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Inoculation of seeds and soil with basidiospores of mycorrhizal fungi
INOCULATION OF SEEDS AND SOIL WITH BASIDIOSPORES OF MYCORRHIZAL FUNGI C. THEODOK~U and G. D. BOWEN C.S.I.R.O.
Division
of Soils. Glen Osmond, (Accepfed
South Australia.
5064
30 April 1973)
Summary-It was demonstrated that basidiospores of the fungus Rhizopogon lute&s, mycorrhizal for Pinus radiara, could be used successfully as seed inoculum after freeze-drying and storage for 3 months at 22”C, provided the inoculum level was increased lOO-fold. Spore inoculum applied to seed could be held dry for at least 2 days before planting provided inoculum was increased IO-fold. On sowing freshly inoculated seed to sterile soil. 3 x lo3 basidiospores/seed were adequate for infection but maximum mycorrhizal infection occurred with 3 x IO4 spores/seed. A doseeresponse curve was obtained for mycorrhizal infection when basidiospores were applied to soil. As few as 100 spores/290 cm3 pot were sufficient for mycorrhizal infection although infection increased with greater spore dose to a maximum of 10’ spores/pot. Plant growth response was related to intensity of infection. It is suggested that the percentage germination of basidiospores in the rhizosphere may be considerably greater than those reported in studies with synthetic medium. A rhizosphere effect on germination of basidiospores was demonstrated and a method developed to facilitate studies of spore germination in the rhizosphere.
INTRODUCTION THEODOROU(1971) demonstrated inoculation of seeds of Pinus radium D. Don with freshly harvested basidiospores of Rhizopogon luteolus Fr. and Nordh to be an easy and effective way of introducing mycorrhizal fungi into both sterile and non-sterile soils. The development of such methods is appropriate to inoculation with selected mycorrhizal fungi for increasing plant growth in areas with an existing population of naturally occurring mycorrhizal fungi (Theodorou and Bowen, 1970) and for large scale afforestation on previously unplanted areas in which the natural occurrence of mycorrhizal fungi may be sporadic. However, the following aspects of practical inoculation with this method needed further study :(i) Preservation ofinoculum; basidiomycete sporophores are usually produced only over a limited period which may not coincide with planting times, and therefore the possibility of freeze-drying sporophores to store viable spore inoculum was investigated; (ii) Survival of inoculum dried on seed before planting; (iii) The influence of soil drying on inoculum following planting; and (iv) a knowledge of numbers of spores necessary for inoculum establishment. This paper reports studies on these aspects using spores of Rhizopogon luteolus, which previous field inoculation studies (Theodorou and Bowen, 1970) have shown to be efficient in stimulating plant growth in phosphate deficient soils. MATERIALS AND METHODS Spore preparations Fresh spore preparations were made from surface sterilized fruiting bodies in sterile distilled water as previously reported (Theodorou, 1971). Freeze-dried basidiospores were obtained from sporophores which had been previously freeze-dried and stored for 3 months in a closed container in the presence of silica gel at 765
766
C. THEODOROU
AND G. D. BOWEN
22°C. The fresh sporophores had been frozen quickly in a container in a mixture of dry ice in ethanol at -80°C for 10 min and then freeze-dried overnight in a Torvac freeze drier (Gordon F. Mee, Melbourne) at -46°C and 5.33 Pa pressure. Spore application Application on seed. Application of basidiospores on surface sterilized seed of Pinus radiata was in a suspension in sterile distilled water as described by Theodorou (1971). Assessment of spore numbers was made by haemacytometer counts of original spore suspensions and of washings from seed. Application on polyethylene strips. 3.92 x lo6 spores in 0.1 ml of suspension were applied aseptically to a 4.5 x 1 cm polyethylene strip previously sterilized in 70 per cent ethanol. The spore suspension adhered to the strip after it was spread on it with a sterile inoculating wire loop. Drying of spores on seed or polyethylene strips was carried out under a sterile laminar down-flow module at 25°C. Subsequent storage was at 20°C. Application to soil. Spore suspensions containing the required number of spores were aseptically pipetted into a 2 x 0.6 cm hole in moist gamma-irradiated soil (5 Mrad) and seedlings were planted in the inoculation hole. Where the inoculated soil was required to be dried, the holes were covered with moist soil, a sterile gravel layer 0.8 cm thick was placed on the surface, and the soil dried in the growth cabinet with 12 h day at 24°C and 12 h night at 16°C for 18 weeks. The seedlings were subsequently planted to the inoculum holes and in order to evaluate specific stimulation of basidiospore germination and mycelial growth by pine roots inert glass fibres, 10 cm long, were treated in the same way as seedlings. Growth of plants Soils. The soil used in all experiments was the surface 30 cm of a podzolized Mt. Burr sand (Stephens, Cracker, Butler and Smith, 1941). Sterilization of soil was by gammairradiation (5 Mrad) or by autoclaving for 1 h at 121°C. Where autoclaving was used the sterile soil was then inoculated with general soil microflora, by adding 10 ml of @l per cent soil suspension and incubated for 7 days before sowing, to detoxify possible phytotoxins produced by autoclaving (Rovira and Bowen, 1966). Growth conditions. Unless otherwise stated, pots contained 1400 g of soil which had been moistened to 70 per cent field capacity (20 per cent moisture) and the plants were grown in a controlled environment chamber with 12 h day at 16.14 k lx at 24°C and 12 h night at 16°C. Experiments were in quadruplicate; pots were thinned to four plants per pot 1 month after seedling emergence and the soil surface was covered with a 0.8 cm thick layer ofdry sterile gravel. Subsurface watering to constant weight was carried out through a permanently positioned sterile watering tube. Assessment of results Mycorrhizal formation. The intensity of infection (assessed on all plants) was expressed as the percentage of short lateral roots per seedling which had become mycorrhizal (“percentage mycorrhiza”) as indicated by dichotomous root forking or the presence of a fungal mantle. The majority of the unbranched short roots were not infected but all forked roots were infected. R. luteolus (and possibly some other mycorrhizal fungi) produce white mantles on P. radiata mycorrhizas. The percentages of mycorrhizas of this type were also recorded.
