Microb Ecol (1984) 10:187-195
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Pure Culture Growth of Ectomycorrhizal Fungi on Inorganic Nitrogen Sources R. C. France s and C. P. P. Reid 2 qntemational Paper Company,Natchez, Mississippi 39120, USA; and 2Colorado State University,Fort Collins, Colorado80523, USA Abstract. Four ectomycorrhizal fungi were tested for their ability to grow (i.e., mycelial mat radial extension and fungal biomass) on nutrient media either supplemented with ammonium-nitrogen or nitrate-nitrogen or in the absence of an inorganic nitrogen source. Pisolithus tinctorius, Cenococcum geophilum and Thelephora terrestris exhibited greater growth on ammonium-nitrogen. Suillus granulatus grew better on the nitrate-nitrogen nutrient medium. Regardless of inorganic nitrogen form preference (i.e., ammonium-nitrogen or nitrate-nitrogen), all 4 species showed some growth on each of the 3 nutrient media. Growth rate maxima varied by fungal species as well as by inorganic nitrogen source. Maximum growth rate for T. terrestris exceeded rates exhibited by the other 3 fungi by 2-5 times. Introduction Mycorrhizae play an integral role in the nitrogen relations of trees. Nitrogen studies have shown an enhanced utilization of the element by the mycorrhizal (both ecto- and endo-) plant when compared with a nonmycorrhizal plant [ 11, 25, 27, 29-31]. The ability of the ectomycorrhiza to absorb and assimilate greater levels of nitrogen has been attributed to a greater surface area for absorption [11]. The fungus is predominantly responsible for this increased absorption ability through sheath formation, expansion of the root cortical cylinder, and emanation of extramatrical hyphae [4, 28]. The preference of specific host-fungus associations for particular nitrogen forms is dependent upon the ability of the individual symbionts to utilize these chemical forms. The fungus exerts a significant effect on the nitrogen relations of the ectomycorrhiza and may possibly alter the normal nitrogen absorption patterns for the nonmycorrhizal short root through sheath thickness and density. Ectomycorrhizal fungi vary in their ability to utilize various nitrogen forms, both organic and inorganic [14, 24, 26]. In forest ecosystems, inorganic forms predominate, and ammonium-nitrogen is usually the major ionic chemical species available to the ectomycorrhiza [13]. Forest soil characteristics, including pH and reductive capacity, usually preclude nitrate-nitrogen as an important source of nitrogen for trees. Pure culture studies [1, 14, 18, 19, 23] have shown ammonium-nitrogen to be the preferred form for growth of many
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e c t o m y c o r r h i z a l fungi. A m a j o r i t y o f t h e h i g h e r b a s i d i o m y c e t e s a p p e a r u n a b l e to u t i l i z e n i t r a t e - n i t r o g e n [7], a n a t t r i b u t e g e n e r a l l y c o n s i s t e n t w i t h o b s e r v a t i o n s o n g r o w t h o f e c t o m y c o r r h i z a l fungi. The purpose of this study was to compare the pure culture growth perform a n c e o f e c t o m y c o r r h i z a l fungi e x p o s e d t o a r t i f i c i a l m e d i a c o n t a i n i n g (1) a m m o n i u m - n i t r o g e n o n l y , (2) n i t r a t e - n i t r o g e n o n l y , a n d (3) n o n i t r o g e n . F u n g a l s p e c i e s i n v e s t i g a t e d i n c l u d e d Pisolithus tinctorius ( P e t s . ) C o k e r a n d C o u c h ( i s o l a t e 133, D. H . M a r x ) , Thelephora terrestris E h r h . ex F t . ( i s o l a t e 201, D. H . M a r x ) , Cenococcum geophilum Fr. ( i s o l a t e M 3 4 6 , B. Z a k ) , a n d Suillus granulatus (L. ex Fr.) O. K u n t z e ( i s o l a t e 7 5 - 2 0 , L. S. G i l l m a n ) .
Methods Mycelial pure cultures of the ectomycorrhizal fungi were grown on sterile, modified Melin-Norkrans (MMN) nutrient agar [ 15, 22]. Modifications of the standard MMN agar medium were utilized to yield 3 nitrogen treatments: ammonium-nitrogen (as ammonium chloride), nitrate-nitrogen (as potassium nitrate), and no added inorganic nitrogen (i.e., no inorganic nitrogen added). Levels of.ammonium and nitrate were maintained at 53 ppm nitrogen which is typical of standard MMN nutrient medium. The no inorganic nitrogen added treatment, as well as the ammonium-nitrogen and nitrate-nitrogen treatments, contained nitrogen in bound forms (e.g., thiamine hydrochloride, 20 ppb nitrogen; malt extract, 1.5 ppm nitrogen, proteinaceous). Concentrations of all other nutrients were maintained as in standard MMN medium to allow comparisons between treatments. Adjustments were accomplished by altering concentrations of normal salts and incorporating additional salts. Medium pH was adjusted to 6.0 +_ 0.2 and maintained throughout the study by means of a sodium citrate buffer additive [32]. Fresh mycelial discs, originally grown on MMN nutrient agar, of 7 mm diameter were aseptically transferred to Petri plates containing agar media (30 ml volume) of the various treatments. Five replications of each treatment for each of 4 species of fungi were used. Fungal cultures were incubated at 23.0 + 0.5"C in the absence of light. The concentric growth nature of these fungi in pure culture allowed periodic determination of radial extension of the mycelial mat. Extension was derived as a mean value for mat diameter determined by measurements at 2 predetermined positions separated perpendicularly to each other. Growth was allowed to occur until the agar surface was covered with hyphae or until stagnation of growth was evidenced by no significant increase in mycelial mat extension for at least 72 hours. Upon termination of growth, mycelial mat and agar medium were steam-sterilized at 121 ~ and 18 psi for 15 rain to separate mycelium and medium. Mycelial mats were then rinsed in warm tap water, frozen, freeze-dried at -50~ and 5/z Hg for 48 hours and then weighed.
