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Evidence for differential responses between host-fungus combinations of vesicular-arbuscular mycorrhizas from a grassland
415
Mycol. Res. 96 ( 6 ) :415-419 (1992) Printed in Great Britain
Evidence for differential responses between host-fungus combinations of vesicular-arbuscular mycorrhizas from a grassland
I. R. SANDERS* A N D A. H. FITTER Department of Biology, University of York, Heslington, York Y O 1 5 0 0 , U.K.
The increase in spore density over that in the soil of an indigenous vesicular-arbuscular (VA) mycorrhizal community was measured in five plant species, after inoculation with the same soil (Experiment I), and in pots containing individuals of the same species, which had been removed from the field site (Experiment 2). In Experiment 1, significant increase in spore density was observed for Glomw constricturn with Plantago lanceolata although increases in density were not observed for this fungus with other host species. Results of Experiment 2 were consistent for the combination of Plantago lanceolata and Glomw constricturn and the numbers of Acaulospora laevis spores, in pots containing Holcw lanatw or Rumex acetosa, were significantly higher than in the soil of the field site. The results do not support or oppose the hypotheses that either multiple infection or selectivity of VA mycorrhizas occur between certain combinations of host-fungus in co-existing plants of a natural community. The results indicate that VA mycorrhizal fungi respond differentlyaccording to the host species and thus such differences could result in selection pressures which favour certain host-fungus combinations.
Vesicular-arbuscular (VA) mycorrhizas are abundant in natural ecosystems (Harley & Smith, 1983) and although they have been presumed to be beneficial to plant growth in the field, very few investigations have clearly demonstrated such benefits (see McGonigle, 1988; Fitter, 1990). The lack of an observable effect on plant growth and nutrient status under field conditions has been attributed to many factors, including low or differing effectiveness of fungal strains (Fitter, 1985) and the presence of different types of VA mycorrhizal fungi within plant roots (Rosendahl, Rosendahl & Sachting, 1990). However, very little information exists on whether multiple infection by VA mycorrhizal fungi, that is the infection of one plant root system by more than one VA mycorrhizal fungus, occurs. Furthermore, there is little evidence regarding differential responses of VA mycorrhizal fungi in different host plants and whether host plants are preferentially infected by certain species or isolates of VA mycorrhizal fungi. In most instances VA mycorrhizal fungi can infect any plant capable of forming mycorrhizas (Nicolson, 1967; Gerdemann & Trappe, 1974; Harley & Smith, 1983) which suggests a potential for multiple infection in the field. McGonigle & Fitter (1990) observed both coarse and fine endophytes growing in the roots of Holctts lanatus L. and Rosendahl et al. (1990) have characterized up to four morphological types of VA mycorrhizal fungus co-existing in the roots of Hieracium pilosella L., Trifolium repens L., Agrostis capillaris L. and Plantago
lanceolata L. ' Current address: Department of Biology, The Pennsylvania State University, University Park, PA 16802,U.S.A.
Harley & Smith (1983) argue that some 'ecological specificity', the preferential infection of plants by certain endophytes, could occur. Whilst there is little evidence with which to test this hypothesis, it is likely that ecological factors influence compatibility in the field, since plants which are normally non-mycorrhizal in the field will often develop mycorrhizas when grown in pots (Harley & Smith, 1983). In the field, VA mycorrhizal fungi would be selectively favoured if they had an ability to infect certain plant species quickly, subsequently inducing a large growth response or other benefit in the host, or influencing the host genome so as to exclude other species. Interspecific differences in both the intensity of infection by VA mycorrhizal fungal host plants (Molina, Trappe & Strickler, 1978; Koske, 1981) and the growth response in different hosts (Hetrick, Kitt & Wilson, 1988; Hetrick, Wilson & Hartnett, 1989; Hetrick, Wilson & Todd, 1990) have been observed. However, only recently has new information been obtained on genome expression in different VA mycorrhizal combinations (see review by Gianinazzi, 1991). The aims of this investigation were to assess whether multiple infection by VA mycorrhizal-forming fungi occurs in plants of a semi-natural grassland and to establish whether differential responses occur between host-fungus combinations when different plant species are exposed to the same mixed inoculum of VA mycorrhizal fungal species. If so, this would support the hypothesis that multiple infections obscure any direct relationships between VA mycorrhizal infection and plant growth in the field. In addition, differential responses of VA m~corrhizaswould demonstrate that the potential for
Differential responses between host-fungus combinations of VA mycorrhizas Table 1. Mean VAM spore density in soil from the field site Mean spore density (spores g-I of soil)
Glomw etunicatum Glomw consfricfum Acaulospora laevis Scutellospora calospora
0 2 8a DlOab O.1Zab O.lOab 0 0 3b
Sporocarps (miscellaneous)
Values followed by a different letter are significantly different at
P = < 005.
Table 2. F ratios from one-way ANOVA on spore density in soil from the field site and soil from pots containing the five plant species, either grown from seed (Experiment I ) or having previously been removed from the field site (Experiment 2) Experiment 1
G. etunicatum G. constricturn A . laevis s . ca/ospora Sporocarps
Harvest I
Harvest 2
Experiment 2
1.63 n.s. 3.10' 2.75' 1.01n.s. 3.38'
098n.s. 2.80" 1.86 n.s. 15.36*'* 0 4 5 n.s.