BASIDIOSPORES
AS MYCORRHIZAL
161
INOCULUM
Basidiospore germination and mycelial growth. Examinations for spore germination and mycelial growth on glass fibres, polyethylene strips, and roots were made under a dissecting microscope with reflected light at 40 x magnification after staining with 1% lactophenol cotton blue for 1 min. RESULTS
Inoculation withfreeze-dried basidiospores Freeze-dried inoculum stored for 3 months was effective in producing heavy mycorrhizal infection (Table 1) when inoculated at the rate of approx 7 x lo6 spores/seed. In both sterile soil and sterilized soil reinoculated with general soil organisms all mycorrhizas were due to the spore inoculum. In the non-sterilized soil inoculation resulted in 44 per cent of the mycorrhizas being similar to those produced by R. luteolus but in the absence of inoculation only 14 per cent of the mycorrhizas resembled this highly efficient type (significantly different at 5 per cent level). TABLE 1. MYCORRHIZAINFECTIONOF Pinusradiata INOCULATEDWITHFREEZE-DRIEDBASIDIOSPORES*.GROWTHFOR 4 MONTHSINCONTROLLEDENVIRONMENT(SeeteXt) Total no. short lateral
Treatment Sterilized soil 1 Inoculated 2 Uninoculated Sterilized soil + non-sterile suspension 3 Inoculated 4 Uninoculated Non-sterilized soil 5 Inoculated 6 Uninoculated
Mycorrhizat
White
roots/seedling
(%)
(%I
313 221
45 0
100
262 219
43 2
100
258 242 LSD 110 P = 0.05 151 P = 0.01
49 42 LSD 14 P = 0.05 23 P = 0.01
44 14 LSD 26 P = 0.05 36 P = 0.01
soil
* Sporophores were freeze-dried and stored for 3 months spores/seed. t Means of four replicates with four plants per replicate.
before
spores
used
for inoculation--6.9
x lo6
Attempted isolation from freeze-dried sporophores was negative after 14 days whereas isolation from fresh sporophores was positive after 2 days. If spore germination occurs in laboratory media it is very slow (Fries, 1966) but growth from viable mycelia is very rapid. We infer from the absence of growth from freeze-dried material that all mycelia had been killed and that subsequent mycorrhiza formation was from spores. A 30 per cent reduction in numbers of short lateral roots on mycorrhizal plants occurred when a general microflora was present (significant at the 5 per cent level), but the general microflora did not lower infection percentage by the applied inoculum (treatment 1 vs treatments 3 and 5, Table 1). Bowen and Rovira (1961) reported that the general soil microflora could reduce lateral root formation by a number of plant species. Effect of drying on survival
qf basidiospores
Drying of spores on seed. Table 2 shows the mycorrhizal formation on seedlings from seed inoculated and either sown immediately in sterile soil (4.4 x lo6 spores/seed) or first
768
C. THEODOROU
AND
G. D. BOWEN
dried for 2 days (3.7 x lo6 spores/seed). In both cases good mycorrhizal formation occurred and no infection occurred with uninoculated seed. Therefore, provided a sufficient number of spores is used, seed may be held dry for up to 2 days before sowing. TABLE 2. EFFECT ON MY~ORRHIZA FORMATIOK OF DRYING Rhizopogon lutrolus BASIDIOSPORESON PINE SEED BEFORE sow~~o.G~ow~~ FOR 4 MONTHSIN CONTROLLEDENVIR~NMFNT(S~~~~~~) Total no. short lateral roots/seedling*
Treatment Uninoculated
Mycorrhiza;seedling* (““)
336
0
with basidiospores
34x
66
Inoculated with basidiospores Dried before sowing
404
64
LSD 89 P = 0.05
LSD 14 P = 0.05 20 P = 0.01
Inoculated Sown wet
* Mean of four replicates of four plants each. Seed sown wet had 4.4 x IO6 spores/seed dried before sowing had 3.7 x IO“ spores/seed.
