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Mycorrhizal fungi of Eucalyptus in the North Island of New Zealand
00384717,82/020133-05103.00:0 Copyright 0 1982 Pergirmon PressLtd
Soil Bid. Biwhum. Vol. 14. pp. 133 to 137. IOR? Printed in Great Britain. All rights raerved
MYCORRHIZAL NORTH
FUNGI ISLAND
OF EUCALYPTUS IN THE OF NEW ZEALAND
MYRA CHU-CHOU and
LYNETTE
J. GRACE
Forest Research Institute. Private Bag. Rotorua, New Zealand (Accepted
10 August
1981)
Summary-Fungal sporocarps of Hydnangium carneum Wallr., Laccaria laccata (Stop. ex Fr.) Berk. ex Br., Sckroderma spp. Hymenogaster spp., Hysterangium spp, Cortinarius spp. etc. were found to be associated with five species of eucalypts (Eucalyptus delegatensis R.T. Bak., E. regnans F. Muell., E. nitens Maiden, Efastigara Deane et Maiden, and E. saligna Sm.) in nurseries or stands in the North Island of New Zealand. A total of 5345 mycorrhizal pieces were plated out in isolation studies. More than 47% of these pieces remained free of fungal outgrowth, 160/i yielded dark sterile mycelia, 7% yielded hyaline sterile fungi, 177; produced sporing Fungi Imperfecti, 5.8% were basidiomycetous fungi identified as H. carneum, Sc/eroderma spp, Paxillus sp. and Hymenogasrer a/bus (Klotzsch) Berk. et Br., and 5.9% were unidentified basidiomycetes. Mycorrhizas were synthesized on eucalypt seedlings by 12 identified basidiomycetes which form sporocarps in eucalypt stands and 12 unidentified basidiomycetes isolated from the mycorrhizas. Under the test conditions two Cortinarius spp and one unidentified basidiomycete did not form mycorrhizas. The results indicate a possible importance of H. carneum as a mycorrhizal fungus of eucalypts in the North Island of New Zealand.
INTRODUCTION In recent years the potential of Eucalyptus
spp as important exotic timber species in this country has received increasing attention, and accordingly there is a need for a better understanding of the biology and silviculture of this genus. Many aspects of eucalypt mycorrhizas have been studied (Ashford et al., 1975; Ashton. 1976; Chilvers, 1968, 1973; Mullett, 1976), but little is known of the fungal component. The only reported study in New Zealand was by Rawlings (1951) who noted the asSociation of Lycoperdon perlaturn Batsch and Scleroderma bovista Fr. with Eucalyprus spp. World-wide there is also a lack of information on the mycorrhiza-formers of eucalypts: fungi reported or suspected to be mycorrhizal with eucalypts include Pisolithus tinctorius (Pers.) Coker & Couch (Chilvers 1973; Marx 1977; Mullett 1976), Scleroderma spp (Levisohn, 1958; Thapar et al., 1967; Bakshi, 1966; Pryor, 1956), Hydnangium carneum Wallr. and Hysterangium sp. (Marx, 1975), and Mesophelliu arenaria Berk. (Ashton, 1976). Recently, Warcup (1971) reported the isolation of 25 fungi from sporocarps collected in eucalypt forests in South Australia and found to be capable of forming mycorrhizas with E. obliqua L’Herit. in pure culture synthesis. So far the names of these fungi have not ,been published. We report observations of some possible mycorrhizal associations of several eucalypt species in the North Island of New Zealand, the isolation of fungal symbionts from eucalypt mycorrhizas, and results of synthesis tests using cultures derived from mycorrhizas and spores from fresh sporocarps. MATERIALSAND METHODS Field
obserwtions
From 1978 to 1980 sporocarps were collected dur-
ing autumn, early winter, and spring from six nurseries growing eucalypts and from North Island plantations of several species of eucalypt of differing ages in Kaingaroa, Rotoehu, Tairua and Whakarewarewa State Forests, and the private forest of N.Z. Forest Products Ltd near Tokoroa. The main species examined in this study were Eucalyptus delegatensis R.T. Bak., E. jhsrigata Deane et Maiden, E. nirens Maiden, E. regnans F. Muell., and E. saligna Sm.; these are among the more important species currently being grown in New Zealand forests. Sporocarps were identified to genus based on Cunningham (1942). Lange and Hora (1975) and Singer (1962). Pure cultures of each kind of sporocarp were obtained (ChuChou, 1979). Isolation
study
Mycorrhizal roots were collected from the nurseries and forests in which the sporocarp observations were made, the method of collection being that used by Chu-Chou (1979). Fungi were isolated by the surface sterilization method used in a study of Pinus radiutu D. Don mycorrhizas by Chu-Chou (1979), with some modifications. After root samples had been washed free of visible soil particles, well-developed mycorrhizal clusters were cut off and placed in a piece of glass tubing (30 x 130 mm). One end of the tubing was covered with nylon gauze and the other plugged with a rubber bung through which a small glass dropper was fitted. This dropper was attached to a high pressure tap and the mycorrhizal roots were washed vigorously with a jet of water. After 4-6 h washing, the mycorrhizal roots were surface sterilized in 0.7% calcium hypochlorite for 1 min. On average four to five sets of mycorrhizal roots were sampled from each age group of trees of each eucalypt species. Over 95 sets of isolations were car-
133
MYRA CHU-CHOW and LYNETTEJ. GRACE
134
ried out and a total of 5345 pieces of mycorrhizal root were plated on to both Hagem medium (Modes% 1941) and modified Melin Norkrans medium (Marx. 1969). Colonies of suspected mycorrhizal fungi were subcultured and identified by matching cultural characters with fungi isolated from identified fungal sporocarps.