1.5I* 3.92"" 3.30' 18.45'** 1.64n.s.
' P < 0 0 5 ; *' P < 0.01; *'" P < 0001. All F values have 5 and 3 1 degrees of freedom.
Table 3. F ratios from one-way ANOVA on increase in spore density for each VA mycorrhizal fungal species with each of five plant species in pots, either grown from seed (Experiment 1) or having previously been removed from the field site (Experiment 2) Experiment 1
P. lanceolata R. acefosa 7. pratense H. lanatus F. rubra * P < 0 0 5 ; ** P of freedom.
Harvest I
Harvest 2
Experiment 2
3.441.64n.s. 0 4 5 n.s. 417* 1.76n.s.
2732*** 070n.s. 0 8 2 n.s. 2.05 n.s. 066n.s.
2.91. 4.25" 0.27n.s. 4.43'" 1'23 n.s.
< 0.01; *** P < 0001.All F values have 4 and 26 degrees
the selection of host-fungus combinations in the field exists. The study involves the use of spore characteristics to ensure certain identification of VA mycorrhizal fungal species. Other methods of VA mycorrhizal identification, using morphological characteristics of the fungi, can be unreliable for studying endophytes in the roots of different plant species (Lackie ef al., 1987; Brundrett & Kendrick, 1990).
MATERIALS A N D M E T H O D S Experiment 1
Surface-sterilized seeds (10% sodium hypochlorite, 10 min) of Planfago lanceolafa, Rumex acefosa L., Trifolium prafense L., Holcus lanafus and Fesfuca rubra L. were germinated in Petri
416
dishes. The seeds had been collected during the summer of 1988 from the field site, Wheldrake Ings, a species-rich meadow (North Yorkshire, England, national grid reference SE 702443). After 10 d individual seedlings were transplanted into 10 cm diam. pots containing sterile sand (autoclaved for 20 min at 120 OC) combined with 10 % (by volume) unsterilised soil. Transplantation took place on 16 May 1989, the soil having been removed from the field site on the previous day. There were six replicates of each species. Plants were kept in a heated glasshouse, with 16 h supplementary lighting per day, watered daily and given phosphate-free Rorison's solution (Hewitt, 1966) once a week. At two harvests, 120 and 180 d after transplantation, 20 g of soil was removed from each pot for spore extraction. After the first harvest the area from which soil was removed was marked so that the soil was not removed from the same region in the second harvest. The volume of soil removed in the first harvest was replaced with an equal volume of sterile sand. At each harvest a small portion of the root system was also taken in order to check whether the plants were mycorrhizal. Roots were cleared in 10% KOH and stained with 0.1 % acid fuchsin (Kormanik & McGraw, 1982). Spore extraction was carried out using the sucrose centrifugation method of Walker, Mize & McNabb (1982) and the number of spores of each VA mycorrhizal fungal species was recorded for each pot. At the first harvest (13 September, 1989) VA mycorrhizal spores were also extracted from six 20 g replicates of the field soil (collected 15 May 1989), in order to obtain a background count of spore numbers present in the original inoculum. In order to compare the change in spore numbers occurring in each treatment and for each VA m~corrhizal species recorded, a measure of the increase in spore density was used. This was calculated by subtracting the mean density of spores in the original inoculum (spores g-' soil) from the mean density of spores in each treatment, allowing for the 1:10 dilution of original inoculum. The increase in spore density is expressed as spores g-l of soil. One-way ANOVA was used to establish whether differences occurred in the change of spore density of each of the different plant species and whether there were significant increases in spore density from that present from the original inoculum.
Six individual plants of P. lanceolafa, R. acefosa, T. prafense, H. lanafus and F. rubra were randomly selected and removed from the same field site on 17 September 1989 using a 6 cm diam. x 16 cm depth soil corer. The root system of each plant was carefully washed to remove all soil debris and other plant root systems. Each plant was placed individually in a pot containing a 10 % (by volume) sterilized mixture of soil from . plants the field site and sand (autoclaved 20 min at 1 2 0 ~ )The were kept in the glasshouse under the same conditions as described above. After 160 d 20 g of soil from each pot was removed for spore extraction. Spore extraction, subsequent calculation of increase in spore density and statistical analysis followed that of Experiment 1.
