradation of macromolecules by microfungi isolated from different podzolic soil horizons. Canadian Journal of Botany 58,422-425. BAJWA, R. & READ, D. J. (1986). Utilization of mineral and amino N sources by the ericoid mycorrhizal endophyte Hymenoscyphus ericae and by mycorrhizal and non-mycorrhizal seedlings of Vaccinium. Transactions of the British Mycological Society 87, 269-277. BLOOMFIELD, B. J. & ALEXANDER, M. (1967).Melanins and the resistance of Fungi to lysis. Journal of Bacteriology 93, 1276-1280. BURNETT, J.H. (1979). Aspects of the structure and growth of hyphal walls. In Fungal Walls and Hyphal Growth (ed. J.H. Bumett & A. P. J. Trinci). Cambridge: Cambridge University Press. LEAKE, J. R. & READ, D. J. (1989). Biology of mycorrhiza in the Ericaceae. XIII. Some characteristics of the extracellular proteinase activity of the ericoid endophyte Hymenoscyphus ericae. New Phyfologist 112, 69-76. MULLER, P.E. (1884). Studier over Skovjord som bidrag ti1 Skovdyrkningens Theori. 11. Om Muld og Mor i Egeskove og paa Heder. Tidsskrift for Skogbruk 7,1-12.
Short Communications NELSON, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. lournal of Biological Chemistry 153, 375-380.
NORKRANS, B. (1949). Some mycorrhiza forming Tricholoma species. Svensk Botanisk Tidskrift 4 3 , 485-490. SEWELL, G. W . F. (1959).Studies of fungi in a Calluna heathland soil. 11. By the complementary use of several isolation methods. Transactions of the British Mycological Society 4 2 , 354-369. SKINNER, C. E. & DRAVIS, F. (1937).A quantitative determination of chitin destroying microorganisms in soil. Ecology 18,391-398.
995 STRIBLEY, D. P. & READ, D. J. (1974). The biology of mycorrhiza in the Ericaceae. IV. The effect of mycorrhizal infection on uptake of 15N from labelled soil by Vaccinium macrocarpon (Ait). New Phytologist 7 3 , 1149-1155.
THORNTON, R. H. (1956).Fungi occurring in mixed oakwood and heath soil profiles. Transactions of the British Mycological Society 39, 485-494.
VELDKAMP, H. (1955). A study of the aerobic decomposition of chitin by microorganisms. Mededelingen van de Landbouwhogeschool te Wageningen (Nederland) 55, 127-174.
(Received for publication 31 August 1989 and in revised form 1 1 December 1989)
Disinfecting vesicular-arbuscular mycorrhizas P. G.WILLIAMS* School of Biological Science, University of New South Wales, P.O. Box I , Kensington, N.S.W. 2033, Australia
Disinfecting vesicular-arbuscular mycorrhizas. Mycological Research 94 ( 7 ) :995-997 (1990). A procedure for disinfecting root pieces of onion (Alliurn cepa) and white clover (Trifolium repens) infected with the vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum is described. In 26 experiments over a 5-yr-period an average of 22% (range 4 4 4 % ) of the 4350 root pieces treated according to the method were uncontaminated and formed regrowth hyphae of the endophyte. Key words: VA mycorrhiza, Disinfection technique, Axenic culture. The development of a technique for disinfecting vesiculararbuscular (VA) mycorrhizas taken directly from the soil would be an important step towards growing these fungi in axenic culture. Experience with rust fungi, which are also obligate biotrophs, has shown that pieces of surface-sterilized tissue containing intercellular mycelia are more effective inocula for axenic cultures than are spores (Williams, 1984). Plant tissues containing intercellular mycelia of VA mycorrhizal fungi can be obtained by establishing monoxenic cultures. Several methods are available based on Mosse's (1962) original procedures. The methods are complex, timeconsuming and in this laboratory have not proved effective. Strullu & Romand (1986) reported a method for decontaminating VA mycorrhizas in strawberry roots but did not indicate the method's success rate or its application to other plants. Their use of a series of strong disinfectants makes the method unsuitable for treating mycorrhizas in delicate roots. I report here a comparatively mild disinfection procedure which gives high yields of uncontaminated sections of VA mycorrhizas from pot cultures of onion and white clover. Roots for disinfection were obtained from pot cultures of Glornus fasciculaturn (Thaxter sensu Gerd.) Gerd. & Trappe propagated on onion (Alliurn cepa L.) and white clover (Trifolium repens L.). The plants were grown in a glasshouse in a sand-and-soil mixture treated with steam:air at 60 OC for 1 h. To minimize damage, roots were exposed to view by shaking out some of the sand and soil from a pot culture. White, apparently sound roots (diam 0.6-1.2 mm) were Permanent address: 6 Undercliff Street, Neutral Bay, N.S.W. 2089.