(2) microscopic examination of T.S. of roots for ectomycorrhiza structure: (3) re-isolation of the inoculated fungi was attempted whenever there was sufficient material.
RESULTS
Synthesis of mycorrhizas
Field observations
The method of synthesis test was that of Chu-Chou. (1979) with slight modifications. Two-week-old eucalypt seedlings (E. saiigna, E. regnans, E. defegatensis) were planted individually in glass vials (25 x 150 mm) with autoclaved nursery potting mixture (soil:peat: pumice = 3: 3: 1). At 6 weeks after planting the seedlings were inoculated with test fungi. Each fungus was tested on only one of the three eucalypt species. Pure cultures were used as inocula for the 12 culturally distinct unidentified basidiomycetes isolated from eucalypt mycorrhizal roots. Spore suspensions, (Chu-Chou, 1979) were used as inocula for the 12 identified basidiomycetes because some of these fungi were not culturable (e.g. Cortinarius spp) and the remainder grew extremely poorly on artificial media. After 4 months the substrate was washed from the roots and the inoculation results were determined by the following criteria:
Table 1 shows a list of fungal species associated with Eucaf~~ptu.~spp of different ages. ~ydna~iarn carneum, Laccaria laccata. and Scferoderma spp were the only fungi found in both nurseries and plantations. Hymenogaster spp sporocarps were frequently found in the plantations but never in nurseries. A large number of sporocarps of Cortfnurius spp (more than 12 species) were found associated with trees older than 6 yr. Sporocarps of inocyhe spp. ~esophellia arenaria, Lycoperdon spp, and Trichoioma spp were also only collected from tree stands older than 6 yr. Sporocarps of ascomycetes were usually found as single specimens and were not commonly found in these forests. Attempts to obtain pure cultures from sporocarps of Cortinarius spp and ~noc~lhe spp were unsuccessful. Pure cultures were obtained from the other kinds of sporocarps although they all grew extremely poorly on media used.
(1) the formation of characteristic mycorrhizas:
Table 1. Fungat sporocarps associated with Etr~uf~ptusspecies of different ages Nursery seedlings
Fungi
2--5
Age of trees (yr) I l-20
&IO
31-50
21-30
Hydnunyium carneum Luccaria fuccutu
d
d
f
r
r
dfnrsdfnrsdnrsodfrsdfso sdfnr sdfnrs
dnrsodfrsdfso
Sefero~erma ~u~~durn and S. uerrucosum Hymenogaster alhus Hymenogaster spp Ahstoma sp. Hpsterangium in&turn Hysterang~um spp Paxillus sp.
d n r
s d d
f
d
r
nrsdfnrsd s n r
d
n
r
dfnr
n
r
d
f
n
r
s
d
sdnrso
f
n
df
s r
0
r
r
0 o
f r
d
d
d 0
r
Ascomycetes Cortinarius cinnaharinus C. cinnamomeus Cortinurius spp Inocjh spp
0 0 o
r dn f
s
fr:dfso
r
d
d d d
d
d
r
d
f n
r
f
dnis
d
f
o d rs
d
r
s
df df d
o o f
rsdfs:
~esop~e~~i~ d d
arenariu Lycoperdon spp Tricholomu pessundatum T. saponaceum Octuoiania sp. Ramaria spp Gauteria sp. d = f = n = r = s = o =
E. delegatensis. E. fastiguta (no trees available 1 l-20 yr). E. nitens (no trees available over 20 yr). E. regnans (no trees available over 30yr). E. suligna. Other species (no trees available under
df
r
r r
f r
d
r
r
f
0 0
d
df d
o 0
d
d
0 0
r
d
s
s
r d r
I1 yr).
126 154 207 119
Nursery seedlings 2-10 11-20 21-50
Nursery seedlings 2- 10 1l-20
E. saligna
E. nitens
* Include E.
All
Other species*
botryoides
140 133 112 225
Nursery seedlings 2--10 21-30 31-50
E. fastigato
Sm..
E. pilularis
18-49
331 391 134 362 94
Nursery seedlings 2-5 6-10 11-20 21-30
E. regnuns
Sm.,
E. sieheri.
5345
514
104 193 153
584 126 380 137 307 319
Nursery seedlings 2-S 6-10 11-20 21-30 3’1-50
E. delegatensis
Species
Total no. mycorrhizal pieces
Age of tree fyrl
E. pulchella
4.01
Desf., and
Miq.