I. R. Sanders and A. H. Fitter Plantago lanceolata
RESULTS
a
Spores of seven VA mycorrhizal species, most closely resembling Acaulospora laevis Gerd. & Trappe, Glomus consfrictum Trappe, Glomus efunicafum Beck. & Gerd., Glomus invermaius Hall, Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, Sclerocystis rubifomis Gerd. & Trappe and Scufellospora calospora (Nicol. & Gerd.) Gerd. & Trappe, were identified in soil from the field site. Results are presented only for the four species which occurred consistently in all samples. The three sporocarp-forming species, G . invermaium, G. mosseae and S. rubiformis, were so infrequent that the data for these species were summed in order to present the densities of sporocarps. Mean overall spore density in the soil from Wheldrake Ings was 0.63 spores g-l soil. There were differences in mean spore densities between VA mycorrhizal fungal types (one-way ANOVA, F,,,, = 3.62, P < 0.05), G. efunicafum spores being more abundant than sporocarps (Table I). All plants in Experiment 1 were infected after I20 d. The density of spores of G . consfricfum,A. laevis and of sporocarps differed between the field soil and the pots containing the five species at harvest 1. Only G . consfricfum differed between the field and pot soil at both harvests (Table 2). Virtually no sporocarps were found at the first harvest and no S. calospora spores were found at the second. This accounted for the significant differences observed in spore density of these species (Table 2). There were no differences in the density of G . efunicatum spores according to treatment at either harvest in Experiment 1. Significant differences between the increase in spore densities, i.e. density in pot minus that in the original soil, of each VA mycorrhizal species occurred with P. lanceolafa at both harvests and in H. lanutus at the first harvest (Table 3). In pots containing P. hnceolafa the increase in spore density of G . consfricfum was higher than that of A. laevis and S. calospora at the first harvest and greater than all other spore types by the second (Fig. 1). With H. lanatus the increase in the density of G . efunicafum was significantly greater than that of G . constricfum and the sporocarp forming fungi (Fig. 1)at the first harvest. No significant differences occurred in the densities of the spores of each of the VA mycorrhizal fungal species in pots containing R. acefosa, T. pratense and F. rubra (Fig. 1).
Rumex acetosa
1
-
Trifolium pratense
IM
C
m
5
1.0,
~ o ~ c lanutus us
Spore densities recorded in Experiment 2 were consistent with Experiment I in that the density of G . consfricfum spores was different between treatments, i.e. the field soil and each of the five different plant species. However, significant differences in spore density were also observed for G . efunicafum, A . laevis and S. calospora (Table 2). As in Experiment I, the low spore density of S. calospora in all pots (zero in most cases) compared
Festuca rubra
Fig. 1. Increase in mean VA mycorrhizal spore density with each of
-0 5
Ge
Gc Al Sc VA mycorrhizal species
SP
five plant species grown in soil from the field site (Experiment I). Different letters above bars within one harvest indicate a significant difference, where P = < 0.05. Harvest 1 (m), harvest 2 (a). Glomw etunicatum (Ge), Glomw constricturn (Gc), Acaulospora laevis (Al), Scutellospora calospora (Sc), sporocarps (Sp).
Differential responses between host-fungus combinations of VA mycorrhizas Plantago lanceolata
1 0.5
.
Ge
Gc
A1
Sc
SP
418
to the original density (0.1 spores g1of soil) accounted for the highly significant treatment effect. In pots containing P. lanceolafa the density of G. consfricfum was significantly higher than in the soil from the field site and some of the other VA mycorrhizal species (Table 3 , Fig. 2). Significant differences between increases in spore density occurred in pots containing R. acefosa, in which A. laevis was especially promoted, and H. lanafus, in which A. laevis and G. efunicafumincreased most strongly (Table 3 , Fig. 2). In pots containing F. rubra or T. prafense there were no differences in the densities of the different VA mycorrhizal fungi.
DISCUSSION
Holcus lanatus
h
VA mycorrhizal species
These experiments were designed to determine whether plant species caused differential propagation within a community of VA mycorrhizal species, as measured by spore production. Any significant increases in the spore production of a particular type recovered from the pots would be interpreted as evidence for growth of the fungus. Whilst in some cases the densities of spores of several VA mycorrhizal species were higher in Experiment 1 than in the original inoculum these increases were only significantly higher for two VA mycorrhizal fungi and two hosts. There is, therefore, no evidence from these results that multiple infection occurred. However, multiple infection cannot be discounted because an increase in spore production by one VA mycorrhizal species is not a clear indication of infection by a single fungus: the roots of one plant species could be infected by more than one VA rnycorrhizal species, only one of which has sporulated during the experimental period. Temporal differences occur in sporulation between VA mycorrhizal species (Wilson & Trinick, 1982). Different results might therefore have been expected from the two harvests of Experiment 1 and between Experiment 1 and Experiment 2 since the plants in Experiment 1 developed from seed within 180 d, while those from the field (Experiment 2) were of unknown age. This could explain why significant increases in the density of A. laevis in H. b n a f u s and R. acefosa were observed in the second experiment and not in the first (Figs 1, 2). Similarly, the increase in density of G . consfricfum spores observed with P. lanceolafa could be due to differences in compatibility of this combination allowing more rapid spore production than the fungus could achieve with other plant species under these growth conditions. In general however, the results of the two investigations were remarkably similar. Trifolium prafense and Fesfuca rubra produced no increases in density of any spore type in either experiment and Planfago lanceolafa promoted Glomus consfricfum in both. Holcus lanafus and Rumex acetosa appeared to produce different results in the two experiments, but inspection of Figs 1 and 2 reveals that the pattern of increase is very similar: it is notable that Acaulospora laevis was significantly Fig. 2. Increase in mean VA mycorrhizal spore density in pots containing one of five plant species which had previously been removed from the field site (Experiment 2). Different letters above bars indicate a significant difference, where P = < 0.05. Glomw etunicatum (Ge), Glomw constrictum (Cc), Acaulospora laeuis (Al), Scufellospora calospora (Sc), sporocarps (Sp).