and seed
Drying of spores in soil. Drying of spores in the soil over 10 weeks and leaving them for a further 2 months at 24°C day temperature and 16°C night temperature did not affect their viability. The mycorrhiza infection on seedlings in inoculated pots was 26 per cent at 12 weeks after sowing and 41 per cent at 16 weeks after sowing whilst on the uninoculated seedlings there was no infection. The roots and soil in the inoculated pots were well colonized with mycelial strands. In the pots with inoculated glass fibres there was no evidence of spore germination or of mycelial growth on either the fibres or in the soil, thus showing the influence of growing roots on germination of spores. A subsidiary experiment with serial harvesting ofinoculated seedlings showed that 1 month from inoculation with spores no mycorrhizas were formed, at 2 months there was 8 per cent infection and at 3 months infection increased to 28 per cent. In a related experiment, at 5 weeks there was no germination of spores inoculated on a polyethylene strip placed beside roots but at 10 weeks there was both mycorrhiza formation (from the inoculated strips) and mycelial growth on the strips. We conclude that spore germination in the rhizosphere of young seedlings takes between 5 and 10 weeks. Concentration
of basidiospore
inoculurn
necessary,for
establiskment
Spores irz soil. Indications of the effects of spore numbers in the root zone or in soil generally on mycorrhiza infections were obtained by spore inoculations of soil. Nil, 1 x IO’, 1 x 103, 1 x IO4 or 1 x 10’ spores were applied per 9.8 x 14.5 cm pot either by placement in two holes (2 cm deep), each subsequently planted with two sterile germinated seedlings, or by including the inoculum in the water used for wetting of the soil. Table 3 shows as few as 100 spores/1400 g soil or 50 spores in the surface 2 cm of each rhizosphere were adequate for some infection in sterilized soil but that mycorrhiza formation increased markedly with inoculum level. It is surprising that so little difference occurred between the two methods of inoculation as the dispersed inoculum could be expected to be at very low concentration in the rhizosphere.
BASIDIOSPORES
AS MYCORRHIZAL
TABLE 3. EFFECT OF LEVELOF Rhizopogon luteohs SPORE INOCULUMON IN STERILIZEDSOIL.GROWTH
FORK MONTHSIN
Uninoculated 100 spores localized* 100 spores dispersed? in pot 1000 spores localized 1000 spores in pot 10,000 spores localized 10,000 spores in pot 100,000 spores localized 100,000 spores in pot
Mycorrhiza/seedling (%)
361 460 434 455 430 442 428 488 465 LSD 78 P = 0.05
769
INTENSITY OF INFECTION OF Pinus radiata
CONTROLLEDENVIRONMENT
No. of lateral short roots/seedling
No. of spores
INOCULUM
0
;g
1 1
Total dry wt/seedling (8) 1.6 1.7 1.7 1.8 1.9 2.1
;2 1 ;;
(See text)
16 41
;;
58 1 LSD P = 0.05 18 13 P = 0.01 24 17
2.3 2.3 LSD 0.2 P = 0.05 0.3 P = 0.01
* 100 spores localized in 2 holes (2 cm deep). t 100 spores dispersed in whole pot.
Figure 1, based on the data of Table 3, indicates the effect of inoculum level on infection. The growth of seedlings increased as inoculum size and mycorrhiza numbers increased (Table 3). In pots inoculated with 1000 spores or more mycelial strands colonized the whole pot and fruiting bodies of R. luteolus were formed in the pot. The higher inoculum produced greater intensity of mycelial strands in soil. Seed inoculation. Seeds were inoculated with nil, 3 x 103, 3 x 104, 3 x lo5 and 1 x lo6 spores/seed and sown immediately or held dry for 2 days before sowing. Inoculation with freeze-dried spores at similar concentrations was also performed, this and the drying treatment being designed to indicate the loss of viability of fresh spores on air-drying or freeze-drying. Twenty seeds were sown in 15.7 x 17 cm (3600 g) of soil at 70 per cent field capacity.
0
I
I
I
I
I
2
3
4
5
log "0. of spores
FIG. I. Intensity of mycorrhizal infection of Pinus radiata in response to inoculation with different numbers of Rhizopogon luteohs basidiospores in soil. (Conditions as in Table 3.)
770
C. THEODOROU
AND
G. D. BOWEN
Table 4 shows 3 x lo3 spores/seed was adequate for infection with freshly inoculated seed although there are indications that more infection could result from increased inoculum level. However it was necessary to increase the inoculum concentration by some 10 times with spores air-dried for 2 days and approximately 100 times with freeze-dried spores for equivalent mycorrhiza formation. TAWLE
4. NUMBERSOF
Rhizopogon
Spore treatment Sown wet Dried before sowing Freeze-dried sown wet
* Mean
luteolus SPORES REQUIRED FOR EFFECTIVE SITHIINOCULATION ON Pinus GR~WTHFOR 4 MONTHSIN THE~LASSHOUSE
Nil spores 0 ._
3 x lo3 spores/seed 29 10 0 LSD 9 P = 0.05 13 P = 0.01
Mycorrhiza* 3 x 10” spores/seed 52 38 0
(“J 3 x IO5 spores/seed 31 41 32
radiata.