Results P u r e c u l t u r e g r o w t h p e r f o r m a n c e is p r e s e n t e d f o r P. tinctorius (Fig. 1), C. geophilum (Fig. 2), T. terrestris (Fig. 3), a n d S. granulatus (Fig. 4). G r o w t h c u r v e f o r m w a s s i m i l a r f o r all 4 s p e c i e s , a n d e a c h c u r v e c o n s i s t e d o f 3 d e f i n e d g r o w t h stages: a n i n i t i a l l a g p e r i o d , a l o g a r i t h m i c g r o w t h stage, a n d a s t a t i o n a r y p h a s e . T i m e f o r e a c h s t a g e v a r i e d b e t w e e n s p e c i e s as well as b e t w e e n n i t r o g e n t r e a t m e n t s f o r e a c h species. G r o w t h w a s first o b s e r v e d b e t w e e n 2 a n d 5 d a y s following study initiation. Pisolithus tinctorius e x h i b i t e d a m a r k e d p r e f e r e n c e for a m m o n i u m - n i t r o g e n f o l l o w e d b y n i t r a t e - n i t r o g e n (Fig. 1). M y c e l i a l g r o w t h w a s s u b s t a n t i a l e v e n i n medium receiving no added inorganic nitrogen. However, growth attained at
Ectomycorrhizal Fungal Growth on Nitrogen Sources
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TIME(DAYS) Fig. 1. Pure culture growth of the ectomycorrhizal fungus Pisolithus tinctorius on nutrient medium containing ammonium-nitrogen (Q), nitrate-nitrogen (O), or no added inorganic nitrogen (n). Each data point represents the mean of 5 replications. Variability about the mean, as determined by 1 SE of the estimate, was less than 5% of the mean mycelial diameter in all cases.
the stationary phase for b o t h the nitrate-nitrogen and no inorganic nitrogen a d d e d treatments was still less than that attained by the fungus in the amm o n i u m - n i t r o g e n m e d i u m . G r o w t h attained after a c o m p a r a t i v e time period o f 30 days for the nitrate-nitrogen and no inorganic nitrogen added treatments, respectively, was 76 and 45% o f that exhibited with a m m o n i u m - n i t r o g e n . Greater variability a m o n g replications occurred with nitrate-nitrogen as compared with the other 2 treatments. Mycelial growth stagnated on nitrate-nitrogen and no inorganic nitrogen added m e d i a before total coverage o f m e d i u m was attained. Peak growth rate with a m m o n i u m - n i t r o g e n (4.75 r a m / d a y ) occurred at 15 days. M a x i m u m growth rates ranged 2.0-2.5 m m / d a y with nitrate-nitrogen and 1.1-1.4 m m / d a y for the no inorganic nitrogen added treatment. M e a n mycelial mat dry weight determinations for P. tinctorius at study t e r m i n a t i o n did not corroborate mycelial culture diameter growth due to the longer time period allowed for growth in the nitrate-nitrogen m e d i u m (Table 1). Later growth o f P. tinctorius on nitrate-nitrogen allowed for rnycelial mat densification as a result o f extensive hyphal branching growth within a fixed colony diameter, thus increasing the total hyphal length and fungal dry weight o v e r a small surface area. T h e growth performance o f C. geophilum followed similar trends as with P. tinctorius in that a m m o n i u m - n i t r o g e n served as the best source o f nitrogen for the fungus (Fig. 2). Preference for a particular form was variable until approximately 28 days into the growth period. B e y o n d this point, a m m o n i u m - n i t r o g e n served as a superior source o f inorganic nitrogen for mycelial growth. Data for the no inorganic nitrogen added t r e a t m e n t were unavailable after this time
R . C . France and C. P. P. Reid
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Table 1.
Mycelial mat dry weight at termination of growth period"
Inorganic nitrogen source Nitrate-nitrogen
No added inorganic nitrogen
114 _+ 10mg (30 days)
211 + 28 (47 days)
50 - 7 (52 days)
314 + 18 (52 days)
177 +__ 10 (57 days)
38 +-- 1 (28 days)
Thelephora terrestris a
226 +_ 32 (12 days)
73 +__ 11 (12 days)
54 +_ 11 (12 days)
Suillus granulatus ~
126 _+ 8 (52 days)
149 + 11 (57 days)
52 _ 7 (52 days)
Fungal species Pisolithus tinctorius b
Cenococcum geophilum c
Ammoniumnitrogen
a Table values represent mean mycelial mat dry weight _ 1 SE of estimate for 5 replications b Mycelial dry weight values for this species represent maximum coverage of agar medium surface area with mycelium (i.e., maximum radial extension o f mycelial mat attained) in the ammonium-nitrogen treatment. Mycelial dry weight values in the nitrate-nitrogen and no added inorganic nitrogen treatments represent some value less than maximum coverage o f agar medium surface area with mycelium (i.e., maximum radial extension of mycelial mat not attained) c Myeelial dry weight values for this species represent some value less than maximum coverage ofagar medium surface area with mycelium (i.e., maximum radial extension o f mycelial mat not attained) d Mycelial dry weight values for this species represent maximum coverage ofagar medium surface area with mycelium (i.e., maximum radial extension of mycelial mat attained)
period. Thus, a comparison of nitrate-nitrogen and no added inorganic nitrogen could not be made after 28 days. However, prior to this time, mycelial growth was greater on the no inorganic nitrogen added medium than on nitrate-nitrogen, suggesting the possibility of an inhibitory effect of nitrate-nitrogen on the growth of C. geophilum. Significant growth was obtained on all 3 media. Variability among replications was low for each treatment up to 28 days. Subsequent sampling times showed greater variation among replications in ammoniumnitrogen growth response than nitrate-nitrogen. Maximum growth rates after 28 days were generally low: 2.0-2.2 mm/day with ammonium-nitrogen, less than 1.0 mm/day with nitrate-nitrogen, and 1.4-1.5 mm/day for the no inorganic nitrogen added treatment. Dry weight results confirmed the pattern established by mycelial mat growth (Table 1). High dry weight values for C. geophilum indicate that growth into the media was masked by surface growth, which was generally slowest for all 4 species. Mycelial growth on ammoniumnitrogen and nitrate-nitrogen stagnated before total coverage of the media surface was attained. Thelephora terrestris grew equally well and very rapidly on all 3 nitrogen treatments (Fig. 3). Total time required to cover the available media was 12 days. Due to this short time period, a preference for a particular nitrogen form could not be observed from radial extension information. Variability was low except in the no inorganic nitrogen added treatment. Peak growth rate values exceeded 10 mm/day and occurred at approximately 6 days for all nitrogen
Ectomycorrhizai Fungal Growth on Nitrogen Sources
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Fig. 3. Pure culture growth of the ectomycorrhizal fungus Thelephora terrestris on nutrient medium containing the same as in Fig. 1. Data points and variability about the mean same as in Fig. 1.