419
I. R. Sanders and A. H. Fitter greater for Rumex acefosa in ExDeriment 2 and also numericallv the most abundant in Experiment I. in this The differences in 'pore production investigation indicate that VA mycorrhizal fungi respond differently according to the host species. Differential responses of some sort between host-fungus combinations must be manifested in order for selection of VA mycorrhizas to occur. However, we cannot predict from these results, firstly whether these different responses actually occur in the field (although they do demonstrate that the potential exists for a differential response), and secondly whether selection of VA mycorrhizas would result in the field as a cause of these differential responses. Differences in the response of VA mycorrhizal fungi within hosts suggests that under certain conditions selection should occur to favour certain host-fungus combinations. However. phenomena such as multiple infection and degree of specificity can be investigated under when new methods have been devised which mycorrhizal fungi to be identified with certainty in roots taken from the field. In this wav it would be to detect the presence of more than one fungus within a host and to determine whether specificity or changes in the relative abundance of different fungi occur in plant communities. u
u
The authors thank Dr Chris Walker for his time and help in the initial identification of VA mycorrhizal spores in the soil inoculum. I.R.S. was in receipt of a studentship from the Natural Environment Research Council, whose support is gratefully acknowledged.
REFERENCES Brundrett, M. & Kendrick, B. (1990). The roots and mycorrhizas of herbaceous woodland plants. 11. Structural aspects of morphology. New Phytologist 114, 469-479. Fitter, A. H. (1985). Functioning of vesicular-arbuscular mycorrhizas under field conditions. N m Phytologist 99, 257-265. Fitter, A. H. (1990). The role and ecological significance of vesicular-arbuscular mycorrhizas in temperate ecosystems. Agriculture, Ecosystems and Environment 29, 137-151. (Accepted 5 November 1991)
Gerdemann, J. W. & TraPPe, 1. M. (1974). Endogonaceae in the pacific northwest. Mycologia, memoir 5 , 76 pp. Gianinazzi, S. (1991). Vesicular-arbuscular (endo-) mycorrhizas: cellular, biochemical and genetic aspects. Agriculfure, Ecosystems and Environment 35, 105-119, Harley, J. L. & Smith, S. E. (1983). Mycowhizal Symbiosis. Academic Press: London. Hetrick, B. A. D., Kitt. D. G. & Wilson, G. T. (1988). Mycorrhizal dependence and growth habit of warn-season and cool-season tallgrass prairie plants. Canadian Journal of Botany 6 6 , 137&1380. Hetrick, B. A. D., Wilson, G. W. T. & Hartnett, D. C. (1989). Relationship between mycorrhizal dependence and competitive ability of two tallgrass prairie grasses. Canadan Journal of Botany 6 7 , 2608-2615. Hetrick, B. A. D., Wilson, G. W. T. & Todd, T. C. (1990). Differential responses of C, and C, grasses to mycorrhizal symbiosis, phosphorus fertilization and soil microorganisms. Canadian Journal of Botany 6 8 , 461-467. Hewitt, E. J. (1966). Sand and Water Culture Methods for Use in the Study of Plant Nutrition. Technical communication no. 22, revised 2nd edn, pp. 535. Commonwealth Agricultural Bureaux: 'amham Kornanik, P. P. & McGraw, A. L. (1982). Quantification of vesiculararbuscuiar mycorrhizae in plant roots. In Methods and principfes of Mycowhizal Research (ed. N. C. Schenk), pp. 37-46. The American Phytopathological Society: s t Paul, Minnesota. Koske. R. (1981). A preliminary study of interactions between species of vesicular-arbuscular mycorrhizal fungi in a sand dune. Transactions of the British Mycological Society 79, 411-416. Lackie, S. M., Garriock, M. L., Peterson, R. L. & Bowley, S. R. (1987).Influence of host plant on the morphology of the vesicular-arbuscular mycorrhizal fungus GIomus vesifome (Daniels & Trappe) Berch. Symbiosis 3, 147-158. McGonigle, T. P. (1988). A numerical analysis of published field trials with vesicular-arbuscular mycorrhizal fungi. Functional Ecology 2, 473-478. McGonigle, T. P. & Fitter, A. H. (1990). Ecological specificity of vesiculararbuscular mycorrhizal associations. Mycological Research 94, 120-122. Molina, R. 1.. Trappe, J. M. & Strickler, G. S. (1978). Mycorrhizal fungi associated with Festuca in western United States and Canada. Canadian Journal of Botany 5 6 , 1691-1695. Nicolson, T. H. (1967). Vesicular-arbuscular mycorrhiza - a universal plant symbiosis. Science Programme, Oxford 55, 561-581. Rosendahl, S., Rosendahl, C. N. & Sochting, U. (1990). Distribution of VA mycorrhizal endophytes amongst plants of a Danish grassland community. Agriculture, Ecosystems and Environment 29, 329-336. Walker, C., Mize, C. W. & McNabb Jr, H. S. (1982). Populations of endogonaceous fungi at two locations in central Iowa. Canadian Journal of Botany 6 0 , 2518-2529. Wilson, J. M. & Trinick, M. J. (1982). Factors affecting the estimation of numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi by the most probable number method. Australian Journal of Soil Research 21, 73-81.