I x 10h spores/seed 60 56 29
of three replicates. DISCUSSION
The studies reported here have implications in practical forestry and form a basis of further study of the physiology and ecology of infection by mycorrhizal fungi in soil. The practical aspects are: (i) the demonstration that freeze-dried basidiospores can be used as inoculum obviates the need for fresh sporophore collection for inoculation thus removing what could be an obstruction to inoculation programmes; (ii) The survival of freeze-dried spores and spore inoculum dried on the seed facilitates transport and overcomes possible deleterious effects of having to hold inoculated seed due to unforeseen planting difficulties. Indeed, inoculated seed has been successfully held in cold storage (2°C) for a month after inoculation. Their survival in soil following planting also augurs well for spore inoculation programmes, while the use of spore inoculum has mechanical advantages over using more bulky mycelial forms of inoculum in that planting spore-inoculated seed through normal seed drills is no problem; (iii) The studies have shown that spore numbers must be increased by up to 100 times with freeze-dried spores and up to 10 times with spores air-dried for 2 days to get equivalent results to those with fresh spores, i.e. considerable death of spores occurs with drying. With its ease of handling and resistance of spores to adverse conditions spore inoculation has some obvious advantages. However, it has some disadvantages, as one is restricted to using fungi which readily fruit, and field collection of inoculum lacks the control and ease of laboratory production. A need for further studies on production of fruiting bodies by mycorrhizal fungi under controlled conditions is apparent. As well as this, the desirability of further studies on the use of resistant vegetative spores produced by fungal mycelia is indicated. Aspects of this and colonization of tree roots by mycorrhizal fungi have been reviewed by Bowen and Theodorou (1973). Amplification of the present studies is needed along at least three lines, viz: (i) the compatibility of spore inoculum with fungicidal seed dressings (this is currently under study); (ii) basidiospore inoculation with different species of mycorrhizal fungi. Variation between and within species in percentage spore germination (Bowen and Theodorou, 1973) and resistance to deleterious soil effects may necessitate modification in levels of inoculum needed; and (iii) numbers of spores needed to successfully compete with antagonistic microorganisms (Bowen and Theodorou, 1973) or naturally occurring mycorrhizal fungi
BASIDIOSPORES
AS MYCORRHIZAL
INOCULUM
771
of low efficiency in tree stimulation (see Bowen, 1973); Theodorou (1971) showed that 4.3 x lo5 spores/seed were sufficient for establishment of Rhizopogon lutrolus in nonsterilized soil but intensity of infection was not as heavy as in soil fumigated by methyl bromide. Hence more spores/seed may be necessary for good establishment in nonsterilized field soil than in pots with sterile soil. The data of Table 3 and Fig. 1 indicate increased infection with increased inoculum concentration up to the highest concentration used and that plant growth response was related to intensity of infection. The continual increase in mycorrhiza production with increasing numbers of spores and the relatively low numbers of mycorrhizas with lower concentrations of inoculum suggest most mycorrhizas in these young plants were formed by separate infection rather than from spread from existing mycorrhizas. Although the soils were initially sterile, they almost certainly did not remain so for long and would have been colonized by other microorganisms. These almost certainly would have retarded ectotrophic growth of the fungus (Bowen and Theodorou, 1973) and this is consistent with our hypothesis that mycorrhizas were formed mainly from new infections in young plants. It is somewhat surprising that as few spores as 100 dispersed in a pot of 290 cm3 of soil would lead to nine infections/seedling as pine roots are usually relatively far apart. This suggests a much higher rate of spore germination in the presence of roots than the 0.1 per cent often obtained in germination under laboratory media (Fries, 1966) and that the rhizosphere effect on germination of basidiospores may be extensive, i.e. a root may not have to actually touch a basidiospore. In these studies we have noted .a rhizosphere stimulation of spore germination; studies on germination of basidiospores of mycorrhizal fungi in the rhizosphere are almost non-existent (Bowen and Theodorou, 1973), but are necessary to an understanding of the dynamics of colonization of tree roots by mycorrhizal fungi in soil. The technique developed here, in which spores are inoculated on to polyethylene strips and planted near roots offers possibilities for detailed studies ofeffects on basidiospore germination of root age, soil factors and proximity to the root. The low numbers of spores needed for infection also suggest the practicability of counting mycorrhizal spore populations of natural soils by modification of existing techniques of dilution to extinction. Ackno&dgrnzmts-We are grateful to Mrs. SUE BRIGGS for competent technical assistance. Some financial assistance was made available from a fund provided by a number of Australian forestry organizations. REFERENCES BOWEN G. D. (1973) Mineral nutrition of ectomycorrhizae. In Ectomycorrhizae: Their Ecology and Physiology (G. C. Marks and T. T. Kozlowski, Eds) pp. 151-205. Academic Press, New York. BOWEN G. D. and ROVIRA A. D. (1961) The e&ts of microorganisms on plant growth. I. Development of roots and root hairs in sand and agar. PI. Soil 15, 16688. BOWEN G. D. and THEODOROU C. (1973) Growth of ectomycorrhizal fungi around seeds and roots. In: Ectomycorrhizae: Their Ecology and Physiologp (G. C. Marks and T. T. Kozlowski, Eds) pp. 107-150. Academic Press, New York. FRIES N. (1966) Chemical factors in the germination of spores of Basidiomycetes. In The Fungus Spore (M. F. Madelin, Ed.) pp. 189-99. Butterworth, London. ROV~RA A. D. and BOWEN G. D. (1966) The effects of microorganisms on plant growth. II. Detoxication of heat sterilized soils by fungi and bacteria. PI. Soil 25, 129-142. STEPHENS C. G., CROCKER R. L., BUTLER B. and SMITH R. (1941) A soil and land use survey of the Hundreds of Riddock, Hindmarsh. Grey, Young and Nangwarry, County Grey, South Australia. Council scient. ind. Res. Amt. Bull. No. 142. THEODOROU C. (1971) Introduction of mycorrhizal fungi into soil by spore inoculation of seed. Amt. For. 35, 23-26. THEODOROUC. and BOWEN G. D. (1970) Mycorrhizal responses of radiata pine in experiments with different fungi. Aust. For. 34, 183-191.