gently excized 10-15 cm from the apex, briefly washed in running water, placed in a dish of tap water and examined with a dissecting microscope for mycorrhizal infection units. Infection units with extramatrical hyphae and internal vesicles were readily located. Infection units with many arbuscules had a slightly water-soaked appearance when viewed against a dark background with axial illumination. However, infection units with few arbuscules lacked any visible marker and together with uninfected regions were trimmed off. A trimmed length of infected root (30-60 mm) was brushed in sterile distilled water with a pair of camel-hair brushes, rinsed and observed with a dissecting microscope at x 100 magnification. While holding the root with a yoke made from a wooden toothpick, pieces about 1mm in length were cut with a surgical blade, placed in a nylon sieve and infiltrated in sterile distilled water under vacuum for 2 min. The root pieces were transferred in the sieve to a laminarflow cabinet and immersed in a 2 % solution of household bleach ('Xixo', nominally 4 % available chlorine), briefly agitated immediately and at 30 s intervals for 2 min. After rinsing in three changes of sterile distilled water each root piece was placed in a drop (about 0.03 ml) of freshly prepared filter-sterilized incubation medium containing penicillin 500 g 1-', streptomycin 500 mg I-' and bovine albumin (Commonwealth Serum Laboratories) 20 g 1-'. The drops of the incubation medium were arrayed on the inside of the inverted lid of a plastic Petri dish. The bottom of the dish contained a layer of agar (8 g I-') to prevent the drops drying out. The dishes, each containing about 35 incubation drops, were sealed with wax film and incubated in an inverted position in the dark at 23O.
SHORT COMMUNICATIONS
Chitin as a nitrogen source for mycorrhizaI fungi
J. R. LEAKE A N D D. J. R E A D Department
of
Animal and Plant Biology, University of Shefield, Shefield S r o ZTN,U.K.
Chitin as a nitrogen source for mycorrhizal fungi. Mycological Research
94 (7):993-995 (1990).
Three ericoid and two ectomycorrhizal fungi were tested for their abilities to utilize chitin as a sole source of nitrogen. All of the ericoid fungi readily degraded chitin. The ecto-fungi, in contrast, used it only sparingly. The results extend the range of polymeric N sources known to be utilized by mycorrhizal fungi and imply that some rnycorrhizal fungi may be involved in recycling of N from this structural component of hyphal walls in soil. The possible role of chitinase activity in influencing fungal interactions in heathland ecosystems is discussed. Key words: Ericoid mycorrhiza, Ectomycorrhiza, Chitin, Chitinase, Nitrogen nutrition, Heathland Recent studies have increased our awareness of the potential of mycorrhizal fungi to provide access to organic sources of nitrogen (Bajwa & Read, 1986; Abuzinadah & Read, 1986; Leake & Read, 1989). These studies have used pure proteins as substrates and there is now a need to determine the ability of these fungi to mobilize other naturally occurring organic macro-molecules which could represent significant sources of nitrogen in soils. One such compound is chitin, a homopolymer of N-acetylglucosamine units linked by P(1-4) glucosidic bonds, which forms around 10% by weight of fungal cell walls and typically contains 6 % nitrogen. While it has been shown that the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Keman can use N-acetyl glucosamine as a sole N source (Bajwa & Read, 1986), the ability of mycorrhizal fungi to use the polymer has not been examined. Because of its importance as a component of the fungal wall it is likely that chitin will be present in considerable quantities, especially in ecosystems which are dominated by plants supporting prolific mycorrhizal mycelial networks. The ability of three ericoid endophytes (two isolates of Hyrnenoscyphus ericae (100 and 101) and one of Oidiodendron griseum Robak), and of two ectomycorrhizal fungi (Paxillus involutus (Batsch) Fr. and Rhizopogon roseolus Fr.) to use chitin was determined by growing the fungi in liquid cultures containing pure chitin as the sole source of nitrogen. The substrate used was purified crystalline a chitin (Sigma Chemical Company) obtained from crustacean shells. O n the basis of Xray powder analysis it has been concluded that this material is structurally similar to that found in fungal cell walls (Aronson, 1965 ; Bumett, 1979). The basal nutrient solution was full-strength MelinNorkrans (MN) solution (Norkrans, 1949) from which the yeast extract and nitrogen source were omitted, but to which was added carbon in the form of D-glucose at a concentration of 10 g I-'. This solution was divided into two equal volumes,