Mycol. Res. 96 ( 6 ) :415-419 (1992) Printed in Great Britain
Evidence for differential responses between host-fungus combinations of vesicular-arbuscular mycorrhizas from a grassland
I. R. SANDERS* A N D A. H. FITTER Department of Biology, University of York, Heslington, York Y O 1 5 0 0 , U.K.
The increase in spore density over that in the soil of an indigenous vesicular-arbuscular (VA) mycorrhizal community was measured in five plant species, after inoculation with the same soil (Experiment I), and in pots containing individuals of the same species, which had been removed from the field site (Experiment 2). In Experiment 1, significant increase in spore density was observed for Glomw constricturn with Plantago lanceolata although increases in density were not observed for this fungus with other host species. Results of Experiment 2 were consistent for the combination of Plantago lanceolata and Glomw constricturn and the numbers of Acaulospora laevis spores, in pots containing Holcw lanatw or Rumex acetosa, were significantly higher than in the soil of the field site. The results do not support or oppose the hypotheses that either multiple infection or selectivity of VA mycorrhizas occur between certain combinations of host-fungus in co-existing plants of a natural community. The results indicate that VA mycorrhizal fungi respond differentlyaccording to the host species and thus such differences could result in selection pressures which favour certain host-fungus combinations.
Vesicular-arbuscular (VA) mycorrhizas are abundant in natural ecosystems (Harley & Smith, 1983) and although they have been presumed to be beneficial to plant growth in the field, very few investigations have clearly demonstrated such benefits (see McGonigle, 1988; Fitter, 1990). The lack of an observable effect on plant growth and nutrient status under field conditions has been attributed to many factors, including low or differing effectiveness of fungal strains (Fitter, 1985) and the presence of different types of VA mycorrhizal fungi within plant roots (Rosendahl, Rosendahl & Sachting, 1990). However, very little information exists on whether multiple infection by VA mycorrhizal fungi, that is the infection of one plant root system by more than one VA mycorrhizal fungus, occurs. Furthermore, there is little evidence regarding differential responses of VA mycorrhizal fungi in different host plants and whether host plants are preferentially infected by certain species or isolates of VA mycorrhizal fungi. In most instances VA mycorrhizal fungi can infect any plant capable of forming mycorrhizas (Nicolson, 1967; Gerdemann & Trappe, 1974; Harley & Smith, 1983) which suggests a potential for multiple infection in the field. McGonigle & Fitter (1990) observed both coarse and fine endophytes growing in the roots of Holctts lanatus L. and Rosendahl et al. (1990) have characterized up to four morphological types of VA mycorrhizal fungus co-existing in the roots of Hieracium pilosella L., Trifolium repens L., Agrostis capillaris L. and Plantago
lanceolata L. ' Current address: Department of Biology, The Pennsylvania State University, University Park, PA 16802,U.S.A.
Harley & Smith (1983) argue that some 'ecological specificity', the preferential infection of plants by certain endophytes, could occur. Whilst there is little evidence with which to test this hypothesis, it is likely that ecological factors influence compatibility in the field, since plants which are normally non-mycorrhizal in the field will often develop mycorrhizas when grown in pots (Harley & Smith, 1983). In the field, VA mycorrhizal fungi would be selectively favoured if they had an ability to infect certain plant species quickly, subsequently inducing a large growth response or other benefit in the host, or influencing the host genome so as to exclude other species. Interspecific differences in both the intensity of infection by VA mycorrhizal fungal host plants (Molina, Trappe & Strickler, 1978; Koske, 1981) and the growth response in different hosts (Hetrick, Kitt & Wilson, 1988; Hetrick, Wilson & Hartnett, 1989; Hetrick, Wilson & Todd, 1990) have been observed. However, only recently has new information been obtained on genome expression in different VA mycorrhizal combinations (see review by Gianinazzi, 1991). The aims of this investigation were to assess whether multiple infection by VA mycorrhizal-forming fungi occurs in plants of a semi-natural grassland and to establish whether differential responses occur between host-fungus combinations when different plant species are exposed to the same mixed inoculum of VA mycorrhizal fungal species. If so, this would support the hypothesis that multiple infections obscure any direct relationships between VA mycorrhizal infection and plant growth in the field. In addition, differential responses of VA m~corrhizaswould demonstrate that the potential for
Differential responses between host-fungus combinations of VA mycorrhizas Table 1. Mean VAM spore density in soil from the field site Mean spore density (spores g-I of soil)
Glomw etunicatum Glomw consfricfum Acaulospora laevis Scutellospora calospora
0 2 8a DlOab O.1Zab O.lOab 0 0 3b
Sporocarps (miscellaneous)
Values followed by a different letter are significantly different at
P = < 005.
