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Mycorrhizal fungi of Pseudotsuga menziesii in the North Island of New Zealand
0038-0717 81~030247-03102 W.0 CopyrIght 0 1981 Pergrmon Press Ltd
Sad Bml Bmrhrm Vol 13. pp 247 lo 249. 1981 Prmted ,n Great Brrtam All nghts reserved
MYCORRHIZAL FUNGI OF PSEUDOTSUGA MENZIESIZ IN THE NORTH ISLAND OF NEW ZEALAND MYRACHU-CHOU
and LYNETTEJ.GRACE
Forest Research Institute, Private Bag Rotorua, New Zealand (Accepted 26 August 1980) Summary-Attempts were made to isolate mycorrhizal fungi of a timber species, Pseudotsugamenziesii (Mirb.) Franc0 which is exotic to New Zealand. Over 2000 pieces of mycorrhizal roots were plated out on Hagem medium and modified Melin-Norkrans medium; 14% of these yielded Rhizopogonuinicolor A. H. Smith, 6% gave rise to Amanitamuscariu(L. ex Fr.) S. F. Gray, and 7% to unidentified basidiomycetes, 21% yielded dark sterile mycelia, 13% were sporing Fungi Imperfecti presumed to be contaminants, and 39% remained free of fungal growth. Eleven basidomycetous fungi were tested for their mycorrhiza-forming ability on P. menziesiiseedlings. Under the test conditions R. uinicolor, Hebeloma crustuliniforme (Bull. ex St. Am) Quel., A. muscuria, Sclerodertnabovista Fr.. Laccaria laccata (Stop. ex Fr.) Berk. & Br.. Inocybe corydalina (?), I. jurana (?), and I. maculata (?) formed mycorrhizas.
INTRODUCTION
In some forest nurseries in New Zealand where Douglas fir (Pseudotsuga menziesii (Mirb.) France) has not previously been grown, the first crop often has a “mycorrhizal problem”, i.e. the seedlings, though looking “normal” in top growth, lack mycorrhizal roots. That the top growth is apparently normal is probably due to the high fertility of the nursery soil. But when such seedlings are planted out severe chlorosis and growth retardation may occur. This has been reported in outplantings of Douglas fir that lack mycorrhizas both in New Zealand and elsewhere (Gilmour, 1958; Trappe and Strand, 1969; Wright, 1971). Douglas fir is an exotic timber species which has been widely planted in New Zealand but its mycorrhizal relationship has so far received little attention. Birch (1937) mentioned the association of Scleroderma bouista Fr. with Douglas fir. Rawlings (1951; 1958) reported the association of Suillus (Boletus) lakei (Murr.) Sing., Suillus piperatus (Bull. ex Fr.) 0. Kuntze, Suillus uuriegatus (Swartz ex Fr.) 0. Kuntze, Amanita muscariu (L. ex Fr.) Pers. ex ,Hooker, Tricholoma sp., Laccaria laccata (Stop. ex Fr.) Berk. & Br., Lycoperdon perlatum Batsch, and S. bovista fructifications with Douglas fir stands. We cannot find any information on the kinds of fungi that can be isolated from mycorrhizas of Douglas fir in New Zealand, nor confirmation of the symbiotic relationship by synthesis tests. It is the purpose of this paper to fill this gap in knowledge. MATERIALS AND METHODS Field observations
From 1976 to 1978 Basidiomycete sporocarps were collected during autumn, early winter, and spring from four nurseries (Forest Research Institute, Gwavas, Owhata and Kaingaroa) in the North Island of New Zealand where Douglas fir was grown and from more than 30 stands of different ages (3 yr to 247
over 50 yr) growing in Kaingaroa and Whakarewarewa State Forests, Rotorua. The sporocarps were identified as reported by Chu-Chou (1979). Isolation offungal symbiontsfrom mycorrhizas and synthesis tests
Mycorrhizal root materials were collected from the same nurseries and forests where field observations were carried out. Three collections were made in which whole root systems of l&20 seedlings were collected from each nursery and mycorrhizal root samples were collected from 10 to 15 randomly selected trees from each stand of different age. Over 40 sets of isolations were carried out and a total of 2308 pieces of mycorrhizal roots were plated on to Hagem medium (Modess, 1941) and modified Melin-Norkrans medium (Marx, 1969). The isolation technique, which was similar to the method used by Chu-Chou in a study of Pinus radiata D. Don mycorrhizas (1979), was first to wash and then to surface-sterilize the mycorrhizal roots in 0.7% calcium hypochlorite for 7-10 min. Twenty to thirty mycorrhizal pieces were plated for each collection. As in the previous study (Chu-Chou, 1979), fungi isolated from mycorrhizas were examined and identified by matching cultural and mycelial characters with fungi isolated from sporocarps of known basidiomycetes. In the synthesis tests (Chu-Chou, 1979) sterile soil was the only substrate used to grow seedlings and spore suspension prepared from fresh sporocarps of each fungal species was used as the only inoculum. Only the 10 most commonly collected fungi and Rhizopogon rubescens Tul.-an important mycorrhizal fungus of P. radiata (Chu-Chou, 1979)--were tested for their mycorrhiza-forming ability on Douglas fir seedlings. RESULTS Field observations