Division
of Soils. Glen Osmond, (Accepfed
South Australia.
5064
30 April 1973)
Summary-It was demonstrated that basidiospores of the fungus Rhizopogon lute&s, mycorrhizal for Pinus radiara, could be used successfully as seed inoculum after freeze-drying and storage for 3 months at 22”C, provided the inoculum level was increased lOO-fold. Spore inoculum applied to seed could be held dry for at least 2 days before planting provided inoculum was increased IO-fold. On sowing freshly inoculated seed to sterile soil. 3 x lo3 basidiospores/seed were adequate for infection but maximum mycorrhizal infection occurred with 3 x IO4 spores/seed. A doseeresponse curve was obtained for mycorrhizal infection when basidiospores were applied to soil. As few as 100 spores/290 cm3 pot were sufficient for mycorrhizal infection although infection increased with greater spore dose to a maximum of 10’ spores/pot. Plant growth response was related to intensity of infection. It is suggested that the percentage germination of basidiospores in the rhizosphere may be considerably greater than those reported in studies with synthetic medium. A rhizosphere effect on germination of basidiospores was demonstrated and a method developed to facilitate studies of spore germination in the rhizosphere.
INTRODUCTION THEODOROU(1971) demonstrated inoculation of seeds of Pinus radium D. Don with freshly harvested basidiospores of Rhizopogon luteolus Fr. and Nordh to be an easy and effective way of introducing mycorrhizal fungi into both sterile and non-sterile soils. The development of such methods is appropriate to inoculation with selected mycorrhizal fungi for increasing plant growth in areas with an existing population of naturally occurring mycorrhizal fungi (Theodorou and Bowen, 1970) and for large scale afforestation on previously unplanted areas in which the natural occurrence of mycorrhizal fungi may be sporadic. However, the following aspects of practical inoculation with this method needed further study :(i) Preservation ofinoculum; basidiomycete sporophores are usually produced only over a limited period which may not coincide with planting times, and therefore the possibility of freeze-drying sporophores to store viable spore inoculum was investigated; (ii) Survival of inoculum dried on seed before planting; (iii) The influence of soil drying on inoculum following planting; and (iv) a knowledge of numbers of spores necessary for inoculum establishment. This paper reports studies on these aspects using spores of Rhizopogon luteolus, which previous field inoculation studies (Theodorou and Bowen, 1970) have shown to be efficient in stimulating plant growth in phosphate deficient soils. MATERIALS AND METHODS Spore preparations Fresh spore preparations were made from surface sterilized fruiting bodies in sterile distilled water as previously reported (Theodorou, 1971). Freeze-dried basidiospores were obtained from sporophores which had been previously freeze-dried and stored for 3 months in a closed container in the presence of silica gel at 765
766
C. THEODOROU
AND G. D. BOWEN
22°C. The fresh sporophores had been frozen quickly in a container in a mixture of dry ice in ethanol at -80°C for 10 min and then freeze-dried overnight in a Torvac freeze drier (Gordon F. Mee, Melbourne) at -46°C and 5.33 Pa pressure. Spore application Application on seed. Application of basidiospores on surface sterilized seed of Pinus radiata was in a suspension in sterile distilled water as described by Theodorou (1971). Assessment of spore numbers was made by haemacytometer counts of original spore suspensions and of washings from seed. Application on polyethylene strips. 3.92 x lo6 spores in 0.1 ml of suspension were applied aseptically to a 4.5 x 1 cm polyethylene strip previously sterilized in 70 per cent ethanol. The spore suspension adhered to the strip after it was spread on it with a sterile inoculating wire loop. Drying of spores on seed or polyethylene strips was carried out under a sterile laminar down-flow module at 25°C. Subsequent storage was at 20°C. Application to soil. Spore suspensions containing the required number of spores were aseptically pipetted into a 2 x 0.6 cm hole in moist gamma-irradiated soil (5 Mrad) and seedlings were planted in the inoculation hole. Where the inoculated soil was required to be dried, the holes were covered with moist soil, a sterile gravel layer 0.8 cm thick was placed on the surface, and the soil dried in the growth cabinet with 12 h day at 24°C and 12 h night at 16°C for 18 weeks. The seedlings were subsequently planted to the inoculum holes and in order to evaluate specific stimulation of basidiospore germination and mycelial growth by pine roots inert glass fibres, 10 cm long, were treated in the same way as seedlings. Growth of plants Soils. The soil used in all experiments was the surface 30 cm of a podzolized Mt. Burr sand (Stephens, Cracker, Butler and Smith, 1941). Sterilization of soil was by gammairradiation (5 Mrad) or by autoclaving for 1 h at 121°C. Where autoclaving was used the sterile soil was then inoculated with general soil microflora, by adding 10 ml of @l per cent soil suspension and incubated for 7 days before sowing, to detoxify possible phytotoxins produced by autoclaving (Rovira and Bowen, 1966). Growth conditions. Unless otherwise stated, pots contained 1400 g of soil which had been moistened to 70 per cent field capacity (20 per cent moisture) and the plants were grown in a controlled environment chamber with 12 h day at 16.14 k lx at 24°C and 12 h night at 16°C. Experiments were in quadruplicate; pots were thinned to four plants per pot 1 month after seedling emergence and the soil surface was covered with a 0.8 cm thick layer ofdry sterile gravel. Subsurface watering to constant weight was carried out through a permanently positioned sterile watering tube. Assessment of results Mycorrhizal formation. The intensity of infection (assessed on all plants) was expressed as the percentage of short lateral roots per seedling which had become mycorrhizal (“percentage mycorrhiza”) as indicated by dichotomous root forking or the presence of a fungal mantle. The majority of the unbranched short roots were not infected but all forked roots were infected. R. luteolus (and possibly some other mycorrhizal fungi) produce white mantles on P. radiata mycorrhizas. The percentages of mycorrhizas of this type were also recorded.