Table 2. F ratios from one-way ANOVA on spore density in soil from the field site and soil from pots containing the five plant species, either grown from seed (Experiment I ) or having previously been removed from the field site (Experiment 2) Experiment 1
G. etunicatum G. constricturn A . laevis s . ca/ospora Sporocarps
Harvest I
Harvest 2
Experiment 2
1.63 n.s. 3.10' 2.75' 1.01n.s. 3.38'
098n.s. 2.80" 1.86 n.s. 15.36*'* 0 4 5 n.s.
1.5I* 3.92"" 3.30' 18.45'** 1.64n.s.
' P < 0 0 5 ; *' P < 0.01; *'" P < 0001. All F values have 5 and 3 1 degrees of freedom.
Table 3. F ratios from one-way ANOVA on increase in spore density for each VA mycorrhizal fungal species with each of five plant species in pots, either grown from seed (Experiment 1) or having previously been removed from the field site (Experiment 2) Experiment 1
P. lanceolata R. acefosa 7. pratense H. lanatus F. rubra * P < 0 0 5 ; ** P of freedom.
Harvest I
Harvest 2
Experiment 2
3.441.64n.s. 0 4 5 n.s. 417* 1.76n.s.
2732*** 070n.s. 0 8 2 n.s. 2.05 n.s. 066n.s.
2.91. 4.25" 0.27n.s. 4.43'" 1'23 n.s.
< 0.01; *** P < 0001.All F values have 4 and 26 degrees
the selection of host-fungus combinations in the field exists. The study involves the use of spore characteristics to ensure certain identification of VA mycorrhizal fungal species. Other methods of VA mycorrhizal identification, using morphological characteristics of the fungi, can be unreliable for studying endophytes in the roots of different plant species (Lackie ef al., 1987; Brundrett & Kendrick, 1990).
MATERIALS A N D M E T H O D S Experiment 1
Surface-sterilized seeds (10% sodium hypochlorite, 10 min) of Planfago lanceolafa, Rumex acefosa L., Trifolium prafense L., Holcus lanafus and Fesfuca rubra L. were germinated in Petri
416
dishes. The seeds had been collected during the summer of 1988 from the field site, Wheldrake Ings, a species-rich meadow (North Yorkshire, England, national grid reference SE 702443). After 10 d individual seedlings were transplanted into 10 cm diam. pots containing sterile sand (autoclaved for 20 min at 120 OC) combined with 10 % (by volume) unsterilised soil. Transplantation took place on 16 May 1989, the soil having been removed from the field site on the previous day. There were six replicates of each species. Plants were kept in a heated glasshouse, with 16 h supplementary lighting per day, watered daily and given phosphate-free Rorison's solution (Hewitt, 1966) once a week. At two harvests, 120 and 180 d after transplantation, 20 g of soil was removed from each pot for spore extraction. After the first harvest the area from which soil was removed was marked so that the soil was not removed from the same region in the second harvest. The volume of soil removed in the first harvest was replaced with an equal volume of sterile sand. At each harvest a small portion of the root system was also taken in order to check whether the plants were mycorrhizal. Roots were cleared in 10% KOH and stained with 0.1 % acid fuchsin (Kormanik & McGraw, 1982). Spore extraction was carried out using the sucrose centrifugation method of Walker, Mize & McNabb (1982) and the number of spores of each VA mycorrhizal fungal species was recorded for each pot. At the first harvest (13 September, 1989) VA mycorrhizal spores were also extracted from six 20 g replicates of the field soil (collected 15 May 1989), in order to obtain a background count of spore numbers present in the original inoculum. In order to compare the change in spore numbers occurring in each treatment and for each VA m~corrhizal species recorded, a measure of the increase in spore density was used. This was calculated by subtracting the mean density of spores in the original inoculum (spores g-' soil) from the mean density of spores in each treatment, allowing for the 1:10 dilution of original inoculum. The increase in spore density is expressed as spores g-l of soil. One-way ANOVA was used to establish whether differences occurred in the change of spore density of each of the different plant species and whether there were significant increases in spore density from that present from the original inoculum.
Six individual plants of P. lanceolafa, R. acefosa, T. prafense, H. lanafus and F. rubra were randomly selected and removed from the same field site on 17 September 1989 using a 6 cm diam. x 16 cm depth soil corer. The root system of each plant was carefully washed to remove all soil debris and other plant root systems. Each plant was placed individually in a pot containing a 10 % (by volume) sterilized mixture of soil from . plants the field site and sand (autoclaved 20 min at 1 2 0 ~ )The were kept in the glasshouse under the same conditions as described above. After 160 d 20 g of soil from each pot was removed for spore extraction. Spore extraction, subsequent calculation of increase in spore density and statistical analysis followed that of Experiment 1.