0.02
0
x
0
z.3
0 0
8
Puxillus
E. tenuiramis
1.25
0
::
2.7
0
2.1 9.2
::
0.8 0
::
10.7 0
:
x
4.5
:: 0
6.2 0 0
Sclerodermu
0
9.7 4.8 0
0
4.5 0 13.3
0
7.4 2.2 L.9 7.4
0
6.6 4.4 0.3 14.7
0 0
Hydnangium carneum
Eucalyptus
of different ages
0.54
4.1
1.3
0 0
0 0 0 0
0 0 0 0
4.8 0 0 0 0
0
Hymenogaster albus
5.93
17.3
1.6 11.8
0
0
0.8 5.4 12.5
0
4.6 3.7 3.8 10.6
0
12.5
E 6:5
0 0.8
Unidentified basidiomycetes
Proportion yielding (%)
Table 2. Fungi isolated from mycorrhizas of different species of
16.22
7.2
31.7 30.1 13.7
14.2 16.2 7.7 6.7
7.1 21.1 24.1 14.2
10.3 27.9 26.1 11.0 8.5
13.7 29.4 13.9 17.5 22.5 20.4
Dark sterile mycelia
7.32
9.7
20.1 6.2 5.2
4.0 1.3 7.7 7.6
3.6 4.5 3.6 6.7
10.3 10.7 9.0 6.6 6.4
::1 8.8 4.6 5.3
8.4
Hyaline sterile fungi
16.91
11.5
387.: 157
1.6 9.1 17.9 26.0
5.0 15.0 17.8 12.0
13.9 18.7 17.9 21.3 11.7
16.6 21.4 21.1 23.3 21.5 15.1
Sporing fungi
47.80
47.5
39.4 22.2 43.1
79.4 61.1 57.1 51.3
73.6 54.1 49.1 41.3
61.0 30.7 41.1 55.4 55.4
55.1 38.1 44.5 43.1 44.6 31.7
No growth
c k $ 2 v) z
K : r! z. FL z G. 0
136
MYRA CHU-CHOU and LYNETTEJ. GRACT Table
3. Mycorrhizal
formation
on Eucalyptus seedlings inoculated cutlure (c) of various basidiomycetes
lnoculum
H@rangium ccrme’um(s) Laccaria laccata (s) Hymenogaster u/bus (s) Hystrrungium injatum (s) Sclerodrrma rerruco.wm (s) Absrorna sp. (s) Cortinarius sp. A (s) Hpsterangium sp. (s) Sclerodermu jkidum (s) Tricholamu saponaceum (s) Unidentified basidiomycetes Act. No. 277 313 280 284 325 288 282 291 283 316 257 279 Cortinarius cinnabarinus (s) Cortinarius sp. B (s)
Isolation
No. of seedlings
No. of seedlings
inoculated
inoculated
infected
E. delegatensis E. delegatensis E. saligna
4 4 4 4 IO 6 7 6 10 9
E. sdigna
E. E. E. E. E. E.
solignu saligna salignu regnans regnans regnans
E. E. El E. E. E. E. E. E. E. E. E. E. E.
regnans regnans regnans regnans regnans regnans regnans regnans regnans regnans regnans regnons rrgnans sulignu
offungi
tests
L. laccatu.
H. alhus.
(SI or pure
Time required for infection (months)
6 10 9
2-4 1.5 I-3 2 3 4 4 4 4 4 5
4
2-4
2 2 2
24
(c)
Of the 12 identified basidiomycetes. 10 formed mycorrhizas on seedlings (Table 3). These were H. carneum.
fresh spores
Tree species
Of the 5345 mycorrhizal pieces plated out (Table 2) nearly 485; remained free of fungal growth, 16:,, produced dark sterile mycelia. 17?/; sporing Fungi Imperfecti, 7P; hyaline sterile fungi, 5.8% were fungi which could be identified as H. carneum, Scleroderma spp, Paxillus spp, and Hymenogaster albus, and 5.9’35 were unidentified basidiomycetes (clamp-bearing mycelia) which were separated into 42 culturally-distinct types and kept as stock cultures. These 42 cultures were further simplified and combined into 12 groups. and one representative culture from each group was used in synthesis tests. Among the identified basidiomycetes, H. carneum was the most frequently isolated from each of the five main eucalypts species from which isolations were attempted. This fungus has not been isolated from the mycorrhizas of nursery seedlings. Scleroderma spp were the only basidiomycetes isolated from nursery seedling mycorrhizas and they have never been isolated from trees more than 2yr old. Paxillus sp. was isolated once from E. delegatensis, and H. albus was isolated three times from E. delegatensis. E. nitens, and E. sieberi L. Johnson. The unidentified basidiomycetes were isolated from the mycorrhizas of all the eucalypt species studied. ranging from 2 to 50 yr of age, and their frequency of isolation appeared to increase with tree age. Synthesis
with
H. injlatum.
Scleroderma
4 4 4 4 4 4 4 4 4 4 4 3 12 8
2 2 2 2 2 2 2
0 0 0
jlacidum. S. cerrucosum. Hysterangium sp. and
Abstoma sp., Cortinarius sp.. Tricholomu saponuceum. The two that gave negative results were Cortinarius cinnaharinus and another Cortinarius sp. Eleven of 12 un-
identified basidiomycetes tested also formed mycorrhizas on seedlings. Attempts to reisolate the inoculated fungi from the synthesized mycorrhizas were unsuccessful.