one of which received chitin at a concentration of 0.5 g 1-' while the other remained nitrogen-free. These solutions were adjusted to pH 4.0 and autoclaved. Each solution was dispensed aseptically in 20 ml aliquots into a series of sterile 250 ml Erlenmeyer flasks, thus providing 10 mg chitin per flask in the plus-chitin treatment. Discs of the fungi were cut with a 5 mm diam corkborer from the actively growing colony margins of isolates maintained on plates of half-strength M N agar. These discs were transferred to fresh plates 5 d prior to inoculation of the culture flasks in order to initiate rapid growth of the fungi. They were then carefully floated on the surface of the culture solutions. Harvests were taken 8, 15, 22 and 30 d after inoculation. At every harvest there were three replicate flasks of each fungus. Harvested mycelium was washed in distilled water to remove loosely adhering chitin fragments and ovendried at 80 OC for 24 h prior to weighing. All of the fungi gave higher yields when chitin was present as the nitrogen source than when there was no nitrogen. The ability of the ectomycorrhizal fungi to utilize chitin was, however, very limited. In the case of Paxillus involutus (Fig. I d ) the fungus formed a superficial mat covering the solutions containing chitin whilst on the zero-nitrogen control, growth was submerged and entirely associated with the inoculum disc. A similar but less-marked effect was observed for the growth form of Rhizopogon roseolus (Fig. le). In contrast, the ericoid endophytes were highly effective at utilizing chitin, in each case achieving yields approximately tenfold higher than the weight of added chitin in each flask. However, the rates of growth of the two Hymenoscyphus isolates differed considerably. Both H. ericae (101) (Fig. l a ) and Oidiodendron griseum (Fig. Ic) rapidly colonized the chitin and formed a dense fungal mat which covered the base of the flasks. By the second harvest microscopic examination of these fungal mats confirmed the disappearance of all traces of chitin particles.
Short Communications
994
Fig. 1. Mycelial dry weight yield of ericoid and ectomycorrhizal fungi when grown for 30 d with chitin as sole source of nitrogen (m) and in control treatments with no nitrogen (0). Vertical bars give L.S.D.P = 005. (a) H . er-icae 101 ( h ) H. ericae 100 120,
-
-0
I
( c ) 0.griseum
40
1 /
( d ) P. involutus
capability, have melanized walls. This may reduce their susceptibility to autolysis and provide them with an advantage when growing with mycelia of the ectomycorrhizal type which, as in the case of the two fungi employed in this study, normally have hyaline or only lightly melanized walls. The preponderance of dark-walled hyphae was recognized by Muller (1884) as being a characteristic feature of 'mor' humus soils. It is, however, evident that since some of the chitinolytic fungi which are characteristic of heathland soil, including Morfierella and M u c o r species, do not have melanized walls, additional factors must contribute to the provision of resistance to the enzymes. We envisage that the chitinolytic capability of the large population of ericoid mycorrhizal fungi in heathland ecosystems will be a potent factor contributing to the success of their host plants. Direct benefits will arise as a result of mobilization of nitrogen, and indirect advantages will be gained if chitinase activity leads to inhibition of ectomycorrhizal fungi and hence to the weakening or exclusion of tree species which are dependent upon such fungi. Further studies are investigating chitin and other fractionated components of fungal mycelia as N sources for mycorrhizal fungi and their host plants.
( e ) R. roseolus
We gratefully acknowledge the financial support of the Natural Environment Research Council and the skilled technical assistance of Mr R. Bradley.