I. R. Sanders and A. H. Fitter Plantago lanceolata
RESULTS
a
Spores of seven VA mycorrhizal species, most closely resembling Acaulospora laevis Gerd. & Trappe, Glomus consfrictum Trappe, Glomus efunicafum Beck. & Gerd., Glomus invermaius Hall, Glomus mosseae (Nicol. & Gerd.) Gerd. & Trappe, Sclerocystis rubifomis Gerd. & Trappe and Scufellospora calospora (Nicol. & Gerd.) Gerd. & Trappe, were identified in soil from the field site. Results are presented only for the four species which occurred consistently in all samples. The three sporocarp-forming species, G . invermaium, G. mosseae and S. rubiformis, were so infrequent that the data for these species were summed in order to present the densities of sporocarps. Mean overall spore density in the soil from Wheldrake Ings was 0.63 spores g-l soil. There were differences in mean spore densities between VA mycorrhizal fungal types (one-way ANOVA, F,,,, = 3.62, P < 0.05), G. efunicafum spores being more abundant than sporocarps (Table I). All plants in Experiment 1 were infected after I20 d. The density of spores of G . consfricfum,A. laevis and of sporocarps differed between the field soil and the pots containing the five species at harvest 1. Only G . consfricfum differed between the field and pot soil at both harvests (Table 2). Virtually no sporocarps were found at the first harvest and no S. calospora spores were found at the second. This accounted for the significant differences observed in spore density of these species (Table 2). There were no differences in the density of G . efunicatum spores according to treatment at either harvest in Experiment 1. Significant differences between the increase in spore densities, i.e. density in pot minus that in the original soil, of each VA mycorrhizal species occurred with P. lanceolafa at both harvests and in H. lanutus at the first harvest (Table 3). In pots containing P. hnceolafa the increase in spore density of G . consfricfum was higher than that of A. laevis and S. calospora at the first harvest and greater than all other spore types by the second (Fig. 1). With H. lanatus the increase in the density of G . efunicafum was significantly greater than that of G . constricfum and the sporocarp forming fungi (Fig. 1)at the first harvest. No significant differences occurred in the densities of the spores of each of the VA mycorrhizal fungal species in pots containing R. acefosa, T. pratense and F. rubra (Fig. 1).
Rumex acetosa
1
-
Trifolium pratense
IM
C
m
5
1.0,
~ o ~ c lanutus us
Spore densities recorded in Experiment 2 were consistent with Experiment I in that the density of G . consfricfum spores was different between treatments, i.e. the field soil and each of the five different plant species. However, significant differences in spore density were also observed for G . efunicafum, A . laevis and S. calospora (Table 2). As in Experiment I, the low spore density of S. calospora in all pots (zero in most cases) compared
Festuca rubra
Fig. 1. Increase in mean VA mycorrhizal spore density with each of
-0 5
Ge
Gc Al Sc VA mycorrhizal species
SP
five plant species grown in soil from the field site (Experiment I). Different letters above bars within one harvest indicate a significant difference, where P = < 0.05. Harvest 1 (m), harvest 2 (a). Glomw etunicatum (Ge), Glomw constricturn (Gc), Acaulospora laevis (Al), Scutellospora calospora (Sc), sporocarps (Sp).
Differential responses between host-fungus combinations of VA mycorrhizas Plantago lanceolata
1 0.5
.
Ge
Gc
A1
Sc
SP
418
to the original density (0.1 spores g1of soil) accounted for the highly significant treatment effect. In pots containing P. lanceolafa the density of G. consfricfum was significantly higher than in the soil from the field site and some of the other VA mycorrhizal species (Table 3 , Fig. 2). Significant differences between increases in spore density occurred in pots containing R. acefosa, in which A. laevis was especially promoted, and H. lanafus, in which A. laevis and G. efunicafumincreased most strongly (Table 3 , Fig. 2). In pots containing F. rubra or T. prafense there were no differences in the densities of the different VA mycorrhizal fungi.
DISCUSSION
Holcus lanatus
h
VA mycorrhizal species
These experiments were designed to determine whether plant species caused differential propagation within a community of VA mycorrhizal species, as measured by spore production. Any significant increases in the spore production of a particular type recovered from the pots would be interpreted as evidence for growth of the fungus. Whilst in some cases the densities of spores of several VA mycorrhizal species were higher in Experiment 1 than in the original inoculum these increases were only significantly higher for two VA mycorrhizal fungi and two hosts. There is, therefore, no evidence from these results that multiple infection occurred. However, multiple infection cannot be discounted because an increase in spore production by one VA mycorrhizal species is not a clear indication of infection by a single fungus: the roots of one plant species could be infected by more than one VA rnycorrhizal species, only one of which has sporulated during the experimental period. Temporal differences occur in sporulation between VA mycorrhizal species (Wilson & Trinick, 1982). Different results might therefore have been expected from the two harvests of Experiment 1 and between Experiment 1 and Experiment 2 since the plants in Experiment 1 developed from seed within 180 d, while those from the field (Experiment 2) were of unknown age. This could explain why significant increases in the density of A. laevis in H. b n a f u s and R. acefosa were observed in the second experiment and not in the first (Figs 1, 2). Similarly, the increase in density of G . consfricfum spores observed with P. lanceolafa could be due to differences in compatibility of this combination allowing more rapid spore production than the fungus could achieve with other plant species under these growth conditions. In general however, the results of the two investigations were remarkably similar. Trifolium prafense and Fesfuca rubra produced no increases in density of any spore type in either experiment and Planfago lanceolafa promoted Glomus consfricfum in both. Holcus lanafus and Rumex acetosa appeared to produce different results in the two experiments, but inspection of Figs 1 and 2 reveals that the pattern of increase is very similar: it is notable that Acaulospora laevis was significantly Fig. 2. Increase in mean VA mycorrhizal spore density in pots containing one of five plant species which had previously been removed from the field site (Experiment 2). Different letters above bars indicate a significant difference, where P = < 0.05. Glomw etunicatum (Ge), Glomw constrictum (Cc), Acaulospora laeuis (Al), Scufellospora calospora (Sc), sporocarps (Sp).