Sporocarps of the following Basidiomycetes (suspected mycorrhizal fungi) have been collected from
248
MYRACHU-CHOUand LYNETTE J. GRACE Table 1. Fungi isolated from mycorrhizas of Douglas fir of different ages
Age of tree Yr
Total no. of mycorrhizal pieces plated out
Rhizopogon vinicolor %
Amanita muscaria %
2-3 (nursery) 46 7-12 13-20 21-30 31-50 >51 All
360 185 260 367 411 336 389 2308
10.8 37.8 11.9 6.8 15.3 15.2 8.0 13.5
0 0 0 13.1 2.9 11.3 7.5 5.5
Proportion yielding* Other Dark sterile basidiomycetes mycelia 0, % ,O 6.4” 0 20.8b 8.7b 7.1b 8.0b 0.5b 1.2
31 7 3.8 28.9 24.0 23.8 17.9 11.8 21.1
Sporing fungi 0, 0 17.8 9.2 13.1 12.3 13.1 59 18.7 13.3
No growth
0,
!3.3 49.2 25.3 35.1 37.8 417 53 5 39.4
*Based on total mycorrhizal pieces plated out for any one tree-age group. “Mainly Hebelomu~plus some-unidentified basidiomycetes “Unidentified basidiomycetes.
Douglas fir nurseries (n) and forests (f): Amanita muscuria (f), Suillus lakei (f), Clitopilus sp. (f), Hebeloma crustuliniforme (Bull. ex St. Am) Quel. (n), Inocybe spp. (f), Laccaria laccata (n, f), Laccaria amethystina (Bolt. ex Hooker) Murr. (f), Lycoperdon gemmatum Batsch (f), L. perlatum (f), Rhizopogon uinicolor A. H. Smith (n. f), Russula sp. (f), Scleroderma bouista Fr. (f), S. uerrucosum Vaill. ex Pers. (f), and Tricholoma sp. (f). Although a detailed quantitative assessment of the sporocarp population of each fungal species was not attempted, marked differences in relative abundance and distribution of each fungus with respect to tree age were noted. Rhizopogon vinicolor was the only fungus consistently associated with Douglas fir in all the age groups investigated. Hebeloma crustuliniforme occurred only in the nurseries, and most of the other species were found only in the forests. Amanita muscaria was found only in stands over 12 yr of age. It is interesting to note that R. vinicolor, as an important component of the mycorrhizal associates, has never previously been reported in New Zealand. The basidiocarps are l-5 cm in size, globose to subglobose, and ovoid or angular from external pressures. The colour of the basidiocarp is white at first, soon becoming stained pink, and finally becoming vinaceous all over. The colour of the gleba is white to ochraceous becoming olivaceous. Species identification was based on Smith and Zeller (1966). Except for Inocybe spp, pure cultures were obtained from all the above-mentioned sporocarps for identification of the fungi isolated from the mycorrhizas. Isolation of fungi
From over 40 root samples of Douglas fir of different ages collected during 2 yr, a total of 2308 mycorrhizal pieces were plated out. About 13.5% of the total mycorrhizal pieces plated out gave rise to R. vinicolor, which was isolated from mycorrhizas of trees of all ages (Table 1). Nearly 6% yielded A. muscaria but it was isolated only from mycorrhizas of trees older than 13 yr of age. Hebeloma crustuliniforme was isolated only from the mycorrhizas of nursery plants. Seven culturally-distinct isolates of unidentified clamp-bearing basidiomycetes were also isolated from the mycorrhizas. The proportion of mycorrhizal pieces plated out that gave rise to R. vinicolor (13.5%)
was greater than all the other mycorrhizal fungi combined (12.7%). The same two types (65 and 76) of dark sterile mycelia which were isolated by Chu-Chou (1979) in a study of P. radiata mycorrhiza were frequently isolated (Table 1). Synthesis
Positive results were shown by R. uinicolor, H. crustuliniforme, L. laccata, Inocybe corydalina (?), I. jurana (?), 1. maculata (?), S. bouista, and A. muscaria. Three fungi-R. rubescens, S. verrucosum, and L. amethystina failed to form mycorrhizas. All fungi inoculated were successfully isolated from mycorrhizas except Inocybe spp.