Time (days)
REFERENCES The H. ericae (100) (Fig. 1b) isolate initially grew very slowly and formed discrete subcolonies which took until the final harvest to merge into a single mycelium. Measurement of residual glucose concentration in the culture filtrates at the final harvest by the method of Nelson (1944) confirmed that the cessation of growth of the two most active endophytes was due to exhaustion of the glucose carbon source. Previous studies have demonstrated an ability of bacteria and actinomycetes to degrade chitin (Veldkamp, 1955; Ames, Mihara & Bayne, 1989). There have been relatively few reports of such activity in fungi, though Skinner & Dravis (1937) isolated 42 species of fungi capable of decomposing chitin. BHIth & Soderstrom (1980) examined the ability of 60 non-mycorrhizal fungi isolated from acidic coniferous forest soils to decompose chitin. They found that while the majority were unable to degrade the polymer, a few, notably members of the genera Morfierella, Verticilliurn and Mucor, had some chitinolytic capability. Both Morfierella and Mucor are ubiquitous in heathland soils (Thomton, 1956; Sewell, 1959). Since in mor humus most of the nitrogen is present in polymeric organic forms (Stribley & Read, 1974), there will be selection for fungi which have an ability to mobilize nitrogen from such sources, and which are themselves resistant to the hydrolytic enzymes required in these processes. Melanization of hyphal walls is known to contribute to this resistance (Bloomfield & Alexander, 1967). It is of interest that the ericoid fungi, shown here to have significantly chitinolytic
ABUZINADAH, R. A. & READ, D. J. (1986).The role of proteins in the nitrogen nutrition of ectomycorrhizal plants. I. Utilization of peptides and proteins by ectomycorrhizal Fungi. New Phytologist 103, 481-493. AMES, R. N., MIHARA, K. L. & BAYNE, H. G. (1989). Chitin decomposing actinomycetes associated with a vesicular-arbuscular mycorrhizal fungus from a calcareous soil. New Phyfologist 111, 67-71. ARONSON, J. M. (1965). The cell wall. In The Fungi 1 (ed. G. C. Ainsworth & A. S. Sussman). New York: Academic Press. B ~ ~ T E. H& , SODERSTROM, B. (1980). Degradation of macromolecules by microfungi isolated from different podzolic soil horizons. Canadian Journal of Botany 58,422-425. BAJWA, R. & READ, D. J. (1986). Utilization of mineral and amino N sources by the ericoid mycorrhizal endophyte Hymenoscyphus ericae and by mycorrhizal and non-mycorrhizal seedlings of Vaccinium. Transactions of the British Mycological Society 87, 269-277. BLOOMFIELD, B. J. & ALEXANDER, M. (1967).Melanins and the resistance of Fungi to lysis. Journal of Bacteriology 93, 1276-1280. BURNETT, J.H. (1979). Aspects of the structure and growth of hyphal walls. In Fungal Walls and Hyphal Growth (ed. J.H. Bumett & A. P. J. Trinci). Cambridge: Cambridge University Press. LEAKE, J. R. & READ, D. J. (1989). Biology of mycorrhiza in the Ericaceae. XIII. Some characteristics of the extracellular proteinase activity of the ericoid endophyte Hymenoscyphus ericae. New Phyfologist 112, 69-76. MULLER, P.E. (1884). Studier over Skovjord som bidrag ti1 Skovdyrkningens Theori. 11. Om Muld og Mor i Egeskove og paa Heder. Tidsskrift for Skogbruk 7,1-12.
Short Communications NELSON, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. lournal of Biological Chemistry 153, 375-380.
NORKRANS, B. (1949). Some mycorrhiza forming Tricholoma species. Svensk Botanisk Tidskrift 4 3 , 485-490. SEWELL, G. W . F. (1959).Studies of fungi in a Calluna heathland soil. 11. By the complementary use of several isolation methods. Transactions of the British Mycological Society 4 2 , 354-369. SKINNER, C. E. & DRAVIS, F. (1937).A quantitative determination of chitin destroying microorganisms in soil. Ecology 18,391-398.
995 STRIBLEY, D. P. & READ, D. J. (1974). The biology of mycorrhiza in the Ericaceae. IV. The effect of mycorrhizal infection on uptake of 15N from labelled soil by Vaccinium macrocarpon (Ait). New Phytologist 7 3 , 1149-1155.