419
I. R. Sanders and A. H. Fitter greater for Rumex acefosa in ExDeriment 2 and also numericallv the most abundant in Experiment I. in this The differences in 'pore production investigation indicate that VA mycorrhizal fungi respond differently according to the host species. Differential responses of some sort between host-fungus combinations must be manifested in order for selection of VA mycorrhizas to occur. However, we cannot predict from these results, firstly whether these different responses actually occur in the field (although they do demonstrate that the potential exists for a differential response), and secondly whether selection of VA mycorrhizas would result in the field as a cause of these differential responses. Differences in the response of VA mycorrhizal fungi within hosts suggests that under certain conditions selection should occur to favour certain host-fungus combinations. However. phenomena such as multiple infection and degree of specificity can be investigated under when new methods have been devised which mycorrhizal fungi to be identified with certainty in roots taken from the field. In this wav it would be to detect the presence of more than one fungus within a host and to determine whether specificity or changes in the relative abundance of different fungi occur in plant communities. u
u
The authors thank Dr Chris Walker for his time and help in the initial identification of VA mycorrhizal spores in the soil inoculum. I.R.S. was in receipt of a studentship from the Natural Environment Research Council, whose support is gratefully acknowledged.
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Gerdemann, J. W. & TraPPe, 1. M. (1974). Endogonaceae in the pacific northwest. Mycologia, memoir 5 , 76 pp. Gianinazzi, S. (1991). Vesicular-arbuscular (endo-) mycorrhizas: cellular, biochemical and genetic aspects. Agriculfure, Ecosystems and Environment 35, 105-119, Harley, J. L. & Smith, S. E. (1983). Mycowhizal Symbiosis. Academic Press: London. Hetrick, B. A. D., Kitt. D. G. & Wilson, G. T. (1988). Mycorrhizal dependence and growth habit of warn-season and cool-season tallgrass prairie plants. Canadian Journal of Botany 6 6 , 137&1380. Hetrick, B. A. D., Wilson, G. W. T. & Hartnett, D. C. (1989). Relationship between mycorrhizal dependence and competitive ability of two tallgrass prairie grasses. Canadan Journal of Botany 6 7 , 2608-2615. Hetrick, B. A. D., Wilson, G. W. T. & Todd, T. C. (1990). Differential responses of C, and C, grasses to mycorrhizal symbiosis, phosphorus fertilization and soil microorganisms. Canadian Journal of Botany 6 8 , 461-467. Hewitt, E. J. (1966). Sand and Water Culture Methods for Use in the Study of Plant Nutrition. Technical communication no. 22, revised 2nd edn, pp. 535. Commonwealth Agricultural Bureaux: 'amham Kornanik, P. P. & McGraw, A. L. (1982). Quantification of vesiculararbuscuiar mycorrhizae in plant roots. In Methods and principfes of Mycowhizal Research (ed. N. C. Schenk), pp. 37-46. The American Phytopathological Society: s t Paul, Minnesota. Koske. R. (1981). A preliminary study of interactions between species of vesicular-arbuscular mycorrhizal fungi in a sand dune. Transactions of the British Mycological Society 79, 411-416. Lackie, S. M., Garriock, M. L., Peterson, R. L. & Bowley, S. R. (1987).Influence of host plant on the morphology of the vesicular-arbuscular mycorrhizal fungus GIomus vesifome (Daniels & Trappe) Berch. Symbiosis 3, 147-158. McGonigle, T. P. (1988). A numerical analysis of published field trials with vesicular-arbuscular mycorrhizal fungi. Functional Ecology 2, 473-478. McGonigle, T. P. & Fitter, A. H. (1990). Ecological specificity of vesiculararbuscular mycorrhizal associations. Mycological Research 94, 120-122. Molina, R. 1.. Trappe, J. M. & Strickler, G. S. (1978). Mycorrhizal fungi associated with Festuca in western United States and Canada. Canadian Journal of Botany 5 6 , 1691-1695. Nicolson, T. H. (1967). Vesicular-arbuscular mycorrhiza - a universal plant symbiosis. Science Programme, Oxford 55, 561-581. Rosendahl, S., Rosendahl, C. N. & Sochting, U. (1990). Distribution of VA mycorrhizal endophytes amongst plants of a Danish grassland community. Agriculture, Ecosystems and Environment 29, 329-336. Walker, C., Mize, C. W. & McNabb Jr, H. S. (1982). Populations of endogonaceous fungi at two locations in central Iowa. Canadian Journal of Botany 6 0 , 2518-2529. Wilson, J. M. & Trinick, M. J. (1982). Factors affecting the estimation of numbers of infective propagules of vesicular-arbuscular mycorrhizal fungi by the most probable number method. Australian Journal of Soil Research 21, 73-81.