treatments. A comparison of mycelial mat dry weight at 12 days showed that the fungus grew far better on ammonium-nitrogen than on the other 2 media (Table 1). Dry weight growth on nitrate-nitrogen and no added inorganic nitrogen was 33 and 24%, respectively, of that attained on ammonium-nitrogen.
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Differences in growth of T. terrestris between nitrogen treatments could be attributed to mycelial mat densification, as previously noted with P. tinctorius. Nitrate-nitrogen served as the best source of nitrogen for the growth of S. granulatus (Fig. 4) as opposed to results with P. tinctorius, C. geophilum;and T. terrestris. Growth on ammonium-nitrogen was similar to that attained with nitrate-nitrogen for approximately the first 28 days. Beyond 28 days, nitratenitrogen served as a superior nitrogen source for mycelial growth. Variability within each treatment was somewhat higher for S. granulatus than for the other fungi, especially with ammonium-nitrogen and nitrate-nitrogen treatments. Growth rate maxima were approximately 2.0 m m / d a y with the ammoniumnitrogen and nitrate-nitrogen treatments and about 1.7 m m / d a y in the no inorganic nitrogen added treatment. Dry weight results verified that growth trends were similar between nitrogen treatments (Table 1). The somewhat longer growth period in the nitrate-nitrogen treatment was inconsequential to overall dry weight, as can be seen by minimal growth, if any, in the late stages of the growth period (Fig. 4). Mycelial growth stagnated on all 3 media before total medium surface area coverage could be attained.
Discussion
Current information indicates that mycorrhizal fungi alone do not possess the ability to decompose organically bound nitrogen in the soil [ 14, 16] unless the humus is first hydrolyzed and glucose is added to initiate degradationr [8]. However, when mycorrhizal fungi are combined in association with short feeder roots, nitrogen may perhaps be mineralized from the humus complex [ 14]. In many forest environments, inorganic nitrogen forms may be most important
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in terms of ectomycorrhizal nutrition, and ammonium-nitrogen is often the most abundant and readily available source [5, 6, 11]. Pure culture growth results for ectomycorrhizal fungi showed that mycelial mat diameter was generally more extensive in the presence of ammoniumnitrogen than in either nitrate-nitrogen or in the absence of readily utilizable inorganic nitrogen. This trend was apparent for P. tinctorius, C. geophilum, and T. terrestris. Dry weight production information supported these findings and, in fact, was necessary to segregate treatment results for 7". terrestris. Similar results for several different isolates of ectomycorrhizal fungi were reported in the works of Bakshi [1], Lundeberg [14], Melin and Mikola [18], Mikola [20], and Norkrans [23]. Of the 3 isolates reported in this work that grew best on ammonium-nitrogen, only C. geophilum had been examined in the aforementioned literature. The growth patterns of T. terrestris were especially interesting. Diameter results indicated no preference for nitrogen substrate including the fact that this fungus grew as well without added inorganic nitrogen as with either ammonium-nitrogen or nitrate-nitrogen. Dry weight findings revealed that ammonium-nitrogen was far superior to either remaining nitrogen treatment. This suggested a densification of mycelium throughout the 12 days with the cationic nitrogen form. Suillus granulatus deviated from the other isolates in that this species attained its greatest diameter growth on nitrate-nitrogen. Dry-weight production supported this finding, but significant growth was also attained on ammoniumnitrogen. These results parallel those of Lundeberg [14] to some extent for 2 isolates of this fungus. One isolate grew much better on ammonium-nitrogen, but a second exhibited only slightly greater dry-weight growth on nitrate-nitrogen as compared with ammonium-nitrogen. Several reports have shown nitrate-nitrogen to be a good source for the growth of ectomycorrhizal fungi, but ammonium-nitrogen proved to be even better [1, 9, 10, 12, 17, 19, 21, 23]. Even though each of these 4 fungal species showed preference for a particular nitrogen form, all 3 nitrogen treatments provided a suitable medium for growth. In no case did any of the nitrogen treatments prohibit growth, as has been shown with nitrite-nitrogen by Lundeberg [ 14]. The no inorganic nitrogen added treatment contained nitrogen in bound forms (i.e., organic) which may account for the growth attained on this medium. The demand for thiamine by the fungus for other metabolic processes and the low nitrogen content of malt extract compared with the levels of ammonium-nitrogen and nitrate-nitrogen added to the media preclude these nitrogen sources as being significant in supporting mycelial growth. However, the substantial fungal growth attained on the no inorganic nitrogen added medium may have been due to organic nitrogen supplied from the agar base, which was a common factor among all 3 treatments. Ammonium-nitrogen would appear to serve as a more readily utilizable source of the element than nitrate-nitrogen based on the additional energy expenditure required in reducing the latter to ammonium-nitrogen prior to incorporation in fungal metabolites [2, 3, 20]. It is apparent that these 4 species perform differently when exposed to the same nutrient environments. Such differences are probably genetically based