THORNTON, R. H. (1956).Fungi occurring in mixed oakwood and heath soil profiles. Transactions of the British Mycological Society 39, 485-494.
VELDKAMP, H. (1955). A study of the aerobic decomposition of chitin by microorganisms. Mededelingen van de Landbouwhogeschool te Wageningen (Nederland) 55, 127-174.
(Received for publication 31 August 1989 and in revised form 1 1 December 1989)
Disinfecting vesicular-arbuscular mycorrhizas P. G.WILLIAMS* School of Biological Science, University of New South Wales, P.O. Box I , Kensington, N.S.W. 2033, Australia
Disinfecting vesicular-arbuscular mycorrhizas. Mycological Research 94 ( 7 ) :995-997 (1990). A procedure for disinfecting root pieces of onion (Alliurn cepa) and white clover (Trifolium repens) infected with the vesicular-arbuscular mycorrhizal fungus Glomus fasciculatum is described. In 26 experiments over a 5-yr-period an average of 22% (range 4 4 4 % ) of the 4350 root pieces treated according to the method were uncontaminated and formed regrowth hyphae of the endophyte. Key words: VA mycorrhiza, Disinfection technique, Axenic culture. The development of a technique for disinfecting vesiculararbuscular (VA) mycorrhizas taken directly from the soil would be an important step towards growing these fungi in axenic culture. Experience with rust fungi, which are also obligate biotrophs, has shown that pieces of surface-sterilized tissue containing intercellular mycelia are more effective inocula for axenic cultures than are spores (Williams, 1984). Plant tissues containing intercellular mycelia of VA mycorrhizal fungi can be obtained by establishing monoxenic cultures. Several methods are available based on Mosse's (1962) original procedures. The methods are complex, timeconsuming and in this laboratory have not proved effective. Strullu & Romand (1986) reported a method for decontaminating VA mycorrhizas in strawberry roots but did not indicate the method's success rate or its application to other plants. Their use of a series of strong disinfectants makes the method unsuitable for treating mycorrhizas in delicate roots. I report here a comparatively mild disinfection procedure which gives high yields of uncontaminated sections of VA mycorrhizas from pot cultures of onion and white clover. Roots for disinfection were obtained from pot cultures of Glornus fasciculaturn (Thaxter sensu Gerd.) Gerd. & Trappe propagated on onion (Alliurn cepa L.) and white clover (Trifolium repens L.). The plants were grown in a glasshouse in a sand-and-soil mixture treated with steam:air at 60 OC for 1 h. To minimize damage, roots were exposed to view by shaking out some of the sand and soil from a pot culture. White, apparently sound roots (diam 0.6-1.2 mm) were Permanent address: 6 Undercliff Street, Neutral Bay, N.S.W. 2089.
gently excized 10-15 cm from the apex, briefly washed in running water, placed in a dish of tap water and examined with a dissecting microscope for mycorrhizal infection units. Infection units with extramatrical hyphae and internal vesicles were readily located. Infection units with many arbuscules had a slightly water-soaked appearance when viewed against a dark background with axial illumination. However, infection units with few arbuscules lacked any visible marker and together with uninfected regions were trimmed off. A trimmed length of infected root (30-60 mm) was brushed in sterile distilled water with a pair of camel-hair brushes, rinsed and observed with a dissecting microscope at x 100 magnification. While holding the root with a yoke made from a wooden toothpick, pieces about 1mm in length were cut with a surgical blade, placed in a nylon sieve and infiltrated in sterile distilled water under vacuum for 2 min. The root pieces were transferred in the sieve to a laminarflow cabinet and immersed in a 2 % solution of household bleach ('Xixo', nominally 4 % available chlorine), briefly agitated immediately and at 30 s intervals for 2 min. After rinsing in three changes of sterile distilled water each root piece was placed in a drop (about 0.03 ml) of freshly prepared filter-sterilized incubation medium containing penicillin 500 g 1-', streptomycin 500 mg I-' and bovine albumin (Commonwealth Serum Laboratories) 20 g 1-'. The drops of the incubation medium were arrayed on the inside of the inverted lid of a plastic Petri dish. The bottom of the dish contained a layer of agar (8 g I-') to prevent the drops drying out. The dishes, each containing about 35 incubation drops, were sealed with wax film and incubated in an inverted position in the dark at 23O.