DISCUSSION
Although a large proportion of fungal species suspected to be mycorrhizal because of sporocarp association with eucalypt stands gave positive results in synthesis tests, only a small proportion of them could be isolated directly from the mycorrhizas. This low recovery rate could be due to the same reasons as discussed by Chu-Chou (1979). Some of the fungi probably do not commonly form mycorrhizas with eucalypts under natural conditions, though they can do so under artificial conditions. These are some of the difficulties that impose a limitation at present on a full understanding of the mycorrhizal relationships in eucalypt species. A large proportion of the mycorrhiza-formers isolated were unidentified basidiomycetes: some of them may have been hypogeous Gasteromycetes and hence have escaped detection in the usual field survey for sporocarps. There is a possibility that some of the hyaline sterile fungi isolated from the mycorrhizal roots may be mycorrhiza formers (non-clamp bearing basidiomycetes or ascomycetes). So far we have not tried to
Mycorrhizal fungi of identify these fungi and none of them has been tested for mycorrhiza-forming ability on eucalypt seedlings. Despite these problems our study suggests that some importance should be given to H. carneum as a eucalypt mycorrhiza-former in this country. H. carneum was first recorded in Britain in association with a species of Eucalyptus by Berkeley and Broome (1875). Trappe and Guzman (1971) collected this fungus in eucalypt forest in Mexico and in 1975 Marx reported its association with E. g/oh&s Labill. in Peru. In 1977 we found this fungus associated with E. delegatensis and E. regnnns seedlings in a forest nursery in the North Island of New Zealand (Chu-Chou and Grace, 1981). There is some evidence that fungi that form mycorrhizas with eucalypts often do not with Pinus radiata (Chilvers, 1973; Warcup, 1971). We have observed that Tricholoma pessundatum (Lyophyllum sp.), common in both P. radiatu and eucalypt stands, has been isolated from P. radiuta mycorrhizas (Chu-Chou, 1979) but not from eucalypt mycorrhizas. Conversely, Scleroderma spp have been isolated from the eucalypt mycorrhizas but not from those of P. radiata. L. laccata, though easily culturable from sporocarp tissues and commonly seen in stands of both species, showed positive results in synthesis tests on both tree species but has never been isolated from mycorrhizas of either. Sporocarps of H. curneum and H. a/bus have been observed to be associated with seedlings and young trees respectively of New Zealand beech (Nothofigus spp) in the Forest Research Institute nursery and H. carneum was isolated from the mycorrhizas of mountain beech (N. solandri var. clifirtioides (Hood. f.) Poole) seedlings. The relationship between mycorrhizal fungi of eucalypts and New Zealand native beech and other native tree species warrants further investigation. REFERENCES
ASHFORDA. E.. LING LEE M. and CHILVERSG. A. (1975) Polyphosphate in eucalypt mycorrhizas: a cytochemical demonstration. New Phytologist 74, 447453. ASHTOND. H. (1976) Studies on the mycorrhizae of EucoIyptus regnans F. Muell. Australian Journal of Botany 24, 723-741.
BAKSHIB. K. (1966) Mycorrhiza in Indian
Forester
Eucalyptus
in India.
92, 19-20.
BERKELEY M. J. and BROOME C. E. (1875) Notices of British Fungi. Annals und Magazine of Narurul History, Ser. 4 15, 2841.
Euculyptus
spp
137
CHILVERSG. A. (1968) Some distinctive types of eucalypt mycorrhiza. Austrulian Journul of Botany 16.49-70. CHILVERSC. A. (1973) Host range of some eucalypt mycorrhizal fungi. Ausrraliun Journal of Botuny 21. 103-111. CHU-CHOUM. (1979) Mycorrhizal fungi of Pinus radiutu in New Zealand. Soil Biology & Biochemistry 11, 557-562. CHU-CHOUM. and GRACEL. J. (1981) Mycorrhizal fungi of Pseudotsuga menziesii in the North Island. New Zealand. Soil Biology & Biochemistry 13, 247-249. CHU-CHOU M. and GRACE L. J. (1981) Hydnangium carneum-A mycorrhizal fungus of Euculyprus in New Zealand. Transactions of British Mycologicul Society. In press. CUNNINGHAM G. H. (1942) The Gasteromycetes of Australia und New Zealand. Mclndoe Printer, Dunedin. LANGEM. and HORAB. (1975) Co//ins Guide to Mushrooms and Toadstools. Collins, London. LEVI~~HNI. (1958) Mycorrhizal infection in Euculyptus. Empire Forestry Review 37, 237-241. MARX D. 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. MARXD. H. (1975) Mycorrhizae of exotic trees in the Peruvian Andes and synthesis of ectomycorrhizae on Mexican pines. Forest Science 21, 353-358. MARXD. H. 11977) Tree host range and world distribution . of the ectomycorrhizal fungus kolithus tincrorius. Canadiun Journal
of Microbiology
23, 217-223.