the
synthesized
DISCUSSION
Results from field observation, isolation studies, and synthesis tests indicate that R. vinicolor is one of the important mycorrhizal fungi of Douglas fir in New Zealand. The role of R. vinicolor as a mycorrhizal fungus of Douglas fir was fully confirmed by Zak (1971a) in the United States. Zak’s observation indicated that the mycorrhizas formed by R. oinicolor in nature were of the tuberculate form, but in synthesis tests only the pinnate was formed. In our study mycorrhizas formed by R. vinicolor in synthesis tests were a distinctive type of compact-pinnate, both simple and complex. This type of mycorrhiza was always present in the mycorrhizal root samples we collected for isolation and R. uinicolor was consistently isolated from this type of mycorrhiza. The tuberculate form of mycorrhiza was found only associated with trees older than 30 yr and R. vinicolor was also isolated from this form. Zak (1971b) reported the close relationship between the morphology of the mycorrhizas and identity of the fungal symbionts. We noticed some correlations between some morphological types of mycorrhizas and their symbiotic fungi and this relationship is being investigated in more detail. Amanita muscaria seems to be another important mycorrhizal symbiont of Douglas fir. Its sporocarps occur in stands older than 12yr of age and it was isolated only from trees older than 13 yr. This age
Mycorrhizal fungi of Pseudotsuga menziesii relationship was also found in P. radiata (Chu-Chou, 1979). Cenococcum graniforme (Sow.) Ferd. & Winge, a common mycorrhizal associate of conifers (Trappe, 1964) in the United States, was not obtained in this study, nor were the jet-black mycorrhizas typical of Cenococcum ever observed in the root samples collected, though some black mycorrhizas, not typical of Cenococcum, were present in our samples. When these black mycorrhizas were plated out they remained free of fungal growth, except on one occasion a black basidiomycete was isolated. Overall 39% of the mycorrhizal pieces plated out remained free of fungal growth, an additional 13% yielded sporing Fungi Imperfecti presumed to be contaminants, and 21% yielded dark sterile mycelia; therefore isolation was apparently unsuccessful in over 70% of the samples plated out. This low recovery rate could be because the fungal symbionts may be fastidious in growth requirement, they may have been in a moribund state, or they may be sensitive to the surface sterilant used. Rhizopogon vinicolor, Amanita muscaria, and Hebeloma crustuliniforme were the only fungi isolated from the mycorrhizas and shown to be positive in synthesis tests. Therefore these fungi should be considered as confirmed mycorrhiza-formers on Douglas fir in New Zealand. Although Laccaria laccata and Scleroderma bouista are readily culturable on the media used, they were never isolated from the mycorrhizas but they did form mycorrhizas in the synthesis tests. Inocybe spp were not successfully cultured in our study but all three species tested formed mycorrhizas on Douglas fir seedlings. The importance of these fungi as mycorrhiza-formers on Douglas fir in New Zealand needs further investigation. Judging from Trappe’s list (1962), Douglas fir and P. radiata may have some common mycorrhizal fungi, e.g. A. muscaria, C. graniforme, L. laccata, Lactarius deliciosus (L. ex Fr.) S. F. Gray, R. rubescens, and B. piperatus. In our study A. muscaria, H. crustuliniforme, and L. laccata were the only three fungi proved to be mycorrhiza-formers of these tree species (Chu-Chou, 1979). Sporocarps of R. rubescens were not seen associated with Douglas fir, the fungus was not isolated from mycorrhizas of Douglas fir and the synthesis
249
tests were also negative, therefore its symbiotic relationship with Douglas fir is very doubtful. Acknowledgement-Thanks are due to Mr G. D. Bowen for reading and commenting on the manuscript.