BASIDIOSPORES
AS MYCORRHIZAL
161
INOCULUM
Basidiospore germination and mycelial growth. Examinations for spore germination and mycelial growth on glass fibres, polyethylene strips, and roots were made under a dissecting microscope with reflected light at 40 x magnification after staining with 1% lactophenol cotton blue for 1 min. RESULTS
Inoculation withfreeze-dried basidiospores Freeze-dried inoculum stored for 3 months was effective in producing heavy mycorrhizal infection (Table 1) when inoculated at the rate of approx 7 x lo6 spores/seed. In both sterile soil and sterilized soil reinoculated with general soil organisms all mycorrhizas were due to the spore inoculum. In the non-sterilized soil inoculation resulted in 44 per cent of the mycorrhizas being similar to those produced by R. luteolus but in the absence of inoculation only 14 per cent of the mycorrhizas resembled this highly efficient type (significantly different at 5 per cent level). TABLE 1. MYCORRHIZAINFECTIONOF Pinusradiata INOCULATEDWITHFREEZE-DRIEDBASIDIOSPORES*.GROWTHFOR 4 MONTHSINCONTROLLEDENVIRONMENT(SeeteXt) Total no. short lateral
Treatment Sterilized soil 1 Inoculated 2 Uninoculated Sterilized soil + non-sterile suspension 3 Inoculated 4 Uninoculated Non-sterilized soil 5 Inoculated 6 Uninoculated
Mycorrhizat
White
roots/seedling
(%)
(%I
313 221
45 0
100
262 219
43 2
100
258 242 LSD 110 P = 0.05 151 P = 0.01
49 42 LSD 14 P = 0.05 23 P = 0.01
44 14 LSD 26 P = 0.05 36 P = 0.01
soil
* Sporophores were freeze-dried and stored for 3 months spores/seed. t Means of four replicates with four plants per replicate.
before
spores
used
for inoculation--6.9
x lo6
Attempted isolation from freeze-dried sporophores was negative after 14 days whereas isolation from fresh sporophores was positive after 2 days. If spore germination occurs in laboratory media it is very slow (Fries, 1966) but growth from viable mycelia is very rapid. We infer from the absence of growth from freeze-dried material that all mycelia had been killed and that subsequent mycorrhiza formation was from spores. A 30 per cent reduction in numbers of short lateral roots on mycorrhizal plants occurred when a general microflora was present (significant at the 5 per cent level), but the general microflora did not lower infection percentage by the applied inoculum (treatment 1 vs treatments 3 and 5, Table 1). Bowen and Rovira (1961) reported that the general soil microflora could reduce lateral root formation by a number of plant species. Effect of drying on survival
qf basidiospores
Drying of spores on seed. Table 2 shows the mycorrhizal formation on seedlings from seed inoculated and either sown immediately in sterile soil (4.4 x lo6 spores/seed) or first
768
C. THEODOROU
AND
G. D. BOWEN
dried for 2 days (3.7 x lo6 spores/seed). In both cases good mycorrhizal formation occurred and no infection occurred with uninoculated seed. Therefore, provided a sufficient number of spores is used, seed may be held dry for up to 2 days before sowing. TABLE 2. EFFECT ON MY~ORRHIZA FORMATIOK OF DRYING Rhizopogon lutrolus BASIDIOSPORESON PINE SEED BEFORE sow~~o.G~ow~~ FOR 4 MONTHSIN CONTROLLEDENVIR~NMFNT(S~~~~~~) Total no. short lateral roots/seedling*
Treatment Uninoculated
Mycorrhiza;seedling* (““)
336
0
with basidiospores
34x
66
Inoculated with basidiospores Dried before sowing
404
64
LSD 89 P = 0.05
LSD 14 P = 0.05 20 P = 0.01
Inoculated Sown wet
* Mean of four replicates of four plants each. Seed sown wet had 4.4 x IO6 spores/seed dried before sowing had 3.7 x IO“ spores/seed.