MODESS0. (1941) Zur Kenntnis der Mykorrhizabildner von kiefer und Fichte. Symholae Boranicae Upsulienses 5, 1-147.
MULLETTK. J. (1976) Studies of eucalypt mycorrhizas. 1. A method of mycorrhiza induction in Eucalyptus gummifera (Gaertn. & Hochr.) by Pisolithus tinctorius (Pers.) Coker & Couch. Australiun Journal of Botony 24, 193-200.
PRYORL. S. (1956) Chlorosis and lack of vigour in seedlings of renantherous species of Eucalyptus caused by lack of mycorrhiza. Proceedings of Linneon Society of New South Wales 81, 91-96. RAWLINGSG. B. (1951) The mycorrhizas of trees in New Zealand forests. New Zealand Forest Service. Forestry Research Notes 1, 15-17. SINGERR. (1962) The Ayaricules in Modern Taxonomy. Cramer, New York. THAPARH. S., SINGHB. and BAKSHIB. K. (1967) Mycorrhizae in Eucalyptus. Indian Forester 93, 756759. TRAPPEJ. M. and GUZM~NG. (1971) Notes on some hypogeous fungi from Mexico. Mycologia 63, 317-332. WARCUPJ. H. (1971) Mycorrhizas of eucalypts and pines. Biennial Report of the Waite Agricultural Research Institute, p. 78.
Soil Bid. Biwhum. Vol. 14. pp. 133 to 137. IOR? Printed in Great Britain. All rights raerved
MYCORRHIZAL NORTH
FUNGI ISLAND
OF EUCALYPTUS IN THE OF NEW ZEALAND
MYRA CHU-CHOU and
LYNETTE
J. GRACE
Forest Research Institute. Private Bag. Rotorua, New Zealand (Accepted
10 August
1981)
Summary-Fungal sporocarps of Hydnangium carneum Wallr., Laccaria laccata (Stop. ex Fr.) Berk. ex Br., Sckroderma spp. Hymenogaster spp., Hysterangium spp, Cortinarius spp. etc. were found to be associated with five species of eucalypts (Eucalyptus delegatensis R.T. Bak., E. regnans F. Muell., E. nitens Maiden, Efastigara Deane et Maiden, and E. saligna Sm.) in nurseries or stands in the North Island of New Zealand. A total of 5345 mycorrhizal pieces were plated out in isolation studies. More than 47% of these pieces remained free of fungal outgrowth, 160/i yielded dark sterile mycelia, 7% yielded hyaline sterile fungi, 177; produced sporing Fungi Imperfecti, 5.8% were basidiomycetous fungi identified as H. carneum, Sc/eroderma spp, Paxillus sp. and Hymenogasrer a/bus (Klotzsch) Berk. et Br., and 5.9% were unidentified basidiomycetes. Mycorrhizas were synthesized on eucalypt seedlings by 12 identified basidiomycetes which form sporocarps in eucalypt stands and 12 unidentified basidiomycetes isolated from the mycorrhizas. Under the test conditions two Cortinarius spp and one unidentified basidiomycete did not form mycorrhizas. The results indicate a possible importance of H. carneum as a mycorrhizal fungus of eucalypts in the North Island of New Zealand.