REFERENCES
BIRCHT. T. C. (1937) A synopsis of forest fungi of significance in New Zealand. New Zealand Journal of Forestry 4, 109-125. CHU-CHOUM. (1979) Mycorrhizal fungi of Pinus radiata in New Zealand. Soil Bioloav & Biochemistrv 11. 557-562. GILMOIJRJ. W. (1958) di;lorosis of Do&& fir. New Zealand Journal of Forestry 7, 94-106. MARXD. H. (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163.
MODESS0. (1941) Zur Kenntnis der Mykorrhizabildner von Kiefer und Fichte. Symbolae botanicae upsalienses 5. 1-147.
RAWLINGSG. B. (1951) The mycorrhizas of trees in New Zealand forests. New Zealand Forestrv Research Notes 1, 15-17. RAWLINGSG. B. (1958) Some practical aspects of forest mycotrophy. New Zealand Societv_ of Soil Science. ProI
cbedings-3.414%.
SMITH A. H. and ZELLER S. M. (1966) A preliminary account of the North American species of Rhizopogon. Memoirs of the New York Botanical Garden 14, 1-178. TRAPPE J. M. (1962) Fungus associates of ectotrophic mycorrhizas. Botanical Review 28, 538-606. TRAPPEJ. M. (1964) Mycorrhizal hosts and distribution of Cenococcum graniforme. Lloydia 21, 100-106. TRAPPEJ. M. and STRANDR. F. (1969) Mvcorrhizal deficiency m a Douglas fir region n&sery: Foiesr Science 15, 381-389. WRIGHTE. (1971) Mycorrhizae on Douglas fir and Ponderosa pine seedlings. Research Bulletin of the Oregon Forest Research Laboratory No. 13.
ZAK B. (1971a) Characterization and classification of mycorrhizas of Douglas fir. II Pseudotsuga menziesii + Rhizopogon vinicolor. Canadian Journal of Botany 49, 1079-1084.
ZAK B. (1971b) Characterization and identification of Douglas fir mycorrhizae. Mycorrhizae, Proceedings 1st North American Conference on Mycorrhizae, 38-53.
Sad Bml Bmrhrm Vol 13. pp 247 lo 249. 1981 Prmted ,n Great Brrtam All nghts reserved
MYCORRHIZAL FUNGI OF PSEUDOTSUGA MENZIESIZ IN THE NORTH ISLAND OF NEW ZEALAND MYRACHU-CHOU
and LYNETTEJ.GRACE
Forest Research Institute, Private Bag Rotorua, New Zealand (Accepted 26 August 1980) Summary-Attempts were made to isolate mycorrhizal fungi of a timber species, Pseudotsugamenziesii (Mirb.) Franc0 which is exotic to New Zealand. Over 2000 pieces of mycorrhizal roots were plated out on Hagem medium and modified Melin-Norkrans medium; 14% of these yielded Rhizopogonuinicolor A. H. Smith, 6% gave rise to Amanitamuscariu(L. ex Fr.) S. F. Gray, and 7% to unidentified basidiomycetes, 21% yielded dark sterile mycelia, 13% were sporing Fungi Imperfecti presumed to be contaminants, and 39% remained free of fungal growth. Eleven basidomycetous fungi were tested for their mycorrhiza-forming ability on P. menziesiiseedlings. Under the test conditions R. uinicolor, Hebeloma crustuliniforme (Bull. ex St. Am) Quel., A. muscuria, Sclerodertnabovista Fr.. Laccaria laccata (Stop. ex Fr.) Berk. & Br.. Inocybe corydalina (?), I. jurana (?), and I. maculata (?) formed mycorrhizas.