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a n d m a y p r o v i d e a n ecological a d v a n t a g e for a f u n g u s i n its n a t u r a l h a b i t a t . F o r e x a m p l e , t h e r a p i d i t y w i t h w h i c h T. terrestris a b s o r b s a n d a s s i m i l a t e s n i t r o g e n m a y c o n t r i b u t e to its a b i l i t y to r a p i d l y i n v a d e f u m i g a t e d soil a n d c o l o n i z e p i n e r o o t s y s t e m s i n n u r s e r i e s i n the s o u t h e a s t e r n U n i t e d States, t h u s a c t i n g as a " p i o n e e r " e c t o m y c o r r h i z a l f u n g u s . T h e a b i l i t y o f S. granulatus to r e a d i l y u t i l i z e b o t h a m m o n i u m - n i t r o g e n a n d n i t r a t e - n i t r o g e n m a y offer t h i s f u n g u s c e r t a i n s u r v i v a l o p p o r t u n i t i e s u n d e r d y n a m i c forest soil n u t r i t i o n a l c o n d i t i o n s w h e r e n i t r o g e n flushes are s p o r a d i c d u e to f e r t i l i z a t i o n practices.
Acknowledgments. Research was conducted under National Science Foundation Grant No. BMS 75-02878.
References 1. Bakshi BK (1974) Mycorrhiza and its role in forestry. Forest Research Institute and Colleges. Dehra Dun, India 2. Beevers L, Hageman RH (1969) Nitrate reduction in higher plants. Ann Rev Plant Physiol 20:495-522 3. Bonner J, Varner JE (eds) (1976) Plant biochemistry. Academic Press, New York 4. Bowen GD (1973) Mineral nutrition of ectomycorrhizae. In: Marks GC, Kozlowski TT (eds) Ectomycorrhizae: their ecology and physiology. Academic Press, New York pp 151-205 5. Carrodus BB (1966) Absorption of nitrogen by mycorrhizal roots of beech. I. Factors affecting the assimilation of nitrogen. New Phytol 65:358-371 6. Carrodus BB (1967) Absorption of nitrogen by mycorrhizal roots of beech. II. Ammonium and nitrate as sources of nitrogen. New Phytol 66:1-4 7. Cochrane VW (1958) Physiology of fungi. John Wiley and Sons, New York 8. Eglite AK (1958) Slightly soluble minerals and organic substances as nutrients of mycorrhizal fungi. Tr Inst Mikrobiol Acad Nauk Latv SSR 7:67-75 (Transl. from Russian, Israel Program Sci. Transl. Jerusalem, 1963) 9. Griffin DM (1972) Ecology of soil fungi. Syracuse University Press, Syracuse, New York 10. Hacskaylo E, Lilly V, Barnett H (1954) Growth of fungi on three sources of nitrogen. Mycologia 46:691-701 11. Hatch AB (1937) The physical basis of mycotrophy in the genus Pinus. Black Rock Forest Bulletin 6:168 12. How JE (1940) The mycorrhizal relations of larch. I. A study ofBoletus elegans Schum. in pure culture. Ann Bot Lond N.S. 4:135-150 13. Keeney DR (1980) Prediction of soil nitrogen availability in forest ecosystems: a literature review. For Sci 26:159-171 14. Lundeberg G (1970) Utilisation of various nitrogen sources, in particular bound nitrogen, by mycorrhizal fungi. Stud suec 79:1-95 15. Marx DH (1969) The influence ofectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonisms of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopath 59:153-163 16. McArdle RE (1932) The relations of mycorrhizae to conifer seedlings. J Agric Res 44: 287-316 17. Melin E (1925) Untersuchungenfiber die Bedentung der Baummykorrhiza. Fisher-Verlag. Jena 18. Melin E, Mikola P (1948) Effect of some amino acids on the growth ofCenococcum graniforme. Physiol Plant 1:109-112 19. Middleton KR, Smith GS (1979) A comparison of ammoniacal and nitrate nutrition of perennial ryegrass through a thermodynamic model. Plant Soil 53:487-504 20. Mikola P (1965) Studies on the ectendotrophic mycorrhiza of pines. Acta For Fenn 79:1-56 21. Nicholas DJD (1965) Utilization of inorganic nitrogen compounds and amino acids by fungi. In: Ainsworth GC Sussman AS (eds) The fungi, Vol. 2. Academic Press, New York, pp 349376
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22. Norkrans B (1949) Some mycorrhiza-forming Tricholoma species. Svensk Bot Tidskr 43:485490 23. Norkrans B (1950) Studies in growth and cellulolytic enzymes of Tricholoma with special reference to mycorrhiza formation. Symb Bot Ups 11:1-126 24. Norkrans B (1953) The effect of glutamic acid, aspartic acid and related compounds on the growth of certain Tricholoma species. Physiol Plant 6:584-593 25. Possingllam JV, Groot Obbink J (197 I) Endotrophic mycorrhiza and the nutrition of grape vines. Vitis 10:120-130 26. Rawald W (1960) Untersuchungen zur Stickstoffernahrung der h~Sheren pilze. Internationales Mykorrhizasymposium. Weimer. Fischer-Verlag, Jena 27. Read DJ, Stribley DP (1973) Effect of mycorrhizal infection on nitrogen and phosphorus nutrition of ericaceous plants. Nature (London) 244:81-82 28. Russell RS, Newbould P (1969) The pattern of nutrient uptake in root systems. In: Whittington WJ (ed) Root growth. Butterworth, London, pp 148-169 29. Smith SE, Daft MJ (1977) Interactions between growth, phosphate content and N 2 fixation in mycorrhizal and non-mycorrhizal Medicago sativa. Aust J Plant Physiol 4:403-413 30. Stribley DP, Read DJ (1976) The biology of mycorrhiza in the Ericaceae. VI. The effects of mycorrhizal infection and concentration of ammonium nitrogen on growth of cranberry (Vaccinium macrocarpon Air.) in sand culture. New Phytoi 77:63-72 31. Stribley DP, Read DJ, Hunt R (1975) The biology of mycorrhiza in the Ericaceae. V. The effects of mycorrhizal infection, soil type and partial soil sterilization (by gamma irradiation) on the growth of cranberry (Vaccinium macrocarpon Ait.). New Phytol 75:119-130 32. Weiss JB (1968) Haemoglobins. In: Smith Ivor (ed) Chromatographic and electrophoretic techniques. Vol. II. Zone electrophoresis. Interscience Publishers, New York pp 43-55.