and seed
Drying of spores in soil. Drying of spores in the soil over 10 weeks and leaving them for a further 2 months at 24°C day temperature and 16°C night temperature did not affect their viability. The mycorrhiza infection on seedlings in inoculated pots was 26 per cent at 12 weeks after sowing and 41 per cent at 16 weeks after sowing whilst on the uninoculated seedlings there was no infection. The roots and soil in the inoculated pots were well colonized with mycelial strands. In the pots with inoculated glass fibres there was no evidence of spore germination or of mycelial growth on either the fibres or in the soil, thus showing the influence of growing roots on germination of spores. A subsidiary experiment with serial harvesting ofinoculated seedlings showed that 1 month from inoculation with spores no mycorrhizas were formed, at 2 months there was 8 per cent infection and at 3 months infection increased to 28 per cent. In a related experiment, at 5 weeks there was no germination of spores inoculated on a polyethylene strip placed beside roots but at 10 weeks there was both mycorrhiza formation (from the inoculated strips) and mycelial growth on the strips. We conclude that spore germination in the rhizosphere of young seedlings takes between 5 and 10 weeks. Concentration
of basidiospore
inoculurn
necessary,for
establiskment
Spores irz soil. Indications of the effects of spore numbers in the root zone or in soil generally on mycorrhiza infections were obtained by spore inoculations of soil. Nil, 1 x IO’, 1 x 103, 1 x IO4 or 1 x 10’ spores were applied per 9.8 x 14.5 cm pot either by placement in two holes (2 cm deep), each subsequently planted with two sterile germinated seedlings, or by including the inoculum in the water used for wetting of the soil. Table 3 shows as few as 100 spores/1400 g soil or 50 spores in the surface 2 cm of each rhizosphere were adequate for some infection in sterilized soil but that mycorrhiza formation increased markedly with inoculum level. It is surprising that so little difference occurred between the two methods of inoculation as the dispersed inoculum could be expected to be at very low concentration in the rhizosphere.
BASIDIOSPORES
AS MYCORRHIZAL
TABLE 3. EFFECT OF LEVELOF Rhizopogon luteohs SPORE INOCULUMON IN STERILIZEDSOIL.GROWTH
FORK MONTHSIN
Uninoculated 100 spores localized* 100 spores dispersed? in pot 1000 spores localized 1000 spores in pot 10,000 spores localized 10,000 spores in pot 100,000 spores localized 100,000 spores in pot
Mycorrhiza/seedling (%)
361 460 434 455 430 442 428 488 465 LSD 78 P = 0.05
769
INTENSITY OF INFECTION OF Pinus radiata
CONTROLLEDENVIRONMENT
No. of lateral short roots/seedling
No. of spores
INOCULUM
0
;g
1 1
Total dry wt/seedling (8) 1.6 1.7 1.7 1.8 1.9 2.1
;2 1 ;;
(See text)
16 41
;;
58 1 LSD P = 0.05 18 13 P = 0.01 24 17
2.3 2.3 LSD 0.2 P = 0.05 0.3 P = 0.01
* 100 spores localized in 2 holes (2 cm deep). t 100 spores dispersed in whole pot.
Figure 1, based on the data of Table 3, indicates the effect of inoculum level on infection. The growth of seedlings increased as inoculum size and mycorrhiza numbers increased (Table 3). In pots inoculated with 1000 spores or more mycelial strands colonized the whole pot and fruiting bodies of R. luteolus were formed in the pot. The higher inoculum produced greater intensity of mycelial strands in soil. Seed inoculation. Seeds were inoculated with nil, 3 x 103, 3 x 104, 3 x lo5 and 1 x lo6 spores/seed and sown immediately or held dry for 2 days before sowing. Inoculation with freeze-dried spores at similar concentrations was also performed, this and the drying treatment being designed to indicate the loss of viability of fresh spores on air-drying or freeze-drying. Twenty seeds were sown in 15.7 x 17 cm (3600 g) of soil at 70 per cent field capacity.
0
I
I
I
I
I
2
3
4
5
log "0. of spores
FIG. I. Intensity of mycorrhizal infection of Pinus radiata in response to inoculation with different numbers of Rhizopogon luteohs basidiospores in soil. (Conditions as in Table 3.)
770
C. THEODOROU
AND
G. D. BOWEN
Table 4 shows 3 x lo3 spores/seed was adequate for infection with freshly inoculated seed although there are indications that more infection could result from increased inoculum level. However it was necessary to increase the inoculum concentration by some 10 times with spores air-dried for 2 days and approximately 100 times with freeze-dried spores for equivalent mycorrhiza formation. TAWLE
4. NUMBERSOF
Rhizopogon
Spore treatment Sown wet Dried before sowing Freeze-dried sown wet
* Mean
luteolus SPORES REQUIRED FOR EFFECTIVE SITHIINOCULATION ON Pinus GR~WTHFOR 4 MONTHSIN THE~LASSHOUSE
Nil spores 0 ._
3 x lo3 spores/seed 29 10 0 LSD 9 P = 0.05 13 P = 0.01
Mycorrhiza* 3 x 10” spores/seed 52 38 0
(“J 3 x IO5 spores/seed 31 41 32
radiata.