INTRODUCTION
In some forest nurseries in New Zealand where Douglas fir (Pseudotsuga menziesii (Mirb.) France) has not previously been grown, the first crop often has a “mycorrhizal problem”, i.e. the seedlings, though looking “normal” in top growth, lack mycorrhizal roots. That the top growth is apparently normal is probably due to the high fertility of the nursery soil. But when such seedlings are planted out severe chlorosis and growth retardation may occur. This has been reported in outplantings of Douglas fir that lack mycorrhizas both in New Zealand and elsewhere (Gilmour, 1958; Trappe and Strand, 1969; Wright, 1971). Douglas fir is an exotic timber species which has been widely planted in New Zealand but its mycorrhizal relationship has so far received little attention. Birch (1937) mentioned the association of Scleroderma bouista Fr. with Douglas fir. Rawlings (1951; 1958) reported the association of Suillus (Boletus) lakei (Murr.) Sing., Suillus piperatus (Bull. ex Fr.) 0. Kuntze, Suillus uuriegatus (Swartz ex Fr.) 0. Kuntze, Amanita muscariu (L. ex Fr.) Pers. ex ,Hooker, Tricholoma sp., Laccaria laccata (Stop. ex Fr.) Berk. & Br., Lycoperdon perlatum Batsch, and S. bovista fructifications with Douglas fir stands. We cannot find any information on the kinds of fungi that can be isolated from mycorrhizas of Douglas fir in New Zealand, nor confirmation of the symbiotic relationship by synthesis tests. It is the purpose of this paper to fill this gap in knowledge. MATERIALS AND METHODS Field observations
From 1976 to 1978 Basidiomycete sporocarps were collected during autumn, early winter, and spring from four nurseries (Forest Research Institute, Gwavas, Owhata and Kaingaroa) in the North Island of New Zealand where Douglas fir was grown and from more than 30 stands of different ages (3 yr to 247
over 50 yr) growing in Kaingaroa and Whakarewarewa State Forests, Rotorua. The sporocarps were identified as reported by Chu-Chou (1979). Isolation offungal symbiontsfrom mycorrhizas and synthesis tests
Mycorrhizal root materials were collected from the same nurseries and forests where field observations were carried out. Three collections were made in which whole root systems of l&20 seedlings were collected from each nursery and mycorrhizal root samples were collected from 10 to 15 randomly selected trees from each stand of different age. Over 40 sets of isolations were carried out and a total of 2308 pieces of mycorrhizal roots were plated on to Hagem medium (Modess, 1941) and modified Melin-Norkrans medium (Marx, 1969). The isolation technique, which was similar to the method used by Chu-Chou in a study of Pinus radiata D. Don mycorrhizas (1979), was first to wash and then to surface-sterilize the mycorrhizal roots in 0.7% calcium hypochlorite for 7-10 min. Twenty to thirty mycorrhizal pieces were plated for each collection. As in the previous study (Chu-Chou, 1979), fungi isolated from mycorrhizas were examined and identified by matching cultural and mycelial characters with fungi isolated from sporocarps of known basidiomycetes. In the synthesis tests (Chu-Chou, 1979) sterile soil was the only substrate used to grow seedlings and spore suspension prepared from fresh sporocarps of each fungal species was used as the only inoculum. Only the 10 most commonly collected fungi and Rhizopogon rubescens Tul.-an important mycorrhizal fungus of P. radiata (Chu-Chou, 1979)--were tested for their mycorrhiza-forming ability on Douglas fir seedlings. RESULTS Field observations
Sporocarps of the following Basidiomycetes (suspected mycorrhizal fungi) have been collected from
248
MYRACHU-CHOUand LYNETTE J. GRACE Table 1. Fungi isolated from mycorrhizas of Douglas fir of different ages
Age of tree Yr
Total no. of mycorrhizal pieces plated out
Rhizopogon vinicolor %
Amanita muscaria %
2-3 (nursery) 46 7-12 13-20 21-30 31-50 >51 All
360 185 260 367 411 336 389 2308
10.8 37.8 11.9 6.8 15.3 15.2 8.0 13.5
0 0 0 13.1 2.9 11.3 7.5 5.5
Proportion yielding* Other Dark sterile basidiomycetes mycelia 0, % ,O 6.4” 0 20.8b 8.7b 7.1b 8.0b 0.5b 1.2
31 7 3.8 28.9 24.0 23.8 17.9 11.8 21.1
Sporing fungi 0, 0 17.8 9.2 13.1 12.3 13.1 59 18.7 13.3
No growth
0,
!3.3 49.2 25.3 35.1 37.8 417 53 5 39.4
*Based on total mycorrhizal pieces plated out for any one tree-age group. “Mainly Hebelomu~plus some-unidentified basidiomycetes “Unidentified basidiomycetes.