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Pure Culture Growth of Ectomycorrhizal Fungi on Inorganic Nitrogen Sources R. C. France s and C. P. P. Reid 2 qntemational Paper Company,Natchez, Mississippi 39120, USA; and 2Colorado State University,Fort Collins, Colorado80523, USA Abstract. Four ectomycorrhizal fungi were tested for their ability to grow (i.e., mycelial mat radial extension and fungal biomass) on nutrient media either supplemented with ammonium-nitrogen or nitrate-nitrogen or in the absence of an inorganic nitrogen source. Pisolithus tinctorius, Cenococcum geophilum and Thelephora terrestris exhibited greater growth on ammonium-nitrogen. Suillus granulatus grew better on the nitrate-nitrogen nutrient medium. Regardless of inorganic nitrogen form preference (i.e., ammonium-nitrogen or nitrate-nitrogen), all 4 species showed some growth on each of the 3 nutrient media. Growth rate maxima varied by fungal species as well as by inorganic nitrogen source. Maximum growth rate for T. terrestris exceeded rates exhibited by the other 3 fungi by 2-5 times. Introduction Mycorrhizae play an integral role in the nitrogen relations of trees. Nitrogen studies have shown an enhanced utilization of the element by the mycorrhizal (both ecto- and endo-) plant when compared with a nonmycorrhizal plant [ 11, 25, 27, 29-31]. The ability of the ectomycorrhiza to absorb and assimilate greater levels of nitrogen has been attributed to a greater surface area for absorption [11]. The fungus is predominantly responsible for this increased absorption ability through sheath formation, expansion of the root cortical cylinder, and emanation of extramatrical hyphae [4, 28]. The preference of specific host-fungus associations for particular nitrogen forms is dependent upon the ability of the individual symbionts to utilize these chemical forms. The fungus exerts a significant effect on the nitrogen relations of the ectomycorrhiza and may possibly alter the normal nitrogen absorption patterns for the nonmycorrhizal short root through sheath thickness and density. Ectomycorrhizal fungi vary in their ability to utilize various nitrogen forms, both organic and inorganic [14, 24, 26]. In forest ecosystems, inorganic forms predominate, and ammonium-nitrogen is usually the major ionic chemical species available to the ectomycorrhiza [13]. Forest soil characteristics, including pH and reductive capacity, usually preclude nitrate-nitrogen as an important source of nitrogen for trees. Pure culture studies [1, 14, 18, 19, 23] have shown ammonium-nitrogen to be the preferred form for growth of many
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e c t o m y c o r r h i z a l fungi. A m a j o r i t y o f t h e h i g h e r b a s i d i o m y c e t e s a p p e a r u n a b l e to u t i l i z e n i t r a t e - n i t r o g e n [7], a n a t t r i b u t e g e n e r a l l y c o n s i s t e n t w i t h o b s e r v a t i o n s o n g r o w t h o f e c t o m y c o r r h i z a l fungi. The purpose of this study was to compare the pure culture growth perform a n c e o f e c t o m y c o r r h i z a l fungi e x p o s e d t o a r t i f i c i a l m e d i a c o n t a i n i n g (1) a m m o n i u m - n i t r o g e n o n l y , (2) n i t r a t e - n i t r o g e n o n l y , a n d (3) n o n i t r o g e n . F u n g a l s p e c i e s i n v e s t i g a t e d i n c l u d e d Pisolithus tinctorius ( P e t s . ) C o k e r a n d C o u c h ( i s o l a t e 133, D. H . M a r x ) , Thelephora terrestris E h r h . ex F t . ( i s o l a t e 201, D. H . M a r x ) , Cenococcum geophilum Fr. ( i s o l a t e M 3 4 6 , B. Z a k ) , a n d Suillus granulatus (L. ex Fr.) O. K u n t z e ( i s o l a t e 7 5 - 2 0 , L. S. G i l l m a n ) .
Methods Mycelial pure cultures of the ectomycorrhizal fungi were grown on sterile, modified Melin-Norkrans (MMN) nutrient agar [ 15, 22]. Modifications of the standard MMN agar medium were utilized to yield 3 nitrogen treatments: ammonium-nitrogen (as ammonium chloride), nitrate-nitrogen (as potassium nitrate), and no added inorganic nitrogen (i.e., no inorganic nitrogen added). Levels of.ammonium and nitrate were maintained at 53 ppm nitrogen which is typical of standard MMN nutrient medium. The no inorganic nitrogen added treatment, as well as the ammonium-nitrogen and nitrate-nitrogen treatments, contained nitrogen in bound forms (e.g., thiamine hydrochloride, 20 ppb nitrogen; malt extract, 1.5 ppm nitrogen, proteinaceous). Concentrations of all other nutrients were maintained as in standard MMN medium to allow comparisons between treatments. Adjustments were accomplished by altering concentrations of normal salts and incorporating additional salts. Medium pH was adjusted to 6.0 +_ 0.2 and maintained throughout the study by means of a sodium citrate buffer additive [32]. Fresh mycelial discs, originally grown on MMN nutrient agar, of 7 mm diameter were aseptically transferred to Petri plates containing agar media (30 ml volume) of the various treatments. Five replications of each treatment for each of 4 species of fungi were used. Fungal cultures were incubated at 23.0 + 0.5"C in the absence of light. The concentric growth nature of these fungi in pure culture allowed periodic determination of radial extension of the mycelial mat. Extension was derived as a mean value for mat diameter determined by measurements at 2 predetermined positions separated perpendicularly to each other. Growth was allowed to occur until the agar surface was covered with hyphae or until stagnation of growth was evidenced by no significant increase in mycelial mat extension for at least 72 hours. Upon termination of growth, mycelial mat and agar medium were steam-sterilized at 121 ~ and 18 psi for 15 rain to separate mycelium and medium. Mycelial mats were then rinsed in warm tap water, frozen, freeze-dried at -50~ and 5/z Hg for 48 hours and then weighed.
Results P u r e c u l t u r e g r o w t h p e r f o r m a n c e is p r e s e n t e d f o r P. tinctorius (Fig. 1), C. geophilum (Fig. 2), T. terrestris (Fig. 3), a n d S. granulatus (Fig. 4). G r o w t h c u r v e f o r m w a s s i m i l a r f o r all 4 s p e c i e s , a n d e a c h c u r v e c o n s i s t e d o f 3 d e f i n e d g r o w t h stages: a n i n i t i a l l a g p e r i o d , a l o g a r i t h m i c g r o w t h stage, a n d a s t a t i o n a r y p h a s e . T i m e f o r e a c h s t a g e v a r i e d b e t w e e n s p e c i e s as well as b e t w e e n n i t r o g e n t r e a t m e n t s f o r e a c h species. G r o w t h w a s first o b s e r v e d b e t w e e n 2 a n d 5 d a y s following study initiation. Pisolithus tinctorius e x h i b i t e d a m a r k e d p r e f e r e n c e for a m m o n i u m - n i t r o g e n f o l l o w e d b y n i t r a t e - n i t r o g e n (Fig. 1). M y c e l i a l g r o w t h w a s s u b s t a n t i a l e v e n i n medium receiving no added inorganic nitrogen. However, growth attained at
Ectomycorrhizal Fungal Growth on Nitrogen Sources
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TIME(DAYS) Fig. 1. Pure culture growth of the ectomycorrhizal fungus Pisolithus tinctorius on nutrient medium containing ammonium-nitrogen (Q), nitrate-nitrogen (O), or no added inorganic nitrogen (n). Each data point represents the mean of 5 replications. Variability about the mean, as determined by 1 SE of the estimate, was less than 5% of the mean mycelial diameter in all cases.
the stationary phase for b o t h the nitrate-nitrogen and no inorganic nitrogen a d d e d treatments was still less than that attained by the fungus in the amm o n i u m - n i t r o g e n m e d i u m . G r o w t h attained after a c o m p a r a t i v e time period o f 30 days for the nitrate-nitrogen and no inorganic nitrogen added treatments, respectively, was 76 and 45% o f that exhibited with a m m o n i u m - n i t r o g e n . Greater variability a m o n g replications occurred with nitrate-nitrogen as compared with the other 2 treatments. Mycelial growth stagnated on nitrate-nitrogen and no inorganic nitrogen added m e d i a before total coverage o f m e d i u m was attained. Peak growth rate with a m m o n i u m - n i t r o g e n (4.75 r a m / d a y ) occurred at 15 days. M a x i m u m growth rates ranged 2.0-2.5 m m / d a y with nitrate-nitrogen and 1.1-1.4 m m / d a y for the no inorganic nitrogen added treatment. M e a n mycelial mat dry weight determinations for P. tinctorius at study t e r m i n a t i o n did not corroborate mycelial culture diameter growth due to the longer time period allowed for growth in the nitrate-nitrogen m e d i u m (Table 1). Later growth o f P. tinctorius on nitrate-nitrogen allowed for rnycelial mat densification as a result o f extensive hyphal branching growth within a fixed colony diameter, thus increasing the total hyphal length and fungal dry weight o v e r a small surface area. T h e growth performance o f C. geophilum followed similar trends as with P. tinctorius in that a m m o n i u m - n i t r o g e n served as the best source o f nitrogen for the fungus (Fig. 2). Preference for a particular form was variable until approximately 28 days into the growth period. B e y o n d this point, a m m o n i u m - n i t r o g e n served as a superior source o f inorganic nitrogen for mycelial growth. Data for the no inorganic nitrogen added t r e a t m e n t were unavailable after this time
R . C . France and C. P. P. Reid
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Table 1.
Mycelial mat dry weight at termination of growth period"
Inorganic nitrogen source Nitrate-nitrogen
No added inorganic nitrogen
114 _+ 10mg (30 days)
211 + 28 (47 days)
50 - 7 (52 days)
314 + 18 (52 days)
177 +__ 10 (57 days)
38 +-- 1 (28 days)
Thelephora terrestris a
226 +_ 32 (12 days)
73 +__ 11 (12 days)
54 +_ 11 (12 days)
Suillus granulatus ~
126 _+ 8 (52 days)
149 + 11 (57 days)
52 _ 7 (52 days)
Fungal species Pisolithus tinctorius b
Cenococcum geophilum c
Ammoniumnitrogen
a Table values represent mean mycelial mat dry weight _ 1 SE of estimate for 5 replications b Mycelial dry weight values for this species represent maximum coverage of agar medium surface area with mycelium (i.e., maximum radial extension o f mycelial mat attained) in the ammonium-nitrogen treatment. Mycelial dry weight values in the nitrate-nitrogen and no added inorganic nitrogen treatments represent some value less than maximum coverage o f agar medium surface area with mycelium (i.e., maximum radial extension of mycelial mat not attained) c Myeelial dry weight values for this species represent some value less than maximum coverage ofagar medium surface area with mycelium (i.e., maximum radial extension o f mycelial mat not attained) d Mycelial dry weight values for this species represent maximum coverage ofagar medium surface area with mycelium (i.e., maximum radial extension of mycelial mat attained)
period. Thus, a comparison of nitrate-nitrogen and no added inorganic nitrogen could not be made after 28 days. However, prior to this time, mycelial growth was greater on the no inorganic nitrogen added medium than on nitrate-nitrogen, suggesting the possibility of an inhibitory effect of nitrate-nitrogen on the growth of C. geophilum. Significant growth was obtained on all 3 media. Variability among replications was low for each treatment up to 28 days. Subsequent sampling times showed greater variation among replications in ammoniumnitrogen growth response than nitrate-nitrogen. Maximum growth rates after 28 days were generally low: 2.0-2.2 mm/day with ammonium-nitrogen, less than 1.0 mm/day with nitrate-nitrogen, and 1.4-1.5 mm/day for the no inorganic nitrogen added treatment. Dry weight results confirmed the pattern established by mycelial mat growth (Table 1). High dry weight values for C. geophilum indicate that growth into the media was masked by surface growth, which was generally slowest for all 4 species. Mycelial growth on ammoniumnitrogen and nitrate-nitrogen stagnated before total coverage of the media surface was attained. Thelephora terrestris grew equally well and very rapidly on all 3 nitrogen treatments (Fig. 3). Total time required to cover the available media was 12 days. Due to this short time period, a preference for a particular nitrogen form could not be observed from radial extension information. Variability was low except in the no inorganic nitrogen added treatment. Peak growth rate values exceeded 10 mm/day and occurred at approximately 6 days for all nitrogen
Ectomycorrhizai Fungal Growth on Nitrogen Sources
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treatments. A comparison of mycelial mat dry weight at 12 days showed that the fungus grew far better on ammonium-nitrogen than on the other 2 media (Table 1). Dry weight growth on nitrate-nitrogen and no added inorganic nitrogen was 33 and 24%, respectively, of that attained on ammonium-nitrogen.
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Differences in growth of T. terrestris between nitrogen treatments could be attributed to mycelial mat densification, as previously noted with P. tinctorius. Nitrate-nitrogen served as the best source of nitrogen for the growth of S. granulatus (Fig. 4) as opposed to results with P. tinctorius, C. geophilum;and T. terrestris. Growth on ammonium-nitrogen was similar to that attained with nitrate-nitrogen for approximately the first 28 days. Beyond 28 days, nitratenitrogen served as a superior nitrogen source for mycelial growth. Variability within each treatment was somewhat higher for S. granulatus than for the other fungi, especially with ammonium-nitrogen and nitrate-nitrogen treatments. Growth rate maxima were approximately 2.0 m m / d a y with the ammoniumnitrogen and nitrate-nitrogen treatments and about 1.7 m m / d a y in the no inorganic nitrogen added treatment. Dry weight results verified that growth trends were similar between nitrogen treatments (Table 1). The somewhat longer growth period in the nitrate-nitrogen treatment was inconsequential to overall dry weight, as can be seen by minimal growth, if any, in the late stages of the growth period (Fig. 4). Mycelial growth stagnated on all 3 media before total medium surface area coverage could be attained.
Discussion
Current information indicates that mycorrhizal fungi alone do not possess the ability to decompose organically bound nitrogen in the soil [ 14, 16] unless the humus is first hydrolyzed and glucose is added to initiate degradationr [8]. However, when mycorrhizal fungi are combined in association with short feeder roots, nitrogen may perhaps be mineralized from the humus complex [ 14]. In many forest environments, inorganic nitrogen forms may be most important
EctomycorrhizalFungalGrowth on Nitrogen Sources
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in terms of ectomycorrhizal nutrition, and ammonium-nitrogen is often the most abundant and readily available source [5, 6, 11]. Pure culture growth results for ectomycorrhizal fungi showed that mycelial mat diameter was generally more extensive in the presence of ammoniumnitrogen than in either nitrate-nitrogen or in the absence of readily utilizable inorganic nitrogen. This trend was apparent for P. tinctorius, C. geophilum, and T. terrestris. Dry weight production information supported these findings and, in fact, was necessary to segregate treatment results for 7". terrestris. Similar results for several different isolates of ectomycorrhizal fungi were reported in the works of Bakshi [1], Lundeberg [14], Melin and Mikola [18], Mikola [20], and Norkrans [23]. Of the 3 isolates reported in this work that grew best on ammonium-nitrogen, only C. geophilum had been examined in the aforementioned literature. The growth patterns of T. terrestris were especially interesting. Diameter results indicated no preference for nitrogen substrate including the fact that this fungus grew as well without added inorganic nitrogen as with either ammonium-nitrogen or nitrate-nitrogen. Dry weight findings revealed that ammonium-nitrogen was far superior to either remaining nitrogen treatment. This suggested a densification of mycelium throughout the 12 days with the cationic nitrogen form. Suillus granulatus deviated from the other isolates in that this species attained its greatest diameter growth on nitrate-nitrogen. Dry-weight production supported this finding, but significant growth was also attained on ammoniumnitrogen. These results parallel those of Lundeberg [14] to some extent for 2 isolates of this fungus. One isolate grew much better on ammonium-nitrogen, but a second exhibited only slightly greater dry-weight growth on nitrate-nitrogen as compared with ammonium-nitrogen. Several reports have shown nitrate-nitrogen to be a good source for the growth of ectomycorrhizal fungi, but ammonium-nitrogen proved to be even better [1, 9, 10, 12, 17, 19, 21, 23]. Even though each of these 4 fungal species showed preference for a particular nitrogen form, all 3 nitrogen treatments provided a suitable medium for growth. In no case did any of the nitrogen treatments prohibit growth, as has been shown with nitrite-nitrogen by Lundeberg [ 14]. The no inorganic nitrogen added treatment contained nitrogen in bound forms (i.e., organic) which may account for the growth attained on this medium. The demand for thiamine by the fungus for other metabolic processes and the low nitrogen content of malt extract compared with the levels of ammonium-nitrogen and nitrate-nitrogen added to the media preclude these nitrogen sources as being significant in supporting mycelial growth. However, the substantial fungal growth attained on the no inorganic nitrogen added medium may have been due to organic nitrogen supplied from the agar base, which was a common factor among all 3 treatments. Ammonium-nitrogen would appear to serve as a more readily utilizable source of the element than nitrate-nitrogen based on the additional energy expenditure required in reducing the latter to ammonium-nitrogen prior to incorporation in fungal metabolites [2, 3, 20]. It is apparent that these 4 species perform differently when exposed to the same nutrient environments. Such differences are probably genetically based
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a n d m a y p r o v i d e a n ecological a d v a n t a g e for a f u n g u s i n its n a t u r a l h a b i t a t . F o r e x a m p l e , t h e r a p i d i t y w i t h w h i c h T. terrestris a b s o r b s a n d a s s i m i l a t e s n i t r o g e n m a y c o n t r i b u t e to its a b i l i t y to r a p i d l y i n v a d e f u m i g a t e d soil a n d c o l o n i z e p i n e r o o t s y s t e m s i n n u r s e r i e s i n the s o u t h e a s t e r n U n i t e d States, t h u s a c t i n g as a " p i o n e e r " e c t o m y c o r r h i z a l f u n g u s . T h e a b i l i t y o f S. granulatus to r e a d i l y u t i l i z e b o t h a m m o n i u m - n i t r o g e n a n d n i t r a t e - n i t r o g e n m a y offer t h i s f u n g u s c e r t a i n s u r v i v a l o p p o r t u n i t i e s u n d e r d y n a m i c forest soil n u t r i t i o n a l c o n d i t i o n s w h e r e n i t r o g e n flushes are s p o r a d i c d u e to f e r t i l i z a t i o n practices.
Acknowledgments. Research was conducted under National Science Foundation Grant No. BMS 75-02878.
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22. Norkrans B (1949) Some mycorrhiza-forming Tricholoma species. Svensk Bot Tidskr 43:485490 23. Norkrans B (1950) Studies in growth and cellulolytic enzymes of Tricholoma with special reference to mycorrhiza formation. Symb Bot Ups 11:1-126 24. Norkrans B (1953) The effect of glutamic acid, aspartic acid and related compounds on the growth of certain Tricholoma species. Physiol Plant 6:584-593 25. Possingllam JV, Groot Obbink J (197 I) Endotrophic mycorrhiza and the nutrition of grape vines. Vitis 10:120-130 26. Rawald W (1960) Untersuchungen zur Stickstoffernahrung der h~Sheren pilze. Internationales Mykorrhizasymposium. Weimer. Fischer-Verlag, Jena 27. Read DJ, Stribley DP (1973) Effect of mycorrhizal infection on nitrogen and phosphorus nutrition of ericaceous plants. Nature (London) 244:81-82 28. Russell RS, Newbould P (1969) The pattern of nutrient uptake in root systems. In: Whittington WJ (ed) Root growth. Butterworth, London, pp 148-169 29. Smith SE, Daft MJ (1977) Interactions between growth, phosphate content and N 2 fixation in mycorrhizal and non-mycorrhizal Medicago sativa. Aust J Plant Physiol 4:403-413 30. Stribley DP, Read DJ (1976) The biology of mycorrhiza in the Ericaceae. VI. The effects of mycorrhizal infection and concentration of ammonium nitrogen on growth of cranberry (Vaccinium macrocarpon Air.) in sand culture. New Phytoi 77:63-72 31. Stribley DP, Read DJ, Hunt R (1975) The biology of mycorrhiza in the Ericaceae. V. The effects of mycorrhizal infection, soil type and partial soil sterilization (by gamma irradiation) on the growth of cranberry (Vaccinium macrocarpon Ait.). New Phytol 75:119-130 32. Weiss JB (1968) Haemoglobins. In: Smith Ivor (ed) Chromatographic and electrophoretic techniques. Vol. II. Zone electrophoresis. Interscience Publishers, New York pp 43-55.