INTRODUCTION In recent years the potential of Eucalyptus
spp as important exotic timber species in this country has received increasing attention, and accordingly there is a need for a better understanding of the biology and silviculture of this genus. Many aspects of eucalypt mycorrhizas have been studied (Ashford et al., 1975; Ashton. 1976; Chilvers, 1968, 1973; Mullett, 1976), but little is known of the fungal component. The only reported study in New Zealand was by Rawlings (1951) who noted the asSociation of Lycoperdon perlaturn Batsch and Scleroderma bovista Fr. with Eucalyprus spp. World-wide there is also a lack of information on the mycorrhiza-formers of eucalypts: fungi reported or suspected to be mycorrhizal with eucalypts include Pisolithus tinctorius (Pers.) Coker & Couch (Chilvers 1973; Marx 1977; Mullett 1976), Scleroderma spp (Levisohn, 1958; Thapar et al., 1967; Bakshi, 1966; Pryor, 1956), Hydnangium carneum Wallr. and Hysterangium sp. (Marx, 1975), and Mesophelliu arenaria Berk. (Ashton, 1976). Recently, Warcup (1971) reported the isolation of 25 fungi from sporocarps collected in eucalypt forests in South Australia and found to be capable of forming mycorrhizas with E. obliqua L’Herit. in pure culture synthesis. So far the names of these fungi have not ,been published. We report observations of some possible mycorrhizal associations of several eucalypt species in the North Island of New Zealand, the isolation of fungal symbionts from eucalypt mycorrhizas, and results of synthesis tests using cultures derived from mycorrhizas and spores from fresh sporocarps. MATERIALSAND METHODS Field
obserwtions
From 1978 to 1980 sporocarps were collected dur-
ing autumn, early winter, and spring from six nurseries growing eucalypts and from North Island plantations of several species of eucalypt of differing ages in Kaingaroa, Rotoehu, Tairua and Whakarewarewa State Forests, and the private forest of N.Z. Forest Products Ltd near Tokoroa. The main species examined in this study were Eucalyptus delegatensis R.T. Bak., E. jhsrigata Deane et Maiden, E. nirens Maiden, E. regnans F. Muell., and E. saligna Sm.; these are among the more important species currently being grown in New Zealand forests. Sporocarps were identified to genus based on Cunningham (1942). Lange and Hora (1975) and Singer (1962). Pure cultures of each kind of sporocarp were obtained (ChuChou, 1979). Isolation
study
Mycorrhizal roots were collected from the nurseries and forests in which the sporocarp observations were made, the method of collection being that used by Chu-Chou (1979). Fungi were isolated by the surface sterilization method used in a study of Pinus radiutu D. Don mycorrhizas by Chu-Chou (1979), with some modifications. After root samples had been washed free of visible soil particles, well-developed mycorrhizal clusters were cut off and placed in a piece of glass tubing (30 x 130 mm). One end of the tubing was covered with nylon gauze and the other plugged with a rubber bung through which a small glass dropper was fitted. This dropper was attached to a high pressure tap and the mycorrhizal roots were washed vigorously with a jet of water. After 4-6 h washing, the mycorrhizal roots were surface sterilized in 0.7% calcium hypochlorite for 1 min. On average four to five sets of mycorrhizal roots were sampled from each age group of trees of each eucalypt species. Over 95 sets of isolations were car-
133
MYRA CHU-CHOW and LYNETTEJ. GRACE
134
ried out and a total of 5345 pieces of mycorrhizal root were plated on to both Hagem medium (Modes% 1941) and modified Melin Norkrans medium (Marx. 1969). Colonies of suspected mycorrhizal fungi were subcultured and identified by matching cultural characters with fungi isolated from identified fungal sporocarps.
(2) microscopic examination of T.S. of roots for ectomycorrhiza structure: (3) re-isolation of the inoculated fungi was attempted whenever there was sufficient material.
RESULTS
Synthesis of mycorrhizas
Field observations
The method of synthesis test was that of Chu-Chou. (1979) with slight modifications. Two-week-old eucalypt seedlings (E. saiigna, E. regnans, E. defegatensis) were planted individually in glass vials (25 x 150 mm) with autoclaved nursery potting mixture (soil:peat: pumice = 3: 3: 1). At 6 weeks after planting the seedlings were inoculated with test fungi. Each fungus was tested on only one of the three eucalypt species. Pure cultures were used as inocula for the 12 culturally distinct unidentified basidiomycetes isolated from eucalypt mycorrhizal roots. Spore suspensions, (Chu-Chou, 1979) were used as inocula for the 12 identified basidiomycetes because some of these fungi were not culturable (e.g. Cortinarius spp) and the remainder grew extremely poorly on artificial media. After 4 months the substrate was washed from the roots and the inoculation results were determined by the following criteria:
Table 1 shows a list of fungal species associated with Eucaf~~ptu.~spp of different ages. ~ydna~iarn carneum, Laccaria laccata. and Scferoderma spp were the only fungi found in both nurseries and plantations. Hymenogaster spp sporocarps were frequently found in the plantations but never in nurseries. A large number of sporocarps of Cortfnurius spp (more than 12 species) were found associated with trees older than 6 yr. Sporocarps of inocyhe spp. ~esophellia arenaria, Lycoperdon spp, and Trichoioma spp were also only collected from tree stands older than 6 yr. Sporocarps of ascomycetes were usually found as single specimens and were not commonly found in these forests. Attempts to obtain pure cultures from sporocarps of Cortinarius spp and ~noc~lhe spp were unsuccessful. Pure cultures were obtained from the other kinds of sporocarps although they all grew extremely poorly on media used.
(1) the formation of characteristic mycorrhizas:
Table 1. Fungat sporocarps associated with Etr~uf~ptusspecies of different ages Nursery seedlings
Fungi
2--5
Age of trees (yr) I l-20
&IO
31-50
21-30
Hydnunyium carneum Luccaria fuccutu
d
d
f
r
r
dfnrsdfnrsdnrsodfrsdfso sdfnr sdfnrs
dnrsodfrsdfso
Sefero~erma ~u~~durn and S. uerrucosum Hymenogaster alhus Hymenogaster spp Ahstoma sp. Hpsterangium in&turn Hysterang~um spp Paxillus sp.
d n r
s d d
f
d
r
nrsdfnrsd s n r
d
n
r
dfnr
n
r
d
f
n
r
s
d
sdnrso
f
n
df
s r
0
r
r
0 o
f r
d
d
d 0
r
Ascomycetes Cortinarius cinnaharinus C. cinnamomeus Cortinurius spp Inocjh spp
0 0 o
r dn f
s
fr:dfso
r
d
d d d
d
d
r
d
f n
r
f
dnis
d
f
o d rs
d
r
s
df df d
o o f
rsdfs:
~esop~e~~i~ d d
arenariu Lycoperdon spp Tricholomu pessundatum T. saponaceum Octuoiania sp. Ramaria spp Gauteria sp. d = f = n = r = s = o =
E. delegatensis. E. fastiguta (no trees available 1 l-20 yr). E. nitens (no trees available over 20 yr). E. regnans (no trees available over 30yr). E. suligna. Other species (no trees available under
df
r
r r
f r
d
r
r
f
0 0
d
df d
o 0
d
d
0 0
r
d
s
s
r d r
I1 yr).
126 154 207 119
Nursery seedlings 2-10 11-20 21-50
Nursery seedlings 2- 10 1l-20
E. saligna
E. nitens
* Include E.
All
Other species*
botryoides
140 133 112 225
Nursery seedlings 2--10 21-30 31-50
E. fastigato
Sm..
E. pilularis
18-49
331 391 134 362 94
Nursery seedlings 2-5 6-10 11-20 21-30
E. regnuns
Sm.,
E. sieheri.
5345
514
104 193 153
584 126 380 137 307 319
Nursery seedlings 2-S 6-10 11-20 21-30 3’1-50
E. delegatensis
Species
Total no. mycorrhizal pieces
Age of tree fyrl
E. pulchella
4.01
Desf., and
Miq.
0.02
0
x
0
z.3
0 0
8
Puxillus
E. tenuiramis
1.25
0
::
2.7
0
2.1 9.2
::
0.8 0
::
10.7 0
:
x
4.5
:: 0
6.2 0 0
Sclerodermu
0
9.7 4.8 0
0
4.5 0 13.3
0
7.4 2.2 L.9 7.4
0
6.6 4.4 0.3 14.7
0 0
Hydnangium carneum
Eucalyptus
of different ages
0.54
4.1
1.3
0 0
0 0 0 0
0 0 0 0
4.8 0 0 0 0
0
Hymenogaster albus
5.93
17.3
1.6 11.8
0
0
0.8 5.4 12.5
0
4.6 3.7 3.8 10.6
0
12.5
E 6:5
0 0.8
Unidentified basidiomycetes
Proportion yielding (%)
Table 2. Fungi isolated from mycorrhizas of different species of
16.22
7.2
31.7 30.1 13.7
14.2 16.2 7.7 6.7
7.1 21.1 24.1 14.2
10.3 27.9 26.1 11.0 8.5
13.7 29.4 13.9 17.5 22.5 20.4
Dark sterile mycelia
7.32
9.7
20.1 6.2 5.2
4.0 1.3 7.7 7.6
3.6 4.5 3.6 6.7
10.3 10.7 9.0 6.6 6.4
::1 8.8 4.6 5.3
8.4
Hyaline sterile fungi
16.91
11.5
387.: 157
1.6 9.1 17.9 26.0
5.0 15.0 17.8 12.0
13.9 18.7 17.9 21.3 11.7
16.6 21.4 21.1 23.3 21.5 15.1
Sporing fungi
47.80
47.5
39.4 22.2 43.1
79.4 61.1 57.1 51.3
73.6 54.1 49.1 41.3
61.0 30.7 41.1 55.4 55.4
55.1 38.1 44.5 43.1 44.6 31.7
No growth
c k $ 2 v) z
K : r! z. FL z G. 0
136
MYRA CHU-CHOU and LYNETTEJ. GRACT Table
3. Mycorrhizal
formation
on Eucalyptus seedlings inoculated cutlure (c) of various basidiomycetes
lnoculum
H@rangium ccrme’um(s) Laccaria laccata (s) Hymenogaster u/bus (s) Hystrrungium injatum (s) Sclerodrrma rerruco.wm (s) Absrorna sp. (s) Cortinarius sp. A (s) Hpsterangium sp. (s) Sclerodermu jkidum (s) Tricholamu saponaceum (s) Unidentified basidiomycetes Act. No. 277 313 280 284 325 288 282 291 283 316 257 279 Cortinarius cinnabarinus (s) Cortinarius sp. B (s)
Isolation
No. of seedlings
No. of seedlings
inoculated
inoculated
infected
E. delegatensis E. delegatensis E. saligna
4 4 4 4 IO 6 7 6 10 9
E. sdigna
E. E. E. E. E. E.
solignu saligna salignu regnans regnans regnans
E. E. El E. E. E. E. E. E. E. E. E. E. E.
regnans regnans regnans regnans regnans regnans regnans regnans regnans regnans regnans regnons rrgnans sulignu
offungi
tests
L. laccatu.
H. alhus.
(SI or pure
Time required for infection (months)
6 10 9
2-4 1.5 I-3 2 3 4 4 4 4 4 5
4
2-4
2 2 2
24
(c)
Of the 12 identified basidiomycetes. 10 formed mycorrhizas on seedlings (Table 3). These were H. carneum.
fresh spores
Tree species
Of the 5345 mycorrhizal pieces plated out (Table 2) nearly 485; remained free of fungal growth, 16:,, produced dark sterile mycelia. 17?/; sporing Fungi Imperfecti, 7P; hyaline sterile fungi, 5.8% were fungi which could be identified as H. carneum, Scleroderma spp, Paxillus spp, and Hymenogaster albus, and 5.9’35 were unidentified basidiomycetes (clamp-bearing mycelia) which were separated into 42 culturally-distinct types and kept as stock cultures. These 42 cultures were further simplified and combined into 12 groups. and one representative culture from each group was used in synthesis tests. Among the identified basidiomycetes, H. carneum was the most frequently isolated from each of the five main eucalypts species from which isolations were attempted. This fungus has not been isolated from the mycorrhizas of nursery seedlings. Scleroderma spp were the only basidiomycetes isolated from nursery seedling mycorrhizas and they have never been isolated from trees more than 2yr old. Paxillus sp. was isolated once from E. delegatensis, and H. albus was isolated three times from E. delegatensis. E. nitens, and E. sieberi L. Johnson. The unidentified basidiomycetes were isolated from the mycorrhizas of all the eucalypt species studied. ranging from 2 to 50 yr of age, and their frequency of isolation appeared to increase with tree age. Synthesis
with
H. injlatum.
Scleroderma
4 4 4 4 4 4 4 4 4 4 4 3 12 8
2 2 2 2 2 2 2
0 0 0
jlacidum. S. cerrucosum. Hysterangium sp. and
Abstoma sp., Cortinarius sp.. Tricholomu saponuceum. The two that gave negative results were Cortinarius cinnaharinus and another Cortinarius sp. Eleven of 12 un-
identified basidiomycetes tested also formed mycorrhizas on seedlings. Attempts to reisolate the inoculated fungi from the synthesized mycorrhizas were unsuccessful.
DISCUSSION
Although a large proportion of fungal species suspected to be mycorrhizal because of sporocarp association with eucalypt stands gave positive results in synthesis tests, only a small proportion of them could be isolated directly from the mycorrhizas. This low recovery rate could be due to the same reasons as discussed by Chu-Chou (1979). Some of the fungi probably do not commonly form mycorrhizas with eucalypts under natural conditions, though they can do so under artificial conditions. These are some of the difficulties that impose a limitation at present on a full understanding of the mycorrhizal relationships in eucalypt species. A large proportion of the mycorrhiza-formers isolated were unidentified basidiomycetes: some of them may have been hypogeous Gasteromycetes and hence have escaped detection in the usual field survey for sporocarps. There is a possibility that some of the hyaline sterile fungi isolated from the mycorrhizal roots may be mycorrhiza formers (non-clamp bearing basidiomycetes or ascomycetes). So far we have not tried to
Mycorrhizal fungi of identify these fungi and none of them has been tested for mycorrhiza-forming ability on eucalypt seedlings. Despite these problems our study suggests that some importance should be given to H. carneum as a eucalypt mycorrhiza-former in this country. H. carneum was first recorded in Britain in association with a species of Eucalyptus by Berkeley and Broome (1875). Trappe and Guzman (1971) collected this fungus in eucalypt forest in Mexico and in 1975 Marx reported its association with E. g/oh&s Labill. in Peru. In 1977 we found this fungus associated with E. delegatensis and E. regnnns seedlings in a forest nursery in the North Island of New Zealand (Chu-Chou and Grace, 1981). There is some evidence that fungi that form mycorrhizas with eucalypts often do not with Pinus radiata (Chilvers, 1973; Warcup, 1971). We have observed that Tricholoma pessundatum (Lyophyllum sp.), common in both P. radiatu and eucalypt stands, has been isolated from P. radiuta mycorrhizas (Chu-Chou, 1979) but not from eucalypt mycorrhizas. Conversely, Scleroderma spp have been isolated from the eucalypt mycorrhizas but not from those of P. radiata. L. laccata, though easily culturable from sporocarp tissues and commonly seen in stands of both species, showed positive results in synthesis tests on both tree species but has never been isolated from mycorrhizas of either. Sporocarps of H. curneum and H. a/bus have been observed to be associated with seedlings and young trees respectively of New Zealand beech (Nothofigus spp) in the Forest Research Institute nursery and H. carneum was isolated from the mycorrhizas of mountain beech (N. solandri var. clifirtioides (Hood. f.) Poole) seedlings. The relationship between mycorrhizal fungi of eucalypts and New Zealand native beech and other native tree species warrants further investigation. REFERENCES
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