Douglas fir nurseries (n) and forests (f): Amanita muscuria (f), Suillus lakei (f), Clitopilus sp. (f), Hebeloma crustuliniforme (Bull. ex St. Am) Quel. (n), Inocybe spp. (f), Laccaria laccata (n, f), Laccaria amethystina (Bolt. ex Hooker) Murr. (f), Lycoperdon gemmatum Batsch (f), L. perlatum (f), Rhizopogon uinicolor A. H. Smith (n. f), Russula sp. (f), Scleroderma bouista Fr. (f), S. uerrucosum Vaill. ex Pers. (f), and Tricholoma sp. (f). Although a detailed quantitative assessment of the sporocarp population of each fungal species was not attempted, marked differences in relative abundance and distribution of each fungus with respect to tree age were noted. Rhizopogon vinicolor was the only fungus consistently associated with Douglas fir in all the age groups investigated. Hebeloma crustuliniforme occurred only in the nurseries, and most of the other species were found only in the forests. Amanita muscaria was found only in stands over 12 yr of age. It is interesting to note that R. vinicolor, as an important component of the mycorrhizal associates, has never previously been reported in New Zealand. The basidiocarps are l-5 cm in size, globose to subglobose, and ovoid or angular from external pressures. The colour of the basidiocarp is white at first, soon becoming stained pink, and finally becoming vinaceous all over. The colour of the gleba is white to ochraceous becoming olivaceous. Species identification was based on Smith and Zeller (1966). Except for Inocybe spp, pure cultures were obtained from all the above-mentioned sporocarps for identification of the fungi isolated from the mycorrhizas. Isolation of fungi
From over 40 root samples of Douglas fir of different ages collected during 2 yr, a total of 2308 mycorrhizal pieces were plated out. About 13.5% of the total mycorrhizal pieces plated out gave rise to R. vinicolor, which was isolated from mycorrhizas of trees of all ages (Table 1). Nearly 6% yielded A. muscaria but it was isolated only from mycorrhizas of trees older than 13 yr of age. Hebeloma crustuliniforme was isolated only from the mycorrhizas of nursery plants. Seven culturally-distinct isolates of unidentified clamp-bearing basidiomycetes were also isolated from the mycorrhizas. The proportion of mycorrhizal pieces plated out that gave rise to R. vinicolor (13.5%)
was greater than all the other mycorrhizal fungi combined (12.7%). The same two types (65 and 76) of dark sterile mycelia which were isolated by Chu-Chou (1979) in a study of P. radiata mycorrhiza were frequently isolated (Table 1). Synthesis
Positive results were shown by R. uinicolor, H. crustuliniforme, L. laccata, Inocybe corydalina (?), I. jurana (?), 1. maculata (?), S. bouista, and A. muscaria. Three fungi-R. rubescens, S. verrucosum, and L. amethystina failed to form mycorrhizas. All fungi inoculated were successfully isolated from mycorrhizas except Inocybe spp.
the
synthesized
DISCUSSION
Results from field observation, isolation studies, and synthesis tests indicate that R. vinicolor is one of the important mycorrhizal fungi of Douglas fir in New Zealand. The role of R. vinicolor as a mycorrhizal fungus of Douglas fir was fully confirmed by Zak (1971a) in the United States. Zak’s observation indicated that the mycorrhizas formed by R. oinicolor in nature were of the tuberculate form, but in synthesis tests only the pinnate was formed. In our study mycorrhizas formed by R. vinicolor in synthesis tests were a distinctive type of compact-pinnate, both simple and complex. This type of mycorrhiza was always present in the mycorrhizal root samples we collected for isolation and R. uinicolor was consistently isolated from this type of mycorrhiza. The tuberculate form of mycorrhiza was found only associated with trees older than 30 yr and R. vinicolor was also isolated from this form. Zak (1971b) reported the close relationship between the morphology of the mycorrhizas and identity of the fungal symbionts. We noticed some correlations between some morphological types of mycorrhizas and their symbiotic fungi and this relationship is being investigated in more detail. Amanita muscaria seems to be another important mycorrhizal symbiont of Douglas fir. Its sporocarps occur in stands older than 12yr of age and it was isolated only from trees older than 13 yr. This age