I x 10h spores/seed 60 56 29
of three replicates. DISCUSSION
The studies reported here have implications in practical forestry and form a basis of further study of the physiology and ecology of infection by mycorrhizal fungi in soil. The practical aspects are: (i) the demonstration that freeze-dried basidiospores can be used as inoculum obviates the need for fresh sporophore collection for inoculation thus removing what could be an obstruction to inoculation programmes; (ii) The survival of freeze-dried spores and spore inoculum dried on the seed facilitates transport and overcomes possible deleterious effects of having to hold inoculated seed due to unforeseen planting difficulties. Indeed, inoculated seed has been successfully held in cold storage (2°C) for a month after inoculation. Their survival in soil following planting also augurs well for spore inoculation programmes, while the use of spore inoculum has mechanical advantages over using more bulky mycelial forms of inoculum in that planting spore-inoculated seed through normal seed drills is no problem; (iii) The studies have shown that spore numbers must be increased by up to 100 times with freeze-dried spores and up to 10 times with spores air-dried for 2 days to get equivalent results to those with fresh spores, i.e. considerable death of spores occurs with drying. With its ease of handling and resistance of spores to adverse conditions spore inoculation has some obvious advantages. However, it has some disadvantages, as one is restricted to using fungi which readily fruit, and field collection of inoculum lacks the control and ease of laboratory production. A need for further studies on production of fruiting bodies by mycorrhizal fungi under controlled conditions is apparent. As well as this, the desirability of further studies on the use of resistant vegetative spores produced by fungal mycelia is indicated. Aspects of this and colonization of tree roots by mycorrhizal fungi have been reviewed by Bowen and Theodorou (1973). Amplification of the present studies is needed along at least three lines, viz: (i) the compatibility of spore inoculum with fungicidal seed dressings (this is currently under study); (ii) basidiospore inoculation with different species of mycorrhizal fungi. Variation between and within species in percentage spore germination (Bowen and Theodorou, 1973) and resistance to deleterious soil effects may necessitate modification in levels of inoculum needed; and (iii) numbers of spores needed to successfully compete with antagonistic microorganisms (Bowen and Theodorou, 1973) or naturally occurring mycorrhizal fungi
BASIDIOSPORES
AS MYCORRHIZAL
INOCULUM
771
of low efficiency in tree stimulation (see Bowen, 1973); Theodorou (1971) showed that 4.3 x lo5 spores/seed were sufficient for establishment of Rhizopogon lutrolus in nonsterilized soil but intensity of infection was not as heavy as in soil fumigated by methyl bromide. Hence more spores/seed may be necessary for good establishment in nonsterilized field soil than in pots with sterile soil. The data of Table 3 and Fig. 1 indicate increased infection with increased inoculum concentration up to the highest concentration used and that plant growth response was related to intensity of infection. The continual increase in mycorrhiza production with increasing numbers of spores and the relatively low numbers of mycorrhizas with lower concentrations of inoculum suggest most mycorrhizas in these young plants were formed by separate infection rather than from spread from existing mycorrhizas. Although the soils were initially sterile, they almost certainly did not remain so for long and would have been colonized by other microorganisms. These almost certainly would have retarded ectotrophic growth of the fungus (Bowen and Theodorou, 1973) and this is consistent with our hypothesis that mycorrhizas were formed mainly from new infections in young plants. It is somewhat surprising that as few spores as 100 dispersed in a pot of 290 cm3 of soil would lead to nine infections/seedling as pine roots are usually relatively far apart. This suggests a much higher rate of spore germination in the presence of roots than the 0.1 per cent often obtained in germination under laboratory media (Fries, 1966) and that the rhizosphere effect on germination of basidiospores may be extensive, i.e. a root may not have to actually touch a basidiospore. In these studies we have noted .a rhizosphere stimulation of spore germination; studies on germination of basidiospores of mycorrhizal fungi in the rhizosphere are almost non-existent (Bowen and Theodorou, 1973), but are necessary to an understanding of the dynamics of colonization of tree roots by mycorrhizal fungi in soil. The technique developed here, in which spores are inoculated on to polyethylene strips and planted near roots offers possibilities for detailed studies ofeffects on basidiospore germination of root age, soil factors and proximity to the root. The low numbers of spores needed for infection also suggest the practicability of counting mycorrhizal spore populations of natural soils by modification of existing techniques of dilution to extinction. Ackno&dgrnzmts-We are grateful to Mrs. SUE BRIGGS for competent technical assistance. Some financial assistance was made available from a fund provided by a number of Australian forestry organizations. REFERENCES BOWEN G. D. (1973) Mineral nutrition of ectomycorrhizae. In Ectomycorrhizae: Their Ecology and Physiology (G. C. Marks and T. T. Kozlowski, Eds) pp. 151-205. Academic Press, New York. BOWEN G. D. and ROVIRA A. D. (1961) The e&ts of microorganisms on plant growth. I. Development of roots and root hairs in sand and agar. PI. Soil 15, 16688. BOWEN G. D. and THEODOROU C. (1973) Growth of ectomycorrhizal fungi around seeds and roots. In: Ectomycorrhizae: Their Ecology and Physiologp (G. C. Marks and T. T. Kozlowski, Eds) pp. 107-150. Academic Press, New York. FRIES N. (1966) Chemical factors in the germination of spores of Basidiomycetes. In The Fungus Spore (M. F. Madelin, Ed.) pp. 189-99. Butterworth, London. ROV~RA A. D. and BOWEN G. D. (1966) The effects of microorganisms on plant growth. II. Detoxication of heat sterilized soils by fungi and bacteria. PI. Soil 25, 129-142. STEPHENS C. G., CROCKER R. L., BUTLER B. and SMITH R. (1941) A soil and land use survey of the Hundreds of Riddock, Hindmarsh. Grey, Young and Nangwarry, County Grey, South Australia. Council scient. ind. Res. Amt. Bull. No. 142. THEODOROU C. (1971) Introduction of mycorrhizal fungi into soil by spore inoculation of seed. Amt. For. 35, 23-26. THEODOROUC. and BOWEN G. D. (1970) Mycorrhizal responses of radiata pine in experiments with different fungi. Aust. For. 34, 183-191.