Mycorrhizal fungi of Pseudotsuga menziesii relationship was also found in P. radiata (Chu-Chou, 1979). Cenococcum graniforme (Sow.) Ferd. & Winge, a common mycorrhizal associate of conifers (Trappe, 1964) in the United States, was not obtained in this study, nor were the jet-black mycorrhizas typical of Cenococcum ever observed in the root samples collected, though some black mycorrhizas, not typical of Cenococcum, were present in our samples. When these black mycorrhizas were plated out they remained free of fungal growth, except on one occasion a black basidiomycete was isolated. Overall 39% of the mycorrhizal pieces plated out remained free of fungal growth, an additional 13% yielded sporing Fungi Imperfecti presumed to be contaminants, and 21% yielded dark sterile mycelia; therefore isolation was apparently unsuccessful in over 70% of the samples plated out. This low recovery rate could be because the fungal symbionts may be fastidious in growth requirement, they may have been in a moribund state, or they may be sensitive to the surface sterilant used. Rhizopogon vinicolor, Amanita muscaria, and Hebeloma crustuliniforme were the only fungi isolated from the mycorrhizas and shown to be positive in synthesis tests. Therefore these fungi should be considered as confirmed mycorrhiza-formers on Douglas fir in New Zealand. Although Laccaria laccata and Scleroderma bouista are readily culturable on the media used, they were never isolated from the mycorrhizas but they did form mycorrhizas in the synthesis tests. Inocybe spp were not successfully cultured in our study but all three species tested formed mycorrhizas on Douglas fir seedlings. The importance of these fungi as mycorrhiza-formers on Douglas fir in New Zealand needs further investigation. Judging from Trappe’s list (1962), Douglas fir and P. radiata may have some common mycorrhizal fungi, e.g. A. muscaria, C. graniforme, L. laccata, Lactarius deliciosus (L. ex Fr.) S. F. Gray, R. rubescens, and B. piperatus. In our study A. muscaria, H. crustuliniforme, and L. laccata were the only three fungi proved to be mycorrhiza-formers of these tree species (Chu-Chou, 1979). Sporocarps of R. rubescens were not seen associated with Douglas fir, the fungus was not isolated from mycorrhizas of Douglas fir and the synthesis
249
tests were also negative, therefore its symbiotic relationship with Douglas fir is very doubtful. Acknowledgement-Thanks are due to Mr G. D. Bowen for reading and commenting on the manuscript.
REFERENCES
BIRCHT. T. C. (1937) A synopsis of forest fungi of significance in New Zealand. New Zealand Journal of Forestry 4, 109-125. CHU-CHOUM. (1979) Mycorrhizal fungi of Pinus radiata in New Zealand. Soil Bioloav & Biochemistrv 11. 557-562. GILMOIJRJ. W. (1958) di;lorosis of Do&& fir. New Zealand Journal of Forestry 7, 94-106. MARXD. H. (1969) The influence of ectotrophic mycorrhizal fungi on the resistance of pine roots to pathogenic infections. I. Antagonism of mycorrhizal fungi to root pathogenic fungi and soil bacteria. Phytopathology 59, 153-163.
MODESS0. (1941) Zur Kenntnis der Mykorrhizabildner von Kiefer und Fichte. Symbolae botanicae upsalienses 5. 1-147.
RAWLINGSG. B. (1951) The mycorrhizas of trees in New Zealand forests. New Zealand Forestrv Research Notes 1, 15-17. RAWLINGSG. B. (1958) Some practical aspects of forest mycotrophy. New Zealand Societv_ of Soil Science. ProI
cbedings-3.414%.
SMITH A. H. and ZELLER S. M. (1966) A preliminary account of the North American species of Rhizopogon. Memoirs of the New York Botanical Garden 14, 1-178. TRAPPE J. M. (1962) Fungus associates of ectotrophic mycorrhizas. Botanical Review 28, 538-606. TRAPPEJ. M. (1964) Mycorrhizal hosts and distribution of Cenococcum graniforme. Lloydia 21, 100-106. TRAPPEJ. M. and STRANDR. F. (1969) Mvcorrhizal deficiency m a Douglas fir region n&sery: Foiesr Science 15, 381-389. WRIGHTE. (1971) Mycorrhizae on Douglas fir and Ponderosa pine seedlings. Research Bulletin of the Oregon Forest Research Laboratory No. 13.
ZAK B. (1971a) Characterization and classification of mycorrhizas of Douglas fir. II Pseudotsuga menziesii + Rhizopogon vinicolor. Canadian Journal of Botany 49, 1079-1084.
ZAK B. (1971b) Characterization and identification of Douglas fir mycorrhizae. Mycorrhizae, Proceedings 1st North American Conference on Mycorrhizae, 